Hirak Ranjan Dash

Pankaj Shrivastava
Surajit Das

Principles and
Practices of DNA
Analysis: A Laboratory
Manual for Forensic
DNA Typing
SPRINGER PROTOCOLS HANDBOOKS

For further volumes:

http://www.springer.com/series/8623
Principles and Practices of DNA
Analysis: A Laboratory Manual
for Forensic DNA Typing

Hirak Ranjan Dash

DNA Fingerprinting Unit, State Forensic Science Laboratory, Sagar, Madhya Pradesh, India

Pankaj Shrivastava
DNA Fingerprinting Unit, State Forensic Science Laboratory, Sagar, Madhya Pradesh, India

Surajit Das
Department of Life Science, National Institute of Technology, Rourkela, Odisha, India
Hirak Ranjan Dash Pankaj Shrivastava
DNA Fingerprinting Unit DNA Fingerprinting Unit
State Forensic Science Laboratory State Forensic Science Laboratory
Sagar, Madhya Pradesh, India Sagar, Madhya Pradesh, India

Surajit Das
Department of Life Science
National Institute of Technology
Rourkela, Odisha, India

ISSN 1949-2448 ISSN 1949-2456 (electronic)

Springer Protocols Handbooks
ISBN 978-1-0716-0273-7 ISBN 978-1-0716-0274-4 (eBook)
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Preface

DNA fingerprinting is one of the greatest discoveries of science in the century. Since its
discovery, the technique is being widely used in varied fields such as deciphering the genetics
of an organism, cracking ancestral belongingness, neonatal diagnosis, diagnosis of genetic
disorder, and many others. However, it has revolutionized the field of criminal justice system
by its uniqueness and individualization capabilities. Currently, DNA fingerprinting is con-
sidered to be the most irrefutable evidence to be produced before the court of law.
Since its inception, the technique has undergone tremendous advancements, and the
progress is being observed continuously. However, with the advent of time a consensus step-
by-step protocol book describing the technology currently used is lacking. Most of the
books available in the same field either describe the obsolete technique of RFLP for DNA
examination or manual DNA extraction procedures. Currently, the technological advance-
ment has seen DNA extraction through automation, and RAPID DNA technology has also
arrived. Hence, we felt the need of a timely updated laboratory manual in the current field.
In this context, the laboratory manual is structured to clear all doubts of the examiners
as well as students regarding conventional and advanced forensic DNA typing experiments.
It will be handy for the beginners as well as for the experts in the field of DNA fingerprinting
for smooth conduction of the experiments, interpretation, and analysis of results. The
uniqueness of the manual involves (a) step-by-step protocol for each experiments;
(b) description of each chemicals and reagents and their corresponding principal/functional
role; (c) separate experiments for DNA extraction from varied types of biological samples of
forensic interest; (d) description of advanced automatic and semiautomatic techniques for
DNA extraction; (e) inclusion of real case studies and statistical analysis; and (f) precaution
and troubleshooting for each experiment.
Every possible effort has been made to present the protocol book in a very simpler form
for easy understanding of students, scientists, and DNA examiners. We have tried our best to
incorporate all our experience and expertise to bring out the form of this manual. Through-
out the writing process, we have faced lots of hurdles and problems; however, all of them
have been overcome due to God’s grace, self-belief, and support from family and friends. We
are really thankful to each and every one for their support and encouragement during the
process of writing. We hope the manual will be of great use for the readers in their career.
Wishing the readers all the best for a successful experiment!!!!

Sagar, Madhya Pradesh, India Hirak Ranjan Dash

Sagar, Madhya Pradesh, India Pankaj Shrivastava
Rourkela, Odisha, India Surajit Das

v
Contents

Preface . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . v

PART I OVERVIEW OF FORENSIC DNA ANALYSIS

1 Introduction to Forensic DNA Analysis. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3

1 Genetic Variation. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3
2 Short Tandem Repeat (STR) Markers . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 4
3 Technological Use in Human STR Typing. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 5
4 Challenges in Forensic DNA Typing . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 7
5 Applications of Forensic DNA Typing . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 9
5.1 O.J. Simpson Case in Los Angeles . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 9
5.2 Clinton-Lewinsky Affair . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 10
5.3 Identification of the Bodies in World Trade Center Attack . . . . . . . . . . . 10
5.4 Assassination of Sri Rajeev Gandhi, Prime Minister of India . . . . . . . . . 11

2 Biological Samples: The Target Sources for DNA Typing . . . . . . . . . . . . . . . . . . . . 13

1 The Locard’s Principle . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 13
2 Types of Biological Samples . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 13
3 Whole Blood/Blood Stains. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 14
4 Hairs. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 16
5 Teeth . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 16
6 Bone . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 17
7 Soft Tissues. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 17
8 Semen and Sperm/Vaginal Fluid. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 18
9 Urine . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 18
10 Saliva . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 18
11 Biological Samples and Their DNA Content . . . . . . . . . . . . . . . . . . . . . . . . . . . . 19

3 Collection, Transportation, and Preservation of Biological Evidences

for DNA Analysis . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 21
1 Liquid Blood from Donor . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 21
2 Liquid Blood and Wet Bloodstains from Crime Scene. . . . . . . . . . . . . . . . . . . . 23
3 Dried Bloodstains . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 24
4 Semen, Seminal Stains, and Evidence from Sexual Assault Victims . . . . . . . . 24
5 Soft Tissues and Organs . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 25
6 Teeth and Bone . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 25
7 Hair. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 25

PART II MANUAL EXTRACTION OF DNA FROM BIOLOGICAL SAMPLES

4 Manual Isolation of DNA from Whole Blood . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 31

1 Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 31
2 Principle. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 32
3 Reagents Required and Their Role . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 32
vii
viii Contents

3.1 Tris-EDTA . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 32
3.2 SDS. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 33
3.3 Proteinase-K. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 33
3.4 Phenol . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 33
3.5 Chloroform-Isoamyl Alcohol . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 33
3.6 Sodium Acetate. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 34
3.7 Isopropyl Alcohol. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 34
3.8 TE Buffer . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 34
4 Procedure . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 34
5 Observation . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 35
6 Result Table . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 36
7 Precautions . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 36
8 Troubleshooting . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 37

5 Manual Isolation of DNA from Body Fluid Stains. . . . . . . . . . . . . . . . . . . . . . . . . . . 39

1 Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 39
2 Principle. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 40
3 Reagents Required and Their Role . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 41
3.1 Tris-EDTA . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 41
3.2 SDS. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 41
3.3 Proteinase-K. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 41
3.4 DTT . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 41
3.5 Phenol . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 41
3.6 Chloroform-Isoamyl Alcohol . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 42
3.7 TE Buffer . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 42
4 Procedure . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 42
5 Observation . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 43
6 Result Table . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 44
7 Precautions . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 44
8 Troubleshooting . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 45

6 Manual Isolation of DNA from Soft-Tissue Samples. . . . . . . . . . . . . . . . . . . . . . . . . 47

1 Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 47
2 Principle. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 48
3 Reagents Required and Their Role . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 48
3.1 Tris-EDTA . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 48
3.2 SDS. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 49
3.3 Proteinase-K. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 49
3.4 Liquid Nitrogen . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 49
3.5 Phenol . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 49
3.6 Chloroform-Isoamyl Alcohol . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 49
3.7 TE Buffer . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 50
4 Procedure . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 50
5 Observation . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 51
6 Result Table . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 51
7 Precautions . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 51
8 Troubleshooting . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 52
 Contents ix

7 Manual Isolation of DNA from Rooted Hair Samples . . . . . . . . . . . . . . . . . . . . . . . 53

1 Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 53
2 Principle. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 54
3 Reagents Required and Their Role . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 55
3.1 Tris-EDTA . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 55
3.2 SDS. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 55
3.3 Proteinase-K. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 55
3.4 Phenol . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 55
3.5 Chloroform-Isoamyl Alcohol . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 56
3.6 TE Buffer . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 56
4 Procedure . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 56
5 Observation . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 57
6 Result Table . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 57
7 Precautions . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 58
8 Troubleshooting . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 58

8 Manual Isolation of DNA from Human Teeth . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 61

1 Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 61
2 Principle. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 62
3 Reagents Required and Their Role . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 62
3.1 Sodium Hypochlorite . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 62
3.2 Tris-EDTA . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 63
3.3 SDS. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 63
3.4 Proteinase-K. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 63
3.5 Phenol . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 63
3.6 Chloroform-Isoamyl Alcohol . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 64
3.7 TE Buffer . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 64
4 Procedure . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 64
5 Observation . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 65
6 Result Table . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 66
7 Precautions . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 66
8 Troubleshooting . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 67

9 Isolation of DNA from Bone Samples . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 69

1 Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 69
2 Principle. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 70
3 Reagents Required and Their Role . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 71
3.1 Sodium Hypochlorite . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 71
3.2 0.5 mM EDTA . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 71
3.3 Normal Saline Solution (NSS) . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 71
3.4 Tris-EDTA . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 71
3.5 SDS. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 71
3.6 Proteinase-K. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 72
3.7 DTT . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 72
3.8 Phenol . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 72
3.9 Chloroform-Isoamyl Alcohol . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 72
3.10 TE Buffer . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 72
x Contents

4 Procedure . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 73
5 Observation . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 74
6 Result Table . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 74
7 Precautions . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 75
8 Troubleshooting . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 75

10 Differential Extraction of Sperm and Epithelial Cell DNA . . . . . . . . . . . . . . . . . . . 77

1 Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 77
2 Principle. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 78
3 Reagents Required and Their Role . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 79
3.1 Tris-EDTA . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 79
3.2 SDS. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 79
3.3 Proteinase-K. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 79
3.4 DTT . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 80
3.5 Phenol . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 80
3.6 Chloroform-Isoamyl Alcohol . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 80
3.7 TE Buffer . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 80
4 Procedure . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 80
5 Observation . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 83
6 Result Table . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 83
7 Precautions . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 83
8 Troubleshooting . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 84

PART III AUTOMATIC AND SEMI-AUTOMATIC EXTRACTION OF DNA

FROM BIOLOGICAL SAMPLES

11 Isolation of DNA by Using Magnetic Bead-Based Extraction System. . . . . . . . . . 87

1 Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 87
2 Principle. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 88
3 Reagents Required and Their Role . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 89
3.1 PrepFiler™ Lysis Buffer . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 89
3.2 PrepFiler BTA™ Lysis Buffer . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 89
3.3 LySep™ Column . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 89
3.4 Cartridges . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 90
3.5 Proteinase-K. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 90
3.6 DTT . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 90
4 Procedure . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 91
4.1 Using PrepFiler Express™ Forensic DNA Extraction Kit . . . . . . . . . . . . 91
4.2 Using PrepFiler Express BTA™ Forensic DNA Extraction
Kit . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 91
4.3 Setup and Run for Automated DNA Extraction. . . . . . . . . . . . . . . . . . . . . 92
5 Observation . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 94
6 Result Table . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 95
7 Precautions . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 95
8 Troubleshooting . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 96
 Contents xi

12 Extraction of DNA by Using Anion-Exchange Resin Chelex® . . . . . . . . . . . . . . . . 97

1 Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 97
2 Principle. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 98
3 Reagents Required and Their Role . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 98
3.1 Sodium Hypochlorite . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 98
3.2 Chelex® 100 Resin. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 98
4 Procedure . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 99
5 Observation . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 100
6 Result Table . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 100
7 Precautions . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 100
8 Troubleshooting . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 101

13 Isolation of DNA by Using Column-Based Extraction System. . . . . . . . . . . . . . . . 103

1 Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 103
2 Principle. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 104
3 Reagents and Plasticware Required and Their Role . . . . . . . . . . . . . . . . . . . . . . 105
3.1 QIAamp® DNA Investigator® Kit. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 105
4 Procedure . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 106
5 Observation . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 107
6 Result Table . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 107
7 Precautions . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 107
8 Troubleshooting . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 108

14 Reliable Use of Whatman™ FTA™ Cards for One-Step Collection

and Isolation of DNA . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 109
1 Sample Types and Collection Procedures . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 110
1.1 Collection of Liquid Blood . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 110
1.2 Collection of Buccal Cells . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 110
1.3 Collection of Saliva Samples . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 110
2 DNA Extraction and Usages (Applications) . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 110
2.1 Organic Extraction. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 111
2.2 Using Chelex 100 Resin. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 111
2.3 QIAamp™ DNA Investigator Kit . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 112
2.4 illustra™ Tissue and Cells genomicPrep Mini Spin Kit . . . . . . . . . . . . . . 112
2.5 DNA IQ™ Kit . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 113
3 Available FTA™ Cards and Other Related Products . . . . . . . . . . . . . . . . . . . . . 114

PART IV QUALITATIVE AND QUANTITATIVE ASSESSMENT OF EXTRACTED DNA

15 Quantification of DNA by Using Agarose Gel Electrophoresis Technique . . . . . 119

1 Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 119
2 Principle. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 120
3 Reagents Required and Their Role . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 121
3.1 Agarose . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 121
3.2 Tris-Acetate Buffer. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 122
3.3 Ethidium Bromide . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 122
3.4 Gel-Loading Buffer . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 122
3.5 DNA Size Marker. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 122
3.6 DNA Sample . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 123
3.7 Gel Documentation System and Software for Analysis . . . . . . . . . . . . . . . 123
xii Contents

4 Procedure . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 123
5 Observation . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 124
6 Result Table . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 124
7 Precautions . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 124
8 Troubleshooting . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 125

16 Quantification of DNA by Using UV–Visible Spectrophotometer . . . . . . . . . . . . 127

1 Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 127
2 Principle. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 128
3 Reagents/Materials/Instruments Required and Their Role . . . . . . . . . . . . . . 129
3.1 Spectrophotometer . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 129
3.2 Cuvette . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 129
3.3 TE Buffer . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 129
3.4 DNA Sample . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 130
4 Procedure . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 130
5 Observation . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 131
6 Result Table . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 131
7 Precautions . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 131
8 Troubleshooting . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 132

17 Quantification of DNA by Using qRT-PCR . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 133

1 Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 133
2 Principle. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 134
2.1 Baseline . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 136
2.2 Threshold . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 136
2.3 Threshold Cycle (Ct). . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 136
2.4 Standard Curve . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 136
2.5 Correlation Coefficient (R2). . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 136
2.6 y Intercept. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 136
2.7 Exponential Phase . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 137
2.8 Slope. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 137
2.9 Efficiency. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 137
2.10 Dynamic Range . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 137
3 Reagents/Materials Required and Their Role . . . . . . . . . . . . . . . . . . . . . . . . . . . 137
3.1 Quantifiler™ THP PCR Reaction Mix . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 137
3.2 Quantifiler™ Trio Primer Mix . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 137
3.3 Quantifiler™ THP DNA Dilution Buffer. . . . . . . . . . . . . . . . . . . . . . . . . . . 138
3.4 Quantifiler™ THP DNA Standard . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 138
4 Procedure . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 138
5 Observation . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 143
6 Result Table . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 146
7 Precautions . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 146
8 Troubleshooting . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 147

18 Fluorescence-Based Instant Quantification of DNA . . . . . . . . . . . . . . . . . . . . . . . . . 149

1 Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 149
2 Principle. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 150
 Contents xiii

3 Reagents/Materials Required and Their Role . . . . . . . . . . . . . . . . . . . . . . . . . . . 151

3.1 Qubit™ 1 dsDNA HS Working Solution
(Component A) . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 151
3.2 Qubit™ 1 dsDNA HS Standards #1 and #2
(Components B and C) . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 151
3.3 TE Buffer . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 151
3.4 DNA Sample . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 151
4 Procedure . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 152
5 Observation . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 152
6 Result Table . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 153
7 Precautions . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 153
8 Troubleshooting . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 154

PART V AMPLIFICATION OF VARIOUS STR MARKERS USING MULTIPLEX PCR

19 Amplification of Autosomal STR Markers by Multiplex PCR . . . . . . . . . . . . . . . . . 157

1 Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 157
2 Principle. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 158
3 Reagents/Materials Required and Their Role . . . . . . . . . . . . . . . . . . . . . . . . . . . 159
3.1 PowerPlex® Fusion 6C 5 Master Mix. . . . . . . . . . . . . . . . . . . . . . . . . . . . . 159
3.2 PowerPlex® Fusion 6C 5 Primer Pair Mix . . . . . . . . . . . . . . . . . . . . . . . . 160
3.3 Molecular Biology-Grade Water . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 160
3.4 Template DNA . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 160
3.5 PCR Machine . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 160
4 Procedure . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 161
5 Observation and Result . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 162
6 Precautions . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 162
7 Troubleshooting . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 163

20 Amplification of Y Chromosome STR Markers by Multiplex PCR . . . . . . . . . . . . 165

1 Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 165
2 Principle. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 166
3 Reagents/Materials Required and Their Role . . . . . . . . . . . . . . . . . . . . . . . . . . . 168
3.1 Yfiler™ Plus Master Mix . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 168
3.2 Yfiler™ Plus Primer Set . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 168
3.3 Molecular Biology-Grade Water . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 168
3.4 Template DNA . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 168
3.5 PCR Machine . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 169
4 Procedure . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 169
5 Observation and Result . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 170
6 Precautions . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 170
7 Troubleshooting . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 171

21 Amplification of X Chromosome STR Markers by Multiplex PCR . . . . . . . . . . . . 173

1 Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 173
2 Principle. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 174
3 Reagents/Materials Required and Their Role . . . . . . . . . . . . . . . . . . . . . . . . . . . 175
3.1 Reaction Mix A . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 175
3.2 Primer Mix . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 175
3.3 Multi-Taq2 DNA Polymerase. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 175
xiv Contents

3.4 Molecular Biology-Grade Water . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 176

3.5 Template DNA . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 176
3.6 PCR Machine . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 176
4 Procedure . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 176
5 Observation and Result . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 177
6 Precautions . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 178
7 Troubleshooting . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 178

22 Amplification of Autosomal Mini-STR Markers from Degraded Samples. . . . . . . 179

1 Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 179
2 Principle. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 180
3 Reagents/Materials Required and Their Role . . . . . . . . . . . . . . . . . . . . . . . . . . . 181
3.1 AmpFlSTR™ MiniFiler™ Master Mix . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 181
3.2 AmpFlSTR™ MiniFiler™ Primer Set . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 182
3.3 Molecular Biology-Grade Water . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 182
3.4 Template DNA . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 182
3.5 PCR Machine . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 182
4 Procedure . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 182
5 Observation and Result . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 184
6 Precautions . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 184
7 Troubleshooting . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 184

PART VI GENOTYPING AND ANALYSIS OF RESULTS

23 Separation of Amplified DNA Fragments by Capillary Electrophoresis Using

Genetic Analyzer 3500xL . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 187
1 Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 187
2 Principle. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 188
3 Reagents/Materials Required and Their Role . . . . . . . . . . . . . . . . . . . . . . . . . . . 189
3.1 PCR-Amplified Product . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 189
3.2 Hi-Di™ Formamide . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 190
3.3 Internal Size Standard. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 190
3.4 Allelic Ladder. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 190
3.5 Capillary . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 190
3.6 Buffers . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 191
3.7 Polymer . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 191
4 Procedure . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 191
5 Observation . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 207
6 Precautions . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 208
7 Troubleshooting . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 209

24 Analysis of Capillary Electrophoresis Results by GeneMapper® ID-X v 1.5

Software . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 213
1 Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 213
2 Principle. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 214
3 Non-template Addition . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 218
4 GeneMapper® IDX Software . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 220
5 Procedure . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 221
6 Observation . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 228
7 Precautions . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 232
8 Troubleshooting . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 236
 Contents xv

25 Calculation of Paternity Index in Paternity Dispute and Identification Cases . . . 239

1 Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 239
2 Principle. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 240
3 Requirements . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 241
3.1 Autosomal STR DNA Profiles . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 241
3.2 Allele Frequencies. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 242
3.3 MS Office Excel Platform . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 243
4 Procedure . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 243
5 Observation . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 248
6 Precautions . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 249
7 Troubleshooting . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 251

PART VII CASE STUDIES

26 Solving Paternity Dispute by DNA Fingerprinting: A Case Study . . . . . . . . . . . . . 255

1 Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 255
2 Principle. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 256
3 Brief Case History. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 257
4 Samples . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 257
5 Procedure . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 257
6 Results and Observation . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 257
7 Precautions . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 258
8 Troubleshooting . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 258

27 Solving a Case of Murder by DNA Fingerprinting: A Case Study . . . . . . . . . . . . . 261

1 Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 261
2 Principle. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 262
3 Brief Case History. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 262
4 Samples . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 263
5 Procedure . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 263
6 Results and Observation . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 263
7 Precautions . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 265
8 Troubleshooting . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 265

28 Solving a Case of Sexual Assault by DNA Fingerprinting: A Case Study . . . . . . . 267

1 Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 267
2 Principle. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 268
3 Brief Case History. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 269
4 Samples . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 269
5 Procedure . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 269
6 Results and Observation . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 270
7 Precautions . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 270
8 Troubleshooting . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 273

29 Identification of an Unknown Skeleton by DNA Fingerprinting: A Case

Study. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 275
1 Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 275
2 Principle. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 276
3 Brief Case History. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 277
xvi Contents

4 Samples . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 277
5 Procedure . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 277
6 Results and Observation . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 278
7 Precautions . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 278
8 Troubleshooting . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 280

PART VIII ADVANCED TECHNOLOGIES FOR FORENSIC DNA ANALYSIS

30 Sequencing Control Region of Human Mitochondrial Genome to Assess

Matrilineal Lineage. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 283
1 Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 283
2 Principle. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 284
3 Reagents/Materials Required and Their Role . . . . . . . . . . . . . . . . . . . . . . . . . . . 285
3.1 Template DNA . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 285
3.2 Primers. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 285
3.3 MyTaq™ HS Red Mix 2 . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 286
3.4 illustra™ ExoProStar™ . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 286
3.5 ABI Prism® BigDye™ Terminator v3.1 Cycle Sequencing
Kit . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 286
3.6 Molecular Biology-Grade Water . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 286
3.7 Genetic Analyzer 3500xL . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 286
4 Procedure . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 287
5 Observation and Result . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 289
6 Revised Cambridge Reference Sequence for HVRI . . . . . . . . . . . . . . . . . . . . . . 289
7 Revised Cambridge Reference Sequence for HVRII . . . . . . . . . . . . . . . . . . . . . 290
8 Precautions . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 290
9 Troubleshooting . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 291

31 Application of Next-Generation Sequencing (NGS) in Forensic DNA Analysis

and DNA Phenotyping . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 293
1 Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 293
2 Principle. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 294
3 Reagents/Materials Required and Their Role . . . . . . . . . . . . . . . . . . . . . . . . . . . 295
3.1 ForenSeq™ DNA Signature Prep Kit . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 295
3.2 ForenSeq™ Index Plate Fixture Kit . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 295
3.3 MiSeq® FGx™ Forensic Genomics System . . . . . . . . . . . . . . . . . . . . . . . . . 295
3.4 ForenSeq™ Universal Analysis Software. . . . . . . . . . . . . . . . . . . . . . . . . . . . 295
3.5 Template DNA . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 296
4 Procedure . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 296
5 Observation and Result . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 299
6 Precautions . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 299
7 Troubleshooting . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 302

32 Forensic Trace and Touch DNA Analysis . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 305

1 Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 305
2 Targets of Trace DNA Samples . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 306
3 Collection Strategies for Trace DNA Samples . . . . . . . . . . . . . . . . . . . . . . . . . . . 307
4 Analysis of Trace DNA Samples. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 308
5 Concerns and Limitations of Trace DNA Analysis . . . . . . . . . . . . . . . . . . . . . . . 309
6 Conclusion . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 311
 Contents xvii

33 RAPID DNA Technology: A Boon to Forensic DNA Typing. . . . . . . . . . . . . . . . . 313

1 Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 313
2 The Technology. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 314
3 Currently Available Technologies . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 315
4 Conclusion . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 316

Appendix A: Composition of Reagents, Buffers, and Solutions . . . . . . . . . . . . . . . . . . . . . . . . 317

Appendix B: List of Commercially Available Kits and Instruments for Forensic
DNA Analysis. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 321
Appendix C: Good Laboratory Practices . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 333
Appendix D: Allele Frequency Data for Calculation of Paternity Index . . . . . . . . . . . . . . . . 337
About the Authors

HIRAK RANJAN DASH is currently a Scientific Officer at the DNA Fingerprinting Unit,
Forensic Science Laboratory, in Sagar, Madhya Pradesh, India. He completed his Ph.D. at
the National Institute of Technology, Rourkela, India, in 2015, and has since been working
in the area of microbial ecology, forensic microbiology, and human genetics. In addition, he
is a member of various scientific societies, including the International Society for Forensic
Genetics, Association of Microbiologists of India and Students Association of Microbiology.
He has been engaged in forensic DNA fingerprinting examinations in criminal and civil cases
including the cases related to paternity, child swapping, rape, identity and incest. He has
authored more than twenty-five papers in reputed peer-reviewed international journals and
has published four books.
PANKAJ SHRIVASTAVA has broad interests in several areas of forensic DNA fingerprinting
including population-based DNA analysis, developing protocols in forensic DNA typing,
validating new multiplex kits and optimizing different steps in forensic DNA typing
methodology. He has supervised Ph.D. students and published over 40 research papers
in peer-reviewed international journals. His publications in Forensic Science International
Genetics, Nature Scientific Reports, Legal Medicine, International Journal of Legal
Medicine, Annals in human Biology, etc., have earned him wide acclaim from the
forensic DNA community. He is presently working at the DNA Fingerprinting Unit in
State Forensic Science Laboratory, Govt. of Madhya Pradesh, India. He is a member
of International Society for Forensic Genetics (ISFG), Indian Society of Human Genetics
(ISHG), Association of Microbiologists of India(AMI) and Association for the promotion
of DNA Fingerprinting and other DNATechnologies (ADNAT).
SURAJIT DAS is an Associate Professor at the Department of Life Science at the National
Institute of Technology, Rourkela, India. His field of research includes microbial genomics,
biosynthesized nanoscale materials, marine biotechnology, environmental microbiology and
bioremediation. He is serving as the Editor of Plos One and BMC Microbiology journal. He
has published more than 70 research papers in peer-reviewed national and international
journals and authored 7 books.

xix
 Part I

Overview of Forensic DNA Analysis

 Chapter 1

Introduction to Forensic DNA Analysis

DNA fingerprinting analysis has been proved to be a useful tool for

crime scene investigation to aid the criminal justice system. With
wide media coverage and many high-profile cases solved by DNA
technology, it has become familiar among the common people and
the awareness to this technology is increasing day by day. The
technology not only is helpful in increasing the rate of conviction
but also plays an important role in exonerating the innocence.
Deoxyribonucleic acid (DNA) is the genetic material present in
every nucleated cell of the body which contains the complete
genetic program of the individual for its storage and transmittance.
The major constituent of DNA is the nitrogenous bases called as
nucleotides, i.e., adenine (A), guanine (G), cytosine (C), and thy-
mine (T), which are arranged in specific order in long sequences.
Nucleotides cross-link with each other in a specific manner, i.e.,
A¼T and GC. The nucleotide bases reside on the sugar (deoxyri-
bose) and phosphate backbone and subsequently twist around each
other to generate a double-stranded helix. A human genome con-
stitutes around 3 billion nucleotide bases connected in a chain-like
structure to form a tightly packed chromosome. The number of
chromosomes is specific to an organism, e.g., 46 (humans),
38 (cat), 64 (horse), 24 (rice), 48 (potato), and 40 (mango). In
this context, genes are the segments of DNA molecules that
are responsible for the structure and function of the cell as well
as the whole organism. A typical human cell harbors around
50,000–100,000 genes which are spaced at intervals.

1 Genetic Variation

A person inherits his or her genetic material from his or her parents
through the cumulative processes of gametogenesis, oogenesis, and

H. R. Dash et al., Principles and Practices of DNA Analysis: A Laboratory Manual for Forensic DNA Typing, Springer Protocols
Handbooks, https://doi.org/10.1007/978-1-0716-0274-4_1, © Springer Science+Business Media, LLC, part of Springer Nature 2020

3
4 Introduction to Forensic DNA Analysis

zygote formation. Due to the phenomenon of crossing over and

genetic recombination occurring at prophase I of the cell division,
the genetic variation occurs among individuals as well as siblings.
Half of the diploid set of chromosomes of a child comes from the
mother and another half from the father. The possible combina-
tions for any parent to transfer its chromosomes to the child is 223
and when both the parents are considered the number of possible
combinations further increases to 246 generating a huge set of
genetic variations among individuals. Additionally, other factors
including mutation, addition/deletion, and substitution of nucleo-
tides also contribute to the genetic variation. In this regard, no two
individuals can have the same genetic makeup except the homozy-
gous twins.
Genes, the short DNA fragments of around few to ten thou-
sand base pairs in length, are the coding regions of DNA which
constitute ~5% of the human DNA sequence. These genes produce
different proteins through the process of transcription which carry
out different physiological functions. The protein-coding region of
a gene is called exon and the remaining intervening portion is called
as the intron. These nonprotein-coding regions present either
within a gene or outside the genes are collectively called as junk
DNA which are devoid of any necessary functions. The polymor-
phic variable markers, i.e., loci that are present in the noncoding
regions of the DNA, are mostly targeted for the generation of DNA
profile of an individual. As human chromosomes occur in pairs and
are homologous in nature, a copy of each gene fragment resides at
the same position on each chromosome inherited from the parents.
The possibilities of each gene fragment occurring at a locus are
termed as alleles which may be homozygous (identical alleles) or
heterozygous (different alleles) in nature. In this regard, the alleles
present at a genetic locus are called as genotype, while a DNA
profile is the cumulative genotype found at a set of genetic loci.
The current method of human identity testing by DNA typing
relies on the use of short tandem repeats (STRs) or microsatellites
or simple sequence repeats (SSRs).

2 Short Tandem Repeat (STR) Markers

STRs are repetitive DNA fragments of around 2–7 base pairs which
are found abundantly at around 3% of the total human genome.
They are considered to be the most polymorphic and prone to
mutations due to slippage during DNA replication. Thus, STR
markers are considered to be the most suitable for DNA typing
analysis as they possess a high degree of interindividual variation
and generation of genotypes with greater resolution. Plenty of STR
loci occur on each type of chromosomes, i.e., autosomes, and X or
Y chromosomes. The STR markers are of various types, i.e., simple
 Technological Use in Human STR Typing 5

repeats (e.g., TPOX, CSF1PO, D5S818, D13S317, D16S539),

simple repeats with non-consensus alleles (e.g., TH01, D18S51,
D7S820), compound repeats (e.g., vWA, FGA, D3S1358,
D8S1179), or complex repeats (e.g., D21S11). Simple repeat
STRs contain several units of nucleotides of identical sequence
and length, whereas the compound STRs may comprise two or
more simple repeats present adjacently. However, the complex
STR repeats contain the blocks of several variable unit lengths.
Based on the number of nucleotides, the STRs have been categor-
ized into di-, tri-, tetra-, penta-, or hexa-nucleotide markers, out of
which the tetranucleotide repeat STR markers are of high use in
forensic DNA typing. Additionally, there exists a uniform nomen-
clature for the STR markers commonly used for forensic DNA
typing. For example, if a genetic marker is a part of a gene, the
name of the gene is used for the marker designation, e.g., TH01
(present in 1st intron of tyrosine hydroxylase gene), CSF1P0 (6th
intron of proto-oncogene c-fms), FGA (3rd intron of alpha-
fibrinogen gene), TPOX (10th intron of thyroid peroxidase
gene), or vWA (40th intron of von Willebrand factor gene). Simi-
larly, if a STR marker is present outside the gene region, it is named
according to its chromosomal position. For a marker present on an
autosomal chromosome, e.g., D5S818, “D” refers to DNA, “5” is
the chromosome number harboring the marker, “S” stands for the
single-copy sequence nature of the marker, and “818” is the order
of discovery of the marker on the chromosome. Additionally, for a
marker on Y chromosome, e.g., DYS391, “D” refers to DNA, “Y”
designates the “Y” chromosome harboring the marker, “S” is the
single-copy sequence of the marker, and “391” is the order of
discovery of the marker on Y chromosome.
There are many advantages of using STRs over the earlier used
genetic markers such as variable number tandem repeats (VNTRs).
Generation of small product size due to less number of repeat units
of nucleotides increases the chance of recovery of genetic informa-
tion from degraded samples which is relatively common in forensic
cases. A combination of STR markers can be used simultaneously in
a single set of PCR amplification process, thus increasing the dis-
crimination power of this technology. Additionally, commercially
available STR kits and analysis of a uniform set of core STR loci
increase the usefulness of this technology for interlaboratory shar-
ing of results and creation of DNA databases.

3 Technological Use in Human STR Typing

Technological advancements of forensic DNA analysis in chrono-

logical order have been given in Table 1.1. The sequential events of
forensic DNA analysis involve DNA isolation, quantification, mul-
tiplex PCR, capillary electrophoresis, and analysis of results which
6 Introduction to Forensic DNA Analysis

Table 1.1
The chronological advancements in forensic DNA typing

Year Milestone discoveries and advancements

1980 First polymorphic RFLP marker discovered by Ray White
1985 Multilocus VNTR probes described by Alec Jeffreys
1985 PCR technique discovered by Kary Mullis
1986 First case solved in the USA by DNA typing through RFLP technique (Florida vs. Tommy Lee
Andrews)
1988 DNA case work lunched by Federal Bureau of Investigation (FBI)
1989 Establishment of Technical Working Group on DNA Analysis Methods (TWGDAM)
1991 First report on short tandem repeats (STRs)
1993 Availability of commercial STR kits
1995 Initiation of DNA database Forensic Science Service (FSS), UK
1996 First use of mitochondrial DNA analysis
1998 Launching of Combined DNA Index System (CODIS) database by FBI
1998 Establishment of Quality Assurance Standards for forensic DNA testing laboratories
1999 Validation of multiplex STRs
2003 Successful application of forensic DNA technology in solving identity crisis in Saddam
Hussein case and September 11, 2001, World Trade Center victims
2003 Human Genome Project completed
2003–04 Y STR kits launched
2007 Launching of first kit to analyze samples with degraded or limited DNA
2010 X-STR kit launched
2012 Development of forensic test to predict the hair and eye color of the suspect(s) based on the
DNA trace left at the crime scene
2013 Introduction of the portable system to analyze human DNA within 75 min
2014 Developmental validation on autosomal STR multiplex kits with Y STRs and InDel markers
2016 Launch of NGS-based STR kits
2017 Expanded CODIS for inclusion of 20 STR loci

has been discussed in much details in the subsequent chapters. In a

nutshell, the preliminary requirement of forensic DNA analysis is
the isolation and purification of DNA from the forensic case exhi-
bits. In any cell, DNA occurs in the form of either nuclear DNA or
extranuclear DNA (e.g., mitochondrial DNA or chloroplast DNA
of plants). Though various DNA isolation techniques are available
nowadays the procedure utilized is exclusively dependent on sam-
ple, technique, and analyst’s preference. DNA isolation can be
 Challenges in Forensic DNA Typing 7

carried out by either manual methods using different sets of che-

micals and reagents or automatic or semiautomatic techniques
depending on the nature of the sample. Isolation of DNA is fol-
lowed by both qualitative and quantitative assessment of the
extracted DNA. Input of optimal quality and quantity of DNA
into the PCR reaction is the key to generate an acceptable DNA
profile. Hence, various quantitation techniques such as UV absor-
bance, gel electrophoresis, quantitative real-time PCR, and others
are being used nowadays. Many qRT-PCR-based kits are also com-
mercially available for the simultaneous detection of many useful
parameters such as amount of total human DNA, ratio of amount
of small-to-large autosomal DNA reflecting the degradation index
of the DNA sample, male-to-female DNA ratio in mixed samples,
and presence of PCR inhibitors in the sample.
Quantification of DNA is followed by the amplification of
target loci by polymerase chain reaction (PCR). In this process,
simultaneous amplification of multiple DNA fragments is carried
out using different dye-labeled primers, thus generating PCR pro-
ducts labeled with dyes. Many multiplex kits are available currently
for amplification of desired markers either on autosomal, X, or Y
chromosome. Subsequently, the PCR products are subjected to
capillary electrophoresis for separation and detection of the ampli-
fied STRs. In this process, DNA fragments are separated based on
their molecular size under voltage and subsequently size of the
amplified fragments is designated by comparing them with the
standards. Finally, the results obtained were analyzed using various
analysis software to generate the desired results. The flowchart
describing the sequential events in forensic DNA typing has been
given in Fig. 1.1.

4 Challenges in Forensic DNA Typing

Ideal samples are rarely received in a forensic DNA laboratory for

examination except the reference samples. Most of the samples
contain either degraded DNA or potential inhibitors of PCR pro-
cess. In certain instances, the samples are highly contaminated from
exogenous DNA and pose a great challenge to the process. Other
major problems of forensic DNA typing include the generation and
analysis of mixed DNA profiles, cost-effectiveness, and requisite
expertise. A few of the most important challenges related to foren-
sic DNA typing have been discussed below.
Forensic casework encounters the major chance of examining
degraded biological samples. Primarily, two factors, i.e., time and
condition of exposed environment, influence on the decomposition
as well as degradation of DNA evidences. The process of degrada-
tion increases with time, whereas the environmental conditions
such as temperature, humidity, and pH influence the rate of
8 Introduction to Forensic DNA Analysis

Fig. 1.1 The generalized steps involved in forensic DNA typing

degradation of the samples. In cases related to burning and mass

disaster generally degraded samples are received for examination.
As the biological samples degrade, the DNA becomes more frag-
mented and the chance of generating a complete DNA profile is
minimized. Most of the common forms of DNA damage include
oxidation, hydrolysis, pyrimidine dimers, cytosine deamination,
and formation of cross-linkages between DNA-DNA and
DNA-protein. In forensic samples DNA damage occurs mostly
due to oxidation, hydrolysis, and pyrimidine dimer formation.
In certain instances, despite the presence of adequate amount
of DNA, the amplification failure takes place leading to
non-generation of a DNA profile. Presence of PCR inhibitors
co-extracted with the target DNA commonly include the dyes
from clothing, humic acid from soil, fatty acid from bone, heme
from blood, and melanin from hair. Presence of inhibitors in a
sample may lead to dropout of alleles at some loci. Other problems
associated with the inhibitors include adverse effect on PCR pri-
mers, overlysis of cells during DNA extraction, and/or reduction of
polymerase activity. Some of the common strategies to overcome
PCR inhibitors include dilution of the extracted DNA to reduce
their content, use of robust, inhibitor tolerant Taq polymerase, use
of sample cleanup devices, as well as application of chemicals with
PCR-enhancing properties such as bovine serum albumin and
PCRBoost™. Magnetic bead-based DNA extraction technique
can be used as it possesses the property of highest inhibitor removal
capability.
 Applications of Forensic DNA Typing 9

Forensic DNA typing is prone to low levels of contamination.

Contamination during forensic DNA typing originates from either
primary or secondary sources. In certain instances, contaminants in
the form of either cells or purified or amplified DNA get accumu-
lated on various equipment leading to their transfer to the ques-
tioned samples. The exogenous DNA can be categorized into
four types, i.e., (1) DNA originated from the examiner, (2) DNA
originated from consumables (pipettes, plasticwares or chemicals)
being used (3) DNA from other samples analyzed together,
and (4) DNA from mixing of allelic ladder with the questioned
samples. Contamination plays a limiting role in the analysis and
interpretation of DNA typing results. The presence of unexpected
peaks, occurrence of additional peaks in a single source sample,
and heterozygous imbalance are the most common problems of
exogenous DNA contamination. A little bit of precaution and the
use of certain chemicals may minimize the risk of contamination
during forensic DNA analysis. Thorough cleaning of working areas,
decontamination or autoclaving of the equipment and instruments,
and application of chemically decontaminating solutions such as
sodium hypochlorite (NaOCl) can reduce the chance of contami-
nation from exogenous sources. Additionally, the complete proce-
dure of DNA typing from DNA isolation to result interpretation
involves a huge cost, thus minimizing its usability in developing
nations. Technological complications lead to the examiners to gain
expertise in a very slow pace. Cost involvement in infrastructure
development also creates a hindrance to adoption of DNA typing
technology in many cases, mostly in developing and undeveloped
countries.

5 Applications of Forensic DNA Typing

DNA fingerprinting has revolutionized the process of justice deliv-

ery system. DNA typing is a reliable technique to identify the
criminal suspect(s) and possesses the potential to either implicate
or exonerate a suspect with greater degree of fidelity. The technol-
ogy can be applied to deduce conclusion in a variety of case types
including paternity dispute, identification of mutilated bodies,
murder, abduction, and sexual assault. Hence, currently DNA typ-
ing is routinely used in most of the civil as well as criminal cases.
Some of the important high-profile cases solved through DNA
typing have been discussed below.

5.1 O.J. Simpson On 12th June 1994, Nicole Brown Simpson and Ronald Goldman
Case in Los Angeles were found murdered in the house of Ms. Simpson. During inves-
tigation, O.J. Simpson, the ex-husband of Ms. Simpson, was found
to be the prime suspect of the incidence. As O.J. Simpson was a
successful footballer and celebrity of that time, the case drew
10 Introduction to Forensic DNA Analysis

attention of the global public. Many samples including the blood

droplets and the stains were collected from the spot for examina-
tion. The samples were examined in three different laboratories,
i.e., Los Angeles Police Department (LAPD), the California
Department of Justice (CA DOJ), and a private laboratory for
forensic DNA typing. Both PCR- and RFLP-based techniques
were employed. Finally, a consensus result was obtained from the
three laboratories with no exclusion between the samples collected
from the scene of crime and reference sample collected from
Mr. Simpson. A long hearing courtesy to the advocates of
Mr. Simpson took place in this case on raising the contamination
theory for collection of scientific evidence. However, a random
match and obtaining of such overwhelmingly incriminating results
are not due to mere contamination and random laboratory error.
This case is popularly known as the case of the century and was a
major boost for advocating implementation of DNA typing in
criminal justice system.

5.2 This was an American political sex scandal case which involved the
Clinton-Lewinsky then president of America, Bill Clinton, and an intern Monica
Affair Lewinsky working at the residential office of the president. The
sexual relationship that occurred during 1995–1997 came to public
in 1998 and Linda Tripp, a co-worker of Monica, was familiar with
the situation. When president denied the allegation in public and
Monica almost submitted an affidavit refuting a relationship with
Clinton, Linda made this case public. Earlier, Linda had encour-
aged Monica to save the semen-stained blue gown without dry
cleaning which later turned out to be the most valuable scientific
evidence. Subsequently, a DNA test was carried out and the DNA
profile obtained from seminal stain present on the blue gown
matched with the reference sample obtained from the president.

5.3 Identification The mass catastrophe occurred after attack on the World Trade
of the Bodies in World Center on 11th September 2001 resulted in the generation of
Trade Center Attack many mutilated charred bodies posing a huge challenge for their
identification. There was no definite list of victims. Hence, an
attempt was made to process as many samples recovered separately
assuming them to have a distinguished origin. At the early stage,
the soft tissues were targeted for identification purpose. Cross
contamination of the samples due to the high explosion in the
spot yielded a mixed STR DNA profile which shifted the target of
identification to bone or skeletal remains rather than the soft tis-
sues. In this regard, the intact bones were preferred over the spongy
bones. Out of the samples analyzed the rate of success in generating
a DNA profile was 75%, leading to the identification of the charred
remains.
 Applications of Forensic DNA Typing 11

5.4 Assassination India’s former Prime Minister Mr. Rajiv Gandhi was assassinated in
of Sri Rajeev Gandhi, a public meeting at Sriperumbudur, Tamil Nadu, India, in May
Prime Minister of India 1991 which shocked the nation. This is one of the earliest cases of
Indian history to be solved by DNA fingerprinting technique. The
mastermind of this case was Sivarasan, the “one-eyed jack” from Sri
Lanka. After 3 years of the incidence, Sivarasan killed himself along
with his associates. The identification of Sivarasan was established
by comparing his DNA profile with that of his mother Sivapakkam
and brother Ravichandran. This settled the identification issue of
Sivarasan and gave a breakthrough to the investigating agency.
Another important aspect of this case was that a girl named
Dhanu was used as the human bomb for this assassination purpose.
However, post-incidence the investigating agencies were clueless
about certain questions, e.g., whether various pieces of the body
that were put together during investigation belong to one individ-
ual, whether the denim belt which contained charred muscle pieces
was worn by that individual, and whether it is a male or female
individual. All these questions were answered with great level of
satisfaction through DNA fingerprinting technique which estab-
lished Dhanu as the suicide bomber in this case.
 Chapter 2

Biological Samples: The Target Sources for DNA Typing

Human body contains around 1013 number of cells. All nucleated

cells of the human body contain DNA and hence any biological
material left in the crime scene can be collected and used as a
biological evidence for DNA typing analysis. However, the samples
should be collected properly to avoid contamination or degrada-
tion. With the advent of PCR technology, a trace amount of
biological evidence present in a crime scene can also be useful due
to the amplification process and reaching the detection limit of the
instruments. In this regard, touch DNA technology is also in
practice currently for small amount of DNA present in the samples.

1 The Locard’s Principle

In the twentieth century, a pioneer forensic scientist Dr. Edmond

Locard formulated the theory of trace evidence which states that
“every contact leaves a trace.” These fragmentary evidences can be
used to link between the victim, suspect(s), spot, and the weapon
(if any). In the process of committing a crime, the perpetrator may
leave some biological samples of his/her own in the crime scene
either knowingly or unknowingly. Thus, a proper investigation and
search in the spot as well as the victim’s belongings may generate a
clue in the form of biological evidence to link the suspect(s). Some
of the biological evidences in the form of either primary sources or
secondary sources have been discussed in details subsequently.

2 Types of Biological Samples

Any object or its surface containing nucleated cells can be used a

source sample for DNA typing analysis. These exhibits can be

H. R. Dash et al., Principles and Practices of DNA Analysis: A Laboratory Manual for Forensic DNA Typing, Springer Protocols
Handbooks, https://doi.org/10.1007/978-1-0716-0274-4_2, © Springer Science+Business Media, LLC, part of Springer Nature 2020

13
14 Biological Samples: The Target Sources for DNA Typing

Fig. 2.1 Samples and their source or DNA for forensic analysis

collected either from the crime scene or from the living human
beings with their consent. Biological evidences of forensic use are of
varied nature, i.e., minute objects such as hairs and nails to large
objects such as skeletons and clothing. The physical nature of these
biological evidences may be either solid or liquid. The biological
samples can be collected instantly under proper conditions. On the
contrary, some biological evidences are collected after a long period
of exposure in the environment. Though the core chemical com-
position of every cell of human body is the same, the amount of the
constituents varies according to the requirement of the body parts.
But the vital molecule DNA is present in equal amount in every
body cell except the gametes. The physical and chemical composi-
tion of few biological source samples has been given in Fig. 2.1.
Additionally, the full forensic value of the evidence is achieved
only when it is compared with the reference DNA profile. In this
regard, the reference samples are also to be collected in the form of
either whole blood, buccal swab, or hair. An extensive list of physi-
cal evidences that can be collected as a source of DNA sample has
been provided in Table 2.1.

3 Whole Blood/Blood Stains

Blood is a specialized fluid connective tissue, constituting 7% of

adult body weight which travels through the circulatory system
transporting gases, nutrients, wastes, and other macromolecules
throughout the body. Human blood has a solid phase of formed
elements and a liquid phase, the plasma. Plasma, the straw colored
aqueous solution, is mostly made up of water (90%), proteins
(7–8%), and other solutes (1–2%). Blood cells (formed elements)
are classified as either erythrocytes (RBC) or leukocytes (WBC).
Additionally, a smaller third cellular constituent is also present
referred to as thrombocytes (platelets). There occur approximately
 Whole Blood/Blood Stains 15

Table 2.1
Sources of biological evidences with potential use in forensic DNA typing

Physical evidences Location within the object Source of DNA

Consumed cigarette Cigarette butt Saliva
Tooth pick Tips Saliva
Stamps and envelopes Licked area Saliva
Bottle or sipper Mouthpiece Saliva
Bite mark Person’s skin Saliva
Used condom Both the sides Semen and vaginal cells
Blanket/bedsheets Surface Semen, sweat, hair, saliva, urine
Fingernail Scrapings Blood, skin
Ligature Surface Skin
Bullet Surface Blood
Clothing Surface Blood, sweat, semen
Hat/mask Inner surface Sweat, hair
Knife Outside surface/handle Blood, tissue, sweat
Credit/debit card Surface Sweat, tissue
Gloves Interior and exterior Sweat, tissue
Toothbrush Surface Buccal tissue, saliva
Dandruff Whole Cell debris
Head comb Surface Sweat, hair, tissue
Razor Blades Tissue, hair roots
Eye glasses Ear and nose piece Sweat, tissue

4.5–5 million RBCs, 4500–11,500 WBCs, and 250,000–450,000

platelets per microliter of blood. WBCs, as a part of body’s immune
system, destroy and/or remove cellular debris and infectious for-
eign pathogens. The source of DNA in blood is only the leukocytes,
i.e., WBC.
Bloodstains can be obtained either from the spot, on the weap-
ons, or from the clothing. From forensic point of view, the shape
and pattern of bloodstains give useful information regarding the
origin and nature of the wound occurred during crime. Additional
information regarding the distance of blood fall, bleeding person’s
position, i.e., standing, still, or walking, etc. can also be deduced
from careful observation of the bloodstains. Bloodstains are of two
types, i.e., wet bloodstains and dry bloodstains. However, both of
them act as a source of DNA for forensic DNA typing.
16 Biological Samples: The Target Sources for DNA Typing

4 Hairs

Hairs grow from the follicles found in the dermis which is the
exclusive feature of the mammals. The primary composition of
human hair is a protein, i.e., alpha-keratin. A typical hair has two
structures: the follicle or bulb which is present beneath the dermis
and the shaft which is the hard filamentous structure that is present
above the sin surface. Several layers exist in a hair fiber which
includes the cuticle, cortex, and medulla. Though there exist
many forms of hair in the human body, scalp and pubic hairs are
the routine candidates for DNA examination.
Hairs are considered to be the most overestimated and misin-
terpreted samples for DNA examination. Many investigators
assume hair to be an ideal sample for DNA examination. However,
the extraction of DNA and subsequent analysis depend primarily on
the part of DNA that has been received for examination. Isolation
and characterization of nuclear DNA are possible only in the hairs
with growing roots and adhering tissues. However, forensic mito-
chondrial DNA analysis relies on the DNA content of the hair shaft
rather than hair root. During the process of keratinization, nuclear
cellular material is degraded in the hair shaft and the presence of
multiple numbers of mitochondria makes them a great source of
mitochondrial DNA for further analysis. In this regard, it is highly
recommended for microscopic examination of hairs prior to their
DNA analysis regarding their suitability.

5 Teeth

With the advancements in DNA technology, tooth becomes one of

the most useful sources of DNA for its subsequent analysis. Human
teeth are considered to be the most preferred source of DNA due to
their unique composition and location within the jawbones which
prevents the decaying process from the physical and chemical
agents. The advantages of the DNA extracted from teeth are their
high quality and being less prone to contamination.
Human teeth have two parts, i.e., the crown which is exposed
in the mouth and the root present in the jawbones. Teeth roots are
primarily composed of cementum and pulp which are a rich source
of DNA. It has been documented that the DNA yield from the
teeth root is ten times higher than that of the DNA yield from the
crown. Enamel, the hardest tissue of human body, covers the crown
of the teeth and is acellular in nature, and hence contains no DNA.
The tissues that primarily occur in the teeth pulp are the odonto-
blasts. Other cells that occur in traces in human teeth include the
fibroblasts, histiocytes and macrophages, plasma cells, nerve cells,
and undifferentiated mesenchymal cells.
 Soft Tissues 17

6 Bone

Bones are the sources of DNA in case of the extremely damaged or

degraded human skeletal remains. Many exogenous elements such
as humidity, organic compounds, and microorganisms reduce the
amount of informative DNA on the soft tissues and other parts of
corpse, thus making bones the most suitable samples for genetic
analysis. Bone possesses a complex anatomical structure. The basic
building block of bone includes the collagen fibrils. Additionally,
vascularization also contributes to the microstructure of bones.
Bone constitutes many organic as well as inorganic molecules
such as hydroxyapatite [Ca10(PO4CO3)6(OH)2], collagen, and
non-collagen proteins such as osteonectin and osteocalcin. Often
DNA gets adsorbed to the inorganic constituents like hydroxyapa-
tite for its long-term storage by developing certain crystal struc-
tures. Additionally, DNA binding to the collagens increases the
stability of the DNA. Certain short fragments of DNA get trapped
in the bone fibrils by encapsulation, increasing its stability. Ulti-
mately, the combination of organic as well as inorganic substances
renders bones their hardness and makes them less prone to degra-
dation after extensive environmental exposure. In this regard, bone
samples provide a useful source of DNA in many forensic cases such
as identification of deceased individuals in cases of accidents, mass
disasters, terrorist attacks, and cases involving defragmentation of
the body.

7 Soft Tissues

Though hard tissues of human corpse such as bone and teeth are
considered to be the most suitable samples for DNA typing studies,
soft tissues are also highly useful in many instances. The usefulness
lies in the fact that they contain a huge amount of DNA and it takes
less time to isolate DNA from them in comparison to bone samples.
Soft tissues can be collected from the fresh corpses with minor
decomposition. Soft tissues are also preferred for the identification
of unknown fetus aborted illegally. The most advised soft tissues for
DNA analysis include the brain, muscle (biceps), kidney (from
upper pole), and heart (right ventricle). The tissue samples are
most suitable for DNA isolation due to their syncytial nature
which yields a higher DNA content in comparison to other body
parts. In this regard, the viscera collected in cases of unnatural death
to determine the cause of death are also useful samples for DNA
tests. However, the base modification and the presence of reactive
oxygen species (ROS) in the viscera samples generated due to the
toxic effects may hinder the DNA technology and further down-
stream processing of the samples.
18 Biological Samples: The Target Sources for DNA Typing

8 Semen and Sperm/Vaginal Fluid

Most of the forensic DNA laboratories receive nearly half of their

cases as the sexual assault cases. In such cases, the mixture of
seminal and vaginal fluids is the most likely sample to be received
for DNA testing in the form of either slide, swab, or clothing.
Sperms are considered to be the most useful source of DNA in a
very small quantity. A very small amount of seminal stain can yield a
good DNA profile as millions of sperm cells are present per micro-
liter of human ejaculate. The time elapsed during the coitus and
collection of sample is of utmost importance as semen can last in the
victim’s body for 72 h or more. In addition to the spermatozoa
many epithelial cells are also expected from the sexual assault cases.
Human seminal fluid contains acid phosphatase, citric acid, inosi-
tol, calcium, zinc, magnesium, fructose, ascorbic acid, prostaglan-
dins, and other compounds. Additionally, vaginal fluid is a mixture
of cervical mucus such as albumin, alpha1-antitrypsin, alpha2-hap-
toglobin, alpha2-macroglobulin, beta-lipoprotein, orosomucoid,
and ceruloplasmin. The epithelial cells of both the male and female
contributors also provide the source of DNA in these samples.

9 Urine

Urine samples are the most easy to collect from a person. Though
urine samples are used for analysis of biomarkers for various types of
diseases, they have limited use in forensic DNA applications. Urine
is not being considered for forensic DNA applications due to the
low content of nucleated cells in them. The major constituent of
human urine is water, urea, chloride, sodium, potassium, creati-
nine, and other organic and inorganic compounds. Most of the
scanty nucleated cells found in urine are in the form of either white
blood cells or epithelial cells originated from renal tubular, transi-
tional urothelial, and squamous cells. Urine of females consists of a
larger amount of epithelial cells in comparison to males. Urine
samples may be of useful forensic implications if DNA can be
isolated from the urine-stained clothing in cases of either sexual
assault or murder.

10 Saliva

Saliva is the biological secretion of mouth helpful in the digestion

process. The major constituents of saliva include urea, glucose,
progesterone, various traces of acids, amino acids, creatinine, and
different proteins. It also contains a high amount of buccal cells that
contribute to the source of DNA. Saliva generates a smaller amount
 Biological Samples and Their DNA Content 19

of DNA than blood and other body fluids. However, its ease of
collection, requirement of simpler logistics, chance of self-
collection, and sample mailing make them a suitable candidate for
forensic DNA analysis.

11 Biological Samples and Their DNA Content

The advent of PCR technology has revolutionized the DNA analy-

sis by reducing the requirement of initial amount of DNA template
to a substantial limit. In this regard, an overview on the approxi-
mate DNA content of the biological samples has been given in
Table 2.2.
A diploid human cell contains approximately 6 pg of DNA. For
blood, the source of nuclear DNA is only WBC. Under normal
circumstances, human blood contains 5–10  106 WBCs/ml.
Hence, theoretically, 1 μl of blood yields 30–60 ng of DNA.
However the amount of DNA present in bloodstains varies greatly
with storage condition of the stain and stage of degradation of the
sample. Similarly, sperm cells, being haploid in nature, contain
around 3 pg of DNA. The stains containing the mixture of semi-
nal/vaginal fluid are a great source of DNA for sexual assault cases.
Additionally, DNA content in hairs varies greatly between

Table 2.2
Biological samples of forensic implications and their DNA content

Source samples DNA content (approximately)

Liquid blood 30–60 ng/μl
Blood stain 250–500 ng/cm2
Semen 150,000–300,000 ng/ml
Vaginal fluid Up to 3000 ng/ml
Saliva 1000–10,000 ng/ml
Oral swab 1000–1500 ng/ml
Hair root (plucked) 49–72 ng/μl
Hair (shed) 1.72–1.872 ng/μl
Dandruff 0.8–16.6 ng/particle
Urine 1–20 ng/ml
Bone 3–10 ng/mg
Teeth 21–37 μg/ml
Soft tissues 3–15 μg/mg
Fibroblast cell line 6.5 μg/l
20 Biological Samples: The Target Sources for DNA Typing

individuals as well as different hair types of the same individual. The

approximate amount of DNA content of hair samples varies
between 49 and 72 ng/μl, whereas storage reduces the DNA
content to approximately 1.72–1.87 ng/μl.
The content of total DNA varies greatly among individuals and
teeth types. Other factors influencing the DNA content of a tooth
sample include the tooth type, age of the individual, as well as
health status of the individual. Studies have shown that the teeth
with large pulp volume yield a highest amount of DNA. Thus,
multirooted teeth such as premolars and molars are the suitable
sources to be subjected for DNA test. The number of odontoblasts
in teeth pulp tissues is approximately 11,000/mm2 and that of
fibroblasts is around 1000/mm2.
DNA content of bone samples is really scanty. A fresh bone
sample may contain 3–10 ng/mg of DNA; however, the presence
of high quantity of PCR inhibitors limits their usefulness. With the
increase in duration of exposure in the environment, the DNA
content of a bone is decreased to a substantial level. Despite all
the odds, the usefulness of bone samples for extraction of DNA is
evidenced from its application in archaeological and paleontological
studies. Human urine contains very trace amount of DNA and the
extractable amount of DNA varies greatly with the methodology
used. The average amount of extractable DNA varies from 53 to
200 ng/ml for females and from 3 to 50 ng/ml for males. Similarly,
the DNA content of saliva in children is of lower amounts than in
the adults. Studies have shown the quantity of DNA obtained as
34.91 μg (2.20–122.04 μg) from 3 ml of saliva.
 Chapter 3

Collection, Transportation, and Preservation of Biological

Evidences for DNA Analysis

Application of DNA technology has revolutionized the field of

forensic science. To obtain an appropriate result from any biological
evidence, proper collection, transportation, and preservation/stor-
age strategies should be employed for respective samples without
manipulating the chain of custody (Fig. 3.1). Many factors contrib-
ute to the generation of a good DNA profile from a biological
sample, i.e., the sensitivity of a PCR setup, rate and extent of
degradation, purity, and DNA content of the sample. No biological
evidence is resistant to degradation. Thus, careful collection and
proper storage of the biological samples can provide a useful infor-
mation when subjected to DNA typing analysis. Microbial or envi-
ronmental degradation does not alter the genetic profile of a
subject, but can impact adversely by obtaining a partial DNA profile
from the sample. Evidences suitable for DNA examination are
limited to biological samples; hence, a proper precaution for collec-
tion, transportation, and storage is required for these samples
which has been discussed in details in this chapter.

1 Liquid Blood from Donor

In most of the DNA typing experiments blood samples collected

from the victim as well as the suspect(s) are considered to be the
most suitable samples. Mostly, reference samples are collected in
the form of liquid blood. Liquid blood is collected with the consent
from the donor by a qualified medical practitioner. Certain medical
parameters such as blood or organ transfusion of the donor should
be communicated to the laboratory along with the samples. Blood

H. R. Dash et al., Principles and Practices of DNA Analysis: A Laboratory Manual for Forensic DNA Typing, Springer Protocols
Handbooks, https://doi.org/10.1007/978-1-0716-0274-4_3, © Springer Science+Business Media, LLC, part of Springer Nature 2020

21
22 Collection, Transportation, and Preservation of Biological Evidences. . .

Fig. 3.1 The sequential events unfolded prior to DNA examination of biological
samples

sample of around 2 ml should be collected in duplicates in vacutai-

ners with 5 mM EDTA, sodium citrate, and heparin as anticoagu-
lants. Blood sample can also be collected from the postmortem
dead body in a similar fashion. In such cases, it should be collected
from non-body cavity areas such as heart or major internal blood
vessels till 24 h of last heartbeat. After collection, each tube should
be labeled with date, time, subject’s name, location, collector’s
name, crime and exhibit number, and sealed properly. It should
be immediately refrigerated (not frozen) and reach at the labora-
tory at the earliest.
Reference samples in the form of liquid blood can also be
collected on Whatman FTA® cards with appropriate labeling. Liq-
uid blood can be dropped on the card in a concentric circular
motion inside the marked circular area. In general, <125 μl of
blood should be placed per inch of the circle on FTA card. It is
recommended to dry the card for 3–5 min before placing the cards
separately on individual labeled envelopes. These cards can be
transported to the laboratory at room temperature. The condition
of transportation and storage of biological exhibits has been given
in Fig. 3.2.
 Liquid Blood and Wet Bloodstains from Crime Scene 23

Fig. 3.2 Recommended physiological conditions for transportation and storage of biological evidence for
forensic DNA analysis

2 Liquid Blood and Wet Bloodstains from Crime Scene

In certain instances, prompt visit to crime scene may encounter

the investigating agency with the presence of liquid blood. Liquid
blood can be collected by a sterile syringe and transferred to a
clean tube preferably containing the anticoagulants. Similarly, a
blood clot can be collected with a sterile spatula in a tube. Alter-
natively, the liquid blood present around a blood clot can be
collected by a clean cotton cloth by soaking without touching
the areas containing serum. The collected samples should be
refrigerated and sent to the laboratory at the earliest. Additionally,
the collected samples should be labeled with crime and exhibit
number, date, time and location of sample collection, and name of
the collector. Later seal the samples properly prior to sending
them for laboratory examination. In many cases, liquid blood-
stains can be found on small or large objects at the crime scene,
where the bloodstains should be allowed to dry completely fol-
lowed by their collection without altering their integrity. If the
stain is present on large objects, the bloodstain should be col-
lected using a moistened sterile cotton swab. The swab should be
air-dried completely before packaging followed by proper labeling
and sealing of the object.
24 Collection, Transportation, and Preservation of Biological Evidences. . .

3 Dried Bloodstains

If the dried bloodstains are present on removable items such as

weapons and clothing, the entire object can be collected on separate
containers with proper labeling and sealing. Bloodstains should be
considered to have their distinguished origin and hence they should
be collected separately to avoid contamination. If the stain is present
on solid immovable objects, the pattern of bloodstain should be
documented prior to its collection. The stain can be tape lifted or
scrapped on a clean paper. In such cases, blood crust and tape lifter
should be placed in a druggist fold and sealed in an envelope. If the
condition arises where the bloodstains can be neither lifted nor
scrapped, a sterile cotton swab moistened with saline or water can
be used to elute the bloodstain. After elution, the swab should be
air-dried properly to prevent fungal growth. Additionally, a control
swab collecting sample from the adjacent area without containing
the bloodstains should be sent along with the sample for examina-
tion. If the object having the bloodstain can be cut, the portion of
the item containing the bloodstain should be cut using a sterile sharp
instrument. Additionally, a control piece containing the unstained
portion of the object should also be sent for examination. The
collected samples should be labeled and sealed properly and can be
stored and transported to the laboratory at room temperature.

4 Semen, Seminal Stains, and Evidence from Sexual Assault Victims

Prior to the collection of seminal stains on an object it is always

recommended to document them by either photography, videogra-
phy, or sketching. For the presence of liquid semen, a fresh syringe or
disposable pipette can be used to transfer the liquid to a fresh tube.
The tube containing the liquid semen should be properly labeled,
sealed, and stored under refrigerated condition. In another manner,
the liquid semen can be adsorbed on a sterile cotton cloth, air-dried,
labeled, and sealed properly. Dried seminal stains present on small
movable objects such as clothing, bedsheet, and pillows can be
collected as such. If any of the articles contain stains in wet condition,
it should be thoroughly air-dried prior to its proper labeling and
sealing. If the stain is present on large immovable objects, it should
be either cut using sterile scissor or scrapped using a sterile scalpel,
and kept at sealed container with proper labeling and sealing.
For sexual assault cases, the medical examination of the victim
should be carried out at the earliest. Based upon the condition,
requisite samples such as oral, vaginal, and/or anal swabs should be
collected, air-dried, packed, labeled, and sealed properly. In a simi-
lar way the victim’s clothing can also be preserved. The main reason
to conduct an early medical examination is that victim’s body
initiates breaking down of the seminal fluid through drainage,
enzyme activity, pH, etc. The purpose of air-drying the objects is
 Hair 25

to minimize the growth of microorganisms. Other body fluids such

as urine, saliva, and others should be collected in a similar fashion as
that of the blood and bloodstains.

5 Soft Tissues and Organs

If any tissue or organ sample is collected from the crime scene, it

should be picked up in clean, sterile forceps, and kept separately in a
glass bottle in pre-sterilized normal saline solution without any other
fixatives. Optionally antibiotics (e.g., 0.1 mg/ml gentamicin solution)
can also be added to the normal saline solution to prevent microbial
growth. Each container should be labeled and sealed properly and
stored at room temperature to reach the laboratory at the earliest. If
soft tissues and organs are collected from a postmortem subject with
advanced decomposition state, a portion of deep muscle tissue with
more than one organ should be preserved in the aforementioned
procedure. Preferred organ tissues for collection are heart or brain.
Liver and kidney are generally not recommended samples for DNA
examination. The collected samples should be refrigerated immedi-
ately either with normal saline solution or without any additives. It is
always recommended not to store the tissue samples in formalin.

6 Teeth and Bone

Teeth and bone samples can be preserved from skeletonized struc-

tures after confirming their human origin from the autopsy sur-
geon. 2–4 intact (rooted) molar teeth are recommended for
collection in such cases. Additionally, compact bone (most prefera-
bly femur) should be collected for the purpose of conducting DNA
test. Bone and teeth samples can be stored and transported at room
temperature to the laboratory after proper labeling and sealing.

7 Hair

For examination of hair samples by nuclear DNA analysis, it should

contain the root sheath. If hairs are found in the crime scene, it
should be collected with precaution in any paper packet to be
placed in an envelope. However, it is recommended to submit
15–20 representative hairs to the laboratory as sometimes addi-
tional microscopic examination may be required for the same. The
most important thing to keep in mind is that the hair samples
should be followed by the reference samples (blood/buccal swab)
of the suspect(s) for DNA match.
The guidelines and precautions for collection, transportation,
and preservation of biological samples for DNA examination have
been given in Table 3.1.
Table 3.1
26

Guidelines and precaution for proper collection, transportation, and preservation of biological samples for forensic DNA examination

Biological Physical state/

samples condition Source Collection and preservation Transportation Precaution
Blood Liquid Living person 2–5 ml in duplicates in EDTA vial/FTA Under refrigerated Use of disposable
card condition, to reach syringe
laboratory at the earliest
Liquid Autopsy sample 2–5 ml in duplicates in EDTA vial/
(24–48 h)
FTA card
Liquid Crime scene In duplicates in EDTA vial/FTA card
Wet cloth Crime scene Collect in sterile tube, either add equal Do not handle clothing
volume of normal saline solution or with bare hand
air-dry and transport at room
temperature
Wet Clothing of victim Air-dry thoroughly at room Must reach the laboratory at Do not use direct
and suspect(s) temperature, pack separately the earliest and can be kept sunlight, hot air
at room temperature blower, or heater to
Wet Object such as knife
dry
Dried blood Crust/stain/spatters Crime scene/ Scrap onto a paper and pack in an Document stain
and other unmovable objects envelope; transfer onto a moisten patterns, do not mix
body fluid cotton swab, air-dry and pack in an stains from different
stains envelope, collect proper negative locations
control from adjacent area
Stain Weapon and on small Allow the stains to dry and collect the
Collection, Transportation, and Preservation of Biological Evidences. . .

movable objects whole item

Stain Vehicle, carpet, Cut the stained area, air-dry, collect a
wooden objects negative control from the adjacent
area
Semen Liquid Victim Collect with sterile cotton swab,
air-dry, pack in envelope
Liquid Crime scene
 Tissue/organ Wet/semisolid Dead body/mutilated Tissue samples to be collected in Submit to the laboratory Do not preserve samples
remains normal saline, refrigerate under refrigerated in formalin
condition at the earliest
Bone/teeth Solid Dead body/crime Clean to remove debris, air-dry Must reach the laboratory at Completely charred
scene completely, pack in paper envelope the earliest and can be kept bones are not suitable
at room temperature for DNA examination
Rooted hair Dry Crime scene, weapon, Collect by sterile forceps and pack in Cut hairs are not
victim/suspect(s) envelope suitable for DNA
body, and clothing examination
Source: FSL Bulletin Forensic Science Laboratory, vol. ii, no. iv, June 2008
Hair
27
 Part II

Manual Extraction of DNA from Biological Samples

 Chapter 4

Manual Isolation of DNA from Whole Blood

Objective: To isolate DNA from whole blood (liquid) by manual

isolation method.

1 Introduction

A typical forensic DNA analysis involves the matching of DNA

profile between the subject and the reference samples. Reference
samples are collected from the victim(s) and suspect(s) in terms of
their blood sample with their consent. Hence, ~30–50% of the
samples received in the forensic DNA laboratories constitute the
whole blood samples. Additionally, for paternity dispute cases
blood samples from the alleged father, mother, and child and in
cases involving identification of mutilated remains reference blood
sample from close blood relatives are generally sent for DNA exam-
ination. Whole blood is considered to be the most suitable target
for isolation of nuclear DNA for forensic DNA analysis due to its
high DNA content. Human blood is constituted of solid phase of
formed elements, i.e., erythrocytes (RBC), leukocytes (WBC), and
thrombocytes (platelets) and the plasma. Out of all the constituents
only WBC are the nucleated cells containing the nuclear DNA.
Thus, the amount of nuclear DNA in blood is determined by the
amount of WBCs in a person’s blood. WBC count varies greatly
with the stages of immunosuppression, inflammation, or leukemia
of the individual affecting the DNA content substantially. The
extractable amount of DNA varies greatly with the protocol used
which varies between 30 and 60 ng/μl. Most of the laboratories
receive whole blood in the form of either liquid blood in EDTA
vials or blood spotted on FTA™ cards. If blood has been collected
in EDTA vials, it should be refrigerated (not frozen) and reach the
laboratory at the earliest under refrigerated conditions.

H. R. Dash et al., Principles and Practices of DNA Analysis: A Laboratory Manual for Forensic DNA Typing, Springer Protocols
Handbooks, https://doi.org/10.1007/978-1-0716-0274-4_4, © Springer Science+Business Media, LLC, part of Springer Nature 2020

31
32 Manual Isolation of DNA from Whole Blood

2 Principle

Different techniques are available for isolation of DNA from whole

blood. However, most of the techniques follow the same principle
of separation of WBCs from the whole blood sample and other
cellular debris such as hemoglobin followed by chemical/physical
lysis of cell and nuclear membrane of WBCs to release nuclear DNA
and removal of proteins, carbohydrates, and RNAs from the solu-
tion. The preliminary aim of any DNA isolation technique is to
yield a high quality and quantity of DNA that can be preserved for a
longer period under recommended conditions. The principal steps
of DNA isolation involve the lysis of cell and nucleus which is
achieved by the combining effect of the enzymes and detergents.
Sodium dodecyl sulfate (SDS) is the most common detergent used
in this purpose, whereas proteinase-K is mostly used for enzymatic
degradation of proteins. The commonly used chemical technique
to remove proteins from the DNA involves its denaturation and
extraction into the organic phase when phenol and chloroform are
used. The DNA, insoluble in organic solution, resides in the aque-
ous phase and can be precipitated, washed, and air-dried. Finally it
can be dissolved in aqueous buffer for long-term storage. The
generalized principle of DNA isolation from a eukaryotic cell has
been shown in Fig. 4.1.

3 Reagents Required and Their Role

3.1 Tris-EDTA The combination of Tris and EDTA forms the major constituent of
lysis buffers. Tris acts as the buffering force, whose major role is to
maintain pH of the solution mostly at 8.0. The most common form
of Tris, i.e., Tris(hydroxymethyl) aminomethane with pKa of 8.1,
has its buffering capacity between pH 7 and 9. Tris buffers are
generally temperature sensitive and hence should be stored at
recommended temperature only. Tris also has an additional

Fig. 4.1 Generalized principle of isolation of DNA from a eukaryotic cell

 Reagents Required and Their Role 33

property of interacting with lipopolysaccharides (LPS) in the cell

membrane, thus destabilizing the cell membrane to aid in cell lysis.
Additionally, ethylenediaminetetraacetic acid (EDTA) binds to cer-
tain divalent cations mostly calcium (Ca2+) and magnesium (Mg2+).
These divalent cations are reported to have their key role in the
maintenance of membrane structure and function. Thus, eliminat-
ing these ions by EDTA leads to the lysis of cell membrane.

3.2 SDS Sodium dodecyl sulfate (SDS) is a strong anionic detergent known
to have protein denaturation properties. It helps to remove the
negative ions from proteins to destabilize their structure and con-
firmation. In this process, SDS helps in rupturing the cell as well as
nuclear membrane to release the genetic material into the solution.
Additionally, it helps in releasing DNA from histone and other
DNA-binding proteins. SDS is also known to have an additional
property of removing lipid membranes, thus aiding in the lysis of
cell and nuclear membrane.

3.3 Proteinase-K Peptide bond present near the carboxyl group of amino acids with
blocked alpha amino groups is the cleavage site for the serine
protease, i.e., proteinase-K. It helps in degrading many protein
impurities to yield a good quality of DNA. It also plays an impor-
tant role in the inactivation of nucleases, hence preventing the
isolated DNA from damage. The activity of proteinase-K is greatly
increased by its simultaneous action with SDS and high tempera-
ture. Proteinase-K works optimally at 50–65  C as higher tempera-
ture unfolds certain proteins to ease the function of the enzyme.

3.4 Phenol In most of the cases, phenol is used in Tris-saturated form. Phenol,
being a weak acid, is equilibrated with buffers to make it slightly
alkaline which is the most suitable pH for DNA isolation. These
buffer-saturated phenols contain around 72% of phenol with a
slightly higher density than that of water. Phenol has the property
to denature proteins during their presence in the aqueous solution.
During this process, the nonaqueous component, i.e., protein, gets
separated from the aqueous component, i.e., DNA.

3.5 Chloroform- This is mostly used at a ratio of 24:1. These are detergents that bind
Isoamyl Alcohol to the proteins and lipids to dissolve them in the organic solution.
After dissolving, the solutions help in clumping the protein-lipid
complexes to form a precipitate. Thus, the liquid-liquid extraction
technique involving these chemicals distinguishes between the
physical positions of the cell components, i.e., upper aqueous
phase containing nucleic acids, lower organic phase containing
protein components, and middle phase constituting the lipids.
34 Manual Isolation of DNA from Whole Blood

3.6 Sodium Acetate Salts like sodium or potassium acetate play an important role in
neutralizing the charges on sugar phosphate backbone of DNA,
e.g., sodium acetate breaks into Na+ and CH3COO in solution.
The positive charge of Na+ neutralizes the negative charge of PO3
groups of DNA to make it less hydrophilic and thus less soluble in
water, which leads to dropout of DNA in the solution.

3.7 Isopropyl Alcohol Alcohol precipitation is the most commonly used technique for
desalting DNA followed by its precipitation. DNA is highly insolu-
ble in isopropanol. Isopropanol dissolves in water to form a solu-
tion that causes the DNA in the solution to aggregate and
precipitate. Additionally, isopropanol is used as a better alternative
for ethanol due to its greater potential for DNA precipitation in
lower concentrations. Besides, it takes lesser time to evaporate.

3.8 TE Buffer TE buffer is generally prepared by mixing 50 mM Tris and 50 mM

EDTA at pH 8.0. Tris, the major constituent of TE buffer, acts as a
common pH buffer. Additionally, EDTA chelates cations like Mg2+.
Hence, TE buffer helps to solubilize DNA by protecting it from
degradation.

4 Procedure

1. Take 500 μl of fresh blood or blood preserved in EDTA solu-

tion in a sterilized 2 ml microcentrifuge tube (MCT). Mark the
tube carefully, and cover the marking with cello tape.
2. Add 500 μl of lysis buffer I to the tube containing 500 μl blood,
cover the tube by Parafilm®, and mix thoroughly by vortex.
Freeze the tubes at 70  C for overnight.
3. Take out the sample carefully by keeping it on ice bucket and
incubate the samples immediately in water bath earlier set at
65  C for 10 min (instant heat shock of temperature difference
~100  C lyse the cells).
4. Carefully take out the samples from water bath, and centrifuge
at 10,000 rpm for 10 min at 4  C; discard the supernatant
carefully (at last a jelly-type substance remains).
5. Add 500 μl of lysis buffer II to the pellet, and mix carefully and
properly by vortex. You may use the pipette tip to disrupt the
pellet. Mix till complete mixing of the pellet; otherwise the
yield of DNA may be reduced.
6. Add proteinase-K to make its final concentration as 100 μg/ml
in total volume.
7. Add sodium dodecyl sulfate (SDS) to make its final concentra-
tion as 2% in total volume.
 Observation 35

8. Cover the cap of the tube in Parafilm®, mix thoroughly, and

incubate in water bath at 56  C for 2 h.
9. After 2 h, take the tubes out of the water bath, add 500 μl (1:1
volume) of Tris-saturated phenol (TSP, pH 8.0), and mix
thoroughly for 10 min by gentle shaking.
10. After proper mixing, centrifuge the tube at 10,000 rpm for
10 min at 4  C.
11. Collect the upper aqueous phase by careful pipetting in a
separate MCT (you may use cut tips).
12. Add half of the volume of TSP (250 μl in this case) and
chloroform:isoamyl alcohol (24:1) (250 μl in this case), and
mix thoroughly for 10 min by gentle shaking.
13. After proper mixing, centrifuge the tube at 10,000 rpm for
10 min at 4  C. Transfer the upper aqueous layer to a
fresh MCT.
14. Add 500 μl (1:1 volume) of chloroform:isoamyl alcohol
(24:1), and mix thoroughly for 10 min by gentle shaking.
15. After proper mixing, centrifuge the tube at 10,000 rpm for
10 min at 4  C. Transfer the upper aqueous supernatant to a
fresh MCT.
16. Add 1/30th volume of 3 M sodium acetate (pH 5.5) and equal
volume of chilled isopropyl alcohol.
17. DNA will be precipitated slowly; remove the reagents except
the DNA pellet carefully from the MCT.
18. Wash the DNA pellet three times with 100 μl of 70% ethanol.
19. Vacuum dry the pellet and dissolve the pellet in 50–100 μl of
TE buffer depending on the size of the pellet.
20. Incubate the tube at 56  C for 10 min to remove RNA
contamination.
21. Store the DNA at 20  C for further use.

5 Observation

The quantity of the isolated DNA can be determined at preliminary

level by UV–visible spectrophotometer. For a 1 cm path length, the
optical density at 260 nm (OD260) equals 1.0 for the following
samples:
l 50 μg/ml of double-stranded DNA
l 33 μg/ml of single-stranded DNA
l 20–30 μg/ml of oligonucleotide
l 40 μg/ml of RNA
36 Manual Isolation of DNA from Whole Blood

6 Result Table

Sample DNA content OD260/280 Inference

Control
Sample 1
Sample 2

OD optical density

7 Precautions
l Wear gloves and goggles during manual DNA isolation
procedure.
l The plasticware to be used during the entire procedure should
be pre-sterilized and DNase free.
l Use low-retention plasticware for maximum recovery of the
samples.
l Handle the chemicals carefully, mostly the phenol and chloro-
form. Phenol, being a strong acid, may cause severe burns
whereas chloroform is a known carcinogen.
l Use wide-bore tips by cutting 2–3 mm from the rear end to
prevent mechanical disruption of DNA during pipetting.
l Use filter tips to avoid cross-sample contamination.
l Do not use the TSP solution if the color of the solution has
changed to pink (occurs due to oxidation of phenol). This may
promote nicks in the extracted DNA.
l Pipetting should be done carefully without mixing the two
phases, i.e., upper aqueous phase and lower organic phase, at
any point of time.
l From forensic DNA typing point of view match the name of the
donor, date, crime no., and other details on the tube containing
blood prior to its consumption for DNA isolation.
 Troubleshooting 37

8 Troubleshooting

Problem Tentative cause Possible solutions

Protein More amount of initial Dilute the blood with normal
contamination cells (WBC) in the saline solution prior to the
blood initial step
The extraction step has Repeat the phenol:
not been followed chloroform:isoamyl alcohol
properly extraction step
Blood in coagulated Check the blood for its
condition quality prior to the
extraction steps
RNA High-temperature Incubate the extracted DNA
contamination incubation not given at 56  C for 10 min
to the extracted DNA
RNase not added Add RNase at a final
concentration of 400 μg/
ml to the isolated DNA
sample
No visible DNA DNA quantity very less Incubate the tubes at 20  C
pellet for overnight after the last
obtained step of adding 3 M sodium
acetate and chilled
isopropanol
Repeat the isolation
procedure with increased
amount of initial blood
quantity
Increase the concentration of
proteinase-K and extend
the time of incubation at
56  C
Insoluble pellet Improper drying of the Avoid extended drying under
after DNA precipitate vacuum conditions; DNA
precipitation being acid in nature use a
slightly alkaline solution
such as TE pH 8.0 to
dissolve the precipitate
Degraded DNA Condition of blood not Isolate DNA from coagulated
appropriate blood by treating them as
forensic samples
Blood found
coagulated
Blood stored for longer
time
 Chapter 5

Manual Isolation of DNA from Body Fluid Stains

Objective: To isolate DNA from body fluid stains on clothing.

1 Introduction

Human body fluids are considered to be the major pieces of evi-

dence from forensic analysis point of view as their presence at a
particular location provides useful information to the investigators.
With the advent of DNA technology, the usefulness of body fluid
stains at a crime scene has increased further as DNA typing of these
samples can not only ascertain a suspect, but also exonerate an
innocence. Additionally, the presence of body fluids provides an
indication regarding the sequential events unfolded during the
crime, e.g., presence of bloodstain signifying struggle or murder,
whereas the detection of seminal and/or vaginal fluid indicating
sexual assault.
Biological examination of body fluids involves identification of
body fluids by chemical, enzymatic, and microscopic examination
followed by retrieving the biological materials by either swabbing,
cutting, or scraping. Later, DNA analysis is performed with these
samples to confirm the individual origin of the samples. From
forensic point of view body fluids such as blood can be expected
to be present at crime scene, weapon, and clothing of both
deceased and suspect(s). In this regard, obtaining a consistent
DNA profile from the objects flooded with bloodstains received
from crime scene, deceased, weapon, and suspect(s) can solve a case
of murder. Additionally, for the cases involving sexual assault, semi-
nal stains can be expected at crime scene, victim’s belongings, and
swab of victim’s private parts. In such cases, male DNA profile
obtained from the victim’s belongings can be matched with that
of the suspect(s) to prove the assault. The prerequisite of all the

H. R. Dash et al., Principles and Practices of DNA Analysis: A Laboratory Manual for Forensic DNA Typing, Springer Protocols
Handbooks, https://doi.org/10.1007/978-1-0716-0274-4_5, © Springer Science+Business Media, LLC, part of Springer Nature 2020

39
40 Manual Isolation of DNA from Body Fluid Stains

downstream processing techniques is the isolation of DNA from

these body fluid stains which has been discussed in much details in
this chapter.
Prior to the isolation of DNA from body fluid stains, the
exact nature of origin of the body fluid needs to be ascertained.
Selection of the preliminary examination to be conducted
depends on the nature of the sample and the examiner’s conve-
nience. Some of the presumptive tests of detection of bloodstains
include the oxidation-reduction reactions and colorimetric assays
such as phenolphthalein assay, leucomalachite green (LMG)
assay, and benzidine assay, whereas the confirmatory assays
involve microcrystal assays and other chromatographic and elec-
trophoretic techniques. Similarly, the preliminary tests to be
conducted to ascertain seminal fluid involves acid phosphatase
test, testing of antigens specific to prostate, seminal vesicle,
examination under UV light, or confirmation of the presence of
spermatozoa by microscopic observation.

2 Principle

Dried or wet bloodstains constitute various components of blood

itself along with certain additional chemicals such as humic acid
(if stain is collected from soil or floor) or chemicals constituting the
dye of the clothing on which the stains are present. In a similar
fashion seminal fluids are found along with the mixture of vaginal
fluids and chemicals such as humic acids and dyes. In both the cases,
DNA isolation technique follows the same principle as that of the
DNA isolation from liquid blood by phenol-chloroform extraction
method. The additional step involves the extraction of biological
materials to the liquid medium from the solid adherent surface. In
this process, the clothing harboring body fluid stains can be incu-
bated along with buffer for some time followed by separation of
clothing by squeezing to release the body fluid materials present in
the form of stains into the solution.
Most of the protocols vary in terms of their buffer pH, chemical
compositions, time, and temperature; however, the principle of
DNA isolation by organic extraction method remains the same
for all the samples, i.e., application of SDS to lyse the cell wall
followed by the use of proteinase-K to digest proteins and liquid-
liquid extraction method using phenol-chloroform-isoamyl alco-
hol. As sperm cells are more robust, an additional component
dithiothreitol (DTT) is applied during the step of cell lysis to release
the DNA into the solution.
 Reagents Required and Their Role 41

3 Reagents Required and Their Role

3.1 Tris-EDTA The combination of Tris and EDTA forms the major constituent of
lysis buffers. Tris acts as the buffering force, whose major role is to
maintain pH of the solution mostly at 8.0. The most common form
of Tris, i.e., Tris(hydroxymethyl) aminomethane with pKa of 8.1,
has its buffering capacity between pH 7 and 9. Tris buffers are
generally temperature sensitive and hence should be stored at
recommended temperature only. Tris also has an additional prop-
erty of interacting with lipopolysaccharides (LPS) in the cell mem-
brane, thus destabilizing the cell membrane to aid in cell lysis.
Additionally, ethylenediaminetetraacetic acid (EDTA) binds to cer-
tain divalent cations mostly calcium (Ca2+) and magnesium (Mg2+).
These divalent cations are reported to have their key role in the
maintenance of membrane structure and function. Thus, eliminat-
ing these ions by EDTA leads to the lysis of cell membrane.

3.2 SDS Sodium dodecyl sulfate (SDS) is a strong anionic detergent known
to have protein denaturation properties. It helps to remove the
negative ions from proteins to destabilize their structure and con-
firmation. In this process, SDS helps in rupturing the cell as well as
nuclear membrane to release the genetic material into the solution.
Additionally, it helps in releasing DNA from histone and other
DNA-binding proteins. SDS is also known to have an additional
property of removing lipid membranes and thus aiding in the lysis
of cell and nuclear membrane.

3.3 Proteinase-K Peptide bond present near the carboxyl group of amino acids with
blocked alpha amino groups is the cleavage site for the serine
protease, i.e., proteinase-K. It helps in degrading many protein
impurities to yield a good quality of DNA. It also plays an impor-
tant role in the inactivation of nucleases, hence preventing the
isolated DNA from damage. The activity of proteinase-K is greatly
increased by its simultaneous action with SDS and high tempera-
ture. Proteinase-K works optimally at 50–65  C as higher tempera-
ture unfolds certain proteins to ease the function of the enzyme.

3.4 DTT Dithiothreitol (DTT) prevents the formation of intramolecular and

intermolecular disulfide bonds among cysteine residues. With pro-
tein disulfide bridges being the major constituent of sperm nuclear
membrane, DTT is essential during lysis of sperm cells to release
sperm DNA. Hence, during the lysis of sperm cells, in addition to
proteinase-K and SDS, DTT is also used in the mixture for their
effective lysis.

3.5 Phenol In most of the cases, phenol is used in Tris-saturated form. Phenol,
being a weak acid, is equilibrated with buffers to make it slightly
42 Manual Isolation of DNA from Body Fluid Stains

alkaline which is the most suitable pH for DNA isolation. These

buffer-saturated phenols contain around 72% of phenol with a
slightly higher density than that of water. Phenol has the property
to denature proteins during their presence in the aqueous solution.
During this process, the nonaqueous component, i.e., protein, gets
separated from the aqueous component, i.e., DNA.

3.6 Chloroform- This is mostly used at a ratio of 24:1. These are detergents that bind
Isoamyl Alcohol to the proteins and lipids to dissolve them in the organic solution.
After dissolving, the solutions help in clumping the protein-lipid
complexes to form a precipitate. Thus, the liquid-liquid extraction
technique involving these chemicals distinguishes between the
physical positions of the cell components, i.e., upper aqueous
phase containing nucleic acids, lower organic phase containing
protein components, and middle phase constituting the lipids.

3.7 TE Buffer TE buffer is generally prepared by mixing 50 mM Tris and 50 mM

EDTA at pH 8.0. Tris, the major constituent of TE buffer, acts as a
common pH buffer. Additionally, EDTA chelates cations like Mg2+.
Hence, TE buffer helps to solubilize DNA by protecting it from
degradation.

4 Procedure

1. Cut the clothing containing suspected stains into small pieces

of 0.5 cm  0.5 cm, place them in a properly labeled sterilized
2 ml microcentrifuge tube (MCT), and cover the labeling with
cello tape.
2. Add 500 μl of forensic buffer to the tube containing stain
pieces, and mix thoroughly by vortex.
3. Incubate the samples at 37  C for 3–4 h.
4. Squeeze the clothing into the same tube with the help of a
disposable syringe; now the tube contains the extracts of the
stains in 500 μl of forensic buffer.
5. Add proteinase-K to make its final concentration as 100 μg/ml
in total volume [in this case, add 2.5 μl from the 20 mg/ml
stock solution].
6. Add sodium dodecyl sulfate (SDS) to make its final concentra-
tion as 2% in total volume [in this case, add 500 μl from the 20%
stock solution].
7. If the extract contains suspected seminal fluid, optionally add
DTT as 150 mM in total volume.
8. Cover the cap of the tube in Parafilm®, mix thoroughly, and
incubate in water bath at 56  C for 2–4 h.
 Observation 43

9. After 2–4 h, take the samples out of the water bath, add 500 μl
(1:1 volume) of Tris-saturated phenol (TSP, pH 8.0), and mix
thoroughly for 10 min by gentle shaking.
10. After proper mixing, centrifuge the tube at 10,000 rpm for
10 min at 4  C.
11. Collect the upper aqueous phase by careful pipetting in a
separate MCT (you may use cut tips).
12. Add half of the volume of TSP (250 μl in this case) and
chloroform:isoamyl alcohol (24:1) (250 μl in this case), and
mix thoroughly for 10 min by gentle shaking.
13. After proper mixing, centrifuge the tube at 10,000 rpm for
10 min at 4  C. Transfer the upper aqueous layer to a
fresh MCT.
14. Add 500 μl (1:1 volume) of chloroform:isoamyl alcohol
(24:1), and mix thoroughly for 10 min by gentle shaking.
15. After proper mixing, centrifuge the tube at 10,000 rpm for
10 min at 4  C.
16. Keep an Amicon® Ultra-0.5 ml 30 kDa centrifugal filters into a
2.0 ml MCT.
17. Transfer the upper aqueous supernatant to the filter device and
cap it.
18. Spin the device at 5000  g for 10 min at 20  C. Discard the
flow through.
19. Add 500 μl of Milli-Q to the device for washing and repeat
spinning of the device at 5000  g for 10 min at 20  C.
20. Remove the assembled device and to recover the concentrated
solute place the Amicon® Ultra-0.5 ml 30 kDa centrifugal filter
in a fresh MCT upside down.
21. Counterbalance with a similar device and spin at 1000  g for
2 min at 20  C.
22. Store the DNA at 20  C for further use.

5 Observation

The quantity of the isolated DNA can be determined at preliminary

level by UV–visible spectrophotometer. For a 1 cm path length, the
optical density at 260 nm (OD260) equals 1.0 for the following
samples:
l 50 μg/ml of double-stranded DNA
l 33 μg/ml of single-stranded DNA
l 20–30 μg/ml of oligonucleotide
l 40 μg/ml of RNA
44 Manual Isolation of DNA from Body Fluid Stains

6 Result Table

Sample DNA content OD260/280 Inference

Control
Sample 1 (bloodstain)
Sample 2 (seminal stain)

OD optical density

7 Precautions
l Wear gloves and goggles during manual DNA isolation
procedure.
l The plasticware to be used during the entire procedure should
be sterilized and DNase free.
l Handle the chemicals carefully, mostly the phenol and chloro-
form. Phenol, being a strong acid, may cause severe burns
whereas chloroform is a known carcinogen.
l Use wide-bore tips by cutting 2–3 mm from the rear end to
prevent mechanical disruption of DNA during pipetting.
l Use filter tips to avoid cross-sample contamination.
l Do not use the TSP solution if the color of the solution has
changed to pink (occurs due to oxidation of phenol). This may
promote nicks in the extracted DNA.
l Pipetting should be done carefully without mixing the two
phases, i.e., upper aqueous phase and lower organic phase, at
any point of time.
l Perform the preliminary and confirmatory tests of the body
fluids prior to the DNA isolation from them.
l Do not overincubate the stained clothing for stain extraction. It
may extract dyes from the clothing to the extract.
l Note down the pattern of stains found on the forensic evidence
before cutting them.
 Troubleshooting 45

8 Troubleshooting

Problem Tentative cause Possible solutions

Protein The extraction step has Repeat the
contamination not been followed phenol:chloroform:isoamyl
properly alcohol extraction step
RNA High-temperature Incubate the extracted DNA
contamination incubation not given at 56  C for 10 min
to the extracted DNA
RNase not added Add RNase at a final
concentration of 400 μg/
ml to the isolated DNA
sample
Colored DNA Extended incubation Avoid extended incubation
extract during extraction of during stain extraction
stains
Nature of the clothing Use alternative DNA isolation
methods such as magnetic
bead-based extraction
 Chapter 6

Manual Isolation of DNA from Soft-Tissue Samples

Objective: To isolate DNA from soft-tissue samples.

1 Introduction

Human tissues, either preserved or dried, are the most common

biological evidence for DNA examination. The most common
among the tissue samples received at laboratories include the fetus
of early stage of development or the product of conception aborted
illegally. Other forms of tissue samples received include tissue frag-
ments attached to skeletal remains, formalin-preserved organs and
tissues, and paraffin blocks containing tissues collected earlier for
pathological diagnostics. During autopsy, viscera of the deceased
are collected for toxicological analysis, which can also be used for
DNA examination during nonavailability of other samples if stored
and preserved properly.
Soft tissues are recommended to be preserved at 80  C after
their collection, failure of which may affect the quality as well as
quantity of DNA necessary for DNA analysis. Additionally, putrefac-
tion of human corpse also affects adversely the DNA content of the
preserved tissues which is a major challenge in many varying climatic
conditions. Other factors contributing to the DNA analysis of human
corpse from soft tissues include mutilation of skeletal remains in
runover situations, contamination of mutilated remains, and exposure
of corpse to extreme environmental conditions such as mass disaster.
Formaldehyde is a common preservative for histopathological
analysis of tissue samples. However, formalin can affect adversely
the DNA content of a cell by either fragmentation, nucleotide base
modification, or cross-linking between DNA and protein or DNA
itself. Such fragmented highly damaged DNA poses a huge chal-
lenge for DNA typing experiments. Though many protocols have
been developed for isolation of DNA from formalin-fixed tissues,

H. R. Dash et al., Principles and Practices of DNA Analysis: A Laboratory Manual for Forensic DNA Typing, Springer Protocols
Handbooks, https://doi.org/10.1007/978-1-0716-0274-4_6, © Springer Science+Business Media, LLC, part of Springer Nature 2020

47
48 Manual Isolation of DNA from Soft-Tissue Samples

still it is not considered as a suitable preservative for tissue samples

to be used for forensic DNA typing. In this regard, sterilized
normal saline solution is recommended for tissue sample preserva-
tion. Optionally antibiotics (e.g., 0.1 mg/ml gentamicin solution)
can also be added to the normal saline solution for proper preser-
vation of soft-tissue samples. Tissue samples can also be stored
without any preservative at 70  C till further use.

2 Principle

Fresh soft tissues contain abundant cellular materials of parenchy-

mal cells which are a huge source of DNA. Certain types of soft
tissues such as cervical lymph nodes and muscle are the preferred
samples for DNA extraction. These fresh tissues should be excised
using sterile scalpel from fresh cadavers followed by their homoge-
nization using a sterilized mill to prevent cross contamination.
However, utmost precaution should be taken while dealing with
putrefied soft-tissue samples. These samples may contain a huge
amount of DNA, but care should be taken to eliminate the PCR
inhibitors from these kind of samples.
The first step during DNA fingerprinting analysis involves the
isolation of DNA from tissue sample. Though many commercial
kits and technologies are available for isolation of DNA from tissue
samples, traditional organic extraction method has a huge advan-
tage over other techniques. In this technique, tissue samples are
disaggregated by physical and chemical techniques followed by cell
lysis using detergents and proteinase-K for protein digestion. Later,
a liquid-liquid extraction method is employed by using phenol,
chloroform, and isoamyl alcohol targeting differential solubility of
proteins, lipids, and nucleic acids. Finally, DNA present in the
aqueous phase is concentrated as well as purified by the use of salt
and ethanol, followed by its resolubilization in Tris-EDTA buffer.

3 Reagents Required and Their Role

3.1 Tris-EDTA The combination of Tris and EDTA forms the major constituent of
lysis buffers. Tris acts as the buffering force, whose major role is to
maintain the pH of the solution mostly at 8.0. The most common
form of Tris, i.e., Tris(hydroxymethyl) aminomethane with pKa of
8.1, has its buffering capacity between pH 7 and 9. Tris buffers are
generally temperature sensitive and hence should be stored at
recommended temperature only. Tris also has an additional prop-
erty of interacting with lipopolysaccharides (LPS) in the cell mem-
brane, thus destabilizing the cell membrane to aid in cell lysis.
Additionally, ethylenediaminetetraacetic acid (EDTA) binds to cer-
tain divalent cations mostly calcium (Ca2+) and magnesium (Mg2+).
These divalent cations are reported to have their key role in the
 Reagents Required and Their Role 49

maintenance of membrane structure and function. Thus, eliminat-

ing these ions by EDTA leads to the lysis of cell membrane.

3.2 SDS Sodium dodecyl sulfate (SDS) is a strong anionic detergent known
to have protein denaturation properties. It helps to remove the
negative ions from proteins to destabilize their structure and con-
firmation. In this process, SDS helps in rupturing the cell as well as
nuclear membrane to release the genetic material into the solution.
Additionally, it helps in releasing DNA from histone and other
DNA-binding proteins. SDS is also known to have an additional
property of removing lipid membranes, thus aiding in the lysis of
cell and nuclear membrane.

3.3 Proteinase-K Peptide bond present near the carboxyl group of amino acids with
blocked alpha amino groups is the cleavage site for the serine
protease, i.e., proteinase-K. It helps in degrading many protein
impurities to yield a good quality of DNA. It also plays an impor-
tant role in the inactivation of nucleases, hence preventing the
isolated DNA from damage. The activity of proteinase-K is greatly
increased by its simultaneous action with SDS and high tempera-
ture. Proteinase-K works optimally at 50–65  C as higher tempera-
ture unfolds certain proteins to ease the function of the enzyme.

3.4 Liquid Nitrogen Liquid nitrogen is the prime requirement during mechanical
homogenization of the tissue samples by cryogenic grinding. Dur-
ing this process, the tissue samples are snap frozen by dropping
them on a beaker of liquid nitrogen. After grinding, the samples
turn into small particle generating higher surface area for the action
of chemical reagents for lysis of cell wall. However, the disadvantage
of using liquid nitrogen-based grinding is the chance of sample loss
during grinding of low-quantity samples.

3.5 Phenol In most of the cases, phenol is used in Tris-saturated form. Phenol,
being a weak acid, is equilibrated with buffers to make it slightly
alkaline which is the most suitable pH for DNA isolation. These
buffer-saturated phenols contain around 72% of phenol with a
slightly higher density than that of water. Phenol has the property
to denature proteins during their presence in the aqueous solution.
During this process, the nonaqueous component, i.e., protein, gets
separated from the aqueous component, i.e., DNA.

3.6 Chloroform- This is mostly used at a ratio of 24:1. These are detergents that bind
Isoamyl Alcohol to the proteins and lipids to dissolve them in the organic solution.
After dissolving, the solutions help in clumping the protein-lipid
complexes to form a precipitate. Thus, the liquid-liquid extraction
technique involving these chemicals distinguishes between the
physical positions of the cell components, i.e., upper aqueous
phase containing nucleic acids, lower organic phase containing
protein components, and middle phase constituting the lipids.
50 Manual Isolation of DNA from Soft-Tissue Samples

3.7 TE Buffer TE buffer is generally prepared by mixing 50 mM Tris and 50 mM

EDTA at pH 8.0. Tris, the major constituent of TE buffer, acts as a
common pH buffer. Additionally, EDTA chelates cations like Mg2+.
Hence, TE buffer helps to solubilize DNA by protecting it from
degradation.

4 Procedure

1. Cut solid tissue samples of size 3 mm3 into small pieces of

around 1–2 mm using sterile scalpel.
2. Either process these tissue samples immediately within 2 h or
store them at 80  C till further use.
3. Put the tissue samples in a clean mortar and dip it with liquid
nitrogen. Using a clean pestle, grind the frozen tissue into small
pieces.
4. Allow liquid nitrogen to evaporate leaving dry frozen grinded
tissue in the mortar.
5. Add 500 μl of forensic buffer to the mortar, containing tissue
powder, and wait till the tissue powder to dissolve completely
in the buffer.
6. Resuspend the samples in buffer completely by gentle pipetting
and transfer it to a 2.0 ml MCT.
7. Add proteinase-K to make its final concentration as 100 μg/ml
in total volume.
8. Add sodium dodecyl sulfate (SDS) to make its final concentra-
tion as 2% in total volume.
9. Cover the cap of the tube in Parafilm®, mix thoroughly, and
incubate in water bath at 56  C for 2–4 h.
10. After 2–4 h, take the samples out of the water bath, add 500 μl
(1:1 volume) of Tris-saturated phenol (TSP, pH 8.0), and mix
thoroughly for 10 min by gentle shaking.
11. After proper mixing, centrifuge the tube at 10,000 rpm for
10 min at 4  C.
12. Collect the upper aqueous phase by careful pipetting in a
separate MCT (you may use cut tips).
13. Add half of the volume of TSP (250 μl in this case) and
chloroform:isoamyl alcohol (24:1) (250 μl in this case), and
mix thoroughly for 10 min by gentle shaking.
14. After proper mixing, centrifuge the tube at 10,000 rpm for
10 min at 4  C. Transfer the upper aqueous layer to a fresh MCT.
15. Add 500 μl (1:1 volume) of chloroform:isoamyl alcohol
(24:1), and mix thoroughly for 10 min by gentle shaking.
16. After proper mixing, centrifuge the tube at 10,000 rpm for
10 min at 4  C.
 Precautions 51

17. Keep an Amicon® Ultra-0.5 ml 30 kDa centrifugal filters into a

2.0 ml MCT.
18. Transfer the upper aqueous supernatant to the filter device and
cap it.
19. Spin the device at 5000  g for 10 min at 20  C. Discard the
flow through.
20. Add 500 μl of Milli-Q to the device for washing and repeat
spinning of the device at 5000  g for 10 min at 20  C.
21. Remove the assembled device and to recover the concentrated
solute place the Amicon® Ultra-0.5 ml 30 kDa centrifugal filter
in a fresh MCT upside down.
22. Counterbalance with a similar device and spin at 1000  g for
2 min at 20  C.
23. Store the DNA at 20  C for further use.

5 Observation

The quantity of the isolated DNA can be determined at preliminary

level by UV–visible spectrophotometer. For a 1 cm path length, the
optical density at 260 nm (OD260) equals 1.0 for the following
samples:
l 50 μg/ml of double-stranded DNA
l 33 μg/ml of single-stranded DNA
l 20–30 μg/ml of oligonucleotide
l 40 μg/ml of RNA

6 Result Table

Sample DNA content OD260/280 Inference

Control
Sample 1 ( fresh tissue)
Sample 2 (decomposed tissue)

OD optical density

7 Precautions
l Wear gloves and goggles during manual DNA isolation procedure.
l The plasticware to be used during the entire procedure should
be sterilized and DNase free.
52 Manual Isolation of DNA from Soft-Tissue Samples

l Handle the chemicals carefully, mostly the phenol and chloro-

form. Phenol, being a strong acid, may cause severe burns
whereas chloroform is a known carcinogen.
l Use wide-bore tips by cutting 2–3 mm from the rear end to
prevent mechanical disruption of DNA during pipetting.
l Use filter tips to avoid cross-sample contamination.
l Do not use the TSP solution if the color of the solution has
changed to pink (occurs due to oxidation of phenol). This may
promote nicks in the extracted DNA.
l Pipetting should be done carefully without mixing the two
phases, i.e., upper aqueous phase and lower organic phase, at
any point of time.
l During grinding, cover the mouth of the mortar using a paper
cloth to avoid spillage of tissue fragments.
l While grinding, work inside a hood to protect yourself from
aerosolized powder.
l Handle liquid nitrogen carefully.

8 Troubleshooting

Problem Tentative cause Possible solutions

Protein The extraction step has Repeat the phenol:
contamination not been followed chloroform:isoamyl alcohol
properly extraction step
RNA High-temperature Incubate the extracted DNA
contamination incubation not given at 56  C for 10 min
to the extracted DNA
RNase not added Add RNase at a final
concentration of 400 μg/
ml to the isolated DNA
sample
PCR product Presence of PCR Clean the samples properly; if
not amplified inhibitors possible wash the tissue
samples with sterilized
normal saline solution prior
to their processing
Partial DNA Tissue stored in formalin Do not process tissue samples
profile stored in formalin, and use
obtained alternative protocols
 Chapter 7

Manual Isolation of DNA from Rooted Hair Samples

Objective: To isolate DNA from rooted hair samples.

1 Introduction

Human hair originating from either scalp or pubic region is a great

source of biological evidence for forensic implications. Earlier days,
hair examination was compromising of their morphological and
microscopic analysis for comparison. However, with the advent of
PCR technology, the DNA content of hairs is regarded as the most
useful source of biological evidence from crime scene. The small
quantity of DNA present in the hair has been highly acknowledged
for their application in individualization.
A typical human hair has two parts, i.e., root and shaft. Root
anchors the hair on the skin whereas the shaft grows outside the
skin surface to be seen outside. Medulla is the center position of
hair which is surrounded by cortex and the whole hair is protected
by layers of dead keratinized cells called as cuticle. Hairs are origi-
nated from the follicles which are located at dermis and epidermis.
Many nerves and capillaries supplying nutrients open up at the base
of the hair root known as papilla and are surrounded by hair bulb.
As any cell contains many copies of mitochondrial DNA, they
can be successfully extracted from both hair roots and shafts. Sub-
sequently, the sequence polymorphism of mitochondrial DNA can
be analyzed to discriminate among individuals. However, as mito-
chondrial DNA is inherited matrilineally, they are not a suitable
candidate for analysis in paternity testing as well as individualization
with superior degree of resolution. Hence, analysis of nuclear DNA
from the hairs is of great interest from forensic DNA analysis point
of view. Freshly plucked hairs with roots are of great use in nuclear
DNA analysis as cells of root region contain higher amount of such
DNA. Hair shafts or naturally shed hairs contain very less DNA in

H. R. Dash et al., Principles and Practices of DNA Analysis: A Laboratory Manual for Forensic DNA Typing, Springer Protocols
Handbooks, https://doi.org/10.1007/978-1-0716-0274-4_7, © Springer Science+Business Media, LLC, part of Springer Nature 2020

53
54 Manual Isolation of DNA from Rooted Hair Samples

comparison to the DNA content of root in freshly plucked hairs.

The average DNA content of a single hair and its surrounding cells
is 0.5 μg. Most of the hairs found in crime scene are generally shed
hairs, which contain very less amount of DNA. Thus, it is always
recommended to use multiple number of hairs simultaneously for
DNA isolation. Additionally, many studies have recovered nuclear
DNA from hair shaft; however, the quantity is considerably less in
comparison to the hair roots.
In spite of the presence of high quantity of nuclear DNA in
anagenic (growth phase) hairs and presence of less quantity of DNA
in telogenic (resting phase) hairs, hairs are always useful from
forensic analysis point of view. In certain instances, hairs devoid of
roots, i.e., shaft part found in crime scene, also yield sufficient
amount of nuclear DNA due to the presence of blood on these
hairs. Additionally, a common form of skin alteration, i.e., dan-
druff, can also be used as a source of DNA for DNA typing analysis
as the quantity of DNA varies greatly from 0.8 to 16.6 ng per
dandruff particle.

2 Principle

Hair follicles of plucked hairs contain certain amount of epithelial

cells and hence contain a huge amount of DNA. Hairs can be
transferred by direct contact, and struggle followed by their deposit
on the clothing, on an object, or at the crime scene. Preliminary
microscopic examination of the presence or absence of hair follicles
should be performed prior to their consumption for DNA isola-
tion. Hairs found on the objects such as hat, blanket, and pillow can
also be targeted as forensic evidence. These samples should always
be analyzed along with the reference samples for matching of DNA
profile.
Though hair shaft contains few amount of DNA, isolation of
DNA targets mostly the hair roots which contain a higher amount
of DNA. In the first step of DNA fingerprinting analysis it is
necessary to isolate sufficient quantity as well as quality of DNA
from the hair samples. In traditional method of DNA isolation from
hair samples, preliminarily cells are digested using detergents and
proteinase-K for protein digestion-based cell disruption. Subse-
quently, the liquid-liquid extraction method is followed employing
phenol, chloroform, and isoamyl alcohol targeting differential sol-
ubility of proteins, lipids, and nucleic acids. Lastly, DNA is concen-
trated and purified by using salt and ethanol. Optionally,
resolubilization of the extracted DNA is performed in Tris-EDTA
buffer.
 Reagents Required and Their Role 55

3 Reagents Required and Their Role

3.1 Tris-EDTA The combination of Tris and EDTA forms the major constituent of
lysis buffers. Tris acts as the buffering force, whose major role is to
maintain pH of the solution mostly at 8.0. The most common form
of Tris, i.e., Tris(hydroxymethyl) aminomethane with pKa of 8.1,
has its buffering capacity between pH 7 and 9. Tris buffers are
generally temperature sensitive and hence should be stored at
recommended temperature only. Tris also has an additional prop-
erty of interacting with lipopolysaccharides (LPS) in the cell mem-
brane, thus destabilizing the cell membrane to aid in cell lysis.
Additionally, ethylenediaminetetraacetic acid (EDTA) binds to cer-
tain divalent cations mostly calcium (Ca2+) and magnesium (Mg2+).
These divalent cations are reported to have their key role in the
maintenance of membrane structure and function. Thus, eliminat-
ing these ions by EDTA leads to the lysis of cell membrane.

3.2 SDS Sodium dodecyl sulfate (SDS) is a strong anionic detergent known
to have protein denaturation properties. It helps to remove the
negative ions from proteins to destabilize their structure and con-
firmation. In this process, SDS helps in rupturing the cell as well as
nuclear membrane to release the genetic material into the solution.
Additionally, it helps in releasing DNA from histone and other
DNA-binding proteins. SDS is also known to have an additional
property of removing lipid membranes, thus aiding in the lysis of
cell and nuclear membrane.

3.3 Proteinase-K Peptide bond present near the carboxyl group of amino acids with
blocked alpha amino groups is the cleavage site for the serine
protease, i.e., proteinase-K. It helps in degrading many protein
impurities to yield a good quality of DNA. It also plays an impor-
tant role in the inactivation of nucleases, hence preventing the
isolated DNA from damage. The activity of proteinase-K is greatly
increased by its simultaneous action with SDS and high tempera-
ture. Proteinase-K works optimally at 50–65  C as higher tempera-
ture unfolds certain proteins to ease the function of the enzyme.

3.4 Phenol In most of the cases, phenol is used in Tris-saturated form. Phenol,
being a weak acid, is equilibrated with buffers to make it slightly
alkaline which is the most suitable pH for DNA isolation. These
buffer-saturated phenols contain around 72% of phenol with a
slightly higher density than that of water. Phenol has the property
to denature proteins during their presence in the aqueous solution.
During this process, the nonaqueous component, i.e., protein, gets
separated from the aqueous component, i.e., DNA.
56 Manual Isolation of DNA from Rooted Hair Samples

3.5 Chloroform- This is mostly used at a ratio of 24:1. These are detergents that bind
Isoamyl Alcohol to the proteins and lipids to dissolve them in the organic solution.
After dissolving, the solutions help in clumping the protein-lipid
complexes to form a precipitate. Thus, the liquid-liquid extraction
technique involving these chemicals distinguishes between the
physical positions of the cell components, i.e., upper aqueous
phase containing nucleic acids, lower organic phase containing
protein components, and middle phase constituting the lipids.

3.6 TE Buffer TE buffer is generally prepared by mixing 50 mM Tris and 50 mM

EDTA at pH 8.0. Tris, the major constituent of TE buffer, acts as a
common pH buffer. Additionally, EDTA chelates cations like Mg2+.
Hence, TE buffer helps to solubilize DNA by protecting it from
degradation.

4 Procedure

1. Examine the hairs carefully (may be under microscope) for the

presence of hair root.
2. Wash the hairs with sterilized deionized water to remove the
debris associated with the hairs.
3. Take 2–3 rooted hairs, cut 1 cm portion of the hair from the
root end, and place it in a labeled microcentrifuge tube.
4. Add 500 μl of hair digestion buffer (for composition see the
Appendix section), and make sure that the hairs are dipped
inside the buffer.
5. Add proteinase-K to make its final concentration as 200 μg/ml
in total volume.
6. Cover the cap of the tube in Parafilm®, mix thoroughly, and
incubate in water bath at 56  C for 2–4 h.
7. After 2–4 h, take the samples out of the water bath, add 500 μl
(1:1 volume) of Tris-saturated phenol (TSP, pH 8.0), and mix
thoroughly for 10 min by gentle shaking.
8. After proper mixing, centrifuge the tube at 10,000 rpm for
10 min at 4  C.
9. Collect the upper aqueous phase by careful pipetting in a
separate MCT (you may use cut tips).
10. Add half of the volume of TSP (250 μl in this case) and
chloroform:isoamyl alcohol (24:1) (250 μl in this case), and
mix thoroughly for 10 min by gentle shaking.
11. After proper mixing, centrifuge the tube at 10,000 rpm for
10 min at 4  C. Transfer the upper aqueous layer to a
fresh MCT.
 Result Table 57

12. Add 500 μl (1:1 volume) of chloroform:isoamyl alcohol

(24:1), and mix thoroughly for 10 min by gentle shaking.
13. After proper mixing, centrifuge the tube at 10,000 rpm for
10 min at 4  C.
14. Keep an Amicon® Ultra-0.5 ml 30 K centrifugal filters into a
2.0 ml MCT.
15. Transfer the upper aqueous supernatant to the filter device and
cap it.
16. Spin the device at 5000  g for 10 min at 20  C. Discard the
flow through.
17. Add 500 μl of Milli-Q to the device for washing and repeat
spinning of the device at 5000  g for 10 min at 20  C.
18. Remove the assembled device and to recover the concentrated
solute place the Amicon® Ultra-0.5 ml 30 K centrifugal filter in
a fresh MCT upside down.
19. Counterbalance with a similar device and spin at 1000  g for
2 min at 20  C.
20. Store the DNA at 20  C for further use.

5 Observation

The quantity of the isolated DNA can be determined at preliminary

level by UV–visible spectrophotometer. For a 1 cm path length, the
optical density at 260 nm (OD260) equals 1.0 for the following
samples:
l 50 μg/ml of double-stranded DNA
l 33 μg/ml of single-stranded DNA
l 20-30 μg/ml of oligonucleotide
l 40 μg/ml of RNA

6 Result Table

Sample DNA content OD260/280 Inference

Control
Sample 1 (hair root)
Sample 2 (hair shaft)

OD optical density
58 Manual Isolation of DNA from Rooted Hair Samples

7 Precautions
l Wear gloves and goggles during manual DNA isolation
procedure.
l The plasticware to be used during the entire procedure should
be sterilized and DNase free.
l Handle the chemicals carefully, mostly the phenol and chloro-
form. Phenol, being a strong acid, may cause severe burns
whereas chloroform is a known carcinogen.
l Use wide-bore tips by cutting 2–3 mm from the rear end to
prevent mechanical disruption of DNA during pipetting.
l Use filter tips to avoid cross-sample contamination.
l Do not use the TSP solution if the color of the solution has
changed to pink (occurs due to oxidation of phenol). This may
promote nicks in the extracted DNA.
l Pipetting should be done carefully without mixing the two
phases, i.e., upper aqueous phase and lower organic phase, at
any point of time.
l Check the presence or absence of root hair prior to the isolation
procedure.
l Check for the presence/absence of any body fluids associated
with the hairs prior to DNA isolation.
l Do not extract DNA from hair samples and reference blood
samples simultaneously.

8 Troubleshooting

Problem Tentative cause Possible solutions

Protein The extraction step has Repeat the phenol:
contamination not been followed chloroform:isoamyl alcohol
properly extraction step
RNA High-temperature Incubate the extracted DNA
contamination incubation not given at 56  C for 10 min
to the extracted DNA
RNase not added Add RNase at a final
concentration of
400 μg/ml to the isolated
DNA sample
PCR product Presence of PCR Clean the samples properly; if
not amplified inhibitors possible wash the hairs
repeatedly with sterilized
deionized water prior to
their processing

(continued)
 Troubleshooting 59

Problem Tentative cause Possible solutions

Mixed DNA Presence of body fluids Wash the samples properly
profile along with hairs before the isolation
obtained procedure
Hairs originating from Process individual hair
more than one samples separately
individual
 Chapter 8

Manual Isolation of DNA from Human Teeth

Objective: To isolate DNA from tooth samples.

1 Introduction

Human teeth are chosen to be the most suitable part of highly

degraded human remains for forensic identification purposes.
Human teeth have a unique composition and their location within
the jawbone protects them from environmental degradations over
the years. DNA obtained from the tooth samples is of superior
quantity and quality in comparison to bone samples. Additionally,
DNA is preserved in teeth for a very long time and becomes a useful
tool for ancient DNA examination of samples
100–10,000 years old.
Human embryonic development gives rise to two types of
teeth, i.e., 20 numbers of deciduous or primary teeth, which are
later replaced by 32 numbers of secondary or permanent dentition.
The major structure of a tooth is composed of the mineralized
matrix called as dentin which does not contain any cells. The
crown of the dentin is covered by a layer of tooth enamel consti-
tuted of crystalline form of calcium phosphate. The interior cham-
ber of tooth, i.e., central pulp cavity, is connected with the nerves
and blood vessels through the root canal. It is the major source of
DNA in tooth samples. Human teeth have been divided into four
types, i.e., incisors, canines, premolars, and molars. Both incisors
and canines constitute single roots, whereas premolars have one or
two roots. However, the molars have three or more roots, thus
projecting them to be a huge potential source of DNA for further
analysis.
Characterization of teeth and their alignment inside mouth
provide a useful information regarding the identification of an
unknown person from radiographic and biochemical point of

H. R. Dash et al., Principles and Practices of DNA Analysis: A Laboratory Manual for Forensic DNA Typing, Springer Protocols
Handbooks, https://doi.org/10.1007/978-1-0716-0274-4_8, © Springer Science+Business Media, LLC, part of Springer Nature 2020

61
62 Manual Isolation of DNA from Human Teeth

view. From forensic dentistry point of view, a victim or accused can

be connected by the analysis of dental remains such as X-rays and
dental casts as well as the analysis of bite marks. However, DNA
fingerprinting is the most useful tool for forensic analysis of tooth
in cases of nonavailability of dental records or suitable controls for
comparison. A good quality and quantity of DNA are expected
from the dental pulp tissue. Hence, it is always recommended to
collect rooted, intact teeth mostly with multiple teeth roots, which
can be preserved and transported at room temperature.

2 Principle

DNA is primarily present at the inner surface, i.e., dental pulp of the
teeth. Hence, the preliminary step during isolation of DNA from
tooth samples is to break open the teeth to expose their inner
surface. In this context, various steps have been exploited starting
from sectioning of teeth at cementoenamel junction horizontally or
vertically up to the root tip. Few other techniques involve the
crushing, cryogenic grinding, or retrieval of dental pulp by various
techniques. The technique involved should be examined carefully
for minimal/no introduction of contaminants as well as inhibitors.
Other environmental factors such as time, temperature, humidity,
light, and other chemical substances should also be assessed prior to
the isolation of DNA from the tooth sample.
DNA isolation of tooth sample involves three different stages,
i.e., lysis of the cells followed by denaturation and inactivation of
proteins and finally extraction of DNA. Though many commercial
kits and technologies are available for isolation of DNA from tooth
samples, traditional organic extraction method has a huge advan-
tage over other techniques. The tooth samples with exposed root
canals are subjected to lysis using detergents and proteinase-K by
protein digestion. Later, a liquid-liquid extraction method is
employed by using phenol, chloroform, and isoamyl alcohol target-
ing differential solubility of proteins, lipids, and nucleic acids.
Finally, DNA present in the aqueous phase is concentrated as well
as purified by the use of salt and ethanol, followed by its resolubi-
lization in Tris-EDTA buffer.

3 Reagents Required and Their Role

3.1 Sodium Sodium hypochlorite (NaOCl) is the most widely used disinfectant
Hypochlorite and is known for its excellent cleaning action. However, the effec-
tiveness of the solution is dependent on the concentration and pH
of the solution. pH of the solution effects the formation of the
hypochlorite ion ( OCl) and proton (H+) by dissociation of the
weak acid hypochlorous acid (HOCl). In this case, HOCl plays an
 Reagents Required and Their Role 63

important role in germicidal action whereas the OCl determines

the cleansing efficacy of the solution.

3.2 Tris-EDTA The combination of Tris and EDTA forms the major constituent of
lysis buffers. Tris acts as the buffering force, which major role is to
maintain the pH of the solution mostly at 8.0. The most common
form of Tris, i.e., Tris(hydroxymethyl) aminomethane with pKa of
8.1, has its buffering capacity between pH 7 and 9. Tris buffers are
generally temperature sensitive and hence should be stored at
recommended temperature only. Tris also has an additional prop-
erty of interacting with lipopolysaccharides (LPS) in the cell mem-
brane, thus destabilizing the cell membrane to aid in cell lysis.
Additionally, ethylenediaminetetraacetic acid (EDTA) binds to cer-
tain divalent cations mostly calcium (Ca2+) and magnesium (Mg2+).
These divalent cations are reported to have their key role in the
maintenance of membrane structure and function. Thus, eliminat-
ing these ions by EDTA leads to the lysis of cell membrane.

3.3 SDS Sodium dodecyl sulfate (SDS) is a strong anionic detergent known
to have protein denaturation properties. It helps to remove the
negative ions from proteins to destabilize their structure and con-
firmation. In this process, SDS helps in rupturing the cell as well as
nuclear membrane to release the genetic material into the solution.
Additionally, it helps in releasing DNA from histone and other
DNA-binding proteins. SDS is also known to have an additional
property of removing lipid membranes, thus aiding in the lysis of
cell and nuclear membrane.

3.4 Proteinase-K Peptide bond present near the carboxyl group of amino acids with
blocked alpha amino groups is the cleavage site for the serine
protease, i.e., proteinase-K. It helps in degrading many protein
impurities to yield a good quality of DNA. It also plays an impor-
tant role in the inactivation of nucleases, hence preventing the
isolated DNA from damage. The activity of proteinase-K is greatly
increased by its simultaneous action with SDS and high tempera-
ture. Proteinase-K works optimally at 50–65  C as higher tempera-
ture unfolds certain proteins to ease the function of the enzyme.

3.5 Phenol In most of the cases, phenol is used in Tris-saturated form. Phenol,
being a weak acid, is equilibrated with buffers to make it slightly
alkaline which is the most suitable pH for DNA isolation. These
buffer-saturated phenols contain around 72% of phenol with a
slightly higher density than that of water. Phenol has the property
to denature proteins during their presence in the aqueous solution.
During this process, the nonaqueous component, i.e., protein, gets
separated from the aqueous component, i.e., DNA.
64 Manual Isolation of DNA from Human Teeth

3.6 Chloroform- This is mostly used at a ratio of 24:1. These are detergents that bind
Isoamyl Alcohol to the proteins and lipids to dissolve them in the organic solution.
After dissolving, the solutions help in clumping the protein-lipid
complexes to form a precipitate. Thus, the liquid-liquid extraction
technique involving these chemicals distinguishes between the
physical positions of the cell components, i.e., upper aqueous
phase containing nucleic acids, lower organic phase containing
protein components, and middle phase constituting the lipids.

3.7 TE Buffer TE buffer is generally prepared by mixing 50 mM Tris and 50 mM

EDTA at pH 8.0. Tris, the major constituent of TE buffer, acts
as a common pH buffer. Additionally, EDTA chelates cations like
Mg2+. Hence, TE buffer helps to solubilize DNA by protecting it
from degradation.

4 Procedure

1. Take two intact, rooted molar teeth in a 50 ml conical tube

containing about 20 ml sterile deionized water.
2. Cap the tubes properly, shake them for 10–12 times, decant the
water, and repeat the washing two times.
3. Add 100% ethanol to the tube and repeat the washing as
mentioned in step 2 for two times.
4. Clean the teeth with 10% sodium hypochlorite solution fol-
lowed by 95% ethanol, place the samples on a petri dish, open
the lid of the petri dish, and allow the samples to air-dry in a
laminar airflow.
5. Take a diamond-cutting disk and cut at the middle line of the
tooth, separating the tooth into crown and root.
6. Collect any visible pulp by forceps and place them in a 15 ml
conical tube.
7. Crush the root portion of the tooth with the help of a dental
drill and place the powder in the same 15 ml conical tube used
earlier to collect the pulp.
8. Add 5 ml of forensic buffer to the 15 ml conical tube contain-
ing the dental pulp, and monitor whether all the materials dip
in the buffer properly.
9. Add proteinase-K to make its final concentration as 100 μg/ml
in total volume.
10. Add sodium dodecyl sulfate (SDS) to make its final concentra-
tion as 2% in total volume.
11. Cover the cap of the tube in Parafilm®, mix thoroughly, and
incubate in water bath at 56  C for 2–4 h.
 Observation 65

12. After 2–4 h, take the samples out of the water bath, add 5 ml
(1:1 volume) of Tris-saturated phenol (TSP, pH 8.0), and mix
thoroughly for 10 min by gentle shaking.
13. After proper mixing, centrifuge the tube at 10,000 rpm for
10 min at 4  C.
14. Collect the upper aqueous phase by careful pipetting in a
separate conical tube (you may use cut tips).
15. Add half of the volume of TSP (2.5 ml in this case) and
chloroform:isoamyl alcohol (24:1) (2.5 ml in this case), and
mix thoroughly for 10 min by gentle shaking.
16. After proper mixing, centrifuge the tube at 10,000 rpm for
10 min at 4  C. Transfer the upper aqueous layer to a fresh
conical tube.
17. Add 5 ml (1:1 volume) of chloroform:isoamyl alcohol (24:1),
and mix thoroughly for 10 min by gentle shaking.
18. After proper mixing, centrifuge the tube at 10,000 rpm for
10 min at 4  C.
19. Transfer the supernatant into an Amicon® Ultra-4 ml centrifu-
gal filter.
20. Spin the device at 5000  g for 10 min at 20  C. Discard the
flow through.
21. Add 5 ml of Milli-Q to the device for washing and repeat
spinning of the device at 5000  g for 10 min at 20  C.
22. Recover the concentrated solute by pipetting the solute from
the filter and transfer it to a fresh and labeled MCT.
23. Store the DNA at 20  C for further use.

5 Observation

The quantity of the isolated DNA can be determined at preliminary

level by UV–visible spectrophotometer. For a 1 cm path length, the
optical density at 260 nm (OD260) equals 1.0 for the following
samples:
l 50 μg/ml of double-stranded DNA
l 33 μg/ml of single-stranded DNA
l 20–30 μg/ml of oligonucleotide
l 40 μg/ml of RNA
66 Manual Isolation of DNA from Human Teeth

6 Result Table

Sample DNA content OD260/280 Inference

Control
Sample 1 (molar tooth)
Sample 2 (canine tooth)

OD optical density

7 Precautions
l Wear gloves and goggles during manual DNA isolation
procedure.
l The plasticware to be used during the entire procedure should
be sterilized and DNase free.
l Handle the chemicals carefully, mostly the phenol and chloro-
form. Phenol, being a strong acid, may cause severe burns
whereas chloroform is a known carcinogen.
l Use wide-bore tips by cutting 2–3 mm from the rear end to
prevent mechanical disruption of DNA during pipetting.
l Use filter tips to avoid cross-sample contamination.
l Do not use the TSP solution if the color of the solution has
changed to pink (occurs due to oxidation of phenol). This may
promote nicks in the extracted DNA.
l Pipetting should be done carefully without mixing the two
phases, i.e., upper aqueous phase and lower organic phase, at
any point of time.
l Clean and sterilize all the physical equipment to be used during
mechanical grinding of the tooth samples.
l Choose the tooth sample with intact root for DNA isolation.
l Clean the surface of the teeth carefully prior to mechanical
disruption to avoid contamination as well as PCR inhibitors.
l Use appropriate concentration of the reagents (especially
sodium hypochlorite) as higher concentration may inhibit fur-
ther downstream processing of the samples.
 Troubleshooting 67

8 Troubleshooting

Problem Tentative cause Possible solutions

Protein The extraction step has not Repeat the phenol:
contamination been followed properly chloroform:isoamyl
alcohol extraction step
RNA High-temperature Incubate the extracted
contamination incubation not given to DNA at 56  C for
the extracted DNA 10 min
RNase not added Add RNase at a final
concentration of
400 μg/ml to the
isolated DNA sample
PCR product Presence of PCR inhibitors Clean the surface of tooth
not amplified samples properly
Partial DNA DNA quantity not sufficient Select more than one teeth
profile and preferably select
obtained molar teeth
Mixed DNA Exogenous contamination Select intact tooth for
profile DNA isolation
obtained
Clean surface of the
samples carefully to
remove contaminants
Colored eluent Presence of inhibitors Use alternative DNA
found (generally occurs in case isolation protocols such
of teeth recovered from as magnetic bead-based
tobacco chewers) system
 Chapter 9

Isolation of DNA from Bone Samples

Objective: To isolate DNA from bone samples.

1 Introduction

Tissues of a human cadaver decompose with time. The factors affect-

ing the decomposition process mostly include various environmental
conditions, i.e., climatic conditions, presence of scavengers, and rate
of bacterial growth. Over time, the soft tissues tend to degrade;
however, tissues of bone remain intact, thus making them a suitable
source of DNA for forensic applications. Bone tissues are preferred
for DNA-based identification in circumstances such as mutilated
bodies, mass disaster, and presence of only skeletal remains in cases
of either war, fire explosions, or criminal cases. Though many tech-
niques are used to identify a person from its skeletal remains such as
exploring facial and other body characteristics, comparing with other
premortem X-rays, superimposition technique, and fingerprint com-
parison, identification based on DNA typing is the most fallible
technique and most widely used in the criminal justice system.
A typical human bone is comprised of either compact or spongy
materials due to the presence of connective tissues, protein fibers, and
deposition of calcium salts. Osteocytes, the most abundant cells of the
bone, cannot divide as they get matured. Osteocytes have two impor-
tant functions, i.e., maintenance and repair. Other types of cells, the
osteoblasts present in the bone matrix, are capable of producing new
bone matrix. Similarly, osteoclasts are the large cells having 50 or more
number of nuclei which are responsible for recycling of new bone
matrix. Hence, the major amount of DNA in found is present in the
osteocytes at a magnitude of 20,000–26,000 cells/mm3. However,
degradation and decomposition severely affect the DNA content of
the bone samples making them the toughest samples for DNA
isolation.

H. R. Dash et al., Principles and Practices of DNA Analysis: A Laboratory Manual for Forensic DNA Typing, Springer Protocols
Handbooks, https://doi.org/10.1007/978-1-0716-0274-4_9, © Springer Science+Business Media, LLC, part of Springer Nature 2020

69
70 Isolation of DNA from Bone Samples

Due to the presence of low quality and quantity of DNA

samples present in the bone samples they are considered to be
challenging from forensic DNA analysis point of view. Femur
bone is the preferred sample among the bones of fresh or decom-
posed cadaver due to longest nature among other human bones.
Though any available bone can also be processed, preference should
be given to the intact bones. Hence, suitable isolation techniques
should be employed to deal with such tough samples. The bone
piece should be cleaned properly and carefully prior to the isolation
of DNA to avoid environmental inhibitors, microbial contamina-
tions, as well as cross contamination.

2 Principle

The majority of the DNA content of the bone is present inside

osteocyte cells. These cells are found embedded in calcified matrix
which hinders direct access of the cells during any DNA extraction
procedure. Thus, the matrix substances mostly made up of the
calcium salts containing collagen fibers should be removed to aid in
the final yield of DNA. In this regard, decalcification is the process of
removal of calcium salt from tissues such as bone to soften them by
the removal of mineralized component. The most common decalci-
fication agents include strong acids, weak acids, and chelating agents.
Strong acids such as nitric acid (HNO3) act as the fastest decalcifier;
however, the prolonged exposure to HNO3 may cause damage to
the bone cells. Similarly, formic acid also acts as a decalcifying agent
but acts very slowly. The most common decalcifying agent used
nowadays in various laboratories is the chelating agent, i.e., EDTA.
Many factors influence the decalcification process of the bone,
i.e., the concentration of the decalcifying agent, incubation tem-
perature, and fluid renewal. Use of a larger volume of decalcifying
agent expedites the process, whereas incubation at higher tempera-
ture also enhances the decalcification process. Additionally, renewal
of the decalcifying agent over a period of time increases the rate of
removal of calcium ions from bone samples.
Decalcification is followed by the isolation of DNA from the
bone cells. In this process, the events unfolded include lysis, purifi-
cation of DNA from cell debris, and finally concentrating of the
isolated DNA. The manual organic DNA isolation process involves
the lysis of the cells followed by denaturation and inactivation of
proteins using detergents and proteinase-K. Use of phenol, chloro-
form, and isoamyl alcohol generates the liquid-liquid extraction
system which separates nucleic acid from other cell debris by
exploiting the differential solubility nature of proteins, lipids, and
nucleic acids. Finally, DNA present in the aqueous phase is con-
centrated and purified by the use of salt and ethanol, followed by its
resolubilization in Tris-EDTA buffer.
 Reagents Required and Their Role 71

3 Reagents Required and Their Role

3.1 Sodium Sodium hypochlorite (NaOCl) is the most widely used disinfectant
Hypochlorite and is known for its excellent cleaning action. However, the effec-
tiveness of the solution is dependent on the concentration and pH
of the solution. pH of the solution effects the formation of the
hypochlorite ion ( OCl) and proton (H+) by dissociation of the
weak acid hypochlorous acid (HOCl). In this case, HOCl plays an
important role in germicidal action whereas the OCl determines
the cleansing efficacy of the solution.

3.2 0.5 mM EDTA EDTA is a chelating agent employed for the decalcification of
bones, the prerequisite for the isolation of DNA from bone sam-
ples. The principle behind the functioning of EDTA is that it
captures the calcium ions from the surface of crystals, thereby
reducing their size. The rate of decalcification of bone using
EDTA solution is pH dependent. The action of EDTA is generally
occurred at neutral pH; however, the activity of EDTA is enhanced
when the pH of the solution is increased to 10. However, this pH
adversely affects the bone tissues by damaging them.

3.3 Normal Saline 0.9% Solution of sodium chloride is called as normal saline. It is
Solution (NSS) mostly used for washing of living cells to remove other debris. As
this concentration of salt solution is isotonic to the cells, it does not
cause lysis or disruption of the cells. However, prior to using the
prepared solution should be sterilized by either autoclaving or
passing through a 0.45 or 0.22 μm sterile filter.

3.4 Tris-EDTA The combination of Tris and EDTA forms the major constituent of
lysis buffers. Tris acts as the buffering force, whose major role is to
maintain pH of the solution mostly at 8.0. The most common form
of Tris, i.e., Tris(hydroxymethyl) aminomethane with pKa of 8.1,
has its buffering capacity between pH 7 and 9. Tris buffers are
generally temperature sensitive and hence should be stored at
recommended temperature only. Tris also has an additional prop-
erty of interacting with lipopolysaccharides (LPS) in the cell mem-
brane, thus destabilizing the cell membrane to aid in cell lysis.
Additionally, ethylenediaminetetraacetic acid (EDTA) binds to cer-
tain divalent cations mostly calcium (Ca2+) and magnesium (Mg2+).
These divalent cations are reported to have their key role in the
maintenance of membrane structure and function. Thus, eliminat-
ing these ions by EDTA leads to the lysis of cell membrane.

3.5 SDS Sodium dodecyl sulfate (SDS) is a strong anionic detergent known
to have protein denaturation properties. It helps to remove the
negative ions from proteins to destabilize their structure and con-
firmation. In this process, SDS helps in rupturing the cell as well as
72 Isolation of DNA from Bone Samples

nuclear membrane to release the genetic material into the solution.

Additionally, it helps in releasing DNA from histone and other
DNA-binding proteins. SDS is also known to have an additional
property of removing lipid membranes, thus aiding in the lysis of
cell and nuclear membrane.

3.6 Proteinase-K Peptide bond present near the carboxyl group of amino acids with
blocked alpha amino groups is the cleavage site for the serine
protease, i.e., proteinase-K. It helps in degrading many protein
impurities to yield a good quality of DNA. It also plays an impor-
tant role in the inactivation of nucleases, hence preventing the
isolated DNA from damage. The activity of proteinase-K is greatly
increased by its simultaneous action with SDS and high tempera-
ture. Proteinase-K works optimally at 50–65  C as higher tempera-
ture unfolds certain proteins to ease the function of the enzyme.

3.7 DTT Dithiothreitol (DTT) prevents the formation of intramolecular and

intermolecular disulfide bonds among cysteine residues. With pro-
tein disulfide bridges being the major constituent of sperm nuclear
membrane, DTT is essential during lysis of sperm cells to release
sperm DNA. Hence, during the lysis of sperm cells, in addition to
proteinase-K and SDS, DTT is also used in the mixture for their
effective lysis.

3.8 Phenol In most of the cases, phenol is used in Tris-saturated form. Phenol,
being a weak acid, is equilibrated with buffers to make it slightly
alkaline which is the most suitable pH for DNA isolation. These
buffer-saturated phenols contain around 72% of phenol with a
slightly higher density than that of water. Phenol has the property
to denature proteins during their presence in the aqueous solution.
During this process, the nonaqueous component, i.e., protein, gets
separated from the aqueous component, i.e., DNA.

3.9 Chloroform- This is mostly used at a ratio of 24:1. These are detergents that bind
Isoamyl Alcohol to the proteins and lipids to dissolve them in the organic solution.
After dissolving, the solutions help in clumping the protein-lipid
complexes to form a precipitate. Thus, the liquid-liquid extraction
technique involving these chemicals distinguishes between the
physical positions of the cell components, i.e., upper aqueous
phase containing nucleic acids, lower organic phase containing
protein components, and middle phase constituting the lipids.

3.10 TE Buffer TE buffer is generally prepared by mixing 50 mM Tris and 50 mM

EDTA at pH 8.0. Tris, the major constituent of TE buffer, acts
as a common pH buffer. Additionally, EDTA chelates cations like
Mg2+. Hence, TE buffer helps to solubilize DNA by protecting it
from degradation.
 Procedure 73

4 Procedure

1. Select intact long bones, preferably femur, humerus, radius,

ulna, or skull.
2. Immerse the bone(s) in 3% sodium hypochlorite (NaOCl)
(prepared in molecular biology-grade water) for 15 min.
3. Optionally clean the surface of the bone using sandpaper for
old, ancient, or excavated bones.
4. Place the bone on a fresh aluminum foil.
5. Drill holes in the bone using a mechanical drilling machine
while another person holding the bone sample tightly.
6. Collect the bone powder in a sterile aluminum foil and transfer
it to a sterile 50 ml centrifuge tube.
7. Add 0.5 M EDTA pH 8.0 sufficient enough to immerse the
bone powder.
8. Incubate the tube containing bone powder in 0.5 M EDTA
solution at 37  C for 72 h with repeated shaking.
9. After 72 h, centrifuge the tube at 10,000 rpm for 15 min at
15  C.
10. Discard the supernatant and wash the bone powder with the
same amount of sterile saline solution thoroughly with vortex;
centrifuge the tube at 10,000 rpm for 15 min at 15  C.
11. Repeat the washing step three times for better removal of
debris.
12. Add forensic buffer (composition given in Appendix A) in
appropriate quantity sufficient enough to immerse the bone
powder.
13. Add proteinase K and SDS to a final concentration of 200 μg/
ml and 2%, respectively.
14. Add DTT to make its final concentration as 100 μg/ml in total
volume.
15. Cover the cap of the tube in Parafilm®, mix thoroughly, and
incubate in water bath at 56  C for 2 h to overnight.
16. Take the samples out of the water bath, and decant the lysate to
a fresh tube.
17. Add 1:1 volume of Tris-saturated phenol (TSP, pH 8.0) to the
lysate, and mix thoroughly for 10 min by gentle shaking.
18. After proper mixing, centrifuge the tube at 10,000 rpm for
10 min at 4  C.
19. Collect the upper aqueous phase by careful pipetting in a
separate conical tube (you may use cut tips).
74 Isolation of DNA from Bone Samples

20. Add half of the volume of TSP and chloroform:isoamyl alcohol

(24:1), and mix thoroughly for 10 min by gentle shaking.
21. After proper mixing, centrifuge the tube at 10,000 rpm for
10 min at 4  C. Transfer the upper aqueous layer to a fresh
conical tube.
22. Add 1:1 volume of chloroform:isoamyl alcohol (24:1), and mix
thoroughly for 10 min by gentle shaking.
23. After proper mixing, centrifuge the tube at 10,000 rpm for
10 min at 4  C.
24. Transfer the supernatant into an Amicon® Ultra-4 ml centrifu-
gal filter.
25. Spin the device at 5000  g for 10 min at 20  C. Discard the
flow through.
26. Add 5 ml of Milli-Q to the device for washing and repeat
spinning of the device at 5000  g for 10 min at 20  C.
27. Recover the concentrated solute by pipetting the solute from
the filter and transfer it to a fresh and labeled MCT.
28. Store the DNA at 20  C for further use.

5 Observation

The quantity of the isolated DNA can be determined at preliminary

level by UV–visible spectrophotometer. For a 1 cm path length, the
optical density at 260 nm (OD260) equals 1.0 for the following
samples:
l 50 μg/ml of double-stranded DNA
l 33 μg/ml of single-stranded DNA
l 20–30 μg/ml of oligonucleotide
l 40 μg/ml of RNA

6 Result Table

Sample DNA content OD260/280 Inference

Control
Sample 1 ( femur)
Sample 2 (skull)

OD optical density
 Troubleshooting 75

7 Precautions
l Wear gloves and goggles during manual DNA isolation
procedure.
l The plasticware to be used during the entire procedure should
be sterilized and DNase free.
l Handle the chemicals carefully, mostly the phenol and chloro-
form. Phenol, being a strong acid, may cause severe burns
whereas chloroform is a known carcinogen.
l Use wide-bore tips by cutting 2–3 mm from the rear end to
prevent mechanical disruption of DNA during pipetting.
l Use filter tips to avoid cross-sample contamination.
l Do not use the TSP solution if the color of the solution has
changed to pink (occurs due to oxidation of phenol); this may
promote nicks in the extracted DNA.
l Pipetting should be done carefully without mixing the two
phases, i.e., upper aqueous phase and lower organic phase, at
any point of time.
l Choose intact piece of bone sample (if available) for DNA
isolation.
l Use appropriate concentration of the reagents (especially
sodium hypochlorite) as higher concentration may inhibit the
further downstream processing of the samples.

8 Troubleshooting

Problem Tentative cause Possible solutions

Protein The extraction step has Repeat the phenol:
contamination not been followed chloroform:isoamyl
properly alcohol extraction step
RNA High-temperature Incubate the extracted DNA
contamination incubation not given to at 56  C for 10 min
the extracted DNA
RNase not added Add RNase at a final
concentration of 400 μg/
ml to the isolated DNA
sample
PCR product Presence of PCR Clean the bone surface
not amplified inhibitors properly
Partial DNA DNA quantity not Increase the sample size; take
profile sufficient more amount of bone
obtained powder

(continued)
76 Isolation of DNA from Bone Samples

Problem Tentative cause Possible solutions

Mixed DNA Exogenous Select intact bone for DNA
profile contamination isolation
obtained
Surface clean the samples
carefully to remove
contaminants
Colored eluent Presence of inhibitors Use alternative DNA
found (generally occurs in isolation protocols such as
old/excavated bones) magnetic bead-based
system
Clean the surface of old
bones properly by
sandpaper prior to
processing
 Chapter 10

Differential Extraction of Sperm and Epithelial Cell DNA

Objective: To isolate sperm and epithelial cell DNA differentially

from mixed samples (vaginal fluid slide) of sexual assault victims.

1 Introduction

Sexual assault has become a huge problem globally. Many reasons

determine the fact that a victim could not identify an offender. In
this process, DNA-based identification is left with the only alterna-
tive to identify the perpetrator. In sexual assault cases, it is of utmost
importance to establish the presence of victim’s DNA as well as the
identification of perpetrator from the examined objects. Over the
years, DNA technology has emerged to be the most useful tool for
identification with high discrimination potential and sensitivity. In
sexual assault cases, the most common sample for examination is
victim’s vaginal fluid slide. The glass slide containing sample solves
two purposes, i.e., isolation of DNA and screening of sperm cells via
optical microscopy. The major challenge in processing such samples
is to generate a mixed DNA profile of both victim and perpetrator.
Thus, many a times, a unique DNA extraction step is followed to
simultaneously isolate male and female DNA fragments from the
same sample in a differential manner.
Perpetrator’s DNA is the most likely to be obtained from the
sperm cells present on the victim’s sources routinely collected
during medical examination. However, the majority fraction of
the DNA present on the victim’s belongings is of female/victim
origin constituting the epithelial cells of the vaginal lining. These
epithelial cells should be separated from the sperm cells prior to the
isolation of DNA from the sperm cells using the differential DNA
isolation procedure described in Fig. 10.1.

H. R. Dash et al., Principles and Practices of DNA Analysis: A Laboratory Manual for Forensic DNA Typing, Springer Protocols
Handbooks, https://doi.org/10.1007/978-1-0716-0274-4_10, © Springer Science+Business Media, LLC, part of Springer Nature 2020

77
78 Differential Extraction of Sperm and Epithelial Cell DNA

Fig. 10.1 Principle of differential extraction technique to separate male and female DNA fractions from mixed
samples

2 Principle

The most common evidences collected in sexual assault cases con-

sist of swabs of vagina; oral, rectal, and exposed skin; as well as
clothing and bedding from the spot. These evidences contain stains
constituting both victim’s cells and seminal stains containing sper-
matozoa from the accused. The most common technique for pro-
cessing of these samples involve isolation of DNA based on two
separate cellular fractions, i.e., non-sperm, epithelial cell fractions
of female origin and sperm cell fraction originating from male or
perpetrator.
The first step of DNA isolation involves the isolation of DNA
from the non-sperm cells by using chemical lysis technique involv-
ing buffers, proteinase-K, and SDS. During this step, the
non-sperm epithelial cells are digested to release their DNA to the
solution and the sperm heads remain intact in this process. Further,
the sperm heads are separated from epithelial DNA and other
cellular debris by centrifugation and repeated washing. In the sec-
ond step of DNA isolation, the sperm heads are subjected to
digestion using proteinase-K, SDS, and dithiothreitol (DTT).
DTT, the reducing agent, disrupts the disulfide bond of the struc-
tural proteins present on the sperm cells.
 Reagents Required and Their Role 79

The differential steps involving DNA isolation can be followed

only when there is the presence of sperm cells in the sample which
needs to be confirmed prior to DNA isolation. The preliminary
steps involving the confirmation of the presence of spermatozoa are
either microscopic or immunological tests. The most common
immunologic test for the presence of semen involves the detection
of a protein P30 alternatively called as prostate-specific antigen.
Additionally, staining of the cells by Christmas tree stain followed
by observation under microscope can also confirm the presence of
spermatozoa in the samples. Differential DNA isolation technique
should be performed only after confirming the presence of sperma-
tozoa in the target samples.

3 Reagents Required and Their Role

3.1 Tris-EDTA The combination of Tris and EDTA forms the major constituent of
lysis buffers. Tris acts as the buffering force, whose major role is to
maintain pH of the solution mostly at 8.0. The most common form
of Tris, i.e., Tris(hydroxymethyl) aminomethane with pKa of 8.1,
has its buffering capacity between pH 7 and 9. Tris buffers are
generally temperature sensitive and hence should be stored at
recommended temperature only. Tris also has an additional prop-
erty of interacting with lipopolysaccharides (LPS) in the cell mem-
brane, thus destabilizing the cell membrane to aid in cell lysis.
Additionally, ethylenediaminetetraacetic acid (EDTA) binds to cer-
tain divalent cations mostly calcium (Ca2+) and magnesium (Mg2+).
These divalent cations are reported to have their key role in the
maintenance of membrane structure and function. Thus, eliminat-
ing these ions by EDTA leads to the lysis of cell membrane.

3.2 SDS Sodium dodecyl sulfate (SDS) is a strong anionic detergent known
to have protein denaturation properties. It helps to remove the
negative ions from proteins to destabilize their structure and con-
firmation. In this process, SDS helps in rupturing the cell as well as
nuclear membrane to release the genetic material into the solution.
Additionally, it helps in releasing DNA from histone and other
DNA-binding proteins. SDS is also known to have an additional
property of removing lipid membranes, thus aiding in the lysis of
cell and nuclear membrane.

3.3 Proteinase-K Peptide bond present near the carboxyl group of amino acids with
blocked alpha amino groups is the cleavage site for the serine prote-
ase, i.e., proteinase-K. It helps in degrading many protein impurities
to yield a good quality of DNA. It also plays an important role in the
inactivation of nucleases, hence preventing the isolated DNA from
damage. The activity of proteinase-K is greatly increased by its simul-
taneous action with SDS and high temperature. Proteinase-K works
80 Differential Extraction of Sperm and Epithelial Cell DNA

optimally at 50–65  C as higher temperature unfolds certain proteins

to ease the function of the enzyme.

3.4 DTT Dithiothreitol (DTT) prevents the formation of intramolecular and

intermolecular disulfide bonds among cysteine residues. With pro-
tein disulfide bridges being the major constituent of sperm nuclear
membrane, DTT is essential during lysis of sperm cells to release
sperm DNA. Hence, during the lysis of sperm cells, in addition to
proteinase-K and SDS, DTT is also used in the mixture for their
effective lysis.

3.5 Phenol In most of the cases, phenol is used in Tris-saturated form. Phenol,
being a weak acid, is equilibrated with buffers to make it slightly
alkaline which is the most suitable pH for DNA isolation. These
buffer-saturated phenols contain around 72% of phenol with a
slightly higher density than that of water. Phenol has the property
to denature proteins during their presence in the aqueous solution.
During this process, the nonaqueous component, i.e., protein, gets
separated from the aqueous component, i.e., DNA.

3.6 Chloroform- This is mostly used at a ratio of 24:1. These are detergents that bind
Isoamyl Alcohol to the proteins and lipids to dissolve them in the organic solution.
After dissolving, the solutions help in clumping the protein-lipid
complexes to form a precipitate. Thus, the liquid-liquid extraction
technique involving these chemicals distinguishes between the
physical positions of the cell components, i.e., upper aqueous
phase containing nucleic acids, lower organic phase containing
protein components, and middle phase constituting the lipids.

3.7 TE Buffer TE buffer is generally prepared by mixing 50 mM Tris and 50 mM

EDTA at pH 8.0. Tris, the major constituent of TE buffer, acts as a
common pH buffer. Additionally, EDTA chelates cations like Mg2+.
Hence, TE buffer helps to solubilize DNA by protecting it from
degradation.

4 Procedure

1. Take a properly labeled sterile microcentrifuge tube (MCT)

containing 500 μl of forensic buffer.
2. Transfer the cellular material present in the vaginal slide by the
help of a cell scrapper using this forensic buffer to the previ-
ously marked tube.
3. Repeat the process 2–3 times so that significant number of cells
are present in the buffer (the buffer in the MCT will turn
translucent from transparent when the cellular materials of
the slide come into the solution; check this).
 Procedure 81

4. Add proteinase-K to make its final concentration as 100 μg/ml

in total volume.
5. Add sodium dodecyl sulfate (SDS) to make its final concentra-
tion as 2% in total volume.
6. Cover the cap of the tube in Parafilm®, mix thoroughly, and
incubate in water bath at 56  C for 2–4 h.
7. Centrifuge the digested material of the sample for 5 min at
12,000  g at 15  C.
8. Transfer the liquid portion of the solution into a fresh tube
(tube I) (this solution contains the digested non-sperm cells).
(a) Add 500 μl of Tris-saturated phenol (TSP, pH 8.0) to
tube I, and mix properly for 10 min by gentle shaking.
(b) After proper mixing, centrifuge the tube at 10,000 rpm
for 10 min at 4  C.
(c) Collect the upper aqueous phase by careful pipetting in a
separate MCT (you may use cut tips).
(d) Add half of the volume of TSP (250 μl in this case) and
chloroform:isoamyl alcohol (24:1) (250 μl in this case),
and mix thoroughly for 10 min by gentle shaking.
(e) After proper mixing, centrifuge the tube at 10,000 rpm
for 10 min at 4  C. Transfer the upper aqueous layer to a
fresh MCT.
(f) Add 500 μl (1:1 volume) of chloroform:isoamyl alcohol
(24:1), and mix thoroughly for 10 min by gentle shaking.
(g) After proper mixing, centrifuge the tube at 10,000 rpm
for 10 min at 4  C.
(h) Transfer the supernatant into an Amicon® Ultra-0.5 ml
30 kDa centrifugal filter placed in a 2.0 ml MCT.
(i) Spin the device at 5000  g for 10 min at 20  C. Discard
the flow through.
(j) Add 500 μl of Milli-Q to the device for washing and
repeat spinning of the device at 5000  g for 10 min at
20  C.
(k) Remove the assembled device and to recover the concen-
trated solute place the Amicon® ultra-0.5 ml 30 kDa
centrifugal filter in a fresh MCT upside down.
(l) Counterbalance with a similar device and spin at
1000  g for 2 min at 20  C.
(m) Recover the concentrated solute by pipetting the solute
from the filter and transfer it to a fresh and labeled MCT
( female fraction).
(n) Store the DNA at 20  C for further use.
82 Differential Extraction of Sperm and Epithelial Cell DNA

9. Wash the pellet twice with pre-sterilized normal saline solution.

10. Add 300 μl of forensic buffer to the pellet obtained after
centrifugation and separation of liquid portion from it (tube
II) (the pellet contains the sperm cells or the male fraction).
11. To tube II, add proteinase-K to make its final concentration as
100 μg/ml in total volume.
12. To tube II, add sodium dodecyl sulfate (SDS) to make its final
concentration as 2% in total volume.
13. To tube II, add dithiothreitol (DTT) to make its final concen-
tration as 150 mM in total volume.
14. Cover the cap of the tube with Parafilm®, mix thoroughly, and
incubate in water bath at 50  C for 2–4 h.
(a) After 2–4 h, take the samples out of the water bath, add
300 μl (1:1 volume) of Tris-saturated phenol (TSP,
pH 8.0), and mix thoroughly for 10 min by gentle
shaking.
(b) After proper mixing, centrifuge the tube at 10,000 rpm
for 10 min at 4  C.
(c) Collect the upper aqueous phase by careful pipetting in a
separate conical tube (you may use cut tips).
(d) Add half of the volume of TSP (150 μl in this case) and
chloroform:isoamyl alcohol (24:1) (150 μl in this case),
and mix thoroughly for 10 min by gentle shaking.
(e) After proper mixing, centrifuge the tube at 10,000 rpm
for 10 min at 4  C. Transfer the upper aqueous layer to a
fresh MCT.
(f) Add 300 μl (1:1 volume) of chloroform:isoamyl alcohol
(24:1), and mix thoroughly for 10 min by gentle shaking.
(g) After proper mixing, centrifuge the tube at 10,000 rpm
for 10 min at 4  C.
(h) Transfer the supernatant into an Amicon® Ultra-0.5 ml
30 kDa centrifugal filter placed in a 2.0 ml MCT.
(i) Spin the device at 5000  g for 10 min at 20  C. Discard
the flow through.
(j) Add 450 μl of Milli-Q to the device for washing and
repeat spinning of the device at 5000  g for 10 min at
20  C.
(k) Recover the concentrated solute by pipetting the solute
from the filter and transfer it to a fresh and labeled MCT
(male fraction).
(l) Store the DNA at 20  C for further use.
 Precautions 83

5 Observation

The quantity of the isolated DNA can be determined at preliminary

level by UV–visible spectrophotometer. For a 1 cm path length, the
optical density at 260 nm (OD260) equals 1.0 for the following
samples:
l 50 μg/ml of double-stranded DNA
l 33 μg/ml of single-stranded DNA
l 20–30 μg/ml of oligonucleotide
l 40 μg/ml of RNA

6 Result Table

Sample DNA content OD260/280 Inference

Control
Sample 1 ( female fraction)
Sample 2 (male fraction)

OD optical density

7 Precautions
l Wear gloves and goggles during manual DNA isolation
procedure.
l The plasticware to be used during the entire procedure should
be sterilized and DNase free.
l Handle the chemicals carefully, mostly the phenol and chloro-
form. Phenol, being a strong acid, may cause severe burns
whereas chloroform is a known carcinogen.
l Use wide-bore tips by cutting 2–3 mm from the rear end to
prevent mechanical disruption of DNA during pipetting.
l Use filter tips to avoid cross-sample contamination.
l Do not use the TSP solution if the color of the solution has
changed to pink (occurs due to oxidation of phenol). This may
promote nicks in the extracted DNA.
l Pipetting should be carried out carefully without mixing the two
phases, i.e., upper aqueous phase and lower organic phase, at any
point of time.
l Prior to DNA isolation confirm the presence of spermatozoa in
the vaginal slide by microscopic techniques.
84 Differential Extraction of Sperm and Epithelial Cell DNA

8 Troubleshooting

Problem Tentative cause Possible solutions

Protein The extraction step has Repeat the phenol:
contamination not been followed chloroform:isoamyl
properly alcohol extraction step
RNA High-temperature Incubate the extracted DNA
contamination incubation not given at 56  C for 10 min
to the extracted DNA
RNase not added Add RNase at a final
concentration of 400 μg/
ml to the isolated DNA
sample
PCR product not Spermatozoa not present Confirm the presence of
amplified for spermatozoa prior to
male DNA DNA isolation
Partial DNA DNA quantity not Quantify the sample prior to
profile obtained sufficient PCR amplification and
take optimum quantity of
DNA
Mixed DNA Presence of female DNA The alleles with higher peak
profile obtained in the sample in height are from male
from male addition to male DNA contributor, whereas the
fraction alleles with lower peak
height are from female
contributor
Mixed DNA Presence of male DNA in The alleles with higher peak
profile obtained the sample in addition height are from female
from female to female DNA contributor, whereas the
fraction alleles with lower peak
height are from male
contributor
 Part III

Automatic and Semi-automatic Extraction of DNA from

Biological Samples
 Chapter 11

Isolation of DNA by Using Magnetic Bead-Based Extraction

System

Objective: To isolate DNA from liquid blood and forensic samples

using magnetic bead-based extraction system.

1 Introduction

Isolation of DNA is the utmost important pre-requirement and

fundamental for forensic analysis and criminal justice system by
DNA fingerprinting technique. Since the first extraction of DNA
in 1869 by Friedrich Miescher, many techniques have been discov-
ered so far for effective extraction of DNA from biological samples.
Pioneer extraction was a simple discovery which relied on the fact
that inside the cell there exists some materials which precipitate out
of the acidic solution whereas they dissolve in alkaline solution.
However the material DNA was discovered in 1953.
Traditional method of DNA extraction involves a liquid-liquid
extraction procedure consisting of three basic steps, i.e., lysis of cell,
removal of other cell debris from nucleic acids, and precipitation of
nucleic acids. In this technique physical/chemical disruption of
cells takes place followed by the use of phenol-chloroform organic
solvents to concentrate DNA in hydrophilic phase. Finally, DNA
pellet is collected at the bottom of the solution followed by its
dissolving in water. The methodologies of manual extraction of
DNA involve manyfold manipulation of the samples as well as the
reagents increasing the chance of contamination. Another short-
coming of manual extraction of DNA involves the consumption of
time for the process and requirement of individual’s attention and
labor till completion of the process. Additionally, the yield of DNA
by manual extraction techniques is always dependent on the che-
micals used and the examiner’s proficiency. In this regard, with the
increase in goal to streamline the process of DNA extraction

H. R. Dash et al., Principles and Practices of DNA Analysis: A Laboratory Manual for Forensic DNA Typing, Springer Protocols
Handbooks, https://doi.org/10.1007/978-1-0716-0274-4_11, © Springer Science+Business Media, LLC, part of Springer Nature 2020

87
88 Isolation of DNA by Using Magnetic Bead-Based Extraction System

throughout the globe, other solid-phase-based systems are being

used for isolation and purification of DNA. Magnetic bead-based
DNA isolation technique has been discussed in much details in this
chapter.

2 Principle

The first report of using magnetic particles for extraction of DNA

was in the year 1998. The initial technique of magnetic separation
involves the use of particles constituting iron oxide core coated
with silane. The surface of these particles is bound with free car-
boxylic acid molecules which in turn are bound to the nucleic acids.
In this case, the concentration of the salts determines the strength
of controlled reversible bonding between the DNA/RNA and the
functional groups. Thus, at optimum concentration of the salts, the
magnetic particles are attached to the nucleic acids. Finally, a mag-
netic field is created by placing a magnet outside the tube, enabling
the magnetic particles containing nucleic acids to stick to the outer
edge of the tube. This step is followed by the washing of the
particles with washing buffer to release the nucleic acid from the
magnetic beads to generate a pure nucleic acid extract. Figure 11.1
describes the schematic representation of the principle of DNA
extraction by magnetic bead-based technique.
There are many commercially available kits in the market for
magnetic bead-based extraction of DNA from forensic samples.
Two magnetic bead-based kits with potential application of DNA
isolation from a wider variety of samples have been included in this
chapter, i.e., PrepFiler Express™ and PrepFiler Express BTA™ kits

Fig. 11.1 Schematic presentation of principle of magnetic bead-based DNA extraction

 Reagents Required and Their Role 89

(Applied Biosystems) for extraction of DNA from several forensic

samples including blood, saliva, body fluid stains, hair root, ciga-
rette butt, chewing gum, bone, and tooth in an efficient manner.
The other available kits for isolation of DNA from forensic samples
employing magnetic bead-based principle have been listed in
Appendix B.

3 Reagents Required and Their Role

3.1 PrepFiler™ Lysis The buffer is provided by the manufacturer. This buffer is used for
Buffer the lysis of common forensic samples including liquid blood, liquid
saliva, bloodstains, other body fluid stains, body fluids on swabs,
and hair root.

3.2 PrepFiler BTA™ The buffer is also provided by the manufacturer. This buffer is used
Lysis Buffer for the lysis of challenging samples such as chewing gum, cigarette
butt, and tape lifts as well as other tough samples, i.e., bone and
tooth.

3.3 LySep™ Column LySep™ column provides a useful tool for simultaneous lysis and
purification of the lysate in the same tube. It consists of two parts,
the LySep™ column and the sample tube, and the insertion of the
column to the tube forms the LySep™ column assembly
(Fig. 11.2). The sample as well as the lysis buffer are added to the
LySep™ column assembly sequentially followed by lysis in a
thermo-shaker/mixer. After completion of lysis process, the assem-
bly is centrifuged to deform the LySep™ column allowing the
lysate to pass through the column to the sample tube. The
LySep™ column and the substrate can be discarded and the sample
tube containing sample can be applied to the instrument for further
processing.

Fig. 11.2 Assembling of LySep™ column for lysis of forensic sample

90 Isolation of DNA by Using Magnetic Bead-Based Extraction System

Fig. 11.3 Cartridge containing reagents and magnetic beads for purification of DNA from cell lysate

3.4 Cartridges All the reagents and chemicals required for purification of DNA
from lysed cell debris are stored in the sealed plastic containers
called as cartridges (Fig. 11.3). Mostly, the cartridges contain lysis
buffer, binding solution, elution buffer, washing buffer, and mag-
netic beads in suspension. Additionally, some compartments are left
empty to provide space for heated chamber for elution. The car-
tridges are generally pre-sealed with aluminum sheets and stored at
room temperature. Prior to the automated isolation steps by the
instrument, automated piercing steps are followed which mini-
mizes possible contamination and buffer spout. Finally, mixing of
the beads, reagents, and sample is carried out by the liquid-
handling action of the instrument. Additionally, separation of
DNA is achieved by magnetic attraction as well as liquid handling.
The cartridges are specific to the isolation kits and the instrument
used.

3.5 Proteinase-K Peptide bond present near the carboxyl group of amino acids with
blocked alpha amino groups is the cleavage site for the serine
protease, i.e., proteinase-K. It helps in degrading many protein
impurities to yield a good quality of DNA. It also plays an impor-
tant role in the inactivation of nucleases, hence preventing the
isolated DNA from damage. The activity of proteinase-K is greatly
increased by its simultaneous action with SDS and high tempera-
ture. Proteinase-K works optimally at 50–65  C as higher tempera-
ture unfolds certain proteins to ease the function of the enzyme.

3.6 DTT Dithiothreitol (DTT) prevents the formation of intramolecular and

intermolecular disulfide bonds among cysteine residues. With pro-
tein disulfide bridges being the major constituent of sperm nuclear
membrane, DTT is essential during lysis of sperm cells to release
sperm DNA. Hence, during the lysis of sperm cells, in addition to
proteinase-K and SDS, DTT is also used in the mixture for their
effective lysis.
 Procedure 91

4 Procedure

4.1 Using PrepFiler 1. Check PrepFiler Express™ lysis buffer for the presence of
Express™ Forensic suspected precipitate. If suspected precipitate matter is found,
DNA Extraction Kit heat the solution at 37  C followed by vortex for 5 s.
2. Prepare the lysis solution freshly by mixing 500 μl lysis buffer
with 5 μl freshly prepared 1 M DTT.
3. Insert a LySep™ column into a sample tube followed by careful
transfer of the sample into LySep™ column.
4. Add 500 μl freshly prepared lysis solution to the column assem-
bly containing the sample. Make sure that the entire sample is
submerged in the lysis solution.
Note: For optimum extraction of DNA from the forensic
samples, input appropriate amount of sample, i.e., liquid
blood/saliva (40 μl), blood-stained clothing (25 mm2 cut-
ting/punch), body fluid stains on clothing (25 mm2 cutting),
and hair root (5 mm cutting from the hair root).
5. Carefully close the lid of the assembled column.
6. Incubate the column containing samples in a thermal shaker at
70  C and 750 rpm for 40 min.
7. After incubation, centrifuge the column assembly for 2 min at
10,000  g for transfer of lysate to the sample tube.
8. Carefully remove the LySep™ column from the sample tube
and discard the LySep™ column.
9. Proceed the samples directly to automated extraction system.

4.2 Using PrepFiler 1. Prepare a fresh lysis solution containing 220 μl lysis buffer, 3 μl
Express BTA™ freshly prepared 1 M DTT, and 7 μl proteinase-K.
Forensic DNA 2. Place the sample (bone/teeth) in a PrepFiler™ Bone and tooth
Extraction Kit lysate tube.
3. Add 230 μl of freshly prepared lysis solution to the lysate tube
containing bone/teeth samples.
Note: For optimum extraction of DNA from bone and
teeth samples, input appropriate amount of samples, i.e., up
to 50 g of bone and teeth powder.
4. Cover the lysate tubes containing the samples and the lysis
solution properly and vortex gently for 5 s.
5. Incubate the lysate tubes containing samples in a thermal
shaker at 56  C and 1100 rpm for 2–3 h.
6. Centrifuge the tubes at 10,000  g for 91 s at 4  C.
7. Transfer the clear lysate to a fresh PrepFiler™ sample tube.
8. Proceed the samples directly for automated extraction system.
92 Isolation of DNA by Using Magnetic Bead-Based Extraction System

Fig. 11.4 (a) Card slot of the instrument and (b) protocol card

4.3 Setup and Run 1. Prior to switching on the instrument, insert the appropriate
for Automated DNA protocol card (either PrepFiler Express™ or PrepFiler Express
Extraction BTA™) in the card slot, and close the card slot. For guidance
see Fig. 11.4.
2. Switch on the instrument; the display of the instrument shows
information regarding the instrument version and the main
menu; press “Start.” For guidance see Fig. 11.5.
3. Press after following each prompt on-screen.
4. Open the instrument door to remove the cartridge rack and tip
and tube rack from the instrument.
5. Load the desired numbers of prefilled reagent cartridges
(depending on the number of samples) into the cartridge
rack. Insert the cartridge rack in the instrument. For guidance
see Fig. 11.6.
6. Load the tip and tube rack. For guidance see Fig. 11.7.
7. Place the PrepFiler™ sample tubes containing lysate samples in
Row S corresponding to the position of cartridges.
8. Place the AutoMate Express™ tips inserted in tip holders in
Row T2 corresponding to the position of sample tubes.
9. Place the labeled PrepFiler™ elution tubes in open condition
in Row E corresponding to the position of sample tubes.
10. Position T1 should be left empty.
11. Insert the loaded tip and tube rack into the instrument.
12. Close the instrument door properly.
13. Press after closing the door.
 Procedure 93

Fig. 11.5 “Display” of the instrument showing information regarding the

instrument and main menu

Fig. 11.6 Loading of the prefilled reagent cartridges into the cartridge rack of the
instrument
94 Isolation of DNA by Using Magnetic Bead-Based Extraction System

Fig. 11.7 Correct position of the sample tubes, elution tubes, and tips and tip
holders on the tip and tube rack

14. Press 1, if you want to use protocol for PrepFiler Express™ kit;
else press 2 for the use of PrepFiler Express BTA™ kit.
15. Press “Start”; the screen shows the real-time steps of the
extraction procedure and the remaining time of the extraction
process.
16. After completion of the run, press to return to the
main menu.
17. Open the instrument door, and remove the cartridge rack as
well as the tip and tube rack.
18. Remove the elution tubes containing the purified DNA and
they can be stored at 20  C till further use.
19. Dispose the exhausted reagent cartridges, tips, and sample
tubes properly.

5 Observation

The quantity of the isolated DNA can be determined at the prelim-

inary level by UV–visible spectrophotometer. For a 1 cm path
length, the optical density at 260 nm (OD260) equals 1.0 for the
following samples:
l 50 μg/ml of double-stranded DNA
l 33 μg/ml of single-stranded DNA
l 20–30 μg/ml of oligonucleotide
l 40 μg/ml of RNA
 Precautions 95

6 Result Table

Sample DNA content OD260/280 Inference

Control
Sample 1 (liquid blood)
Sample 2 (bone powder)

OD optical density

7 Precautions
l Follow the manufacturer’s guidelines in each step properly.
l Wear gloves while dealing with the reagents and plasticware.
l Avoid using an expired kit.
l Do not incubate the samples more than the recommended
period of time; it may result in precipitation of salts.
l Load recommended volume of lysates; lower lysate volume may
result in formation of air bubbles in tips resulting in problems of
the liquid-handling system.
l Check for the presence of lysate and elution tube at
corresponding positions.
l Check for the submerging of the entire sample in the lysis
solution.
l Do not process the samples for extraction by PF Express™ and
PF Express BTA™ kit simultaneously, as the liquid-handling
system can process either of the protocol at a given point of time.
l Never remove the protocol card when the instrument is in “on”
condition.
l Load the cartridges, lysates, tips, and elution tubes in their
designated places.
l Do not touch the surface of the heat block as the temperature of
the same could reach 95  C.
l Carefully select the protocol you want to use in the liquid-
handling system.
l Run the machine with proper power backup, as the process
cannot be resumed in case of any power failure.
l Dispose the used cartridges and reagents carefully.
l Do not add acids or bases to the wastes containing lysis buffer as
the acids/bases may react with guanidine thiocyanate in the lysis
buffer and generate toxic gas.
96 Isolation of DNA by Using Magnetic Bead-Based Extraction System

8 Troubleshooting

Problem Tentative cause Possible solutions

Presence of Extended time of Careful pipetting may
precipitates in incubation than the dissolve the precipitate
the liquid after recommended matter in the solution
lysis process conditions
Lysates stored under Vortex the lysate prior to
refrigerated conditions centrifugation
Ambient temperature very Avoid extended incubation
less of the samples during
lysis process
Avoid storing of the
lysates under
refrigerated conditions
No elution volume No sample added to the Rerun with proper
after run sample tube samples and fresh
reagent cartridges
Sample volume lower than Use recommended
the recommended volume of sample as per
volume the protocol used; if
sufficient amount of
lysate is not present
make up the volume
using Milli-Q
Colored DNA Sample contains dyes Perform qRT-PCR and
elute check whether it affects
CT value of IPC or not
DNA contaminated with Minimize the amount
heme samples, i.e., either
blood or blood-stained
clothing
Low yield of DNA Protocol not followed Review the protocol used
properly or reagents not and addition of
added during the lysis appropriate reagents
step
Incomplete lysis Decrease the amount of
input sample
Add appropriate amount
of proteinase-K
Make sure to completely
submerge the samples
in lysis solution
Incubation time may be
increased
 Chapter 12

Extraction of DNA by Using Anion-Exchange Resin Chelex®

Objective: To isolate DNA from liquid blood and forensic samples

using Chelex® 100 resin.

1 Introduction

The primary step of forensic DNA analysis involves the extraction

of high quality and quantity of DNA for their use in many down-
stream applications. Thus, it becomes highly essential to choose the
most suitable technique for DNA extraction which is dependent on
sample, technology, as well as analyst’s preference. Additionally,
other factors to be taken care of in selecting the suitable technique
of DNA extraction include technical requirements, time efficiency,
cost-effectiveness, as well as nature of biological samples to be
processed.
In this regard, Singer-Sam in 1989 reported the use of Chelex
100 to increase the strength of PCR amplification from a low DNA
tissue culture sample by simultaneous boiling. The initial work of
Singer-Sam suggested the use of boiling suspensions of cells in 5%
Chelex solution and use of the supernatant as template of PCR. The
alkaline nature of the Chelex suspension (pH 10–11) and boiling of
the samples at 100  C generate the DNA fragments by disruption
of the cell membranes. However, this method generates the dena-
tured DNA samples which are not suitable for RFLP analysis.
The advantage of using Chelex is that it sequesters the divalent
metals with the potential of introducing DNA damage. Addition-
ally, Chelex beads can be removed from the lysate containing DNA
without interfering the further downstream processing of the sam-
ples. As proteinase-K is not used in this DNA extraction practice,
blood samples do not release heme from globin, thus generating
less amount of free porphyrin compounds. This minimizes the
inhibitor content in the extracted DNA. Less time requirement to

H. R. Dash et al., Principles and Practices of DNA Analysis: A Laboratory Manual for Forensic DNA Typing, Springer Protocols
Handbooks, https://doi.org/10.1007/978-1-0716-0274-4_12, © Springer Science+Business Media, LLC, part of Springer Nature 2020

97
98 Extraction of DNA by Using Anion-Exchange Resin Chelex®

complete the extraction procedure is another advantage of this

procedure. Additionally, requirement of fewer manipulations in
this isolation practice reduces the chance of inadvertent contamina-
tion of samples by extraneous DNAs.

2 Principle

Positively charged chemical substances such as anion-exchange

resins bind to the oppositely charged substances such as DNA
molecules. In this regard, Chelex constitutes the styrene divinyl-
benzene copolymers with iminodiacetate ions which chelates poly-
valent metal ions, i.e., Mg2+. Subsequently, the nucleases are
inactivated and DNA is protected. The technique provides an
added advantage in the purification of small fragments of DNA,
mostly up to 1000 bp size. Hence, non-Chelex methods should be
employed when high amount of DNA is expected from any sample.
Seligson and others in 1985 first described the use of anion-
exchange resin materials for isolation of nucleic acid from varied
sources such as whole blood. The discovery described the protocol
of using a column containing positively charged resins of diethyla-
minoethyl cellulose groups on its surface for proper binding of
negatively charged phosphate backbone of DNA. Additionally,
the strength of binding of DNA as well as other impurities to the
column can be optimized for salt concentration and pH. Other
contaminants such as proteins and RNAs can be removed from the
column by repeated washing using medium salt buffers.

3 Reagents Required and Their Role

3.1 Sodium Sodium hypochlorite (NaOCl) is the most widely used disinfectant
Hypochlorite and is known for its excellent cleaning action. However, the effec-
tiveness of the solution is dependent on the concentration and pH
of the solution. pH of the solution effects the formation of the
hypochlorite ion ( OCl) and proton (H+) by dissociation of the
weak acid hypochlorous acid (HOCl). In this case, HOCl plays an
important role in germicidal action whereas the OCl determines
the cleansing efficacy of the solution.

3.2 Chelex® 100 Chelex® 100 resin (Bio-Rad Laboratories, Hercules, CA, USA)
Resin consists of styrene divinylbenzene copolymers. These polymers
also contain paired iminodiacetate ions. The role of this reagent
during DNA extraction is the action of a chelating ion-exchange
resin which binds polyvalent metal ions such as nucleases. This
reagent has widespread application for DNA extraction with foren-
sic implications.
 Procedure 99

4 Procedure

Preparation of Chelex® Mixture

1. With the help of 10% sodium hypochlorite, sterilize a dry
spatula and a small magnetic bar.
2. Prepare 10% by weight of Chelex® 100 resin in sterilized
HPLC-grade water.
Note: Take a 50 ml sterile Falcon tube, add 5 g of Chelex®
100 resin into it, and make up the volume to 50 ml mark with
sterilized HPLC-grade water.
3. Place the tube containing Chelex® 100 resin in water in a
magnetic stirrer and mix well.
4. Transfer aliquots of 300 μl of 10% Chelex® 100 resin solutions
to 1.5 ml sterile MCT and store in refrigerator.

DNA Extraction Step

5. Take out the required number of tubes containing 10% Che-
lex® 100 resin solutions from the refrigerator.
6. Label three tubes: one for blood, another for tissue sample, and
third one for negative control.
7. Fill the hole of the heating block with water, and preset the
heating block to 95  C.
8. Sterilize forceps by flaming it over the alcohol burner several
times. Using the sterilized forceps, remove a small portion of
tissue (mostly of 0.2 mm  0.2 mm size) from the sample and
place it in the tube containing 10% Chelex® 100 resin solutions
labeled for tissue sample.
9. Mix 100 μl of whole blood into another tube containing 10%
Chelex® 100 resin solutions labeled for tissue sample.
10. Dip the sterilized forceps into the tube containing 10% Che-
lex® 100 resin solutions labeled for negative control.
11. Vortex the tubes containing the Chelex® solution and the
samples.
12. Spin samples for 10–15 s at high speed to ensure that all the
samples are dipped well into the 10% Chelex® 100 resin
solutions.
13. Incubate the samples at 95  C for 20 min in the heating block.
14. After incubation, vortex the samples for 10–15 s.
15. Centrifuge the tubes at 12,000 rpm for 5 min at 15  C.
16. Transfer the supernatant into a fresh MCT and store at 20  C
till further use.
100 Extraction of DNA by Using Anion-Exchange Resin Chelex®

5 Observation

The quantity of the isolated DNA can be determined at the prelim-

inary level by UV–visible spectrophotometer. For a 1 cm path
length, the optical density at 260 nm (OD260) equals 1.0 for the
following samples:
l 50 μg/ml of double-stranded DNA
l 33 μg/ml of single-stranded DNA
l 20–30 μg/ml of oligonucleotide
l 40 μg/ml of RNA

6 Result Table

Sample DNA content OD260/280 Inference

Control
Sample 1 (liquid blood)
Sample 2 (tissue)

OD optical density

7 Precautions
l Wear gloves while dealing with the reagents and plasticware.
l During incubation, make sure that the lids of the tubes are
closed properly to avoid pop off.
l Transfer the supernatant carefully; a little contamination of
Chelex can inactivate Taq polymerase in the PCR setup.
l Always use an extra reaction for the negative control to check the
level of contamination during the extraction process.
l It is recommended to use freshly prepared Chelex solution.
l Label the tubes appropriately.
 Troubleshooting 101

8 Troubleshooting

Problem Tentative cause Possible solutions

No amplification Inhibition of PCR Wait overnight before using
of DNA after reaction by leftover the extracted DNA samples
PCR Chelex solution
Dilute the extracted DNA
sample to minimize the final
content of leftover Chelex in
the template
Sufficient DNA not Repeat the extraction step
extracted
Mixed profile Contamination with Confirm contamination with
observed after exogenous DNA negative control
genotyping
Use sterilized plasticware and
glassware during the
extraction process
 Chapter 13

Isolation of DNA by Using Column-Based Extraction System

Objective: To isolate DNA from blood and forensic samples using

column-based extraction system.

1 Introduction

Extraction of DNA is the fundamental method to be used in

molecular biology and forensic DNA typing. DNA extraction
from any biological material is either of analytical or preparative
purposes. Earlier, many complicated, time-consuming, laborious
techniques were used for isolation of DNA. However, with the
advent of technology, many sophisticated, advanced, and
specialized systems have been developed for extraction and purifi-
cation of these biomolecules such as the solution- and column-
based protocols. Gradually, the manual methods have been
replaced by the semiautomatic and automated techniques which
has not only reduced the time required for DNA extraction but also
increased the quality of DNA obtained by reducing the chance of
contamination.
The conventional DNA extraction steps involve the cell lysis to
disrupt the cellular structure followed by inactivation of nucleic
acid-degrading enzymes (i.e., DNase and RNase) and finally pur-
ifying the desired nucleic acid from the cell debris. This is called as
liquid-liquid extraction of DNA. However, spin column-based
nucleic acid purification can be achieved using a solid-phase extrac-
tion system for rapid purification of nucleic acid. This technique
relies on the fact that nucleic acids bind to silica under specific
conditions. Many column-based DNA extraction kits are available
in the market. The selective use of the kits for forensic DNA typing
application depends on the nature of the sample, type of sample, as
well as analyst’s preference.

H. R. Dash et al., Principles and Practices of DNA Analysis: A Laboratory Manual for Forensic DNA Typing, Springer Protocols
Handbooks, https://doi.org/10.1007/978-1-0716-0274-4_13, © Springer Science+Business Media, LLC, part of Springer Nature 2020

103
104 Isolation of DNA by Using Column-Based Extraction System

2 Principle

Column-based DNA extraction relies on the principle of solid-

phase extraction system enabling the rapid purification of nucleic
acids. The stages of this method involve lysis, binding, washing, and
elution for effective purification of DNA. During lysis, the cells are
broken open from cellular as well as nuclear membrane to release
the nucleic acid into the solution. In the binding step, a binding
solution is applied on the spin column to provide it a specific
condition for categorical binding of nucleic acids. Upon addition
of the lysed product to the spin column followed by centrifugation,
at optimal pH and salt concentration, the nucleic acid binds to the
silica gel membrane and other biomolecules present in the lysis
solution pass through. This step is followed by the addition of
wash buffer which removes the remaining cell debris leaving only
DNA to bind to the silica membrane. Finally, an elution buffer is
added to the column which removes the nucleic acid from the
column for its subsequent collection at the bottom of the tube.
The major composition of these silica membranes is diethyla-
minoethanol (DEAE). Under suitable pH, when H+ ions are added
to the column, it becomes positively charged. As it is a well-known
fact that DNA molecule is negatively charged, it can categorically
bind to the positively charged column and other cell debris pass
through it (Fig. 13.1). This enables the purification of DNA opti-
mally with highest quantity and quality.
There are many commercially available kits in the market for
column-based extraction of DNA from forensic samples. These kits
may be used manually and employed along with the semiautomatic
instruments to minimize repeated pipetting. In this section we have
included automated extraction of DNA from liquid blood sample
using QIAamp® DNA Investigator® kit and QIAcube liquid-
handling system (QIAGEN®). The other available kits with forensic
DNA isolation implications employing column-based principle
have been listed in Appendix B of the current book.

Fig. 13.1 Principle of binding of silica columns to negatively charged DNA for effective purification
 Reagents and Plasticware Required and Their Role 105

3 Reagents and Plasticware Required and Their Role

3.1 QIAamp® DNA The constituents of the kit include but not limited to the following
Investigator® Kit items:
Buffer AL: It is used for isolation of DNA using the aforemen-
tioned kit, mostly for lysis of the cells. Buffer AL may precipi-
tate upon storage, which can be removed by its incubation at
55  C prior to the start of DNA isolation. However, buffer AL
can remain stable at room temperature (15–25  C) till 1 year.
Buffer AW1: This represents the first wash buffer. Buffer AW1
generally comes in the form of concentrated solution which
needs to be diluted prior to use. 19 ml of buffer should be
diluted with 25 ml of absolute ethanol. This reconstituted
buffer can be stored at room temperature and used up to
1 year. However, this buffer should be mixed by shaking
before use.
Buffer AW2: This represents the second wash buffer. Like AW1,
13 ml of AW2 should be reconstituted with 30 ml of absolute
ethanol. The reconstituted AW2 will remain stable at room
temperature for 1 year. Buffer should be mixed by shaking
prior to its use.
Carrier RNA: Carrier RNA is supplied with the kit. It enhances the
binding of DNA to the membrane of QIAamp MinElute®
columns. It is useful in low-DNA samples when less amount
of target molecules are available to bind the column. However,
carrier RNA also gets extracted along with the DNA in the final
elution step. Thus, carrier RNA should be added wisely and for
samples with higher amount of DNA carrier RNA should not
be used.
Proteinase-K: Peptide bond present near the carboxyl group of
amino acids with blocked alpha amino groups is the cleavage
site for the serine protease, i.e., proteinase-K. It helps in
degrading many protein impurities to yield a good quality of
DNA. It also plays an important role in the inactivation of
nucleases, hence preventing the isolated DNA from damage.
The activity of proteinase-K is greatly increased by its simulta-
neous action with SDS and high temperature. Proteinase-K
works optimally at 50–65  C as higher temperature unfolds
certain proteins to ease the function of the enzyme. However,
proteinase-K is being supplied by the kit manufacturer.
QIAamp MinElute® Columns: These columns work on the fast spin
column procedure, where the traditional phenol-chloroform
extraction steps are not required. In this case, the nucleic acid
categorically binds to the membrane of the columns and allows
the other contaminants to pass through. Washing of the
106 Isolation of DNA by Using Column-Based Extraction System

column membranes allows the efficient removal of many PCR

inhibitors such as divalent cations and proteins.
Buffer ATE: This buffer helps in the efficient removal of DNA from
the bound columns and maintains proper storage condition of
the eluted DNA. In an alternative, molecular biology-grade
water can also be used for elution of DNA from the columns.
The elution volume is chosen significantly as per the nature and
condition of sample as well as the requirements of the down-
stream processing of the samples.

4 Procedure

1. Transfer a minimum of 1 μl and a maximum of 100 μl of liquid

whole blood sample to a 1.5 ml microcentrifuge tube.
2. Make up the total volume to 100 μl by adding buffer ATL.
3. Add 100 μl of buffer AL and 10 μl of proteinase-K, vortex for
15 s to prepare a homogenized solution, and incubate the
samples at 56  C for 10 min.
4. Add 50 μl of absolute ethanol to the lysate and mix properly by
vortexing for 15 s.

5. Incubate the tubes at room temperature (15–25 C) for
2–5 min.
6. Prepare the QIAamp MinElute® column by placing it in a 2 ml
collection tube and without wetting its rim transfer the entire
lysate to the column.
7. Centrifuge the column containing the lysate at 6000  g for
1 min at room temperature.
8. Discard the collection tube containing the flow through and
place the QIAamp MinElute® Column in a fresh 2 ml
collection tube.
9. Add 500 μl buffer AW1 to the column and centrifuge at
6000  g for 1 min at room temperature.
10. Discard the collection tube containing the flow through and
place the QIAamp MinElute® Column in a fresh 2 ml
collection tube.
11. Add 700 μl of buffer AW2 to the column and centrifuge at
6000  g for 1 min at room temperature.
12. Discard the collection tube containing the flow through and
place the QIAamp MinElute® Column in a fresh 2 ml
collection tube.
13. Add 700 μl of absolute ethanol to the column and centrifuge at
6000  g for 1 min at room temperature.
 Precautions 107

14. Discard the collection tube containing the flow through and
place the QIAamp MinElute® Column in a fresh 2 ml
collection tube.
15. Centrifuge at full speed for 3 min at room temperature to
remove excess ethanol remaining in the column.
16. Place the QIAamp MinElute® Column in a fresh 2 ml collec-
tion tube and incubate at room temperature for 10 min by
opening the lid of the column.
17. Add 20–50 μl of buffer ATE to the center of the membrane and
incubate for 1 min at room temperature.
18. Centrifuge at full speed for 1 min. Transfer the elute from the
collection tube to a fresh 1.5 ml microcentrifuge tube and store
at 20  C.

5 Observation

The quantity of the isolated DNA can be determined at the prelim-

inary level by UV–visible spectrophotometer. For a 1 cm path
length, the optical density at 260 nm (OD260) equals 1.0 for the
following samples:
l 50 μg/ml of double-stranded DNA
l 33 μg/ml of single-stranded DNA
l 20–30 μg/ml of oligonucleotide
l 40 μg/ml of RNA

6 Result Table

Sample DNA content OD260/280 Inference

Control
Sample 1 (liquid blood)
Sample 2 (coagulated blood)

OD optical density

7 Precautions
l Follow the manufacturer’s guidelines in each step carefully.
l Wear gloves while dealing with the reagents and plasticware.
l Avoid using an expired kit.
108 Isolation of DNA by Using Column-Based Extraction System

l Do not incubate the samples more than the recommended

period of time; it may result in precipitation of salts.
l Load the lysate to the QIAamp MinElute column without wet-
ting its rim.
l Change pipette tips in between two steps.
l Use aerosol-resistant tips to avoid contamination.
l Do not touch the QIAamp MinElute column membrane
with tip.

8 Troubleshooting

Problem Tentative cause Possible solutions

Less/no DNA Carrier RNA not Dissolve carrier RNA to buffer AL
elute added for low DNA sample and repeat
the purification step
Improper storage Avoid repeated freezing and
of samples thawing of samples; use
recommended storage condition
of the samples
Improper lysis of Monitor the storage of proteinase-
samples K; use a new batch of proteinase-
K
Poor quality of pH of water used DNA will not dissolve properly in
DNA for elution was acidic solutions; ensure correct
too low pH of water
Clogged Incomplete lysis of Increase lysis time or proteinase-K
QIAamp sample concentration
MinElute
column
 Chapter 14

Reliable Use of Whatman™ FTA™ Cards for One-Step

Collection and Isolation of DNA

Objective: One-step collection and isolation of DNA from varied

sources using Whatman™ FTA™ cards and the technology.
FTA™ (Flinders Technology Associates) card (GE Whatman,
Maidstone, Kent, United Kingdom) is a specialized filter paper
which is impregnated with reagents promoting cell lysis and protein
denaturation followed by release of nucleic acids that are entrapped
in the card matrix and stable at room temperature for a long period
of time. Though forensic DNA technology has geared up over the
years in terms of automation and many up-gradation and standar-
dized protocols are in practice, DNA extraction and sample collec-
tion steps still remain the analyst’s preference. In this regard,
FTA™ card may prove to be useful for a wide range of forensic
samples.
These cards enable the optimal collection, transportation, puri-
fication, as well as long-term storage of DNA at room temperature
by the use of a single card, which can be used for both forensic and
database applications. The fundamental of FTA™ cards is that it
lyses the eukaryotic cells that are in contact with the cards. After
lysis the proprietary chemical content of the card protects the DNA
from the environmental and enzymatic damages. Additionally,
DNA on FTA™ cards need not be stored at freezing temperature
as DNA on FTA™ cards has been evidenced to remain stable at
room temperature. Many countries have provisions for long-term
storage and retesting of DNA samples after several years such as
post-conviction testing. In such cases storage of DNA on FTA™
cards is highly useful. Additionally, the chemicals present on these
cards also protect the DNA from harmful UV lights. US Postal
Service Mail has marked FTA™ cards to be safe, and thus can be
transported without any hazardous labeling.

H. R. Dash et al., Principles and Practices of DNA Analysis: A Laboratory Manual for Forensic DNA Typing, Springer Protocols
Handbooks, https://doi.org/10.1007/978-1-0716-0274-4_14, © Springer Science+Business Media, LLC, part of Springer Nature 2020

109
110 Reliable Use of Whatman™ FTA™ Cards for One-Step Collection and Isolation of DNA

1 Sample Types and Collection Procedures

The cards have been designed to be used for collection, transport,

and storage of a wide range of samples, viz. buccal cells, saliva,
blood, and other soft tissues. The ideal procedure for collection
of samples has been described below.
1.1 Collection of 1. Wear gloves, and label the cards with appropriate sample
Liquid Blood identification.
2. Drop <125 μl of freshly collected liquid blood (per 1 in. circle)
in a concentric circular motion on the card.
3. Dry the card for 3–5 min.
4. Place the card in a sealed envelope and send it for investigation at
room temperature.

1.2 Collection of 1. Roll the Catch-All sample collection swabs (Epicentre Bio-
Buccal Cells technologies) tightly inside both sides of the cheek for 15 s.
2. Rub the swabs in a concentric circular motion on the card.
3. Dry the card for 3–5 min.
4. Place the card in a sealed envelope and send it for investigation at
room temperature.

1.3 Collection of 1. Ask the sample donor to wash his/her mouth with water and
Saliva Samples wait for 30 s.
2. Tell him/her to spit in the blue container (DNA Genotek).
3. Dip the Catch-All sample collection swabs (Epicentre Bio-
technologies) in the container containing saliva and rub it in a
concentric circular motion on the card.
4. Dry the card for 3–5 min.
5. Place the card in a sealed envelope and send it for investigation at
room temperature.
Note: Store the unused cards in a cool, dry, and clean environ-
ment. Avoid puddling of the samples. Place the sample within the
marked circular area. Do not rub or smear the sample on the card.

2 DNA Extraction and Usages (Applications)

Though many genotyping kits (PowerPlex® ESX, AmpFlSTR Iden-

tifiler Direct PCR Amplification Kit, GlobalFiler® Express, Power-
Plex® Fusion 6C, Investigator® 24plex GO!, and AmpFISTR®
Yfiler® Plus to mention a few) have been developed for direct
amplification of DNA sample on FTA™ cards, DNA can also be
extracted from FTA™ cards using any of the following
methodologies.
 DNA Extraction and Usages (Applications) 111

2.1 Organic 1. Remove a disk of around 2 mm size from the center of a FTA™
Extraction card containing dried blood stains using a Harris Uni-Core
disposable punch.
2. Place the disk in a 1.5 ml DNase/RNase-free microcentrifuge
tube.
3. Add 500 μl of extraction buffer (10 mM Tris–HCl, pH 8.0;
10 mM EDTA, disodium salt, pH 8.0; 100 mM sodium chlo-
ride; and 2% v/v SDS) and 20 μl of proteinase-K (20 mg/ml).
4. Mix by vortexing and incubate at 56  C for 2 h in a water bath
with gentle shaking.
5. Add equal volume of phenol (pH 8.0), mix properly, and
centrifuge at maximum speed for 10 min at 4  C.
6. Transfer the aqueous upper layer to a fresh 1.5 ml microcen-
trifuge tube and add equal volume of chloroform to it.
7. Mix properly and centrifuge at maximum speed for 10 min
at 4  C.
8. Transfer the upper aqueous phase to a fresh 1.5 ml microcen-
trifuge tube and add 50 μl 3 M sodium acetate (pH 5.2) to it,
followed by the addition of 800 μl of 100% ethanol, mixing by
vortexing, and precipitation at 20  C for 1.5 h.
9. Centrifuge for 30 min at maximum speed at 4  C and discard
the supernatant.
10. Add 1 ml of 70% ethanol and centrifuge at maximum speed for
20 min at 4  C.
11. Dry the pellet at room temperature followed by addition of
50 μl of TE buffer for dissolving of pellet.
12. Store the extracted DNA at 20  C till further use.

2.2 Using Chelex 100 1. Remove a disk of around 2 mm size from the center of a FTA™
Resin card containing dried blood stains using a Harris Uni-Core
disposable punch.
2. Place the disk in a 1.5 ml DNase/RNase-free microcentrifuge
tube.
3. Crush the disk using a 20-gauge needle.
4. Wash the crushed disk twice by adding 1 ml of sterile distilled
water followed by incubation at room temperature for 10 min
and removal of water.
5. Centrifuge the tube at maximum speed for 5 min and discard
the supernatant.
6. Add 200 μl of Chelex 100 to the tube and incubate at 56  C for
20 min.
7. Mix by vortexing for 15 s and incubate at 100  C for 8 min
followed by mixing by vortexing for 15 s.
112 Reliable Use of Whatman™ FTA™ Cards for One-Step Collection and Isolation of DNA

8. Centrifuge the tube at maximum speed for 5 min, transfer the

supernatant containing extracted DNA to a fresh tube, and
store at 20  C till further use.

2.3 QIAamp™ DNA 1. Remove a disk of around 2 mm size from the center of a FTA™
Investigator Kit card containing dried blood stains using a Harris Uni-Core
disposable punch.
2. Place the disk in a 1.5 ml DNase/RNase-free microcentrifuge
tube.
3. Add 280 μl of buffer ATL to the tube, add 20 μl of proteinase-
K to it, and mix by vortexing for 30 s.
4. Incubate at 56  C for 2 h in water bath with gentle shaking.
5. Centrifuge the tube at maximum speed for 30 s and add 300 μl
of buffer AL to it.
6. Mix by vortexing and incubate at 70  C for 10 min in a water
bath with gentle shaking.
7. Centrifuge the tube at maximum speed for 30 s and transfer the
supernatant to the minElute column.
8. Centrifuge the column at 6000  g for 1 min at room temper-
ature and discard the flow through.
9. Add 700 μl of buffer AW2 to the column, centrifuge at
6000  g for 1 min at room temperature, and discard the
flow through.
10. Add 700 μl of 100% ethanol to the column, centrifuge at
6000  g for 1 min at room temperature, and discard the
flow through.
11. Centrifuge the column at maximum speed for 3 min and
incubate the column at room temperature for 10 min by open-
ing the lid of the column.
12. Add 50 μl of sterile distilled water/buffer ATE and incubate at
room temperature for 5 min.
13. Centrifuge at maximum speed for 1 min, and store the flow
through containing DNA at a fresh centrifuge tube at 20  C
till further use.

2.4 illustra™ Tissue 1. Remove a disk of around 2 mm size from the center of a FTA™
and Cells genomicPrep card containing dried blood stains using a Harris Uni-Core
Mini Spin Kit disposable punch.
2. Place the disk in a 1.5 ml DNase/RNase-free microcentrifuge
tube.
3. Add 1 ml of sterile phosphate-buffered saline (PBS) to the tube
and centrifuge at 16,000  g for 1 min at room temperature.
 DNA Extraction and Usages (Applications) 113

4. Crush the disk using a 20-gauge needle; centrifuge at 2000  g

for 10 s at room temperature.
5. Add 50 μl of buffer 1 to the tube, add 10 μl of proteinase-K,
and vortex for 15 s.
6. Incubate at 56  C for 1 h.
7. Centrifuge at 2000  g for 10 s; add 5 μl of RNase A (20 mg/ml)
followed by incubation at room temperature for 15 min.
8. Add 500 μl of buffer 4 and mix by vortexing for 15 s, followed
by incubation at room temperature for 10 min.
9. Transfer the whole content (lysate) to a mini column, centrifuge
at 11,000  g for 1 min, and discard the flow through.
10. Add 500 μl of buffer 4 to the column, centrifuge at 11,000  g
for 1 min, and discard the flow through.
11. Place the column on a fresh collection tube, add 500 μl of
buffer 6, and centrifuge at 11,000  g for 3 min.
12. Transfer the column to a fresh 1.5 ml microcentrifuge tube,
add 50 μl pre-warmed elution buffer to the column, and
incubate at room temperature for 1 min.
13. Centrifuge at 11,000  g for 1 min and store the elute containing
DNA at 20  C till further use.

2.5 DNA IQ™ Kit 1. Remove a disk of around 2 mm size from the center of a FTA™
card containing dried blood stains using a Harris Uni-Core
disposable punch.
2. Immerse the punch with 100 μl of lysis buffer and incubate at
70  C for 30 min.
3. Transfer the lysate to a column placed in a 1.5 ml microcen-
trifuge tube and centrifuge for 2 min at maximum speed at
room temperature.
4. Collect the flow through and add 7 μl of premixed resin to it.
5. Mix by vortexing for 3 s followed by incubation at room
temperature for 5 min with repeated vortexing.
6. Place the tube on a magnetic stand, and discard the supernatant
without disturbing the pellet.
7. Add 100 μl of lysis buffer, vortex the tube for 2 s, place the tube
on magnetic stand, and discard the lysis buffer.
8. Add 100 μl of 1 wash buffer, vortex the tube for 2 s, place the
tube on magnetic stand, and discard the wash buffer.
9. Repeat step 8 two more times.
10. Place the tube on a magnetic stand with lid open and air-dry for
10 min.
114 Reliable Use of Whatman™ FTA™ Cards for One-Step Collection and Isolation of DNA

11. Add 100 μl of elution buffer, vortex for 2 min, and incubate at
65  C for 5 min.
12. Vortex for 2 s, place the tube on magnetic stand, and transfer
the elute to a fresh tube.
13. Store the tube containing DNA at 20  C till further use.
FTA or any other commercially available cards can have a wider
application in forensic use which includes but not limited to the
following: DNA extraction, DNA quantitation using real-time
PCR, STR analysis using conventional genotyping kits, analysis of
mitochondrial DNA, as well as SNP analysis.

3 Available FTA™ Cards and Other Related Products

Many types of FTA™ cards are available in the market. Additionally,

similar cards from different manufacturers are also available com-
mercially which has been listed in Table 14.1.

Table 14.1
Commercially available cards for direct collection and extraction of DNA from biological samples and
their nature

Products Feature Manufacturer and address

FTA Classic Card 125 μl/sample area GE Healthcare, UK
Indicating FTA Classic Card 125 μl/sample area GE Healthcare, UK
FTA Mini Card 125 μl/sample area GE Healthcare, UK
Indicating FTA Mini Card 125 μl/sample area GE Healthcare, UK
FTA Micro Card 125 μl/sample area GE Healthcare, UK
Indicating FTA Micro Card 125 μl/sample area GE Healthcare, UK
FTA Gene Card 75 μl/sample area GE Healthcare, UK
PlantSaver™ Card 25 μl/sample area GE Healthcare, UK
CloneSaver™ Card 5 μl/sample area GE Healthcare, UK
EasiCollect 125 μl/sample area GE Healthcare, UK
FTA Elute Micro Card 30 μl/sample area GE Healthcare, UK
FTA Elute Micro Card 30 μl/sample area GE Healthcare, UK
Ahlstrom-Munksjö 125 μl/sample area AHLSTROM-MUNKSJÖ, North America
GenSaver™
Ahlstrom-Munksjö 70 μl/sample area AHLSTROM-MUNKSJÖ, North America
GenSaver™ Color
Ahlstrom-Munksjö 5/125 μl/sample area AHLSTROM-MUNKSJÖ, North America
GenSaver™ 2.0

(continued)
 Available FTA™ Cards and Other Related Products 115

Table 14.1
(continued)

Products Feature Manufacturer and address

Ahlstrom-Munksjö 5/70 μl/sample area AHLSTROM-MUNKSJÖ, North America
GenSaver™ Color 2.0
NUCLEIC-CARD™ N/A COPAN ITALIA S.P.A., Italy
GENOlCARD N/A Hain Lifescience GmbH, Germany
®
NucleoCard <200 μl MACHEREY-NAGEL, Germany
GenSaver™ DNA cards N/A GenTegra®, Pleasanton, CA
NUCLEIC-CARD™ matrix N/A Thermo Fisher Scientific
InstaDNA™ Card 10–200 μl HiMedia, Mumbai, India
 Part IV

Qualitative and Quantitative Assessment of Extracted DNA

 Chapter 15

Quantification of DNA by Using Agarose Gel Electrophoresis

Technique

Objective: To quantify the isolated DNA from blood and forensic

samples using agarose gel electrophoresis technique.

1 Introduction

Downstream processing of samples requires a defined concentra-

tion of DNA, and hence proper quantification of DNA is the must
prerequisite during DNA analysis. DNA content of forensic sam-
ples varies greatly with the nature of sample, storage condition, as
well as their stages of degradation. Hence, proper quantification of
the same is highly essential prior to any further analysis. Though
many techniques are available for suitable quantification of DNA,
agarose gel electrophoresis is considered to be the most suitable
physical technique for the same. In this technique, DNA migrates
along an agarose matrix under the influence of electric current.
Many factors control the migration of DNA on agarose gel which
include size of DNA, concentration of agarose matrix, voltage
applied, presence or absence of ethidium bromide, type and nature
of agarose, as well as strength of buffer used. It is a typical sieving
process where the small-sized DNA fragments migrate faster and
the large fragments migrate slower which is dependent on the
agarose concentration.
The sensitivity of detection of DNA fragments during STR
analysis is dependent on the sensitivity of the DNA quantification
methodology used. In this regard, electrophoresis through agarose
gel provides an added advantage as it can separate, identify, and
purify nucleic acids. The matrix of the agarose gel has adjustable
and regular pore size. Chemically inert nature of agarose gel also
provides an added advantage.

H. R. Dash et al., Principles and Practices of DNA Analysis: A Laboratory Manual for Forensic DNA Typing, Springer Protocols
Handbooks, https://doi.org/10.1007/978-1-0716-0274-4_15, © Springer Science+Business Media, LLC, part of Springer Nature 2020

119
120 Quantification of DNA by Using Agarose Gel Electrophoresis Technique

2 Principle

Agarose is a natural linear polymer which is extracted from the

seaweed. It can form a matrix by hydrogen bonding when cooled
followed by heating in a buffer. These gels are simple in nature and
can be prepared rapidly. Moderate- and large-sized nucleic acids can
be separated using this medium with optimal resolution depending
on the concentration of the gel. The concentration of the agarose
gel to be prepared depends on the fragment size of DNA. Mostly
the gel is prepared with a concentration of 0.2–3% with the funda-
mentals that lower the agarose gel concentration and increase the
migration of DNA fragments. For a proper resolution of banding
patterns of the DNA fragments, a standard concentration of aga-
rose gel should be used as per Table 15.1.
The basic principle of agarose gel electrophoresis includes the
characteristic migration of a charged molecule towards either posi-
tive or negative electrode under the influence of electric field
(Fig. 15.1). Nucleic acids possess a constant negative charge due
to the net charge imparted by their phosphate backbone constitu-
ents and hence migrate towards the anode. A potential gradient
“E” is generated under the influence of the voltage applied which
can be determined by the equation, E ¼ V/d (where “V” is the
amount of voltage applied and “d” is the distance between the
electrodes in centimeter). Under the influence of the electric gradi-
ent “E,” a force is generated on the charged molecule which can be

Table 15.1
Concentrations of agarose gel and their respective power of resolutions for linear DNA molecules

Agarose concentration (in %) Size of DNA fragments (in kb)

0.2 5–40
0.3 5–60
0.4 5–30
0.6 1–20
0.7 0.8–10
0.8 1–7
0.9 0.5–7
1 0.5–5
1.2 0.4–6
1.5 0.2–3
2 0.1–2
3 0.1–1
 Reagents Required and Their Role 121

Fig. 15.1 Migration pattern of charged molecules under the influence of electric
field

expressed by F ¼ E·q (where “q” is the charge of the molecule in

coulombs). This force “F” is responsible for dragging of the DNA
molecule to the corresponding electrode as per the charge of the
molecule. Additionally, a negative force also exists which helps in
slowing down of the migration pattern of the molecule called as
frictional force and is dependent on (a) hydrodynamic size of the
molecule, (b) shape of the molecule, (c) pore size of agarose gel,
and (d) viscosity of the buffer used.
During analysis of DNA fragments on agarose gel, a fluorescent
intercalating dye is added to the gel matrix, i.e., ethidium bromide
or gold view. These dyes intercalate to the DNA molecules as they
migrate in the gel and flourish under UV light. The principle of
quantification of DNA molecules includes the fact that the higher
the amount of DNA molecules, the more the intensity of bands
visualized under UV light. Thus, when a standard concentration of
DNA molecule is run on agarose gel side by side with the unknown
DNA fragment, by comparing the intensity of the bands, the rela-
tive as well as absolute quantification of the DNA molecule can be
carried out.

3 Reagents Required and Their Role

3.1 Agarose Agarose consists of the repeated subunits of agarobiose, i.e., L- and
D-galactose subunits. It is mostly extracted from the seaweeds of
Gelidium and Gracilaria genera. During gelation the polymers of
agarose associate with each other non-covalently to form a macro-
porous matrix with specific pore size for the specific separation of
nucleic acids. It is nontoxic, devoid of free radical involvement
during polymerization, and thermoreversible in nature which are
122 Quantification of DNA by Using Agarose Gel Electrophoresis Technique

the added advantages of agarose for separation of nucleic acids

during electrophoresis.

3.2 Tris-Acetate The mobility of DNA is dependent on the composition and ionic
Buffer strength of the electrophoresis buffer. In this regard, two most
common buffers are employed which include Tris-borate-EDTA
and Tris-acetate-EDTA. The electrophoresis buffer maintains the
pH of the reaction to neutral, and thus net charge of DNA solely
becomes responsible for its migration under the influence of elec-
tric field. Additionally, buffer provides a constant liquid medium to
prevent hydrolysis of DNA molecules besides preventing DNA
from the attack of DNase. It is always recommended to use freshly
prepared 1 TAE buffer during electrophoresis. However, a stock
solution of 10 TAE can be prepared using the following compo-
sition: Tris—400 mM, acetic acid—200 mM, EDTA—10 mM, and
distilled water—up to 250 ml.

3.3 Ethidium Ethidium bromide (EtBr) is a ring-structured hydrophobic com-

Bromide pound which resembles the nucleotide bases of DNA. It is an
intercalating agent and intercalates between the stacked nucleotide
bases of DNA by forming close van der Walls interaction with the
base pairs. Ethidium is fluorescent in nature and it flourishes in
visible range when exposed to UV light. Thus during electropho-
resis, the DNA fragments that contain intercalated EtBr molecules
also flourish without which the DNA fragments would not have
been visualized. EtBr is required in a trace amount (0.5 μg/ml)
during electrophoresis. For this, add 5 μl of stock EtBr solution
(concentration 10 mg/ml) after the molten agarose gel has cooled
down to 60–70  C.

3.4 Gel-Loading Gel-loading buffer comprises dyes and other compounds that help
Buffer in assessing the speed of the DNA sample during electrophoresis as
well as ascertaining that the sample becomes denser than the buffer.
The dye varies with the manufacturer; however, xylene cyanol,
cresol red, bromophenol blue, orange G, and tartrazine are the
common constituents. Additionally, to increase the density of
DNA, another constituent is generally present in the gel-loading
buffer which includes Ficoll, sucrose, or glycerol. Mostly, the com-
mercially available gel-loading buffer comes as 6 concentration
and its final working concentration should be 1. The buffer
should be stored at 4  C to avoid the growth of molds utilizing
sucrose.

3.5 DNA Size Marker Most of the manufacturers provide ready-to-load DNA size mar-
kers. The concentration of size marker varies with the manufacturer
and during loading manufacturer’s recommended guidelines
should be followed. A DNA ladder of 1 kb size generally gives
fragments of various sizes, i.e., 0.5, 1.0, 1.5, 2.0, 3.0, 4.0, 5.0,
 Procedure 123

Fig. 15.2 Different fragments of DNA in a typical DNA size marker along with their corresponding concentra-
tion of DNA

6.0, 8.0, and 10.0 kb. The amount of DNA (in ng) present on each
DNA fragment is given along with the datasheet of the manufac-
turer which is typically as follows (Fig. 15.2):

3.6 DNA Sample Extracted DNA from reference as well as forensic samples can be
used for estimation of DNA quantity. If you expect a higher quan-
tity of DNA, dilute the extract in TE buffer prior to running the
samples on agarose gel. Various known concentrations of control
DNA can be prepared in TE buffer for assessment of measurement
of DNA concentration by check gel technique.

3.7 Gel Many gel documentation systems are available in the market and
Documentation they have their own analysis software for banding pattern analysis.
System and Software For example Bio-Rad™ has the gel documentation system with the
for Analysis analysis software Quantity One®.

4 Procedure

1. Mix 30 mg of agarose in 30 ml of 1 TAE in a conical flask to

prepare 1% mixture.
2. Use a plastic to cover the flask. Pierce a small hole in the plastic
for ventilation.
3. Boil the solution using a microwave, and swirl the container
gently to resuspend the settled agarose.
4. Cool the agarose till you can touch the flask and add ethidium
bromide at the final concentration of 0.5 μg/ml (in this case
add 1.5 ml from 10 mg/ml stock solution).
5. Pour the gel mixture in the gel apparatus using the gel-casting
tray and comb.
6. Carefully remove the comb after solidification.
7. Prepare a written record of the samples loaded on each well.
Mix 10 μl of DNA sample and 2 μl of gel-loading dye and load
the final 12 μl to each well.
124 Quantification of DNA by Using Agarose Gel Electrophoresis Technique

8. Fill the electrophoresis tank with 1 running buffer, cover the

chamber with lid, and connect the electrodes with power
supply.
9. Turn on the power supply; monitor the gel carefully; once the
dye moves through the gel disconnect the electricity.
10. View the DNA sample in the gel documentation system.
11. Using the analysis software compare the intensity of the
unknown DNA fragments with the intensity of known DNA
fragments of the ladder.
12. The software will automatically calculate the DNA concentra-
tion of the unknown sample.

5 Observation

Manually observe the bands on the gel slab under UV light. Pres-
ence of various bands in the lane with DNA ladder confirms no
methodological error. Prepare standard curve using the fluores-
cence intensity vs. DNA amount in each band and calculate the
quantity of the isolated DNA using the same standard curve.

6 Result Table

Sample Fluorescence intensity Amount of DNA

Control
Sample 1 (liquid blood)
Sample 2 (bone powder)

7 Precautions
l Wear actual-fit heat-resistant gloves for preparation and pouring
of molten agarose.
l During manual checking of bands under UV light always use
UV-proof full-face shield.
l Clean the glassware thoroughly before preparing the
agarose gel.
l Do not overboil the agarose in the hot plate or microwave.
l Handle EtBr with extra care as it is highly carcinogenic in nature.
l Always use freshly prepared buffer for preparation of agarose and
running buffer.
l Dispose the used gels carefully.
 Troubleshooting 125

8 Troubleshooting

Problem Tentative cause Possible solutions

Faint of no DNA Increase the loading amount of DNA
band on the concentration in the gel; if problem persists
gel very low re-isolate DNA
Highly degraded Re-isolate DNA with extra precaution
DNA for nuclease contamination
DNA migrated out Decrease the electrophoresis time;
of the slab gel limit electrophoresis voltage to
20 V/cm; temperature during
electrophoresis should be
maintained at <30  C
UV light source Use UV light source of wavelength
not proper 254 nm
No band, only Highly degraded Re-isolate DNA with extra precaution
smear DNA for nuclease contamination
observed
Too much DNA Dilute the sample with TE buffer
prior to loading on agarose gel
Electrophoresis Maintain electrophoresis voltage to
conditions not 20 V/cm and temperature during
proper electrophoresis <30  C
 Chapter 16

Quantification of DNA by Using UV–Visible

Spectrophotometer

Objective: To quantify DNA isolated from blood and forensic

samples using UV–visible spectrophotometer technique.

1 Introduction

Quantification of DNA from the reference as well as the forensic

(questioned) samples is of utmost importance during DNA finger-
printing analysis. Though many advanced techniques are available
nowadays, UV–visible spectrophotometer is the most fundamental
technique of DNA quantification. This is mostly carried out by
measuring the solution containing DNA at 260 nm which is the
absorption maximum for DNA. This technique is very simple and
time saving in nature. However, high interference in result is
observed due to the presence of degraded nucleotides, denatured
nucleic acids, and colored compounds which are common in DNA
extracted from forensic samples.
In spite of all pitfalls, UV–visible spectrophotometer-based
technique is the most widely used technique for DNA quantifica-
tion due to its simplicity in experimentation and analysis of
obtained results. Additionally, this technique allows to assess the
quality of extracted DNA by measuring the protein and/or RNA
contamination in the sample. Compounds used during nucleic acid
preparation can also show absorbance at 260 nm, leading to inac-
curate measurement of DNA quantity. However, this interference
from these non-DNA samples can be avoided by measuring and
calculating the ratio of absorbance at A260/A280.

H. R. Dash et al., Principles and Practices of DNA Analysis: A Laboratory Manual for Forensic DNA Typing, Springer Protocols
Handbooks, https://doi.org/10.1007/978-1-0716-0274-4_16, © Springer Science+Business Media, LLC, part of Springer Nature 2020

127
128 Quantification of DNA by Using UV–Visible Spectrophotometer

2 Principle

The most common method of measurement of DNA concentration

in an aqueous solution is to measure its absorbance at 260 nm, as
the absorption maximum of DNA falls at this UV wavelength. In
this regard, the absorbance is measured using a spectrophotometer
and the DNA concentration is measured using the Beer-Lambert
law. This law deciphers the relationship between absorbance and
concentration of DNA in a linear fashion, which can be described
using the following equation:

A ¼ OD ¼ ε  l  c

where A: absorbance, OD: optical density, ε: molar extinction

coefficient, l: path length of cuvette, and c: concentration of the
solution.
The amount of extract to be used for measurement of DNA
concentration lies with the capacity of cuvette used. When the
capacity of cuvette is <0.2 ml, then 5 μl of extract mixed with
195 μl of water should be used to measure the absorbance. The
absorbance should be measured in triplicates to get the average
result. It is always recommended to discard the value of absorbance
at 260 nm either lower than 0.02 or between 1 and 1.5 due to
chance of error at a higher rate. Finally, the concentration of the
target molecule can be calculated using the following formula:
Concentration ðpmol=μlÞ : A260 =0:027 ½for single‐stranded DNA 
Concentration ðpmol=μlÞ : A260 =0:020 ½for double‐stranded DNA 
Concentration ðpmol=μlÞ : A260 =0:025 ½for single‐stranded RNA

Note: For a cuvette with 1 cm path length, an absorption at

260 nm A ¼ 1 corresponds to approximately 50 μg/ml of double-
stranded DNA, 37 μg/ml of single-stranded DNA, 40 μg/ml of
RNA, or 30 μg/ml of oligonucleotides. Single-stranded DNA
absorbs more UV than double-stranded DNA evidenced by the
increase in OD value. This change in OD value between single- and
double-stranded DNA is called as hyperchromic shift.
In addition to measuring the quantity of DNA, it is equally
important to measure the quality of DNA prior to further down-
stream processing of samples. Proteins, phenols, salts, and other
compounds routinely used during DNA extraction process can also
interfere with the proper quantification of DNA by absorbance
technique. Purity of nucleic acid samples can be determined by
measuring the ratio of absorbance at 260 nm (Amax of DNA) and
280 nm (Amax of protein). Optionally, background correction can
be done by measuring absorbance at 320 nm at which neither DNA
 Reagents/Materials/Instruments Required and Their Role 129

nor protein absorbs any light. The approximate value of purity of

the desired compounds can be determined by using the following
ratios:

Type A260/A280 (approximate)

Pure DNA 1.8
Pure RNA 2.0
Pure protein 0.57

3 Reagents/Materials/Instruments Required and Their Role

3.1 Spectro- Spectrophotometer is used to determine the concentration of a

photometer solute by transmitting light through the solution. The basic princi-
ple of the instrument lies with the fact that when a known wave-
length of light is passed through a cuvette containing sample, the
amount of light transmitted is measured to calculate the absorbance
by using a photocell. The simplest form of a spectrophotometer
involves a light source, a prism to filter a specific wavelength of
light, a sample holder, and a photocell (Fig. 16.1).

3.2 Cuvette The choice of cuvette to be used depends on the amount of nucleic
acid solution to be measured. Generally, the size and capacity of the
cuvette vary from 1 ml to as low as 5–70 μl. Thus, the cuvette
should be selected based on the sample concentration range,
dilution factor, and available sample volume.

3.3 TE Buffer TE buffer is generally prepared by mixing 50 mM Tris and 50 mM

EDTA at pH 8.0. Tris, the major constituent of TE buffer, acts
as a common pH buffer. Additionally, EDTA chelates cations like
Mg2+. Hence, TE buffer is helpful to solubilize DNA by protecting
it from degradation.

Fig. 16.1 Schematic presentation of a spectrophotometer and its parts

130 Quantification of DNA by Using UV–Visible Spectrophotometer

3.4 DNA Sample Extracted DNA from reference as well as forensic samples can be
used for the estimation of DNA quantity and quality. If you expect a
higher quantity of DNA, dilute the extract in TE buffer prior to
taking the absorbance of the sample. Various known concentrations
of calf thymus DNA (CT DNA) prepared in TE buffer can be used
as control DNA for the assessment of measurement of DNA con-
centration by UV–visible spectrophotometer.

4 Procedure

1. Turn on the power of spectrophotometer (Beckman DU64),

and check that all accessories like printer are ready.
2. Turn on the light source (UV light) and allow it to warm for at
least 5 min.
3. Select the absorbance mode using “ABS” key.
4. Press the “SCAN” key.
5. Go to “EDIT” and enter starting wavelength as “280 nm” and
press “Enter.”
6. Similarly, enter ending wavelength at “260 nm” and press
“Enter.”
7. Manage the speed of the sample scan at 750 nm/min by
pressing the “STEP” key and scrolling through the options to
find 750 nm/min followed by pressing of “Enter.”
8. Set the upper limit at 2000 absorbance; press “Enter.”
9. Set the lower limit at 0.000 absorbance; press “Enter.” Now
the starting wavelength will reappear.
10. The instrument now becomes ready for calibration against a
control solution.
11. Place 200 μl of TE buffer in a quartz cuvette, and open the
sample compartment lid of the instrument.
12. Carefully clean the cuvette using a Kimwipe® and place the
cuvette containing TE buffer into the cuvette holder. Make
sure that the quartz sides of the cuvette are in the path of light
source.
13. Close the lid of the sample compartment.
14. Press “CALB.”
15. The absorbance of TE buffer is being stored in the memory as
background and “Bkg” will appear in the display.
16. Press “READ.”
17. Wait till appearing of “Scan,” which shows that the calibration
is complete and the instrument is ready for sample analysis.
 Precautions 131

18. Open the sample compartment, remove cuvette, and discard

200 μl of TE buffer present in the cuvette onto the kimwipe.
19. Place 200 μl of solution containing DNA in EDTA in cuvette.
20. Place the cuvette in the sample holder.
21. Press “READ.”
22. The absorbance of the sample between 260 and 280 nm will be
measured and plotted as a graph.

5 Observation

For each DNA sample analyzed, note down the absorbance at

280 and 260 nm. Additionally, divide the absorbance at 260 nm
with the absorbance at 280 nm to gather the information regarding
the quality of DNA. To determine the value of DNA concentration
multiply the value of absorbance with the constant, i.e., 50 μg/ml.

6 Result Table

Sample DNA content OD260/280 Inference

Control
Sample 1 (liquid blood)
Sample 2 ( forensic sample)

OD optical density

7 Precautions
l Wear gloves while dealing with the reagents and plasticware.
l Do not touch the quartz size of the cuvette.
l Do not look at the light source directly.
l Clean the cuvette thoroughly before and after measurement.
l Use freshly diluted DNA samples for measurement of their
concentrations.
l Always fill the cuvette to 3/4th of its capacity.
l Use low-retention microtips during preparation of DNA
samples.
l Dispose the reagents and consumables carefully after the
examination.
132 Quantification of DNA by Using UV–Visible Spectrophotometer

8 Troubleshooting

Problem Tentative cause Possible solutions

Inappropriate DNA concentration is Dilute the sample in TE
absorbance too high buffer and take the
(OD) value reading again
Quartz side of the Change the orientation
cuvette is not of the cuvette in the
present in the line of sample holder
light source
Instrument not Call the service person of
working properly the instrument
The initialization of the Instrumental failure Click on “Cancel
spectrophotometry button”; power off the
Electricity problem
showing instrument, wait for
>>>FAILED<<<< few seconds, and
power it on again
The machine showing “Blank required” Place the cuvette with
various dialogues: TE filled in it and take
the measurement by
clicking on “BLANK”
icon button
“UV Lamp required” Switch on the UV source
light. Wait for few
minutes till the UV
light is warming up
“Visible Lamp Switch on the visible
required” light source. Wait for
few minutes till the
visible light is
warming up
 Chapter 17

Quantification of DNA by Using qRT-PCR

Objective: To assess the quality and quantity of DNA isolated from

blood and forensic samples using qRT-PCR technique.

1 Introduction

Careful qualitative and quantitative evaluation of DNA is an imper-

ative for forensic DNA analysis. Most of the DNA samples of
forensic origin contain a large number of PCR inhibitors, degraded
samples, as well as various exogenous contaminants, which affect
the downstream processing of DNA adversely. The commonly used
DNA quantitation techniques such as gel electrophoresis method
or UV–visible spectrophotometer technique are less accurate and
time consuming or they consume a substantial amount of the
precious sample. To overcome these problems and to increase the
accuracy and precision of DNA quantitation method, advanced
real-time quantitative PCR (qRT-PCR) technique has been discov-
ered with an increased efficiency to analyze the challenged,
complex-natured forensic DNA samples.
During forensic DNA typing, PCR is the must prerequisite to
amplify the autosomal as well as other STR markers. This sensitive
step requires an optimum input of DNA quantity to avoid/mini-
mize artifacts in the results which may include stochastic amplifica-
tion and/or oversized DNA profile. Thus, a real-time qPCR
technique is preferred over other techniques using 50 -nuclease
fluorogenic or TaqMan® assays. This technique has many advan-
tages over the other techniques such as generation of a linear
response proportional to DNA quantity and its occurrence in a
closed system which minimizes the potential carryover of contami-
nants. The automation associated with this system is an added
advantage by the use of automated fluorescence detection systems.
Currently many human-specific q-RT PCR assays are available

H. R. Dash et al., Principles and Practices of DNA Analysis: A Laboratory Manual for Forensic DNA Typing, Springer Protocols
Handbooks, https://doi.org/10.1007/978-1-0716-0274-4_17, © Springer Science+Business Media, LLC, part of Springer Nature 2020

133
134 Quantification of DNA by Using qRT-PCR

which target autosomal, Alu repeats, Y chromosome, and mtDNA

targets. Multiplex techniques are currently being used for simulta-
neous detection of large and small human telomerase reverse tran-
scriptase gene (hTERT), a synthetic internal positive control, and
male DNA fragment to give rise to information on total human
DNA quantity, male DNA vs. female DNA ratio, degradation
index, as well as information on potential PCR inhibitor. Many
commercially available kits are available (for details see Appendix
B), out of which Quantifiler® Trio DNA Quantification Kit of
Thermo Fisher Scientific has been described in this chapter.

2 Principle

Four 50 nuclease assays are carried out simultaneously in Quantifi-

ler® Trio DNA Quantification, i.e., human-specific short amplicon
assay, human-specific large amplicon assay, human male
DNA-specific assay, and assay for internal positive control (IPC).
The kit works in TaqMan® chemistry, where each PCR primer of
each target possesses specific dye-labeled probes and nonfluores-
cent quenchers as follows (Table 17.1):
The technique relies on the 50 nuclease assay which takes place
in each cycle of PCR process, which does not interfere with the end
product formation (Fig. 17.1). During PCR process, the TaqMan®
probe containing both the reporter dye and quencher compound
binds to the complementary sequence of the target DNA in
between the forward and reverse primer. In this step, the proximity
between the reporter dye and quencher suppresses the fluorescence
level of the reporter dye mostly by Förster-type energy transfer.
During the extension of primer in PCR process, when the growing
strand arrives at the point of attachment of TaqMan® probe due to
action of Taq polymerase, the TaqMan® probe is cleaved due to the
action of Taq polymerase and the reporter dye and quencher

Table 17.1
Targets and their associated dye and quenchers

Sl. Length of amplicon

no. Target (bp) Copy number Dye Quencher
1 Human-specific large 214 Multi-copy ABY™ QSY™
autosomal
2 Human-specific small 80 Multi-copy VIC™ MGB
autosomal
3 Human-specific male DNA 75 Multi-copy FAM™ MGB
4 Internal PCR control 130 Synthetic DNA JUN™ QSY™
template
 Principle 135

Fig. 17.1 Various steps of 50 nuclease assay using TaqMan® probe for fluorescent detection of quantity of DNA
using qRT-PCR. (a) Polymerization. (b) Strand displacement. (c) Polymerization complete

molecules are separated. This results in an increased fluorescence of

the reporter dye. Thus, there occurs an increase in fluorescence
level of the reporter dye only when the target DNA sequence
complementary to the TaqMan® probe is amplified in PCR and
hence the nonspecific amplifications cannot be detected.
The advantage of using qRT-PCR over other techniques is that
it allows real-time detection of amplicon at each PCR cycle which
results in the accurate detection of the amount of starting quantity
DNA, monitoring the real-time progress of PCR process, dynamic
136 Quantification of DNA by Using qRT-PCR

range of detection, and elimination of post-PCR manipulations.

Additionally, multiplexing allows simultaneous detection of
human-specific DNA, male DNA, degraded DNA, as well as pres-
ence/absence of PCR inhibitors. To understand the qRT-PCR
process better, a few terminologies associated with the process
have been described below.

2.1 Baseline Fluorescent signal levels during 3–15 PCR cycles do not vary much
which is referred to as the baseline. It can also be called as the
“noise.” During PCR process, the baseline can be determined
manually or automatically by the instrument. But a proper designa-
tion of baseline gives rise to the accurate determination of threshold
cycle (Ct). During designation of a baseline, one should be careful
not to choose the background noise as well as not to eliminate the
amplification signals.

2.2 Threshold Threshold signifies the statistically significant increase in the fluo-
rescent signal over baseline. Hence, the threshold level distin-
guishes the background noise from amplification signal. Most of
the qRT-PCR software set the threshold level to tenfold standard
deviation value of the baseline value. However, the threshold can be
set at any point of the exponential phase of the PCR process.

2.3 Threshold Cycle The PCR cycle number at which the fluorescent signal level crosses
(Ct) the threshold is called as the threshold cycle (Ct). As Ct value is
inversely proportional to the initial amount of DNA in the reaction,
it can be used to calculate the initial copy number of DNA present.

2.4 Standard Curve For the analysis of qRT-PCR results, a standard curve is required
using the known DNA concentrations. The standard curve is
prepared using the known DNA concentrations at x-axis and their
corresponding Ct values at y-axis. The concentrations of the known
DNA should be chosen carefully to accommodate the expected
DNA concentration of the unknown samples. Performance of
PCR as well as various reaction parameters can be derived from
the standard curve such as slope, y-intercept, and correlation
coefficient.

2.5 Correlation Correlation coefficient describes the authenticity of the standard

Coefficient (R2) curve. The ideal value of the R2 is 1; however, the calculated value
of 0.999 is considered to be excellent for the use of the standard
curve in result interpretation.

2.6 y Intercept The detection limit or sensitivity of the reaction is determined by

the y-intercept. This result corresponds to the expected Ct value if
the lowest amount of DNA present on the x-axis gives rise to a
statistically significant fluorescent signal. Most of the qRT-PCR
applications are capable of detecting a DNA amounting to 210
copy numbers, which is considered as the lowest target level.
 Reagents/Materials Required and Their Role 137

2.7 Exponential At the early part of the exponential phase of amplification, all the
Phase reagents such as DNA polymerase and primers are present in excess
amount. Hence, it is recommended to quantify the DNA concen-
tration at the early exponential phase of the reaction to get a better,
accurate, and sensitive result.

2.8 Slope The efficiency of a q-RT PCR reaction is measured by the slope of
the log-linear phase. A slope of 3.32 corresponds to 100% effi-
ciency of a q-RT PCR process. The slope value between 3.58 and
3.10 can be considered as a good-quality PCR process.

2.9 Efficiency
Efficiency ¼ 10ð1=slopeÞ  1

100% Efficiency of a PCR process is the doubling of the amount

of DNA after each PCR cycle in the exponential phase. Based upon
the above equation, the ideal efficiency of the reaction can only
happen when the slope value will be 3.32. A reaction is considered
to be good at the efficiency range of 90–110%. A reduction in PCR
efficiency below 90% occurs when nonoptimal reagent concentra-
tion and poor enzyme quality are used.

2.10 Dynamic Range The range over which the increase in template DNA concentration
results in the amplification product is called as the dynamic range.
Ideal dynamic range for genomic DNA samples should be in the
3–4 log range.

3 Reagents/Materials Required and Their Role

3.1 Quantifiler™ It contains dNTPs, buffer, enzyme, Mustang Purple™, and stabili-
THP PCR Reaction Mix zers. Mustang Purple™ is used as the passive reference dye. Stabi-
lizers are generally used in a PCR mix to increase the stability of the
PCR components and to increase the efficacy of a PCR process. The
common PCR stabilizer may include trehalose sugars or glycerol.
Upon receipt of this component at 25 to 15  C temperature, it
can be stored away from direct light at 2–8  C till further use.

3.2 Quantifiler™ Trio It contains the target-specific primers; probes labeled with ABY™,
Primer Mix JUN™, VIC™, and FAM™ dyes; as well as template DNA for
internal PCR control (IPC). As this is a multiplex process, the
added primers correspond to the DNA fragments specific to
human-specific large fragment, human-specific small fragment,
and human-specific male DNA and for the internal PCR control
and their corresponding probes the dyes are ABY™, VIC™,
FAM™, and JUN™. Upon receipt of this component at 25 to
15  C temperature, it can be stored away from direct light at
2–8  C till further use.
138 Quantification of DNA by Using qRT-PCR

3.3 Quantifiler™ The dilution buffer mostly contains the low TE buffer to be used
THP DNA Dilution for preparing various dilutions of the DNA standards. Upon receipt
Buffer of this component at 25 to 15  C temperature, it can be stored
at 2–8  C till further use.

3.4 Quantifiler™ It contains genomic DNA standard at a concentration of 100 ng/μ

THP DNA Standard l. This stock concentration is diluted using Quantifiler™ THP
DNA Dilution Buffer to obtain various dilutions to be used for
standard curve preparation. Upon receipt of this component at
25 to 15  C temperature, it can be stored at 2–8  C for
further use.

4 Procedure

Preparation of Standards
1. Label five 1.5 ml microcentrifuge tubes as Std. 1, Std. 2, Std.
3, Std. 4, and Std. 5.
2. Dispense 10 μl Quantifiler™ THP DNA dilution buffer to the
tube marked as Std. 1 and 90 μl of the same to the tubes Std.
2 to Std. 5.
3. Add 10 μl of Quantifiler™ THP DNA Standard to the buffer
containing tube marked as Std. 1. As the concentration of
Quantifiler™ THP DNA Standard is 100 ng/μl, the final
concentration becomes 50.000 ng/μl in Std. 1.
4. Vortex Std. 1 carefully for 3–5 s, and dispense 10 μl from Std.
1 to the buffer-containing tube marked as Std. 2 using a fresh
microtip. The final concentration of DNA in Std. 2 becomes
5.000 ng/μl.
5. Mix Std. 2 carefully by vortex for 3–5 s and dispense 10 μl from
Std. 2 to the buffer-containing tube marked as Std. 3 using a
fresh microtip. The final concentration of DNA in Std.
3 becomes 0.500 ng/μl.
6. Mix Std. 3 carefully by vortex for 3–5 s and dispense 10 μl from
Std. 3 to the buffer-containing tube marked as Std. 4 using a
fresh microtip. The final concentration of DNA in Std.
4 becomes 0.050 ng/μl.
7. Mix Std. 4 carefully by vortex for 3–5 s and dispense 10 μl from
Std. 4 to the buffer-containing tube marked as Std. 5 using a
fresh microtip. The final concentration of DNA in Std.
5 becomes 0.005 ng/μl.

PCR Setup
8. Calculate the requirement of each component of the PCR
process using the following table as per your requirement/
number of samples (Table 17.2).
 Procedure 139

Table 17.2
Amounts of reagents required to prepare master mixture for q-RT PCR

Volume per No. of reactions Total

qRT-PCR components reaction (μl) including standards volume
Quantifiler™ THP PCR Reaction Mix 8 A 8A
Quantifiler™ Trio Primer Mix 10 A 10A
Total volume ¼ (8A + 10A)

9. Thaw the Quantifiler™ THP PCR reaction mix and Quantifi-

ler™ Trio primer mix, vortex for 3–5 s, and centrifuge briefly
before using.
10. Take out the requisite quantity of reaction mix and primer
mix to a fresh microcentrifuge tube using separate fresh
microtips.
11. Mix the reagents of the tubes containing master mixture by
vortex of 3–5 s.
12. Pipette out 10 μl of master mixture into each well of a 96-well
plate or PCR tube.
13. Add 2 μl of standards, +ve/ve control, and unknown sam-
ples to the designated wells/tubes.
14. Use the optical adhesive cover to seal the 96-well plate or the
optical 8-cap strip for the strip tube.
15. Inspect each well of the plate/each tube for the presence of
any air bubbles. If present, tap each well with fingertip to
remove the same.
16. Rotate the plate in a centrifuge at 3000 rpm for 30 s. Now the
plate is ready to be loaded in the machine.

Experimental Setup
17. Switch on the computer by entering the required user name
and password.
18. Press the power button of 7500 Real Time PCR System
present at its lower right front.
19. Wait till the establishment of communication between the
computer and the instrument when the green power indicator
is lit.
20. Go to Start Programs ! Applied Biosystems ! HID Real-
Time PCR Analysis software ! HID Real-Time PCR analysis
software v1.2.
21. Log in to the software using the proper “user name” and
“password” or log in as “guest”.
140 Quantification of DNA by Using qRT-PCR

Fig. 17.2 Home screen of the software shows all the available kits for HID analysis. You have to select the icon
as per your use

22. After logging in to the software, the home screen will appear
as Fig. 17.2. Select the suitable application, i.e., Quantifiler™
Trio for the current application.
23. After selecting the suitable application, the experiment prop-
erties will appear in the screen (Fig. 17.3), where the name of
the experiment is filled. Rest credentials are autofilled by the
software.
24. Click on “Plate Setup.” Targets will be found to be prefilled as
per the selected assay.
25. Go to define samples and click on “Add New Sample”
(Fig. 17.4a, b). Fill the name of the sample. Repeat the
process for the samples as per your requirement.
26. Go to “Assign Targets and Samples” (Fig. 17.5). Click on the
well to select the sample which is present on that well. On the
left side of the plate layout select the desired sample in “Assign
sample(s) to the selected wells” for which you just selected the
well. Each sample is to be selected by default.
 Procedure 141

Fig. 17.3 “Experimental properties” showing credential to be filled, i.e., “Experiment name”

Fig. 17.4 Define samples by clicking on the “Add new samples” icon as per your requirement and fill the
sample name. (a) Default icons. (b) Addition of samples to be analyzed
142 Quantification of DNA by Using qRT-PCR

Fig. 17.5 Assign the samples and their corresponding targets in the well

Fig. 17.6 Display of Run Method for the run protocol of Quantifiler™ Trio kit

27. Select the “Run Method.” The run protocol for Quantifiler™
Trio kit has been set earlier which will be displayed (Fig. 17.6).
28. Click on “save” to save the protocol.
 Observation 143

Fig. 17.7 Click on “Start Run” after loading the plate in the instrument

Loading of the Plate in the Instrument

29. Press the “door” of the 7500 Real Time PCR instrument to
open the tray/plate holder.
30. Keep the plate on the plate holder with correctly aligned
manner and close the door.
31. Open the software, open the protocol that has been saved
earlier, and click on “Start Run” (Fig. 17.7).

Analysis of Result
32. After completion of run, check for “Amplification Plot,”
“Standard Curve,” “Multicomponent Plot,” “Raw Data
Plot,” “QC Summary,” and “Multiple Plots view” in the
section “Analysis” (Fig. 17.8).
33. To export the results, in the experiment menu, select “Analy-
sis” and “view plate layout,” select the wells to export and
select data to export, and click on “Start Export” (Fig. 17.9).

5 Observation

To access the quality of qRT-PCR result, the following parameters

should be taken care of:
l Amplification plot for small autosomal, large autosomal, and
internal PCR control should be present above Ct value.
l Amplification plot should not be present for NTC, i.e., no
template control.
l All the replicates of the sample/standard should show the same
or near-similar results.
l Standard curve should be linear when 5 pg/μl to 100 ng/μl of
samples are used.
144 Quantification of DNA by Using qRT-PCR

Fig. 17.8 Various parameters for accessing the qRT-PCR results, i.e., (a) amplification plot, (b) standard curve,
(c) multicomponent plot, and (d) raw data plot

l The R2 value should be 0.999.

l The slope should be close to 3.3.
l An increase in template DNA concentration may increase the Ct
value of the internal PCR control due to competition of PCR
components.
l A negative result is observed in case when no human DNA is
present. In such cases, amplification signals from small and large
autosomal and Y-specific targets are not present, but normal
amplification from internal PCR control is observed.
 Observation 145

Fig. 17.9 Export and save qRT-PCR results to the folder as per your requirement

l A complete failure in PCR process results in the absence of

results for all targets, i.e., small and large autosomal DNA,
Y-specific target, as well as internal PCR control.
l Low or weak amplification of IPC is an indication of the pres-
ence of potential PCR inhibitors in the reaction.
l The degradation index is calculated as small autosomal target
DNA concentration (ng/μl)/large autosomal target DNA con-
centration (ng/μl). A degradation index of <1 signifies a good
quality of sample, whereas the value in between 1 and 10 indi-
cates that the sample is moderately degraded. However, when
the value becomes >10 it indicates a highly degraded DNA
sample.
146 Quantification of DNA by Using qRT-PCR

6 Result Table

N
Sample Target M:F Degradation IPCC TCC
name name Ct Quantity ratio index T T
Std. 1 IPC
Large
autosomal
Small
autosomal
Y
Std. 2
Std. 3
Std. 4
Std. 5
Blood
sample
Bone
Vaginal
swab
Hair
Teeth

7 Precautions
l Wear gloves while dealing with the reagents and plasticware.
l Do not store the reagents under a light source.
l Always use freshly prepared dilutions of the standards for the
preparation of standard curve.
l Always use filtered microtips to prevent any possible
contaminations.
l First switch on the computer and then switch on the instrument
followed by switching on the software.
l Always use the standards in triplicates to obtain results with
higher R2 value.
l As qRT-PCR is a very sensitive technique, always obey the three-
room rule for extraction, setup, and qRT-PCR setup.
 Troubleshooting 147

l Use freshly calibrated pipettes for pipetting of accurate small

volumes. Small pipetting error may give rise to ambiguity in
results.
l Always use an optimized protocol. Use the manufacturer’s
guidelines properly.

8 Troubleshooting

Problem Tentative cause Possible solutions

Slope of the standard Incorrect quantity of Update with correct
curve is outside the standards has been values and reanalyze
normal range or R2 applied
value is <0.98
A stored dilution of the Prepare fresh standards
standards was used and rerun the
samples
Standards were not Exclude a failed/
loaded flagged standard in
standard curve
analysis, and
reanalyze the samples
Replicates of the Inappropriate amount Check the result in
samples and/or of Quantifiler™ THP multicomponent plot
standards showing PCR reaction mix was
Compare the volume of
inconsistent ΔRn and added
the reaction mix in
Ct results
the well with
discrepancy in result
with the nearby wells
High Ct value and low Presence of high Take a chance by
ΔRn value quantity of PCR diluting the samples
inhibitors before multiplex
PCR or purify the
sample again and
reanalyze
Positive result observed Improper baseline Change the baseline
in negative control selection detects small manually
samples amount of
fluorescence signals as
amplifications
Contamination to Rerun with fresh batch
consumables and/or of kits, consumables,
reagents and plasticware
 Chapter 18

Fluorescence-Based Instant Quantification of DNA

Objective: To quantify DNA isolated from blood and forensic

samples using fluorometric technique by Qubit™ Fluorometer.

1 Introduction

Measurement of DNA concentration by fluorometric technique

relies on the use of dyes that selectively bind to DNA. Any of the
DNA extraction protocol does not eliminate RNA completely.
Though many of the protocols recommend the use of RNase, it
can only help in reducing the chance of errors. Due to the presence
of RNA contamination in extracted DNA, UV–visible
spectrophotometer-based techniques do not yield an accurate
result of DNA concentration. In this regard, fluorescence-based
technique is more useful as it allows the measurement of double-
stranded DNA-dye complex selectively. On binding with the
double-stranded DNA, the dye undergoes a conformational
change and subsequently a fluorescent energy is emitted which is
detected by the instrument.
The examples of such dyes include ethidium bromide, SYBR®
Green, Hoechst (bis-benzimide) dyes, and PicoGreen®. In this
case, the amount of fluorescence generated by the dyes is directly
proportional to the concentration of DNA in the sample. Thus, the
concentration of DNA is measured indirectly by estimating the
fluorescence level of the dye in the solution. There are many
advantages of the use of fluorescence-based DNA quantification
technique. The main advantage includes the selective binding of the
dyes to DNA and nonbinding to other possible co-extracts during
DNA isolation, i.e., protein or RNA. Additionally, the strong affin-
ity between the DNA and the dye allows detection of DNA at a very
low concentration. The detection of low signal generated due to
trace amount of DNA sample makes this technique more sensitive

H. R. Dash et al., Principles and Practices of DNA Analysis: A Laboratory Manual for Forensic DNA Typing, Springer Protocols
Handbooks, https://doi.org/10.1007/978-1-0716-0274-4_18, © Springer Science+Business Media, LLC, part of Springer Nature 2020

149
150 Fluorescence-Based Instant Quantification of DNA

than spectroscopic technique with the highest degree of accuracy

and affinity. Some of the disadvantages of this technique include the
cost of the fluorescent dyes and the associated health risks of the
fluorescent molecules.

2 Principle

Many fluorescent dyes are available which specifically bind to

double-stranded DNA specifically. Some of these dyes include ethi-
dium bromide, SYBR® Green, Hoechst (bis-benzimide) dyes, and
PicoGreen®. After binding to the double-stranded DNA, these
dyes undergo conformational changes which lead to the generation
of fluorescent energy to be detected by an instrument. In this case,
the amount of fluorescence generated is directly proportional to the
DNA present in the solution. First a standard curve/calibration
curve is prepared (Fig. 18.1) by measuring the fluorescence level
through a fluorometer corresponding to various concentrations of
DNA. Finally, the fluorescence level of an unknown sample is
measured and its corresponding DNA concentration is calculated
based on the calibration curve.
For better understanding a commonly used dye Hoechst
33258 is described below. Hoechst 33258 is a common nucleic
acid stain that binds to double-stranded DNA categorically and
emits blue fluorescence light upon binding. The dye is excited by
the wavelength 352 nm, whereas its emission maximum is at the
wavelength of 461 nm. This dye can be excited by using mercury or
xenon lamp or using UV source. The dye is soluble in both water
and organic solvents such as DMSO or dimethylformamide.

Fig. 18.1 Calibration curve of control DNA (calf thymus DNA) when PicoGreen® is
used as the double-stranded DNA quantitation reagent
 Reagents/Materials Required and Their Role 151

Hoechst 33258 binds to the minor groove of double-stranded

DNA. DNA sequences rich in adenine and thymine have a prefer-
ential high binding with the dye. Another advantage of this dye is
that it is permeable to cell membrane and can bind to the DNA of
both live and fixed cells. To prepare 1 mg/ml Hoechst 33258
solution, add 10 mg of the dye powder to 10 ml of distilled
water, and do not filter. The prepared dye solution can be used
within 6 months by proper storage at 4
C.
Though many kits are available in the market, Qubit™ 1
dsDNA HS Assay Kit has been described in this chapter for
fluorescence-based DNA quantification using Qubit™ 4 Fluorom-
eter model.

3 Reagents/Materials Required and Their Role

3.1 Qubit™ 1 This component of the kit contains the fluorescent dye at 1
dsDNA HS Working concentration. It should be stored at 2–8
C under protection
Solution from light. The dye present in component A is highly selective for
(Component A) double-stranded DNA. It can work for a wide range of DNA
concentrations, i.e., 10 pg/μl to 100 ng/μl, or 0.1–100 ng. The
fluorescent signal generated during this reaction is highly stable till
3 h when it is protected from exposure to light. Common con-
taminants such as salts, free nucleotides, solvents, detergents, or
proteins do not affect this assay of measurement of DNA
concentration.

3.2 Qubit™ 1 Both component B and component C are required for calibration
dsDNA HS Standards of the instrument prior to measurement of unknown DNA concen-
#1 and #2 tration. Standard #1 and Standard #2 contain 0 and 10 ng/μl of
(Components B and C) DNA concentration in TE buffer, respectively. They should be
stored at 2–8
C.

3.3 TE Buffer TE buffer is generally prepared by mixing 50 mM Tris and 50 mM

EDTA at pH 8.0. Tris, the major constituent of TE buffer, acts as a
common pH buffer. Additionally, EDTA chelates cations like Mg2+.
Hence, TE buffer is helpful to solubilize DNA by protecting it from
degradation.

3.4 DNA Sample Extracted DNA from reference as well as forensic samples can be
used for the estimation of DNA. If you expect a higher quantity of
DNA, dilute the extract in TE buffer prior to taking the absorbance
of the sample. Various known concentrations of calf thymus DNA
(CT DNA) prepared in TE buffer can be used as control DNA for
assessment of measurement of DNA concentration by fluorometric
technique.
152 Fluorescence-Based Instant Quantification of DNA

4 Procedure

Preparation of Standards and Samples

1. Label as many number of 500 μl microcentrifuge tubes as your
number of samples on their lids and two additional tubes for
two standards.
2. Add 10 μl of both the standards and samples to the
corresponding tubes.
3. Add Qubit™ 1 dsDNA 1 buffer to each tube to make up
the final volume to 200 μl.
4. Mix both the solutions by vortexing for 3–5 s.
5. Incubate the tubes at room temperature for 2 min.

Measurement of DNA Concentration

6. Switch on the Qubit™ 4 Fluorometer.
7. Press “DNA,” and then select the assay type as “1 dsDNA
HS.”
8. Then the display will show “Read Standards.” Press on the
same to proceed.
9. Insert the tube containing Standard #1 in the sample chamber
followed by closing of the lid and press “Read Standard.”
10. When reading is complete (which takes around 3 s), remove
Standard #1.
11. Insert the tube containing Standard #2 in the sample chamber
followed by closing of the lid and press “Read Standard.”
12. When reading is complete (which takes around 3 s), remove
Standard #2.
13. Press “Run Samples.”
14. Adjust the sample volume between 1 and 20 μl.
15. Select the units for output sample concentration from the drop-
down menu, i.e., ng/μl, ng/ml, μg/μl, μg/ml, or mg/ml.
16. Insert the tube containing unknown DNA concentration to
the sample chamber, close the lid, and press “Read tube.”
17. Wait till reading of the sample (which takes around 3 s) is over.
18. Remove the sample and repeat for any other samples.

5 Observation

The DNA quantity is displayed on the screen. The displayed value

of DNA concentration is related to the concentration of the sample
after dilution in the assay tube. The original concentration of the
 Precautions 153

sample is calculated automatically in Qubit™ 3 and 4 Fluorometers,

whereas for Qubit™ 2.0, the original DNA concentration should
be calculated manually as follows:
Concentration of the sample ¼ QF value  200=x
where QF ¼ value given by the Qubit™ 2.0 Fluorometer and
x ¼ amount of sample added to the assay tube (in μl).

6 Result Table

Sample DNA concentration

Control
Sample 1 (liquid blood)
Sample 2 (bone powder)

7 Precautions
l Follow the manufacturer’s guidelines in each step properly.
l Wear gloves while dealing with the reagents and plasticware.
l Avoid using an expired kit.
l Do not incubate the samples more than the recommended
period of time.
l Do not hold the assay tubes in your hand because it warms the
solution and may give rise to a different reading.
l Avoid keeping the sample directly in light.
l Component A may contain potential mutagens; hence handle it
with care and dispose it with proper regulation.
l Store all the kit components at the recommended temperature
condition.
l Never keep/install the Qubit™ instrument in a wet/humid
place.
l Do not use wet hands to operate the Qubit™ instrument.
l Carefully operate the Qubit™ instrument so that the sample
does not spill in the sample chamber.
l Make sure that the tube is clean and dry from outside while
taking readings.
l Carefully handle the samples prior to taking the readings so that
bubbles are not present in the sample.
154 Fluorescence-Based Instant Quantification of DNA

8 Troubleshooting

Problem Tentative cause Possible solutions

In appropriate The fluorescence dye is Use the fresh reagents
reading degraded due to
inappropriate storage
Bubbles present in the Slightly tap on the tube wall or
sample centrifuge briefly to remove
the bubbles
Outer surface of the tube Wipe the outer side of the
is wet tube using a Kimwipe
Sample chamber is wet Wipe the sample chamber
using a Kimwipe
Improper binding of the Use fresh regents
dye with the sample
Follow recommended
incubation conditions of
the samples and standards
High reading of Sample contains more Dilute the sample and retake
the sample/ DNA the reading
out of range
Lid of the instrument not Make sure to close the lid of
closed while taking the instrument while taking
reading reading
A high/low temperature Make sure that the assay is
of the sample while performed at room
taking readings temperature/the
recommended temperature
Low reading of Sample contains less Use a sample with lower
the sample DNA dilution
Standard and working Use recommended conditions
solutions are not to prepare the standard and
prepared correctly working solutions and
reread the samples
Total volume is less Make sure that the total
volume of working solution
+ standards/samples is
200 μl
Degraded component A Protect the component A
from light, use a fresh set of
reagents, and retake the
reading
A high/low temperature Make sure that the assay is
of the sample while performed at room
taking readings temperature/the
recommended temperature
 Part V

Amplification of Various STR Markers Using Multiplex PCR

 Chapter 19

Amplification of Autosomal STR Markers by Multiplex PCR

Objective: To amplify the autosomal STR markers using multiplex

PCR technique.

1 Introduction

Human identification and individualization are key in criminal

justice system. Discovery of DNA fingerprinting technique has
given rise to molecular level of identification with the highest
degree of fidelity. The technology has provided results for many
types of criminal and civil cases such as identification, murder,
paternity dispute, as well as identification of sexual assault culprits.
The beauty of this technique is that besides convicting the offen-
ders, it can exonerate the innocence.
Human genome is comprised of 23 pairs of chromosomes,
which include 22 pairs of autosomes and 1 pair of sex chromosome.
Half of the autosomes are inherited from both the parents. The
genomic complexity of an individual’s autosomes is a result of
chromosomal recombination and hence it is considered that no
two individuals’ genome is similar except the homozygous twins.
This can be explored by autosomal STR analysis. Another well-
known fact is that the two human beings share 99.9% of their
genome and the genetic variation exists only in 0.1% of the genome.
It was further found that the 0.1% variation occurs in the noncoding
region of the genome. The interpersonal variation of the genome
can be analyzed by determining the number of repeat units of the
STRs, which are a key component in the noncoding region of the
genome.
Short tandem repeats (STRs), also known as the microsatellites,
are the short sequences of 2–7-nucleotide lengths which are repeated
tandemly several times. Though many STR markers are present in

H. R. Dash et al., Principles and Practices of DNA Analysis: A Laboratory Manual for Forensic DNA Typing, Springer Protocols
Handbooks, https://doi.org/10.1007/978-1-0716-0274-4_19, © Springer Science+Business Media, LLC, part of Springer Nature 2020

157
158 Amplification of Autosomal STR Markers by Multiplex PCR

human genome, a few set of markers are being analyzed for forensic
DNA purpose. To generate a coherent DNA profile, a common set
of 20 markers are recommended to be analyzed called as the
expanded CODIS markers which include CSF1PO, D3S1358,
D5S818, D7S820, D8S1179, D13S317, D16S539, D18S51,
D21S11, FGA, TH01, TPOX, vWA, D1S1656, D2S441,
D2S1338, D10S1248, D12S391, D19S433, and D22S1045.
Thus, multiplex PCR is a key step in forensic DNA analysis, where
primers for all the STR markers are used to amplify the said STR loci
simultaneously. As biological materials available in forensic samples
are very less, PCR process plays a useful role in recovering genetic
information from these samples.
The product size of a multiplex PCR is around 100–500 bp
which is separated based on its size as well as fluorescent signals by
capillary electrophoresis. Multiplex PCR process enables a high
discrimination power of the test with a low input of DNA concen-
tration, i.e., around 1 ng. Though many autosomal STR kits are
available in the market (list of which has been given in Appendix B),
amplification of autosomal STR markers using PowerPlex® Fusion
6C System has been described in this chapter.

2 Principle

During amplification of multiple targets in a single PCR setup

multiplexing technique is applied. It varies from the conventional
PCR by the use of multiple set of primers in a single reaction. In
multiplex PCR process, designing of primers plays a useful role
as all the primers should be of appropriate length, and should
have similar melting temperature (Tm), greater specificity, as well
as absence of any by-product formation such as formation of
primer dimer. The greatest challenge in developing a multiplex
reaction is that the primers should not react with each other.
Additionally, it should contain homogenous molecules and a
balanced primer concentration so that each amplicon is present
in equal amounts.
Old-generation STR typing kits take around 2–3 h for com-
pleting the multiplex PCR process. Gradual advancements in tech-
nologies have reduced the time required for the same as well as
improved the throughput of DNA typing. Some rapid polymerases
are available in the market such as PyroStart (Fermentas, Glen
Burnie, MD) and SpeedSTAR (Takara Bio USA, Madison, WI)
which can be used to decrease the time requirement for multiplex
PCR process. In this process, the thermal cycling protocol used for
 Reagents/Materials Required and Their Role 159

the amplification of genetic markers using PowerPlex® Fusion 6C

System is as follows:

PowerPlex® Fusion 6C System is a six-dye multiplex system which

allows co-amplification of 27 genetic markers for their fluorescent
detection. The kit includes all 20 CODIS markers, i.e., CSF1PO,
FGA, TH01, TPOX, vWA, D1S1656, D2S1338, D2S441,
D3S1358, D5S818, D7S820, D8S1179, D10S1248, D12S391,
D13S317, D16S539, D18S51, D19S433, D21S11, and
D22S1045. Along with this, two sex-determining markers, i.e., ame-
logenin and DYS391, are also present in this kit. Penta D, Penta E,
and SE33 have been included in this kit to increase the discrimination
power. Additionally, two rapidly mutating Y-STR markers are also
present in this kit as DYS570 and DYS576. The set of markers present
in this kit satisfy both the expanded Combined DNA Index System
(CODIS) and European Standard Set (ESS) recommendations.
The human specificity of the kit has been examined by amplify-
ing unique exogenous DNA which includes 32 microbial sources,
19 non-primate vertebrate sources, and 4 unique primate sources,
out of which none of the microbial sources yielded significant ampli-
fication. Though the kit showed some amplification for certain
non-primates and primates, it was sufficient to entrust the human
specificity of PowerPlex® Fusion 6C System. The sensitivity of the kit
has been calculated to be as low as 250 pg. Additionally, the kit is
highly useful for forensic applications as it works fine with common
potential inhibitors such as hematin, humic acid, tannic acid, and
EDTA. The precision study of the kit shows its usefulness on 3500,
3500xL, 3130, and 3130xl genetic analyzers. Experiments using this
kit for mixtures, low template concentration, and degraded DNA
have showed its usefulness in real-time forensic case work samples.

3 Reagents/Materials Required and Their Role

3.1 PowerPlex® Master mixture of a PCR generally contains buffer to maintain the
Fusion 6C 5 Master activity of Taq DNA polymerase, and MgCl2 to enhance the activity of
Mix enzyme, mixture of dNTPs, and some other additives to increase the
160 Amplification of Autosomal STR Markers by Multiplex PCR

inhibitor tolerance capacity of the in vitro DNA replication process.

Upon receipt master mix should be stored at 30 to 10  C. How-
ever, after its first use it can be kept at 2–10  C for its stable storage for
6 months.

3.2 PowerPlex® Primer designing is a crucial step of any PCR setup. However, as we
Fusion 6C 5 Primer use the PCR kit, the primers are designed and optimized for the said
Pair Mix reaction by the manufacturer. For multiplexing, a primer set for all
the loci to be amplified is included in the mixture. As the next step is
detection of the amplified fragments by capillary electrophoresis all
the primer sets are labeled with various dye sets. The PowerPlex®
Fusion 6C kit amplifies 27 genetic loci at the same time. Hence,
27 pair of primers are present in the primer mix of the kit. Thus,
upon receipt primer pair mix should be stored at 30 to 10  C.
However, after its first use it can be kept at 2–10  C for its stable
storage for 6 months. As it is light sensitive, it is recommended to be
stored in dark conditions.

3.3 Molecular Molecular biology-grade water is the form of ultrapure high-quality

Biology-Grade Water water which is free from DNase, RNase, and protease contamina-
tion. Additionally, no toxic agents such as DEPC should be present
in this water. Milli-Q water can be produced in the laboratory or it
can be procured from any molecular biology-grade chemical
manufacturer.

3.4 Template DNA Extracted DNA from reference as well as forensic samples can be
used as template DNA for amplification of the genetic markers.
After estimation of quantity, the DNA should be suitably diluted or
concentrated for optimal input in the PCR process, which is ~1 ng.
After dilution, the template DNA should be stored at 20  C till
further use. It is always recommended to prepare fresh dilutions of
the template DNA in TE buffer to prevent rapid degradation
of DNA.

3.5 PCR Machine A PCR machine provides the physical conditions for in vitro replica-
tion of DNA in a tube containing all the requisite chemical agents. In
the first step of PCR process, denaturation of DNA occurs by raising
the temperature to ~95  C. In the next step the temperature is
lowered to as low as ~50  C which allows the binding of primers at
the targeted primer-binding sequence. Further the temperature is
increased to ~72  C for the action of DNA polymerase. These
sequential events are repeated for a number of cycles to increase
the copy numbers of the targeted DNA fragments. There are many
PCR machines available in the market; however, the PCR conditions
should be validated internally under the laboratory condition prior
to its application in forensic DNA analysis.
 Procedure 161

4 Procedure

Preparation of Amplification Mixture

1. Calculate the amount of each component of the PCR process
using the following table as per your requirement/number of
samples (Table 19.1).
2. Thaw PowerPlex® Fusion 6C Master Mix and PowerPlex®
Fusion 6C Primer Pair Mix in ice, vortex for 3–5 s, and centri-
fuge briefly before using.
3. Pipette out the calculated amount of water, master mix, and
primer mix in a 1.5 ml microcentrifuge tube marked as Ampli-
fication Mixture (AM). It is always recommended to add water
first to the tube, followed by PowerPlex® Fusion 6C Master
Mix and PowerPlex® Fusion 6C Primer Pair Mix.
4. As soon as the preparation of AM is over, immediately store the
tubes containing master mix and primer pair mix in the recom-
mended storage condition.
5. Vortex the amplification mixture for 5–10 s, and then dispense
(5 + 5 + x) μl of amplification mixture to each 200 μl PCR tube
pre-marked for each sample.
6. Add 1.0 ng of template DNA to each designated PCR tube
containing the amplification mixture.
7. In one tube add control DNA in the amplification mix and in
another tube add molecular biology-grade water or TE buffer
in place of template DNA which will be considered as positive
and negative control, respectively.
8. Close the tubes properly and briefly centrifuge the tubes so that
all its contents will be present at the bottom of the tube.

Table 19.1
Recommended amounts of reagents required to prepare amplification mixture for PCR

No. of
PCR components Volume per reaction (μl)a reactions Total volume
PowerPlex® Fusion 6C Master Mix 5.0 A 5A
®
PowerPlex Fusion 6C Primer Pair Mix 5.0 A 5A
Molecular biology-grade water Up to a final volume of 25 μl A xA
including template DNA (x)
Total volume (5 + 5 + x)A
a
Note: Half volume of the recommended PCR components also gives rise to a good result; however, it should be
standardized under your laboratory conditions
162 Amplification of Autosomal STR Markers by Multiplex PCR

Thermal Cycling
9. Place the tubes in the thermal cycler.
10. Set the ramp rate of the thermal cycler to maximum or 100%.
11. Set the thermal cycling protocol as 96  C for 1 min, followed
by 29 cycles of 96  C for 5 s and 60  C for 1 min, followed by
60  C for 10 min, and hold at 4  C.
12. Wait till completion of the PCR process, which will take around
1–1.25 h.
13. Store the amplified products at 20  C in dark conditions.

5 Observation and Result

The amplification of the samples can be confirmed after performing

capillary electrophoresis and data analysis which has been described
in Module 6.

6 Precautions
l Wear gloves while dealing with the reagents and plasticware.
l Do not store the reagents under a light source.
l Always use filtered microtips or aerosol-resistant microtips to
prevent any possible contaminations.
l Always put positive and negative control along with the test
samples for PCR process.
l As some of the chemicals are hazardous, they should be handled
carefully.
l Perform all the works related to PCR setup in a dedicated room
using an exclusive set of instruments to avoid contamination.
l Always prepare amplification mixture for extra 10% reactions to
avoid any chance of possible pipetting error.
l Make sure not to exceed the volume of template to 20% of the
total volume of PCR setup.
l Always ensure to provide the recommended amount of DNA
template to the PCR setup. In this purpose, it is useful to ensure
the concentration of template DNA by qRT-PCR.
l Do not store the amplification mix for longer time; it may
increase the chance of artifacts. Hence, after preparation of
amplification mix add template DNA at the earliest followed by
immediate thermal cycling.
l Always optimize the reaction setup and thermal cycling condi-
tions as per your laboratory conditions prior to performing any
experiment.
 Troubleshooting 163

7 Troubleshooting

As the results can be evaluated after performing capillary electro-

phoresis, for troubleshooting of amplification you can refer to the
capillary electrophoresis chapter described in Module 6.
 Chapter 20

Amplification of Y Chromosome STR Markers by

Multiplex PCR

Objective: To amplify the Y chromosome STR markers using

multiplex PCR technique.

1 Introduction

Y chromosome and its inheritance from father play a major role in

the sex determination of a fetus. Its specific occurrence in males,
selective inheritance from father, and high mutation rate are imper-
ative to its investigative and evidentiary value. In male and female
mixed samples, Y STR loci typing plays an important role. Some Y
STR markers which are closely linked and do not undergo recombi-
nation are also useful in paternity determination and establishment
of patrilineal relationship. Thus, Y-STR analysis plays an important
role along with autosomal STR analysis and a combination of their
analyses helps in drawing conclusion with greater confidence.
Amelogenin marker is widely used as a sex-determining marker
during forensic analysis. However, the prevalence of deletion at
AMELY region of the Y chromosome throughout the globe has
raised question on the sole use of amelogenin marker for molecular
method of sex determination. Thus, other Y chromosome STR
markers are recommended to be used in addition to AMELY for
molecular basis of sex determination in humans. Though autoso-
mal STR analysis gives proper identification of an individual, it may
not yield a good outcome when mixed-source DNA samples are
used. Mostly in sexual assault cases, the samples of victim’s origin
yield an autosomal profile with excess DNA from the female source
in comparison to the male DNA. This is due to the presence of
higher number of victim’s epithelial cells in the sample than that of
sperm cells. Due to preferential amplification by PCR, it becomes
difficult to identify the male culprit due to possible allele share

H. R. Dash et al., Principles and Practices of DNA Analysis: A Laboratory Manual for Forensic DNA Typing, Springer Protocols
Handbooks, https://doi.org/10.1007/978-1-0716-0274-4_20, © Springer Science+Business Media, LLC, part of Springer Nature 2020

165
166 Amplification of Y Chromosome STR Markers by Multiplex PCR

Fig. 20.1 Patrilineal transfer of Y chromosome

between the two DNA contributors. In such cases, Y chromosome

STR analysis plays an important role by selective amplification of Y
STRs, which is present only in the male culprit’s DNA and not
present in the female’s DNA.
Additionally, as Y chromosome is shared among the paternally
related male individuals (Fig. 20.1), Y STR analysis is helpful in
solving paternity disputes of male offspring, paternal kinship analy-
sis, as well as familial searching. Y chromosome STR analysis is also
useful in drawing inference on geographical origin of an individual
as well as in historical and genealogical research. However, the
rapidly mutating Y STR markers are not useful in paternity and
kinship testing due to their high mutation rates in a generation. In
an inbreed population, distinguishing the male relatives is not pos-
sible by commercially available Y STR kits. In such cases, for indi-
vidualization some rapidly mutating Y STR markers with high
mutation rates have been discovered. Analysis of a complete set of
13 RM YSTR markers (DYS570, DYS576, DYS627, DYS518,
DYS449, DYF387S1ab, DYF399S1, DYF403S1a+b, DYF404S1,
DYS526a+b, DYS547, DYS612, and DYS626) is useful in indivi-
dualizing a suspect which has left his traces in a crime scene than that
of his close or distant patrilineal relatives. Though many Y chromo-
some STR kits are available in the market (list of which has been
given in Appendix B), amplification of Y chromosome STR markers
has been described in this chapter using Yfiler™ Plus System.

2 Principle

Y chromosome, sized about 60 Mb, is one of the smallest among

human chromosomes. About 95% sequences (24 Mb of euchroma-
tin region and 30 Mb of heterochromatin region) of this chromo-
some are considered to be the non-recombining region (NRY) or
male-specific region (MSY). It shows purely paternal inheritance
(~99.99%) and remains unaffected by genetic exchanges from X
chromosome. Y chromosome also contains the pseudo-autosomal
regions (PARs) which are located at the distal end of both p and q
 Principle 167

Fig. 20.2 A generalized structure of Y chromosome and its usefulness in forensic

DNA analysis

arms. These PARs undergo crossing over and segregation like

autosomal loci in one generation to another. It also contains some
sex-determining regions (SRY) at band Yp11.3 and amelogenin
region which are useful in molecular basis of sex determination in
humans (Fig. 20.2).
Initially, a total of 15 Y-STR markers were developed out of
which 7 core loci were used for forensic DNA analysis purpose.
These sets are being called as the “minimal haplotype Y-STR loci.”
These minimal haplotype Y-STR loci can distinguish 91–97% of
unrelated males in European population. Gradually, the number of
STR loci was increased from 7–9 to 17 panels which includes the
7–9 core loci. Most of the STR loci used in this panel are tetra-
nuclear repeats to minimize the stutter products. Gradually various
Y-STR kits were developed and validated for forensic use. In this
regard, Yfiler™ Plus System amplifies a total of 26 loci which include
7 core loci, i.e., DYS576, DYS389I, DYS635, DYS389II, DYS627,
DYS460, DYS458, DYS19, YGATAH4, DYS448, DYS391,
DYS456, DYS390, DYS438, DYS392, DYS518, DYS570,
DYS437, DYS385 a/b, DYS449, DYS393, DYS439, DYS481,
DYF387S1, and DYS533. The included markers also contain three
highly polymorphic STR loci, i.e., DYS460, DYS481, and DYS533
and seven rapidly mutating loci, i.e., DYF387S1a/b, DYS449,
DYS518, DYS570, DYS576, and DYS627, with >1% of mutation
rates to allow differentiation between related males.
A cycle of 30 PCR conditions has been optimized for the use of
Yfiler™ Plus kit when a mixture of 1 ng male DNA and 1 μg female
DNA is used. The kit has been validated to generate a complete
male profile when as low as 250 pg of male DNA is present in a
background of 3 μg of female DNA. Yfiler™ Plus works fine when
the male-to-female DNA ratio is 1:1000. The kit does not show any
168 Amplification of Y Chromosome STR Markers by Multiplex PCR

cross-reactivity with various non-primates studied, i.e., bovine,

chicken, dog, horse, mouse, pig, rabbit, rat, and sheep. A study
on the role of potential PCR inhibitors suggests that hematin and
humic acids at a concentration of 400 μM and 100 ng/μl do not
affect the obtained result. The concordance study also fits well with
both the results of Yfiler™ plus and Yfiler kit in four ethnicities, i.e.,
African American, Caucasian, Hispanic, and Asian. The kit works
on any model of Genetic Analyzer that works on six-dye chemistry
which includes 3130xl, 3500, and 3500xL; the six dyes being used
include 6-FAM™ (blue), VIC™ (green), NED™ (yellow), TAZ™
(red), SID™ (purple), and LIZ™ (orange).

3 Reagents/Materials Required and Their Role

3.1 Yfiler™ Plus It contains MgCl2 and mixture of dNTPs, bovine serum albumin
Master Mix (BSA), polymerase enzyme, and 0.05% sodium azide in buffer and
salts. Upon the receipt of Yfiler™ Plus Master Mix it should be stored
at 25 to 15  C; however, after first use it can be stored at 2–8  C.

3.2 Yfiler™ Plus It contains locus-specific 6-FAM™, VIC™, NED™, TAZ™, and
Primer Set SID™ dye-labeled and -unlabeled primers in buffer. During synthe-
sis of primers for DYS389I/II, DYS635, DYS627, DYS19, YGA-
TAH4, DYS448, DYS391, DYS390, DYS438, DYS391, DYS390,
DYS438, DYS392, DYS518, DYS437, and DYS449 non-nucleotide
linkers are used which are placed between the primers and fluores-
cent dyes that are used during synthesis of respective oligonucleotide
primers. The fundamental behind the use of non-nucleotide linkers
is that they provide reproducible positioning of alleles by facilitating
inter-locus spacing and simultaneous amplification and efficient sep-
aration of the 27 Y-STR loci. Upon the receipt of Yfiler™ Plus
Primer Set, it should be stored at 25 to 15  C; however, after
first use it can be stored at 2–8  C with protection from light.

3.3 Molecular Molecular biology-grade water is the form of ultrapure high-quality

Biology-Grade Water water which is free from DNase, RNase, and protease contamination.
Additionally, no toxic agents such as DEPC should be present in this
water. Milli-Q water can be produced in the laboratory or it can be
procured from any molecular biology-grade chemical manufacturer.

3.4 Template DNA Extracted DNA from reference as well as forensic samples can be
used as template DNA for amplification of the genetic markers.
After estimation of quantity, the DNA should be suitably diluted or
concentrated for optimal input in the PCR process, which is ~1 ng.
After dilution, the template DNA should be stored at 20  C till
further use. It is always recommended to prepare dilutions of the
template DNA in TE buffer to prevent rapid degradation of DNA.
Additionally, the quality of the template DNA can be maintained by
adding 20 μg/ml of glycogen in the TE buffer.
 Procedure 169

3.5 PCR Machine A PCR machine provides the physical conditions for in vitro repli-
cation of DNA in a tube containing all the requisite chemical
agents. In the first step of PCR process, denaturation of DNA
occurs by raising the temperature to ~95  C. In the next step the
temperature is lowered to as low as ~50  C which allows the
binding of primers at the targeted primer-binding sequence. Fur-
ther the temperature is increased to ~72  C for the action of DNA
polymerase. These sequential events are repeated for a number of
cycles to increase the copy numbers of the targeted DNA frag-
ments. Many PCR machines are available in the market; however,
the PCR conditions should be validated internally under the labo-
ratory condition prior to its application in forensic DNA analysis.

4 Procedure

Preparation Amplification Mixture

1. Calculate the amount of each component of the PCR process
using the following table as per your requirement/number of
samples (Table 20.1).
2. Thaw Yfiler™ Plus Master Mix and Yfiler™ Plus Primer set in
ice, vortex for 3–5 s, and centrifuge briefly before using.
3. Pipette out the calculated amount of water, master mix, and
primer set in a 1.5 ml microcentrifuge tube marked as Amplifi-
cation Mixture (AM). It is always recommended to add water
first to the tube, followed by Yfiler™ Plus Master Mix and
Yfiler™ Plus Primer set.
4. As soon as the preparation of AM is over, immediately store the
tubes containing master mix and primer set in the recom-
mended storage condition.
5. Vortex the amplification mixture for 5–10 s, and then dispense
(10 + 5 + x) μl of amplification mixture to each 200 μl PCR
tube pre-marked for each sample.

Table 20.1
Recommended amounts of reagents required to prepare amplification mixture for PCR

PCR components Volume per reaction (μl)a No. of reactions Total volume
Yfiler™ Plus Master Mix 10.0 A 10A
Yfiler™ Plus Primer set 5.0 A 5A
Molecular biology-grade water Up to a final volume of 25 μl A xA
including template DNA (x)
Total volume (10 + 5 + x)A
a
Note: Half volume of the recommended PCR components also gives rise to a good result; however, it should be
standardized under your laboratory conditions
170 Amplification of Y Chromosome STR Markers by Multiplex PCR

6. Add 1.0 ng of template DNA to each designated PCR tube

containing the amplification mixture.
7. In one tube add control DNA in the amplification mix and in
another tube add molecular biology-grade water or TE buffer
in place of template DNA which will be considered as positive
and negative control, respectively.
8. Close the tubes properly and briefly centrifuge the tubes so that
all its contents will be present at the bottom of the tube.

Thermal Cycling
9. Place the tubes in the thermal cycler.
10. Set the ramp rate of the thermal cycler to maximum or 100%.
11. Set the thermal cycling protocol as 95  C for 1 min, followed
by 30 cycles of 94  C for 4 s and 61.5  C for 1 min, followed by
60  C for 22 min, and hold at 4  C.
12. Wait till completion of the PCR process, which will take around
1–1.25 h.
13. Store the amplified products at 20  C in dark conditions.

5 Observation and Result

The amplification of the samples can be confirmed after performing

capillary electrophoresis and data analysis which has been described
in Module 6.

6 Precautions
l Wear gloves while dealing with the reagents and plasticware.
l Do not store the reagents under a light source.
l Always use filtered microtips or aerosol-resistant microtips to
prevent any possible contaminations.
l Always put positive and negative control along with the test
samples for PCR process.
l As some of the chemicals are hazardous, they should be handled
carefully.
l Do all the works relating to PCR setup in a dedicated room
using a dedicated set of instruments to avoid contamination.
l Always prepare amplification mixture for extra 10% reactions to
avoid any chance of possible pipetting error.
l Make sure not to exceed the volume of template to 20% of the
total volume of PCR setup.
 Troubleshooting 171

l Always ensure to provide the recommended amount of DNA

template to the PCR setup. In this purpose, it is useful to ensure
the concentration of template DNA by qRT-PCR.
l Do not store the amplification mix for longer time; it may
increase the chance of artifacts. Hence, after preparation of
amplification mix add template DNA at the earliest followed by
immediate thermal cycling.
l Always optimize the reaction setup and thermal cycling condi-
tions as per your laboratory conditions prior to performing any
experiments.
l For storage of PCR-amplified products for <2 weeks, place
them at 2–8  C; however for long-term storage (>2 weeks)
keep them at 25  C to 15  C.

7 Troubleshooting

As the results can be evaluated after performing capillary electro-

phoresis, for troubleshooting of amplification of Y STR markers
you can refer to the capillary electrophoresis chapter described in
Module 6.
 Chapter 21

Amplification of X Chromosome STR Markers by

Multiplex PCR

Objective: To amplify the X chromosome STR markers using

multiplex PCR technique.

1 Introduction

STR techniques are widely used in forensic DNA typing for solving
various types of cases but not limited to paternity dispute, identifica-
tion, murder, as well as sexual assault cases. However, in certain
complex cases such as kinship analysis as well as cases with limitations
of reference samples, autosomal STR analysis alone cannot give a
definitive conclusion. In such a scenario, analysis of STRs located on
sex chromosomes becomes imperative. Thus, analysis of X-STRs plays
a significant role in forensic DNA typing. As men are hemizygous and
women are dizygous, men receive single X from their mother. This
unique inheritance pattern of X chromosome makes its suitability in
deciphering the deficient paternity cases. For a disputed girl child,
X-STR analysis between the father and daughter can be performed.
However, it cannot be performed for a male child in paternity dispute
case due to non-inheritance of X chromosome from father.
The analysis of X-STR in supplement with autosomal STR
analysis increases the discrimination power besides affecting the
paternity exclusion probability. It is useful in the analysis of complex
kinship cases such as paternal half-sisters, paternal aunt/uncle-niece,
and maternal uncle-nephew. X-STR analysis is highly useful in inces-
tuous relationships involving grandparent, half-sibling, and uncle/
aunt by distinguishing between such relationships. However, the
major limitation of X-STR analysis is that as all X-STR markers are
present on same X-chromosome, it leads to linkage disequilibrium
among them due to linkage. Additionally, the currently available
X-STR kits harbor a limited number of markers and generate a large

H. R. Dash et al., Principles and Practices of DNA Analysis: A Laboratory Manual for Forensic DNA Typing, Springer Protocols
Handbooks, https://doi.org/10.1007/978-1-0716-0274-4_21, © Springer Science+Business Media, LLC, part of Springer Nature 2020

173
174 Amplification of X Chromosome STR Markers by Multiplex PCR

amplicon size. This leads to their inappropriateness in forensic

applications. Though few kits are commercially available till now
(for detailed list see Appendix B), amplification of X-STRs has been
described in this chapter using Investigator Argus X-12 kit.

2 Principle

Human X chromosome is comprised of around 155 million base

pairs in length which contains almost 1100 genes. It constitutes 5%
and 2.5% of the total DNA of a female and male cell, respectively. In
males, the tips of X-chromosome recombine with pseudo-
autosomal region of the Y chromosome so that its segregation is
maintained during cell division. However, both the X chromo-
somes in the female recombine just like any other autosomal chro-
mosome during meiosis. Such unique biological nature and pattern
of inheritance increase the usefulness of X-chromosome STR typing
in various cases, i.e., complex kinship testing involving female
samples, motherless paternity dispute cases, half-sister testing,
comparison of grandparent and grandchildren, and paternity estab-
lishment in incest cases (Fig. 21.1).
In this regard, Argus X-12 kit provides a multiplex system for
simultaneous amplification of 12 X-STR markers, namely DXS7132,
DXS7423, DXS8378, DXS10074, DXS10079, DXS10101,
DXS10103, DXS10134, DXS10135, DXS10146, DXS10148, and
HPRTB, besides amelogenin marker for sex determination. The
X-STRs present in less than 1 cm distance are considered as a linkage
group which are inherited together during recombination. These
12 markers are inherited as four linkage groups (i.e., group 1:

Fig. 21.1 Few types of cases where X-STR analysis is useful: (a) disputed female child with mother
unavailable, (b) disputed male child with father unavailable, (c) disputed female child with father unavailable,
(d) identification of female individual with available reference sample of paternal grandmother
 Reagents/Materials Required and Their Role 175

DXS8378, DXS10135, and DXS10148; group 2: DXS7132,

DXS10074, and DXS10079; group 3: HPRTB, DXS10101, and
DXS10103; group 4: DXS7423, DXS10134, and DXS10146).
This reflects that the result of each set of these three markers can
be treated as a different haplotype in genotyping. Due to the pres-
ence of these linkage groups, the result of X-STR analysis can be used
as an alternative tool for the analysis of opposite-sex siblings with
increased specificity.
The kit requires a template DNA amount of 0.2–0.5 ng for
optimum result. It works on any genotyping platform which can
accommodate five-dye chemistry or more as the primers of the
markers are labeled with four dyes, i.e., 6-FAM™ (blue), BTG
(green), BTY (yellow), and BTR (red). Thus, it can be used on
ABI PRISM® 310 and 3100 and Applied Biosystems 3130 and
3500 genetic analyzers. A simulated study on the effect of various
inhibitors such as humic acid (200 ng/μl), hematin (500 μM), tannic
acid (3000 ng/μl), indigo carmine (6 mM), collagen (150 ng/μl),
and calcium (2 mM) suggests the suitability of the kit in the presence
of various potential PCR inhibitors. The kit works fine in the
degraded samples with as low as 150 bp of fragment-sized DNA.
Besides chimpanzees, bonobos, orangutans, and gorillas, no other
animals give rise to a reproducible peaks using Argus X-12 kit.

3 Reagents/Materials Required and Their Role

3.1 Reaction Mix A It contains dNTPs mixture, MgCl2, and bovine serum albumin (BSA).
dNTPs serve as the building blocks for in vitro DNA synthesis by PCR.
MgCl2 acts as a cofactor and enhances the productivity of Taq poly-
merase. BSA stabilizes the enzyme during storage and provides an
environment free from nucleases. Additionally, BSA also prevents
adhering of Taq polymerase to the wall of the PCR tubes. Upon the
receipt of reaction mix A it should be stored at 25 to 15  C.

3.2 Primer Mix A set of primers for each locus are present in this mixture. Each
primer set is labeled with a specific fluorescent dye, i.e., 6-FAM™
(amelogenin, DXS10103, DXS8378, DXS7132, DXS10134),
BTG (DXS10074, DXS10101, DXS10135), BTY (DXS7423,
DXS10146, DXS10079), and BTR (HPRTB, DXS10148). Upon
the receipt of the primer mix, it should be stored at 25 to 15  C
with protection from light.

3.3 Multi-Taq2 DNA It contains Taq polymerase which attaches the dNTPs to a DNA
Polymerase template by extending the length of primer. Thus, it helps in a sister
strand synthesis. Taq polymerase isolated from a thermophilic bac-
terium Thermus aquaticus is chosen for this purpose as it can
withstand a temperature required for the PCR process. It should
be stored at 20  C for its better performance.
176 Amplification of X Chromosome STR Markers by Multiplex PCR

3.4 Molecular Molecular biology-grade water is the form of ultrapure high-

Biology-Grade Water quality water which is free from DNase, RNase, and protease
contamination. Additionally, no toxic agents such as DEPC should
be present in this water. Milli-Q water can be produced in the
laboratory or it can be procured from any molecular biology-grade
chemical manufacturer.

3.5 Template DNA Extracted DNA from reference as well as forensic samples can be
used as template DNA for amplification of the genetic markers.
After estimation of quantity, the DNA should be suitably diluted or
concentrated for optimal input in the PCR process, which is ~1 ng.
After dilution, the template DNA should be stored at 20  C till
further use. It is always recommended to prepare dilutions of the
template DNA in TE buffer to prevent rapid degradation.

3.6 PCR Machine A PCR machine provides the physical conditions for in vitro repli-
cation of DNA in a tube containing all the requisite chemical agents.
In the first step of PCR process, denaturation of DNA occurs by
raising the temperature to ~95  C. In the next step the temperature
is lowered to as low as ~50  C which allows the binding of primers at
the targeted primer-binding sequence. Further the temperature is
increased to ~72  C for the action of DNA polymerase. These
sequential events are repeated for a number of cycles to increase
the copy numbers of the targeted DNA fragments. There are many
PCR machines available in the market; however, the PCR conditions
should be validated internally under the laboratory condition prior
to its application in forensic DNA analysis.

4 Procedure

Preparation Amplification Mixture

1. Calculate the amount of each component of the PCR process
using the following table as per your requirement/number of
samples (Table 21.1).
2. Thaw reaction mix A and primer mix in ice, vortex for 3–5 s,
and centrifuge briefly before using.
3. Pipette out the calculated amount of molecular biology-grade
water, reaction mix A, primer mix, and multi-Taq2 DNA poly-
merase in a 1.5 ml microcentrifuge tube marked as Amplifica-
tion Mixture (AM). It is always recommended to add water first
to the tube, followed by reaction mix A, primer mix, and multi-
Taq2 DNA polymerase.
4. As soon as the preparation of AM is over, immediately store the
tubes containing reaction mix A, primer mix, and multi-Taq2
DNA polymerase in the recommended storage condition.
 Observation and Result 177

Table 21.1
Recommended amounts of reagents required to prepare amplification mixture for PCR

PCR components Volume per reaction (μl) No. of reactions Total volume
Reaction Mix A 5.0 A 5A
Primer Mix 2.5 A 2.5A
Multi-Taq2 DNA polymerase 0.6 A 0.6A
Molecular biology-grade water Up to a final volume of 25 μl A xA
including template DNA (x)
Total volume (5 + 2.5 + 0.6 + x)A

5. Vortex the amplification mixture for 5–10 s, and then dispense

(5 + 2.5 + 0.6 + x) μl of amplification mixture to each 200 μl
PCR tube pre-marked for each sample.
6. Add 1.0 ng of template DNA to each designated PCR tube
containing the amplification mixture.
7. In one tube add control DNA in the amplification mix and in
another tube add molecular biology-grade water or TE buffer
in place of template DNA which will be considered as positive
and negative control, respectively.
8. Close the tubes properly and briefly centrifuge the tubes so that
all its contents will be present at the bottom of the tube.

Thermal Cycling
9. Place the tubes in the thermal cycler.
10. Set the ramp rate of the thermal cycler to maximum or 100%.
11. Set the thermal cycling protocol as 94  C for 4 min; followed
by 5 cycles of 96  C for 30 s, 63  C for 120 s, and 72  C for
75 s; followed by 94  C for 30 s, 60  C for 120 s, and 72  C for
75 s; and hold at 4  C.
12. Wait till completion of the PCR process.
13. Store the amplified products at 20  C in dark conditions or
immediately process for electrophoresis.

5 Observation and Result

The amplification of the samples can be confirmed after performing

capillary electrophoresis and data analysis which has been described
in Module 6.
178 Amplification of X Chromosome STR Markers by Multiplex PCR

6 Precautions
l Wear gloves while dealing with the reagents and plasticware.
l Do not store the reagents under a light source.
l Always use filtered microtips or aerosol-resistant microtips to
prevent any possible contaminations.
l Always put positive and negative control along with the test
samples for PCR process.
l As some of the chemicals are hazardous, they should be handled
carefully.
l Do all the works relating to PCR setup in a dedicated room
using a dedicated set of instruments to avoid contamination.
l Always prepare amplification mixture for extra 10% reactions to
avoid any chance of possible pipetting error.
l Make sure not to exceed the volume of template to 20% of the
total volume of PCR setup.
l Always ensure to provide the recommended amount of DNA
template to the PCR setup. For this purpose, it is useful to
ensure the concentration of template DNA by qRT-PCR.
l Do not store the amplification mix for longer time; it may
increase the chance of artifacts. Hence, after preparation of
amplification mix add template DNA at the earliest followed by
immediate thermal cycling.
l Always optimize the reaction setup and thermal cycling condi-
tions as per your laboratory conditions prior to performing any
experiments.
l For storage of PCR-amplified products for <2 weeks, place
them at 2–8  C; however for long-term storage (>2 weeks)
keep them at 25 to 15  C.

7 Troubleshooting

As the results can be evaluated after performing capillary electro-

phoresis, for troubleshooting of amplification you can refer to the
capillary electrophoresis chapter described in Module 6.
 Chapter 22

Amplification of Autosomal Mini-STR Markers from

Degraded Samples

Objective: To amplify the autosomal mini-STR markers by multi-

plex PCR technique.

1 Introduction

Most of the biological samples received for DNA examination are

degraded in nature. Due to exposure to varied environmental con-
ditions, the samples also contain many potential PCR inhibitors or
environmental insults. The most common damage to DNA in
forensic and ancient analysis relies on the degradation of DNA by
both endogenous and exogenous nuclease activity as well as nonen-
zymatic hydrolytic cleavage which generates strand breaks. In such
compromised samples, a partial DNA profile is highly expected as
amplification of large-sized STR loci is carried out during PCR
process using routine STR kits. Such partial DNA profiles do not
value much from forensic point of view as they do not provide
sufficient power of discrimination to either include or exclude a
suspect. In such cases, an advanced technique can be exploited for
amplification of STR markers using specially designed primers.
These primers preferentially target the larger STR loci in such a
way to generate a smaller amplification product.
Mini-STR primers possess the capability to magnify the STR
loci which increases the chance of successive amplification from the
degraded samples. Thus, the mini-STR technology can generate a
DNA profile from the compromised samples with increased sensi-
tivity. In this way, analysis of mini-STRs is highly useful in providing
genetic data from degraded samples with extremely low-quantity as
well as -quality DNA. Additionally, the quality of DNA profiling
result is increased manyfold by using the mini-STR technology.
Mini-STR analysis is capable of extracting genetic information

H. R. Dash et al., Principles and Practices of DNA Analysis: A Laboratory Manual for Forensic DNA Typing, Springer Protocols
Handbooks, https://doi.org/10.1007/978-1-0716-0274-4_22, © Springer Science+Business Media, LLC, part of Springer Nature 2020

179
180 Amplification of Autosomal Mini-STR Markers from Degraded Samples

from STR loci with allele size <150 bp more efficiently. The adverse
quality of STR profiles witnessed by high peak height imbalance in
degraded samples can be minimized using mini-STR technology.
This technology may open a new era in forensic DNA typing as
previously unsolvable cases due to nonavailability of suitable DNA
can be resolved. The protocol for amplifying mini-STRs from
degraded samples using AmpFl STR™ MiniFiler™ PCR amplifica-
tion kit has been described in this chapter.

2 Principle

Most of the commonly used STR loci contain a large number of

repeats and are unable to cause amplification when less than 150 bp
size of a DNA fragment is present. In such degraded DNA samples,
there are two ways to produce a result, i.e., (a) application of new
polymorphic markers with smaller repeat units and narrow allele
range or (b) moving forward the forward and reverse primers to
generate a low amplicon size. The fundamental of mini-STRs is to
increase the proximity of the forward and reverse primers to the
STR region (Fig. 22.1). In this way, the amplicon size of STRs
becomes less and genetic information can be recovered from the
degraded samples.
In this regard, the AmpFl STR™ MiniFiler™ PCR amplifica-
tion kit is capable of amplifying eight autosomal loci simultaneously,
i.e., D13S317, D7S820, D2S1338, D21S11, D16S539, D18S51,
CSF1PO, and FGA, besides the sex-determining marker amelo-
genin. This kit utilizes the non-nucleotide linkers to reduce the
amplicon size of large loci which are more prone to allele dropout.
These linkers are capable of positioning the alleles which facilitates
inter-locus spacing. Additionally, combinatorial use of five-dye fluo-
rescent system (6-FAM™, VIC™, NED™, PET™, and LIZ™)
and linkers allows simultaneous amplification and separation of
these STR markers. In comparison to the other commercially avail-
able kits, the markers show average 58% and 48% size reduction in
smallest and largest allele, respectively, except D21S11. Amplicon
length of the markers has been reduced significantly to 129 bp
(D7S820), 99 bp (D13S317), 33 bp (D21S11), 183 bp
(D2S1338), 168 bp (D18S51), 157 bp (D16S539), 87 bp
(FGA), and 201 bp (CSF1PO). In this way, the kit is highly useful
in tough samples such as bone, old stains, hairs, and swabs contain-
ing low level of DNA.
Many population studies have demonstrated the concordance
of the AmpFl STR™ MiniFiler™ PCR amplification kit result with
other commercially available kits. The validation experiments of the
kit as per SWAGDAM guidelines advocate its usefulness in forensic
DNA typing. Though full profile has been obtained using 0.125 ng
of template DNA, the optimal DNA quantity for AmpFl STR™
 Reagents/Materials Required and Their Role 181

Fig. 22.1 Selection of primers for (a) large amplicon size in conventional STR analysis and (b) small amplicon
size in mini-STR analysis

MiniFiler™ PCR amplification kit has been recommended to be

0.5–0.75 ng. A full profile has been generated when a good-quality
DNA is digested with 5 U of DNaseI for 20 min, suggesting its
usefulness in highly degraded sample.

3 Reagents/Materials Required and Their Role

3.1 AmpF lSTR™ It contains polymerase enzyme, MgCl2, dNTPs, carrier proteins
MiniFiler™ Master Mix such as BSA, and sodium azide. dNTPs serve as the building blocks
for in vitro DNA synthesis by PCR. MgCl2 acts as a cofactor and
enhances the productivity of Taq polymerase. Taq polymerase
attaches the dNTPs to a DNA template by extending the length
of the primer. Carrier protein such as BSA stabilizes the enzyme
during storage and provides an environment free from nucleases.
Additionally, BSA also prevents adhering of Taq polymerase to the
wall of the PCR tubes. Sodium azide is a preservative used in buffer
to prevent exogenous contamination. Upon the receipt of reaction
mix A it should be stored at 25 to 15  C and after initial use it
should be stored at 2–8  C.
182 Amplification of Autosomal Mini-STR Markers from Degraded Samples

3.2 AmpF lSTR™ A set of primers for each locus are present in this mixture. This
MiniFiler™ Primer Set contains the forward and reverse primers to amplify the specific
human targets categorically. Each primer set is labeled with a spe-
cific fluorescent dye, i.e., 6-FAM™ (D13S317, D7S820), VIC™
(amelogenin, D2S1338, D21S11), NED™ (D16S539, D18S51),
PET™ (CSF1PO, FGA), and LIZ™ (Size Standard). Upon the
receipt of the primer mix, it should be stored at 25 to 15  C
with protection from light.

3.3 Molecular Molecular biology-grade water is the form of ultrapure high-quality

Biology-Grade Water water which is free from DNase, RNase, and protease contamination.
Additionally, no toxic agents such as DEPC should be present in this
water. Milli-Q water can be produced in the laboratory or it can be
procured from any molecular biology-grade chemical manufacturer.

3.4 Template DNA Extracted DNA from reference as well as forensic samples can be used
as template DNA for amplification of the genetic markers. After
estimation of quantity, the DNA should be suitably diluted or con-
centrated for optimal input in the PCR process, which is 0.5–0.75 ng.
After dilution, the template DNA should be stored at 20  C till
further use. It is always recommended to prepare dilutions of the
template DNA in TE buffer to prevent rapid degradation.

3.5 PCR Machine A PCR machine provides the physical conditions for in vitro repli-
cation of DNA in a tube containing all the requisite chemical agents.
In the first step of PCR process, denaturation of DNA occurs by
raising the temperature to ~95  C. In the next step the temperature
is lowered to as low as ~50  C which allows the binding of primers at
the targeted primer-binding sequence. Further the temperature is
increased to ~72  C for the action of DNA polymerase. These
sequential events are repeated for a number of cycles to increase
the copy numbers of the targeted DNA fragments. There are many
PCR machines available in the market; however, the PCR conditions
should be validated internally under the laboratory condition prior
to its application in forensic DNA analysis.

4 Procedure

Preparation Amplification Mixture

1. Calculate the amount of each component of the PCR process
using the following table as per your requirement/number of
samples (Table 22.1).
2. Thaw AmpFl STR™ MiniFiler™ Master Mix and AmpFl STR™
MiniFiler™ Primer Set in ice, vortex for 3–5 s, and centrifuge
briefly before using.
 Procedure 183

Table 22.1
Recommended amounts of reagents required to prepare amplification mixture for PCR

No. of
PCR components Volume per reaction (μl) reactions Total volume
AmpFlSTR™ MiniFiler™ Master Mix 10.0 A 10A
AmpFlSTR™ MiniFiler™ Primer Set 5.0 A 5A
Molecular biology-grade water Up to a final volume of 25 μl A xA
including template DNA (x)
Total volume (10 + 5 + x)A

3. Pipette out the calculated amount of molecular biology-grade

water, AmpFl STR™ MiniFiler™ Master Mix, and
AmpFl STR™ MiniFiler™ Primer Set in a 1.5 ml microcentri-
fuge tube marked as Amplification Mixture (AM). It is always
recommended to add water first to the tube, followed by
AmpFl STR™ MiniFiler™ Master Mix and AmpFl STR™ Mini-
Filer™ Primer Set.
4. As soon as the preparation of AM is over, immediately store the
tubes containing AmpFl STR™ MiniFiler™ Master Mix and
AmpFl STR™ MiniFiler™ Primer Set in the recommended
storage condition.
5. Vortex the amplification mixture for 5–10 s, and then dispense
(10 + 5 + x) μl of amplification mixture to each 200 μl PCR
tube pre-marked for each sample.
6. Add 0.5–0.75 ng of template DNA to each designated PCR
tube containing the amplification mixture.
7. In one tube add control DNA in the amplification mix and in
another tube add molecular biology-grade water or TE buffer
in place of template DNA which will be considered as positive
and negative control, respectively.
8. Close the tubes properly and briefly centrifuge the tubes so that
all its contents will be present at the bottom of the tube.

Thermal Cycling
9. Place the tubes in the thermal cycler.
10. Set the ramp rate of the thermal cycler to maximum or 100%.
11. Set the thermal cycling protocol as 95  C for 11 min; followed
by 30 cycles of 94  C for 20 s, 59  C for 2 min, and 72  C for
1 min; followed by 60  C for 45 min; and hold at 4  C.
12. Wait till completion of the PCR process.
13. Store the amplified products at 20  C in dark conditions or
immediately process for electrophoresis.
184 Amplification of Autosomal Mini-STR Markers from Degraded Samples

5 Observation and Result

The amplification of the samples can be confirmed after performing

capillary electrophoresis and data analysis which has been described
in Module 6.

6 Precautions
l Wear gloves while dealing with the reagents and plasticware.
l Do not store the reagents under a light source.
l Always use filtered microtips or aerosol-resistant microtips to
prevent any possible contaminations.
l Always put positive and negative control along with the test
samples for PCR process.
l As some of the chemicals are hazardous, they should be handled
carefully.
l Do all the works relating to PCR setup in a dedicated room
using a dedicated set of instruments to avoid contamination.
l Always prepare amplification mixture for extra 10% reactions to
avoid any chance of possible pipetting error.
l Make sure not to exceed the volume of template to 20% of the
total volume of PCR setup.
l Always ensure to provide the recommended amount of DNA
template to the PCR setup. For this purpose, it is useful to
ensure the concentration of template DNA by qRT-PCR.
l Do not store the amplification mix for longer time; it may
increase the chance of artifacts. Hence, after preparation of
amplification mix add template DNA at the earliest followed by
immediate thermal cycling.
l Always optimize the reaction setup and thermal cycling condi-
tions as per your laboratory conditions prior to performing any
experiments.
l For storage of PCR-amplified products for <2 weeks, place
them at 2–8  C; however for long-term storage (>2 weeks)
keep them at 25 to 15  C.

7 Troubleshooting

As the results can be evaluated after performing capillary electro-

phoresis, for troubleshooting of amplification you can refer to the
capillary electrophoresis chapter described in Module 6.
 Part VI

Genotyping and Analysis of Results

 Chapter 23

Separation of Amplified DNA Fragments by Capillary

Electrophoresis Using Genetic Analyzer 3500xL

Objective: To separate the amplified fragments by capillary electro-

phoresis using Genetic Analyzer 3500xL.

1 Introduction

After amplification of genetic loci (STRs) using multiplex process,

the amplified fragments need to be analyzed by electrophoresis. In
this process, the amplified products get separated on the basis of their
fragment sizes and the corresponding alleles can be designated. As
the amplified products are multiple in numbers labeled with different
fluorescent dyes, a sophisticated, automated instrument is required
to separate as well as identify the amplified fragment with great
efficiency. In this regard, since the inception of STR-based forensic
DNA typing, capillary electrophoresis has become an integral part of
the analysis process. It is an advanced version of the electrophoresis
process, where the gel slab has been replaced by the narrow capillaries
for the efficient separation of nucleic acids. The narrow capillary is
capable of performing a versatile analysis at high electric fields, thus
decreasing the run time as well as minimizing the overheating pro-
blems during the application of high voltage. The technique has
become immensely popular among the users due to its capabilities
of automation leading to elimination of preparation of gel slabs as
well as loading the DNA fragments onto a gel slab. Additionally,
consumption of a scanty amount of DNA and software-based detec-
tion and analysis of DNA fragments have popularized the capillary
electrophoresis process.
Coupling of capillary electrophoresis with automated injection
of samples has increased the versatility of the instrument. Invention
of instruments with multiple fluorescence detection capabilities has
increased the efficiency of the instrument in analyzing multiple

H. R. Dash et al., Principles and Practices of DNA Analysis: A Laboratory Manual for Forensic DNA Typing, Springer Protocols
Handbooks, https://doi.org/10.1007/978-1-0716-0274-4_23, © Springer Science+Business Media, LLC, part of Springer Nature 2020

187
188 Separation of Amplified DNA Fragments by Capillary Electrophoresis

genetic fragments in a single run. It has also shown its usefulness in

other applications such as sequencing of human genome, gunshots,
and explosives residue analysis as well as drug analysis. The capillary
number of various instruments varies from 1 to 384, where these
many number of samples can be analyzed simultaneously in a single
run. Many capillary electrophoresis systems are available in the
market (for details see Appendix B); however, ABI Genetic Ana-
lyzer 3500xL instrument, a 24-capillary 6-dye chemistry instru-
ment, has been described in this chapter.

2 Principle

A conventional capillary electrophoresis system involves a high-

voltage power supply, a system for sample injection, a narrow-
width capillary tube, the detector, and the output system
(Fig. 23.1). In most of the instruments, a temperature control
device is also present to maintain the temperature. Maintenance of
temperature is required as an increase in temperature leads to
improper electrophoretic mobility and decrease in viscosity of the
solution. For application of high voltage, two electrodes are
included which initiates the migration of samples from cathode to
anode under the influence of electric current inside the capillary.
Mostly the capillary is made up of silica which is dipped in cathode
and anode buffer in both the sides. Before electrophoresis, the
capillary is filled with the polymer which acts as the matrix for the
transfer of DNA fragments. A laser beam along with a detector is
fitted at the anode end of the capillary to detect the fluorescence of
the amplified fragments. Additionally a photomultiplier is connected
to gather additional information of the separated fragments.
Samples (amplified DNA) are injected into the capillary array by
a technique called as electrokinetic injection. In this process when an
electric field is applied to the liquid sample, the negatively charged
DNA molecules get dragged by the positive voltage into the capil-
lary. Such a type of injection produces narrow injection zones to
allow separation of DNA with high resolution. However, impurities

Fig. 23.1 Schematic diagram of capillary electrophoresis instrument along with its working components
 Reagents/Materials Required and Their Role 189

Fig. 23.2 A view of Genetic Analyzer 3500xL: (a) Polymer pack, (b) outlet buffer reservoir, (c) mechanical
pump, (d) oven door, (e) inlet buffer reservoir, (f) autosampler, (g) inside view of the oven chamber showing
24 capillaries

like chloride ions in DNA solution may compete with the negatively
charged DNA molecules to be injected into the capillary by the
electrokinetic injection process. In this regard, the quantity of
DNA injected into a capillary is dependent on voltage (E), injection
time (t), DNA concentration (DNAsample), capillary opening area
(πr2), ionic strength of sample (λsample) and buffer (λbuffer), electro-
phoretic mobility (μep), and electroosmotic flow (μeof), as follows:
 
 
DNA injected ¼ E  t πr 2 μep þ μeof
 
 DNA sample ðλbuffer Þ=λsample

After completion of electrophoresis, the fluorescently labeled

PCR products are detected by a fluorescent detector either with a
photomultiplier tube (PMT) or charge-coupled device (CCD).
Thus, the amplified STR alleles are marked as peaks on an electro-
pherogram with the light intensity measured as arbitrary unit, i.e.,
relative fluorescence units (RFUs). Genetic Analyzer 3500xL is a
24-capillary instrument with reduced power requirements (110 V)
and capability of 6-dye detection (Fig. 23.2). Additionally, the
monitoring of consumables is carried out by the radio-frequency
identification (RFID) tags for ready-to-use and easy maintenance of
reagents and consumables.

3 Reagents/Materials Required and Their Role

3.1 PCR-Amplified The products of PCR after amplification of autosomal STRs,

Product Y-STRs, X-STRs, or mini-STRs are required for run in Genetic
Analyzer. The PCR products can be immediately processed for
electrophoresis or stored at 20 till electrophoresis. It is always
recommended to store the PCR products under dark conditions.
190 Separation of Amplified DNA Fragments by Capillary Electrophoresis

3.2 Hi-Di™ This is a form of highly deionized formamide used for resuspension
Formamide of PCR products for electrokinetic injection during capillary elec-
trophoresis. Additionally, it acts as a denaturant and provides sta-
bility to the sample during heat denaturation step as well as during
the process of capillary electrophoresis. The commonly used
POP-4 for fragment analysis contains linear, uncross-linked
dimethyl polyacrylamide at a concentration of 4%. Presence of
urea in this solution provides added advantage during capillary
electrophoresis by creating an environment for keeping the DNA
molecules in denatured condition. Before using a polymer pouch,
check for the presence of crystals (if any) which may hamper the
electrophoresis process. It can be stored at 2–8  C for up to 1 week;
however, for long-term storage up to 3 months, it should be stored
at 15 to 25  C. The best way to use formamide is to prepare small
aliquots in 1.5 ml or smaller tubes for routine use and avoiding
repeated freeze-thaw or exposure to air.

3.3 Internal Size Internal size standard is mixed with each sample for processing of
Standard samples as well as separation and detection of STR alleles. It is
useful in the correct detection of size of the amplified fragments.
An additional dye set, mostly the LIZ™, is used for labeling of the
size standard. As different samples are mixed with a set of DNA
fragments of known size, it can correlate the results from run to run
as well as it helps in troubleshooting of the process at the step of
either PCR or capillary electrophoresis. Peak height normalization
can also be performed using internal size standard which leads to
peak height uniformity among capillaries, run, and instruments. It
is kit specific. Hence, size standard supplied with the STR amplifi-
cation kit should be used for electrophoresis of those samples.

3.4 Allelic Ladder It is an artificial mixture of nucleotide fragments that are of same
size to the common alleles available in human population for all the
markers analyzed. This plays a major role in allele designation in a
STR locus after PCR amplification. Allelic ladder acts as a ruler for
each locus, as the amplified fragment will match with the position of
the allele present on the ladder to designate it as an allele. Com-
mercial manufacturers supply allelic ladders along with their kits. As
the allele range of the genetic markers varies, it is always recom-
mended to use the specific allelic ladder of the specific kit for
analysis purpose. The allelic ladder should be transported at 15
to 25  C whereas while in use it should be stored at 2–8  C with
protection from light. As allelic ladder contains the products from
PCR, it should not be stored along with the unamplified DNA.

3.5 Capillary Capillary is the most vital accessory of the instrument. It is com-
monly made up of fused silica (SiO2) and their inner wall contains
the hydroxyl groups which are negatively charged at pH 5.0. When
electrophoresis buffer with pH 8.0 is used, positive ions from buffer
line up with SiO along their wall to create a double layer. Under
 Procedure 191

Fig. 23.3 Internal environment of a capillary tube generating the electroosmotic flow

electric current, the positive ions move towards the negative elec-
trode called as electroosmotic flow (EOF) (Fig. 23.3). The EOF
may create problem to obtain a DNA separation result with higher
reproducibility. Thus, the capillary is chemically modified or dyna-
mically coated to minimize/prevent EOF during electrophoresis.
To avoid problems and increase the separation resolution, the
capillary should be replaced each time after 100–150 injections.

3.6 Buffers For electrophoresis in a Genetic Analyzer, anode and cathode

buffers are necessary. The common constituents of anode and
cathode buffers include 100 mM/l N-tris-(hydroxymethyl)-
methyl-3-aminopropane-sulfonic acid (TAPS) and 1 mM/l
EDTA adjusted to pH 8.0. Additionally to accomplish DNA dena-
turation and maintain temperature during electrophoresis some
additives are also added which include formamide, urea, and
2-pyrrolidinone. After repeated runs the composition of anode
and cathode buffers changes due to migration of buffer ions and
electrolysis. Thus, it should be changed and replenished with fresh
set of buffer within a stipulated time interval.

3.7 Polymer Commercially available poly-dimethylacrylamide POP-4 and

POP-6 polymers are mostly used in Genetic Analyzer 3500xL. It
provides a sieving matrix for efficient separation of single-stranded
DNA during capillary electrophoresis. Additionally as it suppresses
EOF, it increases resolution of amplified fragments. The constitu-
ents of POP-4 include mostly di-methylacrylamide (4%), urea
(8 mol/l), and 2-pyrrolidinone (5%).

4 Procedure

Plate Preparation
1. Thaw PCR-amplified products, Hi-Di™ Formamide, size stan-
dard, and allelic ladder on ice. Vortex size standard and allelic
ladder briefly for 10–15 s for proper mixing of the fragments.
192 Separation of Amplified DNA Fragments by Capillary Electrophoresis

2. Pipette out 247 μl of Hi-Di™ Formamide and 13 μl of size

standard in a 1.5 ml microcentrifuge tube. Vortex for 10–15 s
to mix the reagents.
3. Dispense 10 μl of the mixture in 24 wells of a 96-well plate
from 1A to 1H, 2A to 2H, and 3A to 3H.
4. Add 1 μl of the PCR-amplified product in the wells (you can
prepare a sheet of paper, designating the sample name to be
loaded on each well) from 1A to 1H, 2A to 2H, and 3A to 3G.
5. Add 1 μl of allelic ladder to well 3H of the 96-well plate.
6. Cover the 96-well plate with an appropriate septa.
7. Centrifuge the plate at 5000 rpm for 30 s at room temperature
using a plate rotor.
8. Denature the samples by incubating the plate at 95  C for 3 min
followed by immediate freezing on ice for 3 min. Always dena-
ture samples just before loading of the plate in the instrument.
9. Place the plate containing samples on the plate base and cover it
with plate retainer. Check for the proper alignment of the holes
of the plate retainer with the holes of the septa. Click on “Tray”
button of the instrument, wait till the indicator turns green,
load the plate, and close the door (Fig. 23.4).

Instrument Setup and Preparation of Plate Record

10. Switch on the computer followed by switching on the
instrument.
11. Wait till the computer finishes booting and the connection
between the computer and instrument is established. Comple-
tion of booting can be confirmed by “active” of the status icon
of the 3500 server monitor present at the right lower corner of
the desktop (Fig. 23.5).

Fig. 23.4 (a) A 96-well plate with loaded samples, covered with septa placed on a plate base and covered with
plate retainer; (b) loading of the plate in the instrument
 Procedure 193

Fig. 23.5 “Active icon” confirming the establishment of connection between the instrument and computer as
(a) Active icon and (b) 3500 server. Monitor activation confirming the establishment of connection between the
instrument and computer

12. Launch the 3500 Data collection software by using the desig-
nated “user id” and “password.”
13. The Dashboard is displayed after opening the software
(Fig. 23.6). This Dashboard shows information and status of
the instrument, consumables, and maintenance.
14. From the Dashboard pick “Create New Plate”; enter a “plate
name”; select the number of wells as “96,” plate type as “HID,”
capillary length as “36 cm,” and polymer as “POP-4” (Fig. 23.7).
15. Go to “Assign Plate Contents”; a 96-well format will appear on
the screen (Fig. 23.8).
16. Go to “Assays”; click on “Add from library”; select the suitable
assay you want to perform, for electrophoresis of amplicons
amplified using Global Filer™ kit; select “GF+Norm_POP4-
xl”; click on “Add to Plate”; and then close the window
(Fig. 23.9).
17. Go to “File Name Convention”; click on “Add from library”;
select the suitable file name convention you want to perform;
click on “Add to Plate”; and then close the window
(Fig. 23.10).
18. Go to “Result Groups”; click on “Add from library”; select the
suitable Result Group where you want to keep your results; click
on “Add to Plate,”; and then close the window (Fig. 23.11).
19. Click on the corresponding position on the 96-well format that
appears on the screen and add information of the samples
loaded at that position. Make sure to add information of ladder
loaded at well 3H (Fig. 23.12).
20. Select all the wells with information added to each well and
click on the box present near the selected Assay, File name
convention, and Result Group (Fig. 23.13).
21. Go to customize Sample Info, select all the samples except
ladder, and assign “Sample” from the drop-down menu.
194 Separation of Amplified DNA Fragments by Capillary Electrophoresis

Fig. 23.6 Home screen of the data collection software

 Procedure 195

Fig. 23.7 Assignment of plate name and other attributes

Fig. 23.8 Plate content assignment

Fig. 23.9 Adding information of the assay to be performed

Similarly, select the ladder present at 3H position and assign

“Allelic Ladder” from the drop-down menu (Fig. 23.14).
22. Click on “Link Plate for Run.” Wait for sometime; a window
will be displayed as in Fig. 23.15.
196 Separation of Amplified DNA Fragments by Capillary Electrophoresis

Fig. 23.10 Adding information on the File name Convention

Fig. 23.11 Adding information on the “Result Group”

23. To be in safe side, click on “Preview Run.” Check that all the
information attributed to each well is correct (Fig. 23.16).
24. Go back to “link plate for run” and click on “Start Run”
(Fig. 23.17).
25. When the run starts the green indicator of the machine blinks
and the window displays as follows (Fig. 23.18).
 Procedure

Fig. 23.12 Adding information of samples loaded at the corresponding positions of the 96-well plate
197
 198
Separation of Amplified DNA Fragments by Capillary Electrophoresis

Fig. 23.13 Selection of the wells and to link with the assigned Assay, File name Convention, and Result Group
 Procedure 199

Fig. 23.14 Assigning the customized (a) sample and (b) allelic ladder position to the data collection software

26. When the run ends, blinking of the green indicator stops and
the window displays as follows (Fig. 23.19).

Instrument Calibration and Maintenance

27. There are two types of calibration, i.e., spatial calibration and
spectral calibration. You need to perform spatial calibration
each time after you replace the capillary array, during opening
of detector cell, or after moving the instrument. However,
spectral calibration needs to be performed when using a new
dye set, changing capillary array, and changing polymer type
and during repeated observance of reduced spectral separa-
tion in raw data.
28. For spatial calibration, go to “Maintenance” and select “Spa-
tial Calibration.”
29. Select “Fill” to fill the capillary with polymer prior to
calibration.
30. Select “Start Calibration.” The result will be displayed as the
run progresses (Fig. 23.20).
31. The spatial calibration result should be evaluated as follows:
one sharp peak for each capillary, one “+” mark at the top of
each peak, and all the peaks having an almost equal peak
height.
32. If you are satisfied with the result, select “Accept Results” or
else “Reject Results” and perform troubleshooting as
described later in the chapter.
33. If accepted result is found, select “Export,” enter a name of
the export file, select its type as “CSV,” and click on “Save.”
34. Before performing spectral calibration, make sure that all the
consumables are appropriate and buffer levels are up to
the mark.
35. Before using Global Filer™ kit, use DS-36 Matrix Standard
(dye set J6 which contains DNA fragments labeled with
6-FAM™, VIC™, NED™, SID™, TAZ™, LIZ™) for spec-
tral calibration.
200 Separation of Amplified DNA Fragments by Capillary Electrophoresis

Fig. 23.15 The displayed window after linking the plate for run
 Procedure

Fig. 23.16 Select “Preview Run” to assess the input of correct information to each sample
201
202 Separation of Amplified DNA Fragments by Capillary Electrophoresis

Fig. 23.17 Go back to “Link Plate for Run” and “Start Run”
 Procedure

Fig. 23.18 Display of the window when the run starts

203
204 Separation of Amplified DNA Fragments by Capillary Electrophoresis

Fig. 23.19 Display of the window after completion of the run

 Procedure 205

7500

5500

3500

1500

–500
0 100 200 300 400

Fig. 23.20 An acceptable result of a spatial calibration in a 24-capillary system

2400

2000

1600

1200

800

400

–400
0 1000 2000 3000 4000 5000
Intensity vs Scan Number

Fig. 23.21 A passing six-dye spectral calibration result

36. Vortex the matrix standard briefly and spin in a centrifuge.

37. Prepare the standard by mixing 6 μl of standard into a 294 μl of
Hi-Di™ Formamide. Vortex the mixture and spin briefly in a
centrifuge.
38. Dispense 10 μl of the mixture to the A1 to H1, A2 to H2, and
A3 to H3 wells of a 96-well plate.
39. Cover the plate using appropriate septa.
40. Denature the plate contents at 95  C for 5 min followed by
immediate snap cool on ice.
41. Go to “Maintenance” and select “Spectral Calibration.”
42. Specify the location of the samples placed in the 96-well plate
in the instrument.
43. Select the standard chemistry and the dye set for which you
want to perform the calibration. N.B.: Any new dye set can also
be calibrated on the instrument by creating the custom dye set.
44. Select “Start Run.” The system will perform three injections
and the run data is displayed after the end of each injection
(Fig. 23.21).
45. Examine the result for all the capillaries and the result obtained
among different injections.
206 Separation of Amplified DNA Fragments by Capillary Electrophoresis

46. When all the capillaries yield an acceptable result, select

“Accept Results” or else “Reject Results” and perform trou-
bleshooting as described later in the chapter.
47. During HID applications, three maintenance activities need to
be performed regularly, i.e., replenish polymer, change the
buffers, and replace the old capillary with a new one.
48. For change in buffer, bring the anode buffer container (ABC)
from the storage and equilibrate it to room temperature.
49. Ensure the presence of 1 buffer in the larger side of the
container, verify the content up to the mark, and peel off the
seal of the ABC.
50. Place the ABC at the designated position of the instrument
below the pump. Close the instrument door.
51. Refresh the dashboard of the software to check the updated
status of the ABC.
52. Similarly, bring the cathode buffer container (CBC) from the
storage and equilibrate it to room temperature.
53. Verify the content of CBC up to the mark and peel off the seal
of the CBC.
54. Place appropriate septa on both sides of the CBC and place the
CBC on the autosampler.
55. Close the instrument door and refresh the dashboard of the
software to check the updated status of the CBC.
56. When the polymer is utilized, the dashboard itself shows warn-
ing signal to replenish the polymer pouch. In such cases, the
used polymer pouch needs to be replaced with a fresh pouch.
57. To replenish polymer, bring the polymer pouch from the stor-
age and equilibrate it to room temperature.
58. Select “Replenish Polymer” from the maintenance wizards
screen.
59. Open the door of the instrument and replace the old polymer
pouch with a new one. Close the door and follow instructions
of the maintenance wizard.
60. Refresh in the dashboard to verify the updated status of the
polymer.
61. Check for the presence of any bubbles in the path. If found,
follow the wizard “Remove Bubbles” which will take around
10–15 min to complete.
62. Additionally, to change the capillary array, select “Install Capil-
lary array” from the maintenance wizard.
63. Replace the old capillaries with a set of new capillaries and close
the door carefully.
 Observation 207

64. Follow the instructions given by the maintenance wizard. Refresh

the dashboard to verify the updated status of the capillary.

5 Observation

You can monitor the electrophoresis process during the run as

following parameters can be visualized.
(a) In the instrument Run Views select “EPT” and for an ideal
electrophoresis run the EPT graph should be found as given in
Fig. 23.22.
(b) In the instrument Run Views select “Array” and the result will
appear as given in Fig. 23.23. Presence of all the dye sets in
each capillary suggests the separation of amplified products in
that capillary.
(c) In the instrument Run Views select “Sample” followed by
selecting allelic ladder or a sample; the result will appear as
given in Fig. 23.24. Presence of peaks of all fluorescent colors
suggests proper amplification and electrophoresis.

Fig. 23.22 An ideal EPT curve shown by the instrument

Fig. 23.23 Visualization of samples present in the corresponding capillary arrays

208 Separation of Amplified DNA Fragments by Capillary Electrophoresis

Fig. 23.24 Visualization of peaks for the (a) allelic ladder and (b) sample

6 Precautions
l Wear gloves while dealing with the reagents and plasticware.
l Always use filtered microtips or aerosol-resistant microtips to
prevent any possible contaminations.
l As some of the chemicals are hazardous, they should be handled
carefully.
l Always store the reagents in appropriate storage conditions.
l Store the matrix standard in dark conditions.
l Check for the presence of crystals in the polymer pouch before
using it on the instrument.
l Never mix the pre-PCR and post-PCR area.
l Avoid repeated freezing and thawing of the allelic ladder and size
standard.
l All the reagents, i.e., polymer, ABC, CBC, and conditioning
reagents, should be brought to room temperature prior to
putting them on the instrument.
l Always maintain the room temperature for optimal performance
of the instrument.
l Before performing the experiment always switch on the com-
puter first followed by the instrument and finally the data collec-
tion software.
l Before launching the data collection software always wait for
sometime till the establishment of connection between the
instrument and the system.
l Denature the plate just before putting it in the instrument.
 Troubleshooting 209

l Used septa should be cleaned carefully; otherwise it may lead to

contamination.
l Check the alignment of the 96-well plate before putting it in the
plate base and plate retainer.
l Recheck the samples loaded in the 96-well plate and the samples
assigned to the instrument for electrophoresis.
l Never use the reagents (polymer, ABC, CBC, and conditioning
reagent) without proper seal.
l Never use expired reagents.
l After run is complete do not close the data collection software
immediately as it may take few more minutes to complete data
collection after completion of run.
l Always check the status of electrophoresis by monitoring the
EPT graph prior to data analysis.
l In between runs do not close the data collection software and
open it. It may hamper the life span of the laser present in the
instrument.

7 Troubleshooting

Problem Tentative cause Possible solutions

Continuous blinking Pause during run Select “Resume Run”
of yellow light
Door of the instrument Carefully close the door
remained open of the instrument
Run failure Check for any error
message and rectify
it. Rerun the samples
or conduct another
run
“Start Run” icon is Initialization of the Wait for 10 s till the
not responding instrument is not yellow light turns to
complete green after closing the
instrument door, and
then proceed with
“Start Run”
Run paused Reading of RFID is not Refresh the dashboard;
unexpectedly appropriate in the if the problem
instrument persists, restart the
computer,
instrument, and
software

(continued)
210 Separation of Amplified DNA Fragments by Capillary Electrophoresis

Problem Tentative cause Possible solutions

Run paused Less content in ABC Replace with a fresh
unexpectedly with ABC
error of electric
Leak in the system Run the Bubble
discharge
Removal wizard and
rerun
Polymer crystals present Ensure cleaning of the
in the buffer pin valve buffer pin valve at
regular interval
Run stopped after RFID of the consumables Check the connection
one or more than could not be read by between the
one injections the instrument instrument and
software and proceed
with a new injection
The dashboard does The wizards have not Refresh the dashboard
not update been refreshed or start a new run
automatically after
replenishing the
consumables
Pre-run check Check the dashboard for Replace the
validation failed the status of the ABC/CBC/
consumables polymer/capillary for
which the pre-run
check validation failed
Error during linking No plate in position A If you have loaded the
the plate plate at position B,
link the plate with
position B
No plate detected Place the plate at
position A and follow
the instructions
During spatial No communication Restart the system,
calibration “start” between instrument instrument, and
button is disabled and data collection software; then
software perform the spatial
calibration again
During spatial Detection cell is not Reinstall the capillary
calibration unusual installed properly array, followed by fill
peaks or no peaks array, and perform the
appear experiment again
Stability of the Repeat the experiment
instrument not after sometime
achieved
Capillary is broken Check the capillary; if
found broken replace
it

(continued)
 Troubleshooting 211

Problem Tentative cause Possible solutions

Bad spatial Faulty capillary array Replace the capillary
calibration results array with a new one
and perform the
experiment
Message pops out as Conditioning reagent is Replace the
“spatial calibration present in place of conditioning reagent
error” polymer with polymer pouch
No signal during Sample is not prepared Prepare the samples
spectral calibration properly again with fresh
Hi-Di™ Formamide
and perform the
experiment again
Bubbles present in the Remove the bubbles by
wells containing centrifugation and
samples perform the
experiment again
Tip of the capillary is not Check the volume of the
touching the samples sample loaded; if
properly/capillary recommended
array is not properly volume is applied, call
installed the service personnel
of the instrument
“No spectral files Capillary is blocked First try with refilling
found” error is the capillary array; if
shown the problem persists
install a fresh capillary
array and perform the
experiment again
Incorrect run module/ Correct the
chemistry file/dye set inappropriate files and
selected perform the
experiment again
Expired matrix standard Check the matrix
used standard and if
necessary use a fresh
set of reagents
Peaks found below 1 peaks found below Rerun the spectral
the required the recommended standards with a
amplitude amplitude, i.e., 750 higher amount of
standard

(continued)
212 Separation of Amplified DNA Fragments by Capillary Electrophoresis

Problem Tentative cause Possible solutions

Spikes appeared in Faulty polymer Replace the faulty
the result polymer with a new
Contaminants/crystals
fresh polymer pouch
found in the polymer
and perform the
experiment again
Bubbles present in the Perform the bubble
polymer path removal option using
the wizard and
perform the
experiment again
Baseline is elevated Spectral calibration is Perform the experiment
poor again
 Chapter 24

Analysis of Capillary Electrophoresis Results by

GeneMapper® ID-X v 1.5 Software

Objective: To analyze the capillary electrophoresis results by using

GeneMapper® ID-X v 1.5 software.

1 Introduction

Data interpretation is the most crucial step of forensic DNA analysis

and needs enormous expertise to deal with it. As forensic DNA
typing is a comparative assessment of results between the control/
reference sample and the test/questioned sample, three possible
outcomes may appear for an experiment, i.e., a match, a mismatch
(maybe mutational), or an inconclusive result. All these possible
results should be evaluated carefully by an examiner. With the
advancement of technology, many software have eased the work
of the forensic scientists; however, thorough examination of a true
allele designation can reverse the opinion of a case examined
through the technique.
Multiplex PCR followed by capillary electrophoresis generates
a huge data that cannot be analyzed manually. Data interpretation is
the most laborious step of STR typing. Due to complex nature of
the data generated during capillary electrophoresis, data interpreta-
tion requires 50% or more of the resources. Thus, to minimize
human intervention during data analysis, many software are in
practice nowadays (for comprehensive list see Appendix B); the
GeneMapper® ID-X is widely used across the globe in forensic
DNA analysis. Besides designating alleles to the amplicons gener-
ated in the form of peaks, the software is also capable of distinguish-
ing between a true peak and the possible artifacts, for example dye
blobs, stutters, and spikes. Though GeneMapper® is capable of
generating a DNA profile by analyzing the capillary electrophoresis
data, it still requires manual review. The process quality values

H. R. Dash et al., Principles and Practices of DNA Analysis: A Laboratory Manual for Forensic DNA Typing, Springer Protocols
Handbooks, https://doi.org/10.1007/978-1-0716-0274-4_24, © Springer Science+Business Media, LLC, part of Springer Nature 2020

213
214 Analysis of Capillary Electrophoresis Results

(PQVs) increase the confidence of the allele designation and trou-

bleshooting efforts. The advanced version of GeneMapper® pro-
vides solution to analyze data from single-source samples as well as
for a mixture interpretation.

2 Principle

After completion of electrophoresis, the Genetic Analyzer 3500

system generates .hid (human identity) files that contains electronic
information on the laser power and run current besides information
of the amplified DNA fragments. The overall role of data analysis
software is to convert the time/scan points to the DNA sizes with
reference to the internal size standard encrypted in the electronic
file (.hid file). Further by the utilization of kit-specific bins, panels,
and allelic ladders the amplified fragment is designated as an allele.
When the software generates a DNA profile, analysts’ role becomes
imperative to assess the electrophoretic data as a noise or a proper
peak, distinguishing a proper peak from common artifacts, pairing
alleles to form a genotype, and combining the genotype of all the
loci tested to generate a complete DNA profile. Prior to analysis of a
DNA profile the following terms associated with result interpreta-
tion should be understood carefully to establish sensitivity, repro-
ducibility, and precision of the obtained data as well as correct
assignment of heterozygosity and assessment of mixture samples.
Thresholds: During low-copy-number DNA analysis, it becomes
highly difficult to distinguish between a true peak and the technical
artifacts. Thus, peak height thresholds have been established to
designate a true allele. In such cases, when the relative fluorescent
unit (RFU) value of peak height exceeds the designated threshold
value the peak will be called as an allele (Fig. 24.1). Though there is
no standard rule for establishing a threshold value, it should be a
part of any laboratory’s internal validation procedure. It can be
maintained by observing the signal-noise ratio or the software sets
it arbitrarily. Though the software manufacturer (Thermo Scien-
tific) recommends the peak height threshold to be 150 RFU,
forensic samples as low as 50 RFU can be used for allele calling.
An ideal data: Unlike “bands” of slab gel electrophoresis, the
results of capillary electrophoresis are generated in the form of
“peaks.” A proper peak should start from the baseline and finish
at baseline. At the signal level, the top of the peaks should be well
above the analytical threshold and well differentiated from the
noise. The loci should have well-defined peaks within the
pre-designated shaded beans defined by the allelic ladder. In an
ideal profile, all the peaks for homozygous alleles and all the peaks
for heterozygous alleles should have the same peak height ratio.
The peak height of the homozygous allele should be twice that of
the peak height of a heterozygous allele (Fig. 24.2). An ideal profile
 Principle 215

Fig. 24.1 Role of threshold in allele calling for a sample

Fig. 24.2 An ideal data of alleles from four genetic loci

without any artifact is capable of differentiation of contributor’s

DNA profile from a mixed sample and low DNA sample analysis.
Spikes: Spikes are the known artifacts that appear due to voltage
fluctuation during electrophoresis. The fluctuation in voltage gen-
erally arises due to the presence of minute air bubbles in the
capillary or crystal formation in the polymer or contamination of
polymer by the fluorescent materials. More amounts of spikes are
generally visible in the raw data than the analyzed data. Spikes when
not appearing along the true peak generally appear at the same data
point for all the dye sets. They also do not fall as per the true peaks
as they do not have a broader base (Fig. 24.3). The appearance of
spikes in the result can be minimized by using a fresh set of reagents
and samples. However, the frequent occurrence of spikes is an
indication of system malfunctioning.
Dye blobs: Dye blobs can be clearly distinguished from the true
peaks as they have a broader base and are mostly seen in a specific
dye set for one analysis (Fig. 24.4). Dissociation of fluorescent dyes
from the labeled primers due to improper storage conditions
216 Analysis of Capillary Electrophoresis Results

Fig. 24.3 Occurrence of spikes at the same data point in all the dye sets

Fig. 24.4 Occurrence of dye blob with wider base in the green dye set only

generates dye blobs in an electrophoresis data. To minimize the dye

blobs in result, the primers should be stored in the recommended
storage conditions. Further, purification of the amplified product
using a filtration unit will improve the result by passing through the
dissociated primers and retaining the amplified DNA fragments.
However, in a set forensic laboratory system and use of standar-
dized protocols the filtration step may not be required.
Noise: It is the series of baseline peaks which are
non-reproducible and present as backgrounds to the sample results.
Noise appears due to electronic fluctuations, presence of air bub-
bles and crystals in the polymer, and low-level sample contamina-
tion. Noise does not create problem in the analysis as they are
present in the baseline. However, if they appear close to the thresh-
old value they can be misinterpreted as a true peak. The most useful
way to distinguish between noise and a true allele is that noise is not
reproducible whereas peaks are always reproducible. When the
result is confusing between the allele and noise, the sample should
be subjected to electrophoresis again and the result can be
interpreted.
Pull-up: Pull-up is a major problem when high amount of
template DNA is added to a PCR setup followed by electrophore-
sis. The oversaturated data pulls up one color dye to another dye set
and in such cases the software is unable to distinguish between
different colors. Re-amplification with less amount of input DNA
can minimize the pull-ups. Careful observation of the pull-ups can
 Principle 217

Fig. 24.5 Occurrence of pull-ups in the blue and yellow dye sets due to oversaturated peaks present in the
green and red dye sets

distinguish it from the true peaks as they appear at the above or

below dye set at the same position of a saturated data (Fig. 24.5).
Proper quantification and addition of optimum quantity of DNA in
a PCR process will minimize these artifacts; however if the problem
persists even after adding optimal DNA quantity, a new matrix
should be run and applied to the set of results.
Stutter: Stutter is a PCR artifact which is formed as one repeat
shorter or larger than the true PCR product and accumulates as a
minor product. Though the mechanism of stutter formation is not
completely understood, the bulging of the template strand during
strand breathing results in the formation of a reverse stutter
whereas bulging of the newly synthesized strand results in the
formation of a forward stutter (Fig. 24.6). Due to its specific
appearance and size, stutters can be easily identified in a single-
source sample. However, it is difficult to distinguish between a true
allele and a stutter in a mixed-source sample. Studies have shown
the occurrence of a higher percentage of stutters in low unit
repeats, homogeneity of repeat units, and large alleles. Sometimes
stutter products are also helpful to distinguish between the true
allele from other artifacts such as pull-ups and spikes as only PCR
products contain the stutter peaks.
218 Analysis of Capillary Electrophoresis Results

Fig. 24.6 Formation of N4 and N+4 stutter products during in vitro DNA replication

3 Non-template Addition

It is a known PCR artifact which is generated by the de novo action

of Taq polymerase. Taq polymerase adds nucleotides, mostly ade-
nine, to the 30 end of the PCR product resulting the PCR product
to be one base pair larger than the actual product. The final incu-
bation step at 60 or 72  C favors the non-template addition by PCR
process. During the final incubation step, the degree of adenylation
is dependent on the sequence of the template strand. Amplification
process is always carried out from the 50 end. When the forward
primer, labeled with a fluorescent dye, is used for amplification,
then only the top strand/amplified fragment is detected by its
fluorescence in the instrument (50 –30 ). Additionally, when this
labeled amplified fragment is used as template, primer binds to
the 30 end of the fragment and the terminal nucleotide is dependent
on the 50 end of the reverse primer. Studies have also revealed that
complete addition of adenine by polymerase is favored by the
presence of guanosine at the 50 end of the primer. Thus, different
loci possess different adenylation properties due to differences in
their primer sequences. From the analysis point of view, adenylation
leads to –A peaks or +A peaks, where the peaks become broader and
a split peak appears when the detection system has a poor resolution
(Fig. 24.7). To minimize split peaks arising due to non-template
addition, the final extension step of PCR process should be opti-
mized so that all amplicons result in the same length.
Off-ladder (OL) alleles: Human population may contain some
rare alleles that are different from the common alleles of the genetic
 Non-template Addition 219

Fig. 24.7 Addition of non-template A by Taq polymerase at the end of the terminal nucleotide (X) at the 50 end
which is detected by the terminal nucleotide (Y) of reverse primer; this difference in adenylation pattern leads
to the formation of –A or +A form of the amplified products

markers tested. While using any commercially available kit, the

corresponding allelic ladder contains a mixture of commonly
occurred alleles. When amplification of a rare allele occurs the
peaks fall in between the alleles of the ladder and this is designated
as off ladder (OL). Allele designation of an OL should be carefully
assigned a unique allele or position. In all the instances, the
off-ladder peak should be confirmed by re-PCR.
Microvariant alleles: It is an unusual finding where a microvar-
iant represents an incomplete repeat of a STR marker allele. When
an allele is designated as 15 for D18S51, it represents 15 repeats of
AGAA. However, when a microvariant allele appears as 14.2, it
denotes 14 complete repeat units of AGAA followed by a partial
repeat of two nucleotides. Most of the commonly occurring micro-
alleles are included in the allelic ladder; hence a microvariant allele is
called during analysis. In general, a more polymorphic marker
possesses a higher number of microvariant alleles such as FGA,
D21S11, and D18S51.
Heterozygous imbalance: For stochastic effects such as low DNA
samples, degraded DNA, and mixed samples, heterozygous imbal-
ance is a major concern. If less amount of DNA is introduced to a
PCR process, the heterozygous samples are amplified differentially.
Similarly, for mixed samples, the major contributor generates a
higher peak in comparison to the minor contributor. In such
cases, it becomes highly important to distinguish between the
heterozygote alleles and mixed alleles. It becomes imperative to
calculate the heterozygosity of alleles and when the heterozygosity
220 Analysis of Capillary Electrophoresis Results

is found to be less than 70% it is an indicative of mixture or

stochastic effect.
Dropout: It may be a single-allele dropout for a given locus or
complete locus dropout when both the alleles of a locus are miss-
ing. Dropout occurs due to the stochastic effects when low DNA
template or potential PCR inhibitors are present in the sample. In
some instances, mutation in the primer-binding site may result in
allele dropout by non-amplification. Sometimes allele sizes are
outside the normal calling range of the locus and remain unde-
tected resulting in the dropout.
Tri-allelic pattern: In some instances, three alleles can be
observed at a locus of autosomal STR DNA profile in a single-
source sample, which are generally the copy number variants. Due
to the presence of an extra chromosome fragment or whole chro-
mosome as in case of Down’s syndrome three alleles appear in this
particular locus. Two types of tri-allelic patterns have been estab-
lished, i.e., type 1 where the sum of peak heights of two alleles is
equal to the peak height of the third allele. Type 1 tri-allelic pattern
is highly common in occurrence in comparison to type 2 where
peak heights of the three alleles are balanced in nature (Fig. 24.8).
In some cases, false tri-allelic pattern may be observed when an
extreme off-ladder peak from an adjacent marker is misunderstood.
Additionally, sometimes type 1 tri-allelic pattern may be masked by
high stutters which should be taken care of during data
interpretation.
Mutations: Mutation refers to the alteration in genetic com-
plexion of an individual than that of its parents. Mutational events
during meiosis can complicate the paternity testing of a disputed
child. Thus, mutational events should not be confused as exclusion
and need to be ascertained prior to reporting of any forensic case.

4 GeneMapper® IDX Software

GeneMapper® IDX software (Thermo Scientific) is capable of pro-

cessing .fsa data generated from Genetic Analyzers of 31xx series,
310 series, and 37xx series instruments as well as .hid data gener-
ated from 3500 instruments series. It possesses an upper hand over
the other data analysis software available in the market by providing
improved data interpretation. Other advantages of this software
include minimized edits of pull-ups, first-pass success rate of ana-
lyzed data, and efficient transfer of data for probabilistic genotyp-
ing. Flexibility in plate loading, streamlined workflow, support for
languages other than English, and capability of analyzing six-dye
results are added advantages of using this software.
 Procedure 221

Fig. 24.8 Two types of tri-allelic pattern commonly occurred in population

Fig. 24.9 Home screen of GeneMapper® ID-X software

5 Procedure

1. Open previously installed GeneMapper® ID-X v 1.5 in the

computer with your customized user name and password.
After opening the home screen will appear as in Fig. 24.9.
2. Go to “Add Samples to Project”; select the location of the
results (in the previously assigned result group during plate
setup), select the run, and select “Add to Project” followed
by “Add” (Fig. 24.10).
3. After adding samples to a new project, the project will appear as
in Fig. 24.11.
4. Select the suitable “Analysis Method,” “Panel,” and “Size
Standard” from the drop-down menu (Fig. 24.12).
222 Analysis of Capillary Electrophoresis Results

Fig. 24.10 (a) Adding of samples to the project from (b) the location of the result group for analysis and (c)
selecting ‘Add to project’

Fig. 24.11 Samples added to the project

5. Select the suitable “Analysis Method,” “Panel,” and “Size

Standard” for all the samples, controls, and allelic ladder. You
can select column for all the parameters and press “Ctrl+D” to
fill all the columns with the selected “Analysis Method,”
“Panel,” and “Size Standard” (Fig. 24.13).
6. Select “Analyze,” assign project name, and select “OK”
(Figs. 24.14 and 24.15).
 Procedure 223

Fig. 24.12 Selection of suitable (a) analysis method, (b) panel, and (c) size standard for data analysis

Fig. 24.13 Selection of “Analysis Method,” “Panel,” and “Size Standard” for all the samples
224 Analysis of Capillary Electrophoresis Results

Fig. 24.14 Steps showing “Analyze” and assignment of a project name

7. After completion of analysis, the analysis summary appears

(Fig. 24.16). Check for the status of quality control for allelic
ladder and all the samples. When one or more threshold is not
met for the allelic ladder it fails (shows the red signal besides it)
and further analysis of the samples cannot be performed. Simi-
larly, the samples and controls should be monitored for quality
control before analysis.
8. Select “View” and choose “Raw Data” from the drop-down
menu (Fig. 24.17). The raw data should be checked for the
allelic ladder as well as the samples. Peaks from all the dye set
should be present in the raw data if amplification and electro-
phoresis are proper.
9. Select “View” and choose “EPT data” from the drop-down
menu (Fig. 24.18). The EPT graph should always appear as
given in Fig. 24.19. Any deviation of EPT graph from the given
figure is suggestive of an improper electrophoresis.
10. Select “Tools,” choose “Size Match Editor” from the drop-
down menu, and select “Size Calling Curve” (Fig. 24.19). The
size calling curve should always be a straight line with the R2
value close to 1.
11. After analyzing all quality parameters, select “View” and “Sam-
ples.” Select Allelic Ladder and go to “Display Plots.” The
plots of allelic ladder will appear as in Fig. 24.20.
12. Similarly select any sample and go to “Display Plots” to visua-
lize the sample plots (Fig. 24.21). Select Panes to “6” so that
all the plots of a six-dye chemistry kit appear in a single window.
 Procedure

Fig. 24.15 Display of software during analysis of project

225
226 Analysis of Capillary Electrophoresis Results

Fig. 24.16 Window displaying the analysis summary for the allelic ladder as well as the samples

Here the peaks in the orange dye denote the fragments of size
standard.
13. For autosomal STR analysis the amelogenin marker shows
either “XY” for female or “X” for females. Other autosomal
markers either show two peaks (for heterozygosity) or one peak
(for homozygosity) for a single-source sample.
14. Look for the presence of artifacts if any (as described in Sect.
2), in any of the markers. Two common artifacts, i.e.,
off-ladder (OL) and outside marker range (OML), are shown
in Fig. 24.22.
15. After identification of an artifact, correct the profile by deleting
the artifact through selecting “delete label” and giving appro-
priate reason for deleting the same. Figure 24.23 shows cor-
rection of a common artifact off ladder in the profile while
Fig. 24.24 shows the correction of another common artifact
outside marker range in the profile.
 Procedure 227

Fig. 24.17 Selection and visualization of (a) raw data for (b) allelic ladder and (c) sample

Fig. 24.18 (a) Visualization of “EPT data” during data analysis and (b) a representative EPT data

16. To check heterozygosity imbalance between two peaks, it is

important to determine the peak height. Select “Tools” fol-
lowed by “Plot Settings.” Go to “Labels” and in “Label 2”
select “Height,” and press “OK.” The peaks will be shown with
allele number as well as peak height (Fig. 24.25).
17. Optionally you can modify other parameters of sample analysis
such as “Peak detection threshold,” “marker specific stutter
ratio,” “Peak quality parameters,” and other “SQ and GQ
parameters” by opening the analysis method, modifying the
228 Analysis of Capillary Electrophoresis Results

Fig. 24.19 Visualization of size calling curve

desired parameters, saving the analysis method, and reanalyz-

ing the project (Fig. 24.26).
18. Select sizing table at the top right corner of the window after
selecting a sample. Allele numbers of each marker amplified will
appear as a separate window (Fig. 24.27).
19. Go to “File” and “Export Table” and save the allele table in the
designated place in your system (Fig. 24.28).
20. After completion of analysis and transfer of allele data and other
relevant information save the project prior to ending the Gen-
eMapper® software (Fig. 24.29).

6 Observation

Under normal circumstances:

1. Autosomal STR profile of a single-source sample shows either
two peaks for heterozygous alleles or one peak for homozygous
allele.
2. For a normal sample the amelogenin marker shows either XY
with balanced peak height for female sources, only X peak for
female samples, or an unbalanced XY peak for mixed-source
samples (not true for Y deletion samples).
3. Y chromosome STR profile of a single-source sample shows a
single peak in all the markers due to its hemizygous nature and
 Observation 229

Fig. 24.20 (a) Visualization of plots for allelic ladder and (b) a representative allelic ladder plot

more than one peaks appearing in mixed samples

(in commercially available kits DYS385 and DYF387S1 mar-
kers show two peaks; however, these two markers are dupli-
cated loci and considered as two independent loci).
4. X chromosome STR profile always shows a single peak for male
single-source samples, whereas for single-source female sam-
ples it may show two peaks for heterozygous alleles or one peak
for homozygous alleles.
 230
Analysis of Capillary Electrophoresis Results

Fig. 24.21 Display of peaks in the markers analyzed in all six-dye sets
 Observation 231

Fig. 24.22 Two common artifacts “OL” and “OMR” appearing in a profile

Fig. 24.23 (a) Correction of a common artifact “off-ladder” in the profile and (b) mentioning the reason for the
change
232 Analysis of Capillary Electrophoresis Results

Fig. 24.24 (a) Correction of a common artifact “outside marker range” in the profile and (b) mentioning the
reason for the change

7 Precautions
l Make sure to select the samples, allelic ladder, and controls
correctly prior to sample analysis.
l Check size standard for the presence of any nonspecific peaks.
l Check ladder carefully as all the alleles should be called properly;
none of the alleles should be called as OL.
l Never analyze any sample with less than 50 thresholds.
l Always check the inter-locus and intra-locus variation of peak
height.
l In low DNA samples allele calls above 300 bps should be
checked carefully to avoid any allele dropouts.
l Carefully check the presence of any additional allele beyond the
marker boundary, though rarely this phenomenon can also
occur.
l Analyze only those samples with all threshold parameters passed.
l Population-specific mutation rate of each marker should be
known to the analyzer before working on any human
population.
l Do not remove any labels with potential allele label.
l Extra caution should be adopted for mixed samples.
l Edit only the electrophoresis artifacts.
l If not masked, do not edit the stutter position designating a peak
in mixed samples.
l Document all removal of labels.
Fig. 24.25 Designation of both allele number and peak height from (a) Plot Settings (b) Plot Settings Editor in (c, d) a single electropherogram
Precautions
233
234 Analysis of Capillary Electrophoresis Results

Fig. 24.26 Modification of various parameters of analysis such as (a) peak detection threshold, (b) minus and
plus stutter distance, (c) peak quality parameters, and (d) SQ and GQ settings
 Precautions 235

Fig. 24.27 Window showing (a) sizing table of each marker along with (b) other parameters

Fig. 24.28 (a, b) Window showing how to export sizing tables of the samples analyzed
236 Analysis of Capillary Electrophoresis Results

Fig. 24.29 Window showing how to save the project after completion of all the analyses and export of data

l Always check raw data, EPT, size calling curve, peaks of allelic
ladder, size standard, and positive and negative control before
analyzing any sample data.
l Regularly export your analyzed data to another file in the system.

8 Troubleshooting

Problem Possible cause Possible solutions

Quality Ladder failed Check the raw data of ladder
failed for the presence of peaks; if
(red) raw data shows the presence
of high peaks, increase the
threshold and reanalyze
Check the raw data of ladder
for the presence of peaks; if
raw data shows the presence
of low peaks, decrease the
threshold and reanalyze
Ladder not marked correctly Assign the correct sample to be
allelic ladder and reanalyze
Peaks of ladder are marked as Check the polymer and buffers;
OL due to shift due to rerun with fresh reagents and
mobility issue in repeat analysis
electrophoresis
Sample size too much Dilute the amplicon and rerun
Dilute the sample and re-PCR

(continued)
 Troubleshooting 237

Problem Possible cause Possible solutions

Quality Peaks do not fit to the bin sets Mark allelic ladder properly and
check reanalyze
(yellow)
Sample defined as positive Correctly define positive
control is not correct control and reanalyze
Sample defined as negative Rerun the samples with correct
control shows the presence negative control
of peaks
Matrix not found Locate the missing file or
recreate it and reanalyze
Too much size standard Decrease the quantity of size
loaded standard in subsequent runs
Too much sample loaded Decrease the quantity of
samples in subsequent runs
A difference in peak height in Check the threshold and assign
heterozygosity the correct alleles
No peaks Amplification not occurred Re-PCR the sample with
detected optimum quantity of DNA
Negative control marked as Check the correct sample
sample position
High threshold maintained Decrease the threshold to as
low as 50 and reanalyze
Partial Less DNA input Use optimum quantity of DNA
profile sample for PCR
Degraded DNA sample Use alternative STR kits such as
mini-STRs or a kit
containing mini-STR loci
Presence of inhibitors Modify the DNA extraction
procedure and repeat the
steps
High threshold maintained Decrease the threshold to as
low as 50 and reanalyze
 Chapter 25

Calculation of Paternity Index in Paternity Dispute and

Identification Cases

Objective: To calculate the paternity index of an individual in cases

of paternity dispute and identification.

1 Introduction

Cases related to paternity dispute of both criminal and civil natures

can only be solved by DNA fingerprinting analysis. Genetic identi-
fication of a questioned individual to be the biological father/
mother is the goal of this experiment. Besides paternity dispute
this experiment also allows for reverse paternity testing when iden-
tification of any mutilated body or skeleton needs to be performed
with the availability of parents’ reference samples. Similar to pater-
nity dispute cases, maternity dispute cases can also be solved using
this technique where the father is known to be the biological father
of the questioned child and the mother becomes alleged.
Half of a child’s genetic material is inherited from its father and
another half from its mother. The alleles of all the analyzed genetic
markers of the disputed child are determined and compared with
the alleles of both the parents followed by the application of a series
of statistical equations to determine the probability of paternity. It
is always a practice to calculate the paternity index when all the
alleles of each genetic marker of the child match with the alleles of
the parents. Though theoretically mismatch at a single locus is
sufficient enough to exclude a person to be the biological father
of the questioned child, mutational events cannot be ignored
completely for one or few more allele mismatch. As currently we
use more numbers of autosomal STR markers for paternity dispute
cases, mutational events giving rise to mismatch at two genetic loci
which occurs rarely can be considered to be a common event. In
this regard, a number of software are available nowadays to

H. R. Dash et al., Principles and Practices of DNA Analysis: A Laboratory Manual for Forensic DNA Typing, Springer Protocols
Handbooks, https://doi.org/10.1007/978-1-0716-0274-4_25, © Springer Science+Business Media, LLC, part of Springer Nature 2020

239
240 Calculation of Paternity Index in Paternity Dispute and Identification Cases

calculate the paternity index as well as kinship likelihood. Some of

these computer tools include DNA-view (Charles Brenner, USA),
Easy DNA (Wing Kam Fung, Hong Kong), EasyPat (Michael
Krawczak, Germany), eDNA (eDNA Consortium), familias (Petter
Mostad, Norway), FamLink (David Kling, Norway), GenoProof
(Qualitype, Germany), GeneMarker HID (SoftGenetics, USA),
and KinCalc (Steven Myers, USA). However, this chapter describes
the manual calculation of paternity index in a disputed paternity
case using MS Office Excel sheet.

2 Principle

The fundamental of rules of genetic inheritance of alleles in a

generation involves the following:
(a) Any genetic marker on autosomal chromosome of a child possesses
two alleles, each of them inherited either from the father or the
mother.
(b) Child always possesses haplotype of mitochondrial DNA from the
matrilineal lineage.
(c) Only male child possesses Y chromosome haplotype from patrilin-
eal lineage.
In this regard, autosomal STRs are analyzed to calculate the
paternity index (PI) which describes the probability of paternity of
an individual. Calculation of PI is done as follows:

PI ¼ X=Y

where
X ¼ alleged father is the biological father
Y ¼ any randomly selected man of that population is the biological
father
This calculation gives rise to either inclusion or exclusion of the
alleged father to be the biological father of the child with some
statistical weight on the likelihood ratio in the form of paternity
index (PI). In such cases, with the conventional matching of one-
to-one allele on each marker, the obligate parental allele is deter-
mined and analyzed. As under normal circumstances the child
receives one allele from each parent (Fig. 25.1), the probability of
that allele present in a random man in the population is the basis of
the statistical evaluation.
The must prerequisite of paternity index calculation involves
the detailed population genetics study of the said population before
any calculation. Allele frequency data of all the markers tested
should be available for the population tested. On availability of
DNA profiles from the alleged father, known mother and disputed
 Requirements 241

Fig. 25.1 Overview of a case of disputed paternity

child Punnett square is used to estimate the possibility of alleged

parents’ genotypes to produce the child’s genotype. Further allele
frequency data of each genetic marker comes into play to exclude
the false inclusion of any random men in this case.
As current-day DNA fingerprinting technique involves the typing
of multiple genetic loci simultaneously, it increases the discrimination
power. In such cases individual paternity index (PI) is calculated for all
each locus separately and the product of all the individual PIs gives
rise to combined paternity index (CPI). CPI signifies the chance of
any random, unrelated man of the population to be the biological
father. Using this CPI value the probability of paternity of the alleged
individual can be calculated using the formula:
Probability of paternity ¼ ðCPI=CPI þ 1Þ  100

3 Requirements

3.1 Autosomal STR For a paternity trio experiment to be performed the autosomal STR
DNA Profiles DNA profile is required for the questioned child, known mother,
and alleged father. Genetic markers should be amplified using a
single multiplex kit for all three samples. Additionally, all three
samples need to be run in a single electrophoresis experiment.
The parameters set for analyzing the obtained profiles should be
the same for these three samples. For analysis in this experiment,
the profile obtained for the paternity trio experiment is as follows:

Genetic markers Known mother Male child Alleged father

D3S1358 16,17 16,17 16,17
vWA 14,17 17,19 18,19
D16S539 11,12 11,12 11,13

(continued)
242 Calculation of Paternity Index in Paternity Dispute and Identification Cases

Genetic markers Known mother Male child Alleged father

CSF1PO 11,12 10,12 10,12
TPOX 11,11 8,11 8,9
D8S1179 10,10 10,16 16,16
D21S11 28,33.2 31,33.2 30,31
D18S51 13,20 14,20 14,18
D2S441 11,13 11,13 10,11
D19S433 13,14 13,13.2 13.2,14
TH01 6,9 6,8 7,8
FGA 20,23 22,23 22,22
D22S1045 15,16 15,15 15,16
D5S818 10,11 10,11 11,13
D13S317 8,10 10,12 12,12
D7S820 8,8 8,8 8,8
SE33 19,28.2 28.2,30.2 30.2,31.2
D10S1248 14,17 14,17 14,17
D1S1656 11,11 11,12 12,17
D12S391 19,21 17,19 17,18.3
D2S1338 20,24 20,22 19,22
Amelogenin XX XY XY
DYS391 – 11 11
InDel – 2 2

3.2 Allele Allele frequency data is required for performing the calculation. It
Frequencies describes the proportion of total number of alleles represented by a
particular allele in a population. This is population specific. Due to
polymorphic nature the STR alleles may generate many numbers of
alleles in a population. For the total allele pool in a population,
homozygous individuals contribute two of the same allele whereas
heterozygous individuals contribute one of the particular alleles to
the total allele number of the population; for example, in a popula-
tion of 100 individuals, 15 individuals are homozygous for allele
M (MM), 40 individuals are heterozygous MN, and rest 45 indivi-
duals are heterozygous MO. Thus, 15 homozygous (MM)
individuals contribute two “M” allele each in the population,
40 heterozygous (MN) individuals contribute one “M” allele
each to the population whereas another 45 heterozygous (MO)
individuals also contribute one “M” allele each to the population.
 Procedure 243

Thus, in a three-allele system population, the allele frequency can

be calculated as

Frequency of allele} M} ¼ ½ð2No: of homozygous MM individualsÞ

þðNo: of heterozygous MN individualsÞ
þðNo: of homozygous MO individualsÞ=2n

where n ¼ no. of individuals in the populations.

The sum total of the frequency of all the alleles in a population
for a genetic marker will be one. Allele frequency data calculated
earlier for central Indian population is given in Appendix D which
will be used in this experiment.

3.3 MS Office Excel All the calculations will be performed in MS Office Excel sheet.
Platform Hence, it should be installed in the system prior to the commence-
ment of the PI calculation.

4 Procedure

1. Open a new Excel sheet using MS Office tool (Fig. 25.2).

2. Enter genetic markers and their corresponding genotype for
the known mother and the questioned child side by side
(Fig. 25.3).
3. Calculate “obligate allele” for each marker. Obligate allele is
either two or one in number (Fig. 25.4). It is the allele that
must be present in the alleged father if he is the correct
biological father. In the current example, for genetic marker
“D3S1358” both 16 and 17 can be the obligate alleles, as both
16 and 17 have the possibility of being transmitted from the
known mother. However, for “vWA” 19 becomes the obligate
allele as it is evidenced that 17 is inherited from the mother
to the questioned child and 19 must be present in the
alleged father to be the biological father. Significance of the

Fig. 25.2 A blank MS Office Excel sheet

244 Calculation of Paternity Index in Paternity Dispute and Identification Cases

Fig. 25.3 Genetic profile of the known mother and questioned child

“obligate allele” is that when the obligate paternal allele has

corresponding allele in the alleged father, it is a case of inclu-
sion. In contrary, when the obligate paternal allele does not
have the corresponding allele in the alleged father, it becomes a
case of exclusion.
4. Enter the profile of the alleged father besides the obligate allele
(Fig. 25.5). Check whether the obligate allele is present in the
corresponding genetic marker of the alleged father or not.
5. Enter allele frequency of each obligate allele next to the profile
of the alleged father (Fig. 25.6). As allelic frequency data is
population specific, it should be calculated prior to perfor-
mance of the experiment or the data from any published litera-
ture can be used for this purpose. In this experiment, we will
use the allele frequency data calculated on central Indian pop-
ulation which is given in Appendix D.
6. Calculate the likelihood that the alleged father could transmit
the obligate allele “X” (Fig. 25.7). The value of “X” is either
1 or 0.5. When the father is homozygous (see FGA of the
current example) the value of X becomes 1. Similarly, when
both the obligate alleles are present in the alleged father’s
genotype (see D3S1358 of the current example) the total
value of “X” becomes 1. Thus, the value of “X” becomes 1 as
 Procedure 245

Fig. 25.4 Determination of obligate paternal allele from the profile of known mother and questioned child

the likelihood of transmitting the allele(s) becomes 2/2 ¼ 1. In

contrary, when any one of the two obligate alleles is present in
the alleged father’s genotype (see D16S539 of the current
example) or the alleged father has only one copy of the obligate
allele (see CSF1PO of the current example) the value of X
becomes 0.5. This is due to the fact that the likelihood of
transmitting the obligate allele from the paternal genotype to
the disputed child becomes 1/2 ¼ 0.5.
7. Calculate paternity index (PI) using the formula X/allele fre-
quency, where X is the likelihood of transmitting the paternal
obligate allele by the alleged father. Thus, PI will be either
1/allele frequency or 0.5/allele frequency. However, in certain
instances (as in case of D3S1358 and D16S539) there are two
246 Calculation of Paternity Index in Paternity Dispute and Identification Cases

Fig. 25.5 Genetic profile of the alleged father besides obligate alleles

obligate alleles and hence two corresponding allele frequencies.

In this case, the PI is calculated as 1/(allele frequency of
16 + allele frequency of 17) (for D3S1358) or 0.5/(allele
frequency of 11 + allele frequency of 12) (for D16S539)
(Fig. 25.8).
8. Calculate combined paternity index (CPI). It can be calculated
by multiplication of the individual PIs of the individual genetic
markers. In this case the CPI value is calculated to be
5.25576  1011 (Fig. 25.9).
9. Calculate random man not excluded (RMNE) value. It signifies
the power of a paternity test by determining the proportion of
the population that are capable of contributing to the obligate
allele and hence cannot be excluded or included falsely. It can
be calculated by using the formula RMNE ¼ 1  (1  frequency)2
or (frequency)2 + 2 (frequency) (1  frequency) (Fig. 25.10).
 Procedure 247

Fig. 25.6 Window showing allele frequency data of the corresponding obligate alleles

When more than two allele frequencies are available for the
same locus (as in case of D3S1358 and D16S539 loci), the
calculation should be performed using the formula
RMNE ¼ 1  [1  (frequency 1 + frequency 2)2]. Additionally
calculate the combined random men not excluded (CRMNE)
value (Fig. 25.10). It can be calculated by simply multiplying
the individual calculated RMNE values. Due to very small
values of CRMNE, it is recommended to calculate the recipro-
cal of this number (1/CRMNE), which is referred to as “prob-
ability of exclusion” or exclusion power (EP) or power of
exclusion (PE) (Fig. 25.10). It represents the probability of
excluding a falsely accused man. In this case, the calculated
value of PE is 56,171,746,392. It signifies that the alleged
father is approximately 56,171,746,392 times as likely to be
the father of the questioned child as an unrelated Indian male.
248 Calculation of Paternity Index in Paternity Dispute and Identification Cases

Fig. 25.7 Calculation of likelihood of transmitting paternal obligate allele from the alleged father

10. Calculate the probability of paternity (PP). PP reflects the

probability of the alleged father to be the biological father of
the questioned child. It can be calculated using the formula:
PP ¼ 1/1 + (1/CPI). It has been calculated to be
0.999999999998 for the current analysis.

5 Observation

The probability of paternity in this case is calculated to be

0.999999999998 which is >99.9999%. This suggests that the
alleged father cannot be excluded as the biological father of the
questioned child, under the assumption that the combined pater-
nity index is 5.25576  1011, which results in the probability of
paternity of >99.9999% when compared to an untested, unrelated
individual of the Indian population (assuming the prior probability
of 0.5).
 Precautions 249

Fig. 25.8 Calculation of paternity index (PI) of the individual genetic markers

6 Precautions
l Perform all the calculations carefully.
l Always use population-specific allele frequency data for the
calculation.
l Carefully determine the paternal obligate allele.
l Determine the likelihood value of transmitting the paternal
obligate allele to be either 0.5 or 1.
l While calculating CPI or CRMNE values do not sum the indi-
vidual PI or RMNE values; the correct formula is to multiply the
individual values.
l The calculated CRMNE value is generally very small; be careful
while calculating to obtain a smaller CRMNE value after
calculation.
l For a case of inclusion the calculated probability of paternity
value should be >0.99999.
250 Calculation of Paternity Index in Paternity Dispute and Identification Cases

Fig. 25.9 Spreadsheet showing the calculation of combined paternity index (CPI) value

Fig. 25.10 Calculation of RMNE, CRMNE, and PE values

 Troubleshooting 251

7 Troubleshooting

Problem Possible cause Possible solutions

Calculated probability Case of exclusion Check the presence of all
of paternity is not paternal obligate alleles in
>99.9999 alleged father’s profile
Check all the calculations
carefully before declaring
the case of exclusion
Calculation is Check all the calculations and
not correct formula used carefully
Proper allele Use population-specific allele
frequency data frequency data
is not selected
Excel sheet showing the Decimal places Go to Format Cells:
probability of are selected by Numbers: Select decimal
paternity value to be 1 default places and increase the
decimal places
Paternity trio not Known mother You cannot use the same
complete profile not calculations for this case
available
Mother is also Draw the Punnett square to
alleged see whether the questioned
child inherits alleles from
both the parents or not
First keep the mother’s
genotypes to be known and
calculate paternity index
using the same calculation
Next keep the father’s
genotypes to be known and
calculate maternity index
using the same calculation
 Part VII

Case Studies
 Chapter 26

Solving Paternity Dispute by DNA Fingerprinting: A Case

Study

Objective: To solve a paternity dispute case by DNA fingerprinting.

1 Introduction

Paternity dispute cases are of either criminal or civil in nature.

Sexual assault in pretext of marriage leading to pregnancy can be
solved by identification of fetus or newborn as the biological off-
spring of the victim and the suspect. Similarly, paternity dispute in
civil nature is also common where the biological father denies the
claim of the child. In both the scenario DNA fingerprinting is the
only established technique to draw a conclusive decision to ascer-
tain that the claims and the biological identity of an individual are
established.
In such cases the reference biological samples of the questioned
child, known mother, and alleged father are collected. The refer-
ence sample may include the invasive sample blood in EDTA vial or
on FTA® card or the noninvasive sample such as buccal swab. Each
laboratory has its guidelines for collection, preservation, and trans-
portation of reference samples to the laboratory for examination.
However, in this case maintenance of chain of custody is highly
important. Liquid blood sample should be collected aseptically
from the donor in EDTA vial and stored in ice till arrival in the
laboratory within a stipulated period of time. Similarly, liquid blood
can be spread on the designated area of the FTA® card, dried
completely, and stored and transported at room temperature to
the laboratory. Noninvasive samples such as buccal swab can also
be collected and the cotton swab should be dried completely before
its storage and transportation at room temperature. Additionally,
buccal swab can also be prepared on a FTA® card and processed
similarly to the blood sample on FTA® card.

H. R. Dash et al., Principles and Practices of DNA Analysis: A Laboratory Manual for Forensic DNA Typing, Springer Protocols
Handbooks, https://doi.org/10.1007/978-1-0716-0274-4_26, © Springer Science+Business Media, LLC, part of Springer Nature 2020

255
256 Solving Paternity Dispute by DNA Fingerprinting: A Case Study

2 Principle

Autosomal STR analysis is conducted for solving paternity disputes

cases of both civil and criminal nature. The nature of transfer of
parental genotypes to the offspring is dependent on the allele com-
bination present in the parents. The segregation of alleles in a genetic
locus always follows the laws of Mendelian inheritance. For parent-
age examination the Mendelian rules should be tested as follows:
(a) The allele absent in both the parents cannot be present in the
child.
(b) The pair of alleles is inherited from the parents.
(c) When both the parents have the same allele, there is the
possibility that the child may possess two copies of the same
allele.
(d) The genetic marker present as an identical pair in both the
parents is present in the child.
Thus, in the absence of mutational events, establishment of
parentage depends on the fact that on each genetic locus tested
the alleles of the child match with the corresponding alleles of the
parents at the same genetic marker (Fig. 26.1).

Fig. 26.1 Inheritance of alleles from both the parents to the offspring as per (a) Mendelian pattern of
inheritance and (b) determination of maternal and obligate paternal allele
 Results and Observation 257

For solving paternity dispute cases, the ISFG Paternity Testing

Commission (PTC) has recommended several considerations such
as calculation of likelihood ratio (LR) of the DNA evidences and
use of population-specific genetics for calculation of allele fre-
quency as well as for cases with sample deficiency, extra statistical
evaluations should be carried out before deducing any conclusion.

3 Brief Case History

In the pretext of marriage, the suspect performed sexual activity

with the complainant. When the complainant became pregnant, the
suspect denied of marrying her. A criminal case of sexual assault was
filed in an Indian police station by the complainant. When the child
was born from the complainant, the case was referred for DNA
examination. Subsequently, the DNA fingerprinting laboratory
received liquid blood samples of the complainant, newborn, and
the suspect in EDTA vials through proper channel which were
subjected to examination.

4 Samples

Three liquid blood samples in EDTA vial consisting of Article A

(blood sample of complainant), Article B (blood sample of child),
and Article C (blood sample of the suspect).

5 Procedure

1. DNA was extracted from the liquid blood samples as per the
protocol described in Chap. 4 of the book.
2. Extracted DNA was quantified using the protocol described in
Chap. 18.
3. Amplification of autosomal STRs was performed using the
protocol described in Chap. 19.
4. Capillary electrophoresis/genotyping and subsequent data
analysis were performed using the protocols described in
Chaps. 23 and 24, respectively.

6 Results and Observation

1. Quantification of extracted DNA samples yielded the results

as given in Table 26.1. The samples were further diluted using
TE buffer to obtain the optimum DNA concentration for
multiplex PCR.
258 Solving Paternity Dispute by DNA Fingerprinting: A Case Study

Table 26.1
Estimation of DNA concentration of the extracted blood samples

Article A Article B Article C

DNA content 200 ng/μl 150 ng/μl 350 ng/μl
Dilutions 1:200 1:150 1:350
Final concentration 1 ng/μl 1 ng/μl 1 ng/μl

2. The Punnett square table of the obtained autosomal STR DNA

profile was as given in Table 26.2. Amelogenin marker shows
“XY” suggesting that the questioned child is a male child.
Further, the presence of maternal allele (highlighted) was
found in the DNA profile of the questioned child. Similarly,
the obligate paternal allele (underlined) was also found to be
present in the DNA profile of the suspect. It suggests that the
alleged father is the biological father of the questioned child.
Statistical analysis can be performed to deduce the paternity
index in this case.

7 Precautions
l Label all the tubes containing samples carefully.
l Handle the samples carefully, so that they do not get mixed up in
the subsequent steps of experiment.
l Match the alleles one by one among the obtained profiles.
l Maintain the chain of custody for the samples.
l Maintain your internal laboratory record for case opening, DNA
extraction, quantification, genotyping, and result interpretation.
l Refer to other precautions described in the individual experi-
ments for DNA extraction (Chap. 4), quantification (Chap. 18),
multiplex PCR (Chap. 19), genotyping (Chap. 23), and result
interpretation (Chap. 24).

8 Troubleshooting

Refer to corresponding troubleshooting of the respective experi-

ments used in this case study, i.e., DNA extraction (Chap. 4),
quantification (Chap. 18), multiplex PCR (Chap. 19), genotyping
(Chap. 23), and result interpretation (Chap. 24).
 Troubleshooting 259

Table 26.2
Punnett square table of the samples analyzed

Genetic Article A Article B Article C

markers (complainant) (newborn) (suspect)
D3S1358 15,17 15,15 15,18
D1S1656 11,11 11,14 13,14
D2S441 10,10 10,11 11,14
D10S1248 13,16 13,14 14,14
D13S317 9,12 9,12 8,12
Penta E 7,15 12,15 12,19
D16S539 8,11 8,11 11,11
D18S51 14,14 13,14 13,14
D2S1338 22,23 21,23 17,21
CSF1PO 9,10 10,12 12,12
Penta D 11,12 11,13 13,14
TH01 6,9 6,9 7,9
vWA 16,16 16,16 16,17
D21S11 29,30 30,33.2 32.2,33.2
D7S820 10,12 10,11 11,11
D5S818 11,12 11,12 11,11
TPOX 8,10 8,11 11,11
D8S1179 10,13 13,15 14,15
D12S391 17,20 19,20 17,19
D19S433 13,13.2 13,14.2 14.2,16.2
SE33 30.2,33.2 16,33.2 16,16
D22S1045 15,16 11,16 11,11
FGA 21,24 21,22 21,22
Amelogenin X,X X,Y X,Y
DYS391 – 10 10
DYS576 – 19 19
DYS570 – 19 19
 Chapter 27

Solving a Case of Murder by DNA Fingerprinting: A Case

Study

Objective: To solve a case of murder by DNA fingerprinting.

1 Introduction

Murder cases can be solved by DNA fingerprinting examination of

the biological evidences collected during investigation. Based on the
Locard’s exchange principle, i.e., “with contact between two items,
there will be an exchange of materials,” evidences can be collected
and referred for DNA examination. Evidences collected from the
deceased, suspect, crime scene, murder weapon, as well as the witness
present (if any) can be used for this purpose. Due to the requirement
of low amount of DNA samples in the currently practiced DNA
fingerprinting technology, substances as tiny as cigarette butt or
strands of hairs are sufficient enough to establish a crime.
Certain evidences such as eye witnesses can lose credibility over
time; however, the evidences generated by DNA examination have
the capability to reveal truth even after 20 years of the commitment
of offense. Some of the previously clueless evidences such as blood
left on a broken window, saliva found on the cigarette butt or bear
bottle, and skin cells on the wheels of a theft car are being solved
nowadays with its full potential using DNA technology. Additional
considerations need to be taken care of to minimize the degradation
of evidences as well as to avoid contamination-related issues. Thus,
the on-field analyst should be trained properly regarding the DNA
evidences as well as their collection and preservation techniques.
With the advancement of DNA technology and its requirement of a
very less input of DNA quantity, many secondary evidences present
in the crime scene can also be targeted such as baseball bat, hat or
mask, eyeglasses, dirty laundry, toothpick, cigarette butt, envelope
seal, bottle/glass, bite marks, as well as fingernail scrapings.

H. R. Dash et al., Principles and Practices of DNA Analysis: A Laboratory Manual for Forensic DNA Typing, Springer Protocols
Handbooks, https://doi.org/10.1007/978-1-0716-0274-4_27, © Springer Science+Business Media, LLC, part of Springer Nature 2020

261
262 Solving a Case of Murder by DNA Fingerprinting: A Case Study

2 Principle

For solving a case of murder using DNA technology, the following

considerations need to be followed:
(a) Deposition of suspect’s DNA on the victim’s body or clothing
(b) Deposition of suspect’s DNA on any object
(c) Deposition of suspect’s DNA on a location
(d) Deposition of victim’s DNA on the suspect’s body/clothing
(e) Deposition of victim’s DNA on the object
(f) Deposition of victim’s DNA on a location
(g) Deposition of witness’s DNA (if any) on the victim/suspect
(h) Deposition of witness’s DNA (if any) on an object or location
Accordingly biological evidences may be collected, preserved,
and examined to link between the crime scene, victim/deceased,
murder weapon, and witness (if any). In this regard, swab from the
blood-stained floor or blood-stained soil can be collected from the
crime scene along with the suitable controls. To link the deceased,
clothing of the deceased or its body parts and bone samples for
skeletonized body should be preserved. A murder weapon can be
any hard, sharp, or blunt object capable of causing harm to the
deceased. It may be recovered from the crime scene or from the
memorandum of the suspect or from the body of the deceased. The
common murder weapons may involve the stones/bricks, knife/
sickle, or bullet. During autopsy bullets may be recovered from the
body of the deceased followed by its DNA examination and control
matching with the gun recovered from the suspect can link the
suspect to the crime. Similarly, linking the suspect to the crime is of
utmost importance in a case of murder. In this regard, DNA
technology becomes highly useful by examining the clothing of
the suspect with suspected bloodstains; rooted hairs found on the
deceased body or in the crime scene; presence of cigarette butts,
bear bottles, or glasses at the crime scene; or any other article found
in the crime scene useful to link the suspect.

3 Brief Case History

A dermatologist’s wife was stabbed to death by the suspect accusing

of improper treatment by the doctor. Later when the assailant
denied the crime, the investigation team took help of the DNA
examination to corroborate the crime. In this case, the laboratory
received clothing of the deceased, bloodstain swab from the floor,
clothing of the suspect, and the knife for examination. The prelim-
inary examination of the body fluid identification shows the pres-
ence of bloodstains in all the articles. Further they were subjected
to DNA fingerprinting examination.
 Results and Observation 263

4 Samples

Clothing of the deceased (Article A), bloodstain swab from the

floor (Article B), control swab (Article C), clothing from the sus-
pect (Article D), knife seized from the suspect (Article E), and
blood sample of suspect (Article F) were received in this case. All
the samples were maintained in their recommended storage condi-
tion. Appropriate seal and label were checked properly prior to their
examination.

5 Procedure

1. DNA was extracted from the deceased’s clothing (Article A),

swab of the floor (Article B), control swab (Article C), suspect’s
cloth (Article D), and knife (Article E) as per the protocol
described in Chap. 5 of the book.
2. DNA was extracted from the blood sample of suspect (Article
F) as per the protocol described in Chap. 4 of the book.
3. Extracted DNA was quantified using the protocol described in
Chap. 18.
4. Amplification of autosomal STRs was performed using the
protocol described in Chap. 19.
5. Capillary electrophoresis/genotyping and subsequent data
analysis were performed using the protocols described in
Chaps. 23 and 24, respectively.

6 Results and Observation

1. Quantification of extracted DNA samples yielded the results

as given in Table 27.1. The samples were further diluted using
TE buffer to obtain the optimum DNA concentration for
multiplex PCR.
2. The results of the autosomal STR DNA profiling of the exhibits
are given in Table 27.2. Amelogenin marker of Article A shows

Table 27.1
Estimation of DNA concentration of the extracted blood samples

Article A Article B Article C Article D Article E Article F

DNA content 100 ng/μl 75 ng/μl 0 ng/μl 25 ng/μl 12.5 ng/μl 175 ng/μl
Dilutions 1:100 1:75 – 1:25 1:12.5 1:175
Final concentration 1 ng/μl 1 ng/μl – 1 ng/μl 1 ng/μl 1 ng/μl
264 Solving a Case of Murder by DNA Fingerprinting: A Case Study

Table 27.2
Autosomal STR DNA profile obtained from various examined articles

Article A Article B Article D Article E Article F

Genetic Clothing Swab Article C Clothing Knife Blood
markers (deceased) (floor) Swab (control) (suspect) (suspect) (suspect)
D3S1358 15,18 15,18 DNA profile could 14,15,16,18 15,18 14,16
not be detected
D1S1656 13,16 13,16 13,16 13,16 16,16
D2S441 11,11 11,11 10,11 11,11 10,10
D10S1248 14,14 14,14 14,14 14,14 14,14
D13S317 8,8 8,8 8,8 8,8 8,8
Penta E 10,14 10,14 10,12,14,16 10,14 12,16
D16S539 9,12 9,12 8,9,12 9,12 8,12
D18S51 16,19 16,19 13,14,16,19 16,19 13,14
D2S1338 18,20 18,20 18,20,23,25 18,20 23,25
CSF1PO 12,12 12,12 10,11,12 12,12 10,11
Penta D 10,13 10,13 10,11,13 10,13 10,11
TH01 6,9 6,9 6,8,9 6,9 8,9
vWA 14,15 14,15 14,15 14,15 14,14
D21S11 30,34.2 30,34.2 28,30,31,34.2 30,34.2 28,31
D7S820 10,10 10,10 9,10,11 10,10 9,11
D5S818 11,11 11,11 11,12 11,11 11,12
TPOX 11,11 11,11 8,9,11 11,11 8,9
D8S1179 10,15 10,15 10,15 10,15 15,15
D12S391 18,20 18,20 18,20,24 18,20 20,24
D19S433 15.2,15.2 15.2,15.2 13,15.2 15.2,15.2 13,13
SE33 21,21.2 21,21.2 17,21,21.2,31.2 21,21.2 17,31.2
D22S1045 11,16 11,16 11,16 11,16 11,11
FGA 22,26 22,26 20,22,25.2,26 22,26 20,25.2
Amelogenin X,X X,X X,Y X,X X,Y
DYS391 – – 10 – 10
DYS576 – – 18 – 18
DYS570 – – 19 – 19
 Troubleshooting 265

“XX” suggesting the deceased to be a female individual. No

DNA profile was detected from the Article C suggesting no
experimental error. Further, consistent autosomal STR DNA
profile was detected from the source of Article A, Article B, and
Article E. Article D yielded a mixed profile. All the alleles found
from the victim’s source are present in the mixed DNA profile
obtained from the source of suspect’s clothing, i.e., Article D
(alleles highlighted and underlined). This suggests the transfer
of body fluid of the deceased to the clothing of suspect during
assault. Similarly, the alleles present in the DNA profile of the
suspect’s blood are also found to be present in the mixed DNA
profile which mostly arises due to the regular wearing of the
clothing. Further, statistical analysis can be performed for mix-
ture interpretation as well as calculation of likelihood ratio
between two DNA profiles.

7 Precautions
l Label all the tubes containing samples carefully.
l Handle the samples carefully, so that they do not get mixed up in
the subsequent steps of experiment.
l Match the alleles one by one among the obtained profiles.
l Maintain the chain of custody for the samples.
l Maintain your internal laboratory record for case opening, DNA
extraction, quantification, genotyping, and result interpretation.
l Refer to other precautions described in the individual experi-
ments for DNA extraction (Chap. 4), quantification (Chap. 18),
multiplex PCR (Chap. 19), genotyping (Chap. 23), and result
interpretation (Chap. 24).

8 Troubleshooting

Refer to corresponding troubleshooting of the respective experi-

ments used in this case study, i.e., DNA extraction (Chaps. 4 and
5), quantification (Chap. 18), multiplex PCR (Chap. 19), genotyp-
ing (Chap. 23), and result interpretation (Chap. 24).
 Chapter 28

Solving a Case of Sexual Assault by DNA Fingerprinting:

A Case Study

Objective: To solve a case of sexual assault by DNA fingerprinting.

1 Introduction

Sexual assault cases are more prevalent than any other types of
crimes happening throughout the globe. Though sexual assaults
are of many types, penetration of a body cavity is the most common
form of assault. Intimacy of this assault leads to the transfer/
exchange of body fluids without the presence of any common eye-
witnesses. In many instances, the victim may be unable to provide
the details of the perpetrator for varied reasons. In such instances,
only DNA fingerprinting analysis becomes the masterpiece among
scientific evidences to reach at the culprit.
In sexual assault cases, the timing of filing the complaint,
medicolegal examination, and collection of evidences play impor-
tant roles in establishing the crime by DNA examination. The
medical examination of both the victim and assault should be
carried out as per the local ethical guidance after obtaining the
respective consents for the same. The medicolegal certificate should
contain certain information regarding the details of the allegation
as well as the medical and sexual history of the persons examined.
With the increase in difference in time between the assault and
collection of samples, both mechanical (due to drainage and
hygiene) and biological elimination (degradation) of the evidences
from the body takes place in addition to the physiologic dilution.
Many samples from the victim may be collected based on the
history of the assault such as anal, oral, or vaginal assault. Addition-
ally, reference samples of both the suspect and the victim are
required for DNA fingerprinting analysis.

H. R. Dash et al., Principles and Practices of DNA Analysis: A Laboratory Manual for Forensic DNA Typing, Springer Protocols
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267
268 Solving a Case of Sexual Assault by DNA Fingerprinting: A Case Study

In earlier scenario identification microscopy, comparative

microscopy, serological analyses, and biochemical analyses were
performed for the biological samples. However, with the advent
of DNA fingerprinting technology, it has replaced the conventional
techniques due to its individualization nature. DNA fingerprinting
examination of a sexual assault case reveals three types of results,
i.e., either inclusion, exclusion, or no result. In all instances, the
result becomes conclusive to determine the accused as well as to
exonerate the falsely implicated suspect.

2 Principle

DNA of both the accused and victim is supposed to be present in

the questioned samples collected in sexual assault cases. Due to the
transfer of body fluids, the sexual assault cases can be solved in
either of the two ways, i.e., detection of suspect’s DNA profile on
victim’s belongings or detection of victim’s DNA profile on
belongings of the accused. The latter scenario occurs mostly in
sexual assault cases of prepuberty victims. Sexual assault in most
of the prepuberty victims results in profuse bleeding from the
private parts resulting in the transfer of blood in the accused private
parts in contact. Thus, DNA analysis from the clothing, pubic hair,
penile, and/or scrotal swab may generate the victim’s DNA profile
confirming the sexual assault. However, in sexual assault cases of
victims of postpuberty age, the common practice is to detect the
DNA profile of the accused on the victim’s belongings. In such
cases, the common questioned articles include vagina/cervical
swab, pubic hair, and clothing of the victim.
In all instances, a mixed autosomal STR DNA profile was
expected in the questioned samples. As the current analysis involves
the PCR process, preferential amplification of the DNA occurs. The
articles from the victim’s belongings are expected to contain more
amount of victim’s DNA in comparison to the DNA of the accused.
Hence, after PCR process, a large number of DNA fragments of the
victim’s source mask the amplified DNA fragments of the accused.
Subsequently only DNA from the victim’s source is detected after
genotyping. To overcome this problem, Y chromosome STR anal-
ysis is recommended which gives amplification of male DNA only
present in the mixed sample. Most of the commercially available kits
can successfully generate a male DNA profile when male-to-female
DNA ratio is present as low as 1:1000. Additionally, due to statisti-
cal limitations and chance matching, sexual assault cases involving
more than one accused cannot be interpreted by autosomal STR
analysis. In such a scenario only Y chromosome STR analysis proves
useful.
 Procedure 269

3 Brief Case History

A working lady in her 20s alleged that her manager sexually har-
assed her when working for a firm for over 1 year. She also alleged
that her complaint to the management of the firm was continuously
ignored. As per her allegation, she was denied promotion due to
her complaint. After repeated complaint and continuous sexual
assault, she resigned from her job and complained in the nearby
police station. As per the victim, she was assaulted 1 day before her
complaint at police station. As soon as the complaint was lodged,
the victim was undergone medical test and the medical surgeon
collected vaginal swab, undergarments, and pubic hair from her.
The reference blood samples from both the victim and the suspect
were also collected. Body fluid examination showed the presence of
spermatozoa in the vaginal swab and undergarments of the victim,
whereas the spermatozoa could not be detected from the pubic hair
of the victim. Further all the articles were subjected to DNA
fingerprinting examination.

4 Samples

Vaginal swab (Article A), control swab (Article B), pubic hair
(Article C), undergarment (Article D), and blood sample (Article
E) were collected from the victim. Similarly, reference blood sample
(Article F) was collected from the suspect. All the samples were
maintained in their recommended storage condition. Appropriate
seal and label were checked properly prior to their examination.

5 Procedure

1. DNA was extracted from the victim’s vaginal swab (Article A),
control swab (Article B), pubic hair (Article C), and undergar-
ment (Article D) as per the protocol described in Chap. 10 of
the book.
2. DNA was extracted from the blood sample of victim (Article E)
and suspect (Article F) as per the protocol described in Chap. 4
of the book.
3. Extracted DNA was quantified using the protocol described in
Chap. 18.
4. Amplification of Y chromosome STRs was performed using the
protocol described in Chap. 20.
5. Amplification of autosomal STRs was performed using the
protocol described in Chap. 19.
270 Solving a Case of Sexual Assault by DNA Fingerprinting: A Case Study

6. Capillary electrophoresis/genotyping and subsequent data

analysis were performed using the protocols described in
Chaps. 23 and 24, respectively.

6 Results and Observation

1. Quantification of extracted DNA samples yielded the results as

given in Table 28.1. The samples were further diluted using TE
buffer to obtain the optimum DNA concentration for
multiplex PCR.
2. The results of the Y chromosome STR DNA profile are given in
Table 28.2. All the samples yielded a Y profile except Article B
and Article E confirming no experimental error. No male DNA
profile was detected from the source of Article C. Further,
same/consistent Y chromosome STR DNA profile was
detected from the source of Article A, Article D, and Article F.
3. The results of autosomal chromosome STR DNA profile are
given in Table 28.3. No autosomal STR DNA profile could be
detected from the source of Article B confirming no experi-
mental error. Same/consistent female autosomal STR DNA
profile was obtained from the source of Articles C and E
suggesting their same origin. Additionally, mixed autosomal
STR DNA profile was obtained from the source of Article A
and Article D. All the alleles generated from the suspect’s blood
sample (Article F) were found to be included in the mixed
DNA profile obtained from Article A and Article D. Similarly,
all the alleles generated from the victim’s blood sample (Article
E) were also found to be included in the mixed DNA profile
obtained from Article A and Article D. Further statistical anal-
ysis can be performed for mixture interpretation.

7 Precautions
l Label all the tubes containing samples carefully.
l Handle the samples carefully, so that they do not get mixed up in
the subsequent steps of experiment.

Table 28.1
Estimation of DNA concentration of the extracted blood samples

Article A Article B Article C Article D Article E Article F

DNA content 350 ng/μl – 15 ng/μl 200 ng/μl 450 ng/μl 500 ng/μl
Dilutions 1:350 – 1:15 1:200 1:450 1:500
Final concentration 1 ng/μl – 1 ng/μl 1 ng/μl 1 ng/μl 1 ng/μl
 Precautions 271

Table 28.2
Y chromosome STR DNA profile obtained from various examined articles

Article A
Vaginal Article C Article D Article F
Genetic swab Article B Pubic hair Undergarment Article E Blood
markers (victim) (Control swab) (victim) (victim) Blood (victim) (suspect)
DYS576 19 Y-STR DNA Y-STR DNA 19 Y-STR DNA 19
profile could profile could profile could
DYS389I 14 14 14
not be not be not be
DYS635 21 detected detected 21 detected 21
DYS389II 32 32 32
DYS627 21 21 21
DYS460 10 10 10
DYS458 16 16 16
DYS19 15 15 15
YGATAH4 11 11 11
DYS448 18 18 18
DYS391 10 10 10
DYS456 16 16 16
DYS390 25 25 25
DYS438 10 10 10
DYS392 13 13 13
DYS518 43 43 43
DYS570 15 15 15
DYS437 14 14 14
DYS385 15,20 15,20 15,20
DYS449 33 33 33
DYS393 14 14 14
DYS439 11 11 11
DYS481 23 23 23
DYF387S1 36,37 36,37 36,37
DYS533 10 10 10

l Match the alleles one by one among the obtained profiles.

l Maintain the chain of custody for the samples.
l Maintain your internal laboratory record for case opening, DNA
extraction, quantification, genotyping, and result interpretation.
272 Solving a Case of Sexual Assault by DNA Fingerprinting: A Case Study

Table 28.3
Autosomal STR DNA profile of the examined articles

Article C
Article A Article B Pubic Article D Article E Article F
Genetic Vaginal swab (Control hair Undergarment Blood Blood
markers (victim) swab) (victim) (victim) (victim) (suspect)
D3S1358 15,16,17 DNA profile 15,16 15,16,17 15,16 16,17
could not be
D1S1656 8,12,13,15 12,13 8,12,13,15 12,13 8,15
detected
D2S441 10,11,13 10,11 10,11,13 10,11 10,13
D10S1248 13,14,15,16 13,15 13,14,15,16 13,15 14,16
D13S317 8,10,13 10,13 8,10,13 10,13 8,10
Penta E 7,12,13,19 7,13 7,12,13,19 7,13 12,19
D16S539 10,11,13 10,11 10,11,13 10,11 11,13
D18S51 12,13,17 13,17 12,13,17 13,17 12,13
D2S1338 18,19,23,24 19,23 18,19,23,24 19,23 18,24
CSF1PO 11,12 11,11 11,12 11,11 12,12
Penta D 9,10,11,12 11,12 9,10,11,12 11,12 9,10
TH01 6,8,9 6,8 6,8,9 6,8 8,9
vWA 14,18 14,18 14,18 14,18 14,14
D21S11 28,29,30,32.2 28,29 28,29,30,32.2 28,29 30,32.2
D7S820 8,9,10,11 8,9 8,9,10,11 8,9 10,11
D5S818 11,13 11,11 11,13 11,11 11,13
TPOX 8,11 8,11 8,11 8,11 8,11
D8S1179 13,15,16 15,16 13,15,16 15,16 13,15
D12S391 18,20 18,20 18,20 18,20 18,18
D19S433 13,14 14,14 13,14 14,14 13,13
SE33 20,21.1,25.2,31.2 21.1,25.2 20,21.1,25.2,31.2 21.1,25.2 20,31.2
D22S1045 11,15 15,15 11,15 15,15 11,15
FGA 21,23,24 24,24 21,23,24 24,24 21,23
Amelogenin X,Y X,X X,Y X,X X,Y
DYS391 10 – 10 – 10
DYS576 19 – 19 – 19
DYS570 18 – 18 – 18
 Troubleshooting 273

l Refer to other precautions described in the individual experi-

ments for DNA extraction (Chap. 4), quantification (Chap. 18),
multiplex PCR (Chap. 19), genotyping (Chap. 23), and result
interpretation (Chap. 24).

8 Troubleshooting

Refer to corresponding troubleshooting of the respective experi-

ments used in this case study, i.e., DNA extraction (Chaps. 4 and
10), quantification (Chap. 18), multiplex PCR (Chap. 19), geno-
typing (Chap. 23), and result interpretation (Chap. 24).
 Chapter 29

Identification of an Unknown Skeleton by DNA

Fingerprinting: A Case Study

Objective: To identify an unknown skeleton by DNA

fingerprinting.

1 Introduction

Identification of human remains has been transformed manyfold

since last two decades due to the advent of this fingerprinting
technology. Before the discovery of with technology, other techni-
ques such as dactyloscopy, anthropology, odontology, and medico-
legal examinations were used for identification purposes. These
techniques provide limited information in comparison to the
molecular level of DNA examination which determines the exact
origin of the unknown human remains. The human remains may be
of soft or degraded tissues, bones, teeth, or hairs from which the
genetic information can be extracted. As the fundamental of genet-
ics states that two close relatives share the genetic variants, the DNA
profile of the unknown human remains can be matched with the
DNA profile of the tentative relatives. Based on the result, conclu-
sion on the identification of the unknown deceased can be drawn.
The identification of a missing person can also be carried out
using the medical or personal objects. Medical samples including
the biopsies, umbilical cords, teeth, or other parts of the body can
provide the ideal samples for the identification of a missing person.
Similarly, personal artifacts also provide simple and powerful
matching of DNA. Personal belongings such as a hairbrush, tooth-
brush, and razor can be analyzed to match with the DNA profile of
the missing person. Additionally, database-led identification pro-
grams can be conducted by matching the unknown profile with the
database of population DNA profiles.

H. R. Dash et al., Principles and Practices of DNA Analysis: A Laboratory Manual for Forensic DNA Typing, Springer Protocols
Handbooks, https://doi.org/10.1007/978-1-0716-0274-4_29, © Springer Science+Business Media, LLC, part of Springer Nature 2020

275
276 Identification of an Unknown Skeleton by DNA Fingerprinting: A Case Study

2 Principle

Analysis of DNA in the case of identification of human remains is

comprised of five different steps such as
(a) Extraction of DNA from the human remains
(b) Extraction of DNA from the reference samples
(c) Generating the DNA profile from both the samples, i.e., human
remains and reference sample
(d) Comparison of the DNA profiles of the questioned and the refer-
ence samples
(e) Conclusion on the basis of degree of matching between the ques-
tioned and reference samples
Identification may be required in cases such as burning leading
to death, army conflicts, recovery of skeletonized body, and recov-
ery of body parts. In all the cases, the DNA starts to degrade post-
death of the individual depending on the environment. For exam-
ple, environments with warm and humid conditions increase the
degradation rate, whereas the cold and dry environments help in
preserving the DNA samples. However, in most of the cases, it
becomes a huge challenge for the examiner to generate a DNA
profile from the degraded samples. Thus, when human remains are
recovered in a short time after death, soft-tissue samples such as
muscle tissues can be preserved for DNA examination. When doubt
rises regarding degradation of the soft tissues, hard tissues such as
bones and teeth may be collected for identification purpose. As the
cells present in the hard tissues are embedded in a dense biomineral
matrix, they are least affected by decomposition as well as putrefac-
tion. Though all bones preserve DNA, long bones such as femur
are better sources of DNA so as the teeth.
Correct identification of a skeletal remain depends on the
availability of reference samples. In this regard, presence of a rela-
tionship triangle, i.e., father-mother-son or father-mother-daugh-
ter, is the most suitable sample for conclusive identification of
an individual by autosomal STR typing (Fig. 29.1). In reference

Fig. 29.1 Significance of a triangle of relationships for proper identification of the deceased
 Procedure 277

sample-limited conditions, patrilineal lineage of a missing person

can be established by analyzing the Y chromosome profile. Simi-
larly, mtDNA analysis can also determine the matrilineal lineage
of an individual. Further, matching among the siblings can
also be carried out; however, it may not identify an individual
conclusively.

3 Brief Case History

A group of dacoits kidnapped an individual from his home in a

remote village of India. After 3 days some local people observed
some burnt skeletal remains inside the nearby jungle. They
informed police and the autopsy surgeon confirmed the burnt
skeletal remains to be of human origin. Investigating team sus-
pected this to be the action of the group of dacoits. Due to the
availability of only burnt skeletal remains, phenotypic identification
of the deceased could not be performed. Hence, the half-burnt
skeletal samples along with the reference blood samples of its
parents were received for DNA examination.

4 Samples

Half-burnt bone samples (Article A) were collected after medical

report confirming their human origin. Reference blood samples
from the putative father (Article B) and the putative mother (Arti-
cle C) were collected. All the samples were maintained in their
recommended storage condition. Appropriate seal and label were
checked properly prior to their examination.

5 Procedure

1. DNA was extracted from the blood samples (Article B and

Article C) as per the protocol described in Chap. 4 of the book.
2. DNA was extracted from the bone samples (Article A) as per
the protocol described in Chap. 9 of the book.
3. Extracted DNA was quantified using the protocol described in
Chap. 18.
4. Amplification of autosomal STRs was performed using the
protocol described in Chap. 19.
5. Capillary electrophoresis/genotyping and subsequent data
analysis were performed using the protocols described in
Chaps. 23 and 24, respectively.
278 Identification of an Unknown Skeleton by DNA Fingerprinting: A Case Study

Table 29.1
Estimation of DNA concentration of the extracted blood samples

Article A Article B Article C

DNA content 3 ng/μl 350 ng/μl 200 ng/μl
Dilutions 1:3 1:350 1:200
Final concentration 1 ng/μl 1 ng/μl 1 ng/μl

6 Results and Observation

1. Quantification of extracted DNA samples yielded the results as

given in Table 29.1. The samples were further diluted using TE
buffer to obtain the optimum DNA concentration for
multiplex PCR.
2. The results of autosomal chromosome STR DNA profile are
given in Table 29.2.
3. Amelogenin marker shows “XY” suggesting the deceased to be
a male individual. Further, the maternal allele (highlighted) was
found to be present in the DNA profile of the skeletal remains.
Similarly, the obligate paternal allele (underlined) was also
found to be present in the DNA profile of the putative father.
It suggests that the skeletal remains belong to the biological
offspring of the putative father and the mother. Further, statis-
tical analysis can be performed to deduce the paternity index in
this case.

7 Precautions
l Label all the tubes containing samples carefully.
l Handle the samples carefully, so that they do not get mixed up in
the subsequent steps of experiment.
l Match the alleles one by one among the obtained profiles.
l Maintain the chain of custody for the samples.
l Maintain your internal laboratory record for case opening, DNA
extraction, quantification, genotyping, and result interpretation.
l Refer to other precautions described in the individual experi-
ments for DNA extraction (Chap. 4), quantification (Chap. 18),
multiplex PCR (Chap. 19), genotyping (Chap. 23), and result
interpretation (Chap. 24).
 Precautions 279

Table 29.2
Autosomal STR DNA profile of the examined articles

Article C Article A Article B

Genetic Blood sample (putative Skeletal remains Blood sample (putative
markers mother) (deceased) father)
D3S1358 15,16 14,16 14,15
D1S1656 15,16.3 15,15 15,16
D2S441 10,14 10,14 10,10
D10S1248 16,16 16,16 16,16
D13S317 11,11 11,11 11,12
Penta E 15,21 11,21 11,11
D16S539 12,13 10,12 9,10
D18S51 10,14 10,15 11,15
D2S1338 18,25 18,21 21,25
CSF1PO 11,12 12,12 11,12
Penta D 9,13 9,9 9,11
TH01 6,10 6,9.3 9.3,9.3
vWA 18,20 17,18 14,17
D21S11 29,30 30,31.2 30,31.2
D7S820 8,8 8,8 8,12
D5S818 11,12 11,12 11,12
TPOX 11,11 8,11 8,11
D8S1179 14,16 14,15 11,15
D12S391 20,21 18,21 18,18
D19S433 12.2,16.2 12.2,13 13,15
SE33 19.3,32.2 30.2,32.2 19,30.2
D22S1045 15,15 15,15 15,15
FGA 21,24 21,24 20,24
Amelogenin X,X X,Y X,Y
DYS391 – 10 10
DYS576 – 15 15
DYS570 – 16 16
280 Identification of an Unknown Skeleton by DNA Fingerprinting: A Case Study

8 Troubleshooting

Refer to corresponding troubleshooting of the respective experi-

ments used in this case study, i.e., DNA extraction (Chaps. 4 and
5), quantification (Chap. 18), multiplex PCR (Chap. 19), genotyp-
ing (Chap. 23), and result interpretation (Chap. 24).
 Part VIII

Advanced Technologies for Forensic DNA Analysis

 Chapter 30

Sequencing Control Region of Human Mitochondrial

Genome to Assess Matrilineal Lineage

Objective: To analyze control region of mitochondria by using

Sanger sequencing technique.

1 Introduction

Mitochondrion is the exclusive cell organelle of human cells which

contains extracellular DNA. Uniqueness of mitochondrial DNA
(mtDNA) involves its double-stranded circular and histone-free
structure. The compact mtDNA codes for 13 polypeptides involved
in oxidative phosphorylation as well as 2 rRNAs and 22 tRNAs.
Besides, it contains a control region of around 1100 bp length
which is noncoding in nature. The copy number of mtDNA varies
from 2 to 10 per mitochondrion whereas a cell contains as many as
1000 mitochondria depending on the physiological condition of
the cell (Budowle et al. 2003). However, size of mtDNA is quite
smaller than the genomic DNA as the approximate size of genomic
DNA is around 3 billion bp, whereas the size of circular mitochon-
drial DNA is 16,569 bp (Lutz et al. 2000).
The unique inheritance pattern of mtDNA advocates its wide-
spread application in anthropology, evolution, and forensic ana-
lyses. Unlike to the nuclear DNA, mtDNA is inherited exclusively
from the mother with little or no contribution from the father. Due
to uniparental inheritance, mtDNA does not undergo recombina-
tion; hence it represents matrilineal lineage in generations. From
forensic point of view, mtDNA analysis is of much importance as
they are found in multiple copies per cell contributing to higher
amount of DNA in comparison to nuclear DNA, mostly in com-
promised forensic samples. Robustness and resistance to extreme
environmental conditions make mtDNA useful for ancient DNA

H. R. Dash et al., Principles and Practices of DNA Analysis: A Laboratory Manual for Forensic DNA Typing, Springer Protocols
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283
284 Sequencing Control Region of Human Mitochondrial Genome to Assess Matrilineal Lineage

analysis. Additionally, mtDNA analysis is useful in samples such as

hair shaft, where chance of getting nuclear DNA result is negligible.
mtDNA analysis has a wide variety of applications in history.
Victims of world trade center were identified using mtDNA
sequencing technology. Analysis of remains of a Neanderthal man
(Krings et al. 1997), World War victims (Pajnič et al. 2010), and a
7000-year-old brain tissue (Paabo et al. 1988) through mtDNA
sequencing has reflected the utility of this technique. However,
mtDNA sequencing technology earned a huge appreciation
throughout the globe in case of verification of Tsar Nicholas II’s
bones. mtDNA sequence was successfully compared with the living
maternal relatives and putative bone remains of Tsar Nicholas II
were identified (Gill et al. 1994). However, the analysis procedure
of mtDNA varies from the routine nuclear DNA analysis which has
been described in details in this chapter.

2 Principle

The major goal of forensic DNA analysis is to differentiate among

individuals. In this context, the control regions of mtDNA which
are highly polymorphic in nature become the obvious choice for
analysis. In general, two hypervariable regions are present in
mtDNA called as HVRI and HVRII which show genomic variation
at the highest level. As these regions do not contain any coding
gene sequences, their mutation rate becomes ten times higher than
that of the corresponding coding regions. The length of HVRI
region (342 bp) is more than the HVRII region (268 bp)
(Fig. 30.1). In some instances, a small (137 bp) third hypervariable
region (HVRIII) is also present in human mtDNA which is useful
in resolving samples where HVRI/HVRII analysis does not pro-
vide sufficient differentiation.

Fig. 30.1 An overview of human mitochondrial DNA with specific reference to

control region
 Reagents/Materials Required and Their Role 285

For analysis, the mtDNA is extracted from the biological sam-

ples and the target region; most preferably the hypervariable
regions are amplified using the primer sets followed by sequencing
using BigDye Terminator Cycle Sequencing kit (Thermo Fisher
Scientific). During this process the conventional chain termination
technique developed by Frederick Sanger is used. This technique
involves amplification of the target region by routine PCR method
using dNTPs as well as labeled ddNTPs. When ddNTPs are
incorporated into the strand during amplification process, termina-
tion of template amplification occurs and different DNA fragments
are generated. Thus, finally, DNA fragments of different sizes each
ending with a different nucleotide labeled with different dye are
generated. Finally, capillary electrophoresis is performed and the
software analyzes the sequence of a DNA strand based on the size of
the fragment and the emitted fluorescence.
The resultant DNA sequences are analyzed using specific anal-
ysis software and the sequence similarities and dissimilarities are
calculated. After obtaining mtDNA sequence, it is first compared
with the Cambridge or Anderson reference sequence or the revised
Cambridge sequence (rCRS). Based on the sequence similarities or
dissimilarities, the result can be interpreted as either inclusion,
exclusion, or inconclusive. As per Scientific Working Group on
DNA Analysis (SWGDAM)’s recommendation, difference of two
or more nucleotide sequences between the questioned and refer-
ence samples can be referred as exclusion. However, when both the
sequences are exactly same, they cannot be excluded of having the
same origin. An inconclusive result is obtained when there exists a
single-nucleotide difference between the questioned and reference
sample. Few mtDNA population databases randomly selecting dif-
ferent individuals are present which include FBI mtDNA database,
EMPOP (http://www.empop.org), and mtDNA manager (http://
mtmanager.yonsei.ac.kr/).

3 Reagents/Materials Required and Their Role

3.1 Template DNA Specific DNA extraction protocol should be followed for extraction
of mtDNA from degraded forensic samples. Routine DNA quanti-
fication steps may not solve the purpose as they estimate the total
DNA including microbial DNA and in some instances total human
DNA including nuclear and mitochondrial DNA.

3.2 Primers For amplification of whole control region of mitochondrial DNA,

use the following primer sets, i.e., L15977: 50 CAC CAT TAG CAC
CCA AAG CT 30 (Ward et al. 1991) and H599: 50 TTG AGG AGG
TAA GCT ACA TA 30 (Brandst€atter et al. 2004). If a high quality of
mtDNA is extracted from the forensic samples, the entire control
region is amplified using this primer set which is approximately
286 Sequencing Control Region of Human Mitochondrial Genome to Assess Matrilineal Lineage

1.1 kb in size. If a poor quality of mtDNA is extracted an alternative

strategy known as Midi-Mito strategy is applied amplifying the
control region in multiple overlapping fragments. During sequenc-
ing seven sequencing reactions are performed using primers
L15977, H16175, H16401, L16450, H274, L314, and H599.

3.3 MyTaq™ HS Red MyTaqTM HS Red Mix (BIOLINE) is the ready-to-use mixture
Mix 2 for setting up of hot-start PCR which contains the Taq DNA
polymerase and dNTPS required for the PCR process. The mixture
is powered by the antibody-mediated hot-start technique which
does not allow the nonspecific amplification during the reaction
setup. It is recommended to be stored at 20  C and repeated
freeze-thaw should be avoided to ensure optimum activity of the
reagents.

3.4 illustra™ It is the enzymatic technique used for PCR and sequencing reaction
ExoProStar™ cleanup. It is a double-enzyme reaction mechanism to remove
leftover primers and dNTPs used during a PCR process. The reac-
tion simultaneously uses alkaline phosphatase and exonuclease
I. Exonuclease I digests single-stranded DNA to release deoxyribo-
nucleoside 50 monophosphates (dNMPs) whereas alkaline phos-
phatase catalyzes the dephosphorylation of unincorporated
nucleotides and dNMPs to release nucleosides and Pi.

3.5 ABI Prism® The kit provides useful chemicals and reagents required for
BigDye™ Terminator sequencing a DNA fragment. The kit is useful for sequencing
v3.1 Cycle Sequencing single- or double-stranded DNA templates. The components of
Kit the kit include ready reaction mix, dilution buffer, control DNA,
and control primers. The ready reaction mix, control DNA, and
primers should be stored at 15 to 25  C whereas the sequence
buffer can be stored at 4  C.

3.6 Molecular Molecular biology-grade water is the form of ultrapure high-quality

Biology-Grade Water water which is free from DNase, RNase, and protease contamina-
tion. Additionally, no toxic agents such as DEPC should be present
in this water. Milli-Q water can be produced in the laboratory or it
can be procured from any molecular biology-grade chemical
manufacturer.

3.7 Genetic Analyzer Genetic Analyzer 3500xL instrument is one of the advanced ver-
3500xL sions of sequencer which can accommodate 24 samples in a single
run. The instrument relies on the principle of capillary electropho-
resis where the conventional gel slab has been replaced with thin
capillaries and electrophoresis takes place. The only difference is
that the result of capillary electrophoresis is being generated in the
form of peaks in comparison to the bands of the slab gel electro-
phoresis result. The dedicated data collection software analyzes the
peaks and designates nucleotides to the peaks to generate a com-
plete DNA sequence.
 Procedure 287

Table 30.1
Recommended amounts of reagents required to prepare amplification mixture for PCR

PCR components Volume per reaction (μl) No. of reactions Total volume
MyTaq HS Red Mix 2 10.0 A 10A
L15977 primer (5 μM) 0.8 A 0.8A
H599 primer (5 μM) 0.8 A 0.8A
Molecular biology-grade water 6.4 A 6.4A
Total volume (10 + 0.8 + 0.8 + 6.4)A

4 Procedure

1. Calculate the requirement of each component of the PCR

process using the following table as per your requirement/
number of samples (Table 30.1).
2. Thaw all the PCR components in ice, vortex for 3–5 s, and
centrifuge briefly before using.
3. Pipette out the calculated amount of water, MyTaq HS Red
Mix, and primer sets in a 1.5 ml microcentrifuge tube marked
as Amplification Mixture (AM). It is always recommended to
add water first to the tube, followed by MyTaq HS Red Mix
and primer sets.
4. As soon as the preparation of AM is over, immediately store the
tubes containing PCR components in the recommended stor-
age condition.
5. Vortex the amplification mixture for 5–10 s, and then dispense
(10 + 0.8 + 0.8 + 6.4) μl of amplification mixture to each 200 μl
PCR tube pre-marked for each sample.
6. Add 2.0 μl of the extracted mtDNA to each designated PCR
tube containing the amplification mixture.
7. In one tube add control DNA (2800 M DNA) in the amplifi-
cation mix and in another tube add molecular biology-grade
water or TE buffer in place of template DNA which will be
considered as positive and negative control, respectively.
8. Close the tubes properly and briefly centrifuge the tubes so that
all its contents will be present at the bottom of the tube.
9. Place the tubes in the thermal cycler. Set the thermal cycling
protocol as 95  C for 3 min followed by 36 cycles of 94  C for
15 s, 56  C for 1 min, and 72  C for 2 min, and finally hold at
4  C for 1.
10. The amplified products can be visualized at 1.5% agarose gel
and under UV light.
288 Sequencing Control Region of Human Mitochondrial Genome to Assess Matrilineal Lineage

11. When a proper amplification is evident from the agarose gel

electrophoresis process, perform PCR cleanup using illustra™
ExoProStar™ PCR cleanup kit. Add 1.5 μl of ExoProStar™
1 to the remaining 10–12 μl of PCR product and incubate the
tube at 37  C for 45 min followed by 80  C for 15 min.
12. Perform separate sequencing reaction using ABI Prism® Big-
Dye™ terminator cycle sequencing kit for each primer using
the PCR product as template.
13. Calculate the amount of each reagent using Table 30.2 as per
your requirement.
14. Vortex the amplification mixture for 5–10 s, and then dispense
(0.68 + 1.02 + 0.408 + 3.6) μl of amplification mixture to the
wells of a 96-well plate.
15. Add 1 μl of purified PCR product to the corresponding wells of
the 96-well plate.
16. Seal the plate with a PCR seal.
17. Perform the sequencing reaction in a PCR machine using the
protocol as 96  C for 4 min followed by 25 cycles of 96  C for
15 s, 50  C for 10 s, and 60  C for 2 min.
18. Add 1.7 μl of 125 mM EDTA and 20 μl of 100% ethanol to
each well, vortex briefly, and incubate at room temperature for
15 min.
19. Centrifuge the plate at 2250  g for 30 min to precipitate the
sequencing products.
20. Discard the supernatants by inverting the plate and centrifuge
at inverted position at 4000 rpm for 10 min.
21. Wash the plate twice using 70% ethanol by repeating steps
18 and 19.
22. Incubate the plate at room temperature till the leftover ethanol
is evaporated.

Table 30.2
Recommended amounts of reagents required to prepare amplification mixture for sequencing PCR

Volume per No. of

PCR components reaction (μl) reactions Total volume
Ready reaction 0.68 A 0.68A
Dilution buffer 1.02 A 1.02A
Sequencing primer (5 μM) (L15977/ 0.408 A 0.408A
H16175/H16401/L16450/ H274/
L314/H599)
Molecular biology-grade water 3.6 A 3.6A
Total volume (0.68 + 1.02 + 0.408 + 3.6)A
 Revised Cambridge Reference Sequence for HVRI 289

23. Add 10 μl of deionized formamide to each well followed by

heating at 95  C for 3 min and snap cool in ice for 5 min.
24. Run the plate on the capillary electrophoresis instrument
Genetic Analyzer 3500xL (Thermo Scientific).
25. Align the obtained sequences using the software SeqScape
(Life Technologies) or Sequencer (Gene Codes).
26. Analyze the control region sequence by comparing with the
revised Cambridge Reference Sequence (rCRS). Additionally,
few points need to be considered while analyzing the obtained
mtDNA sequences:
(a) Try to have a complete sequence coverage for the control
region.
(b) Observe the common phenomena of difference in sequence
between the examined and reference mtDNA at position
263 and insertion of “C” following 315 position.
(c) Consider length heteroplasmy while analyzing mtDNA
sequence which generally arises due to insertion or deletion
of a base.
27. Use EMPOP (www.empop.org) to calculate the frequency of
generated mtDNA sequence.

5 Observation and Result

Concordance: When two mtDNA sequences, i.e., one from the

questioned sample and another from the reference sample, are
consistent with each other, two samples cannot be excluded to
be originated from the same matrilineal lineage.
Exclusion: When two mtDNA sequences, i.e., one from the ques-
tioned sample and another from the reference sample, differ by
two or more nucleotides, the samples are excluded to be origi-
nated from the same matrilineal lineage.
Inconclusive: When two mtDNA sequences, i.e., one from the
questioned sample and another from the reference sample,
differ by only one nucleotide, the resulting comparison can be
concluded to be inconclusive.

6 Revised Cambridge Reference Sequence for HVRI

16024: TTCTTTCATG GGGAAGCAGA TTTGGGTACC

ACCCAAGTAT
16064: TGACTCACCC ATCAACAACC GCTATGTATT
TCGTACATTA
16104: CTGCCAGCCA CCATGAATAT TGTACGGTAC
CATAAATACT
290 Sequencing Control Region of Human Mitochondrial Genome to Assess Matrilineal Lineage

16144: TGACCACCTG TAGTACATAA AAACCCAATC

CACATCAAAA
16184: CCCCCTCCCC ATGCTTACAA GCAAGTACAG
CAATCAACCC
16224: TCAACTATCA CACATCAACT GCAACTCCAA
AGCCACCCCT
16264: CACCCACTAG GATACCAACA AACCTACCCA
CCCTTAACAG
16304: TACATAGTAC ATAAAGCCAT TTACCGTACA
TAGCACATTA
16344: CAGTCAAATC CCTTCTCGTC CC-16365

7 Revised Cambridge Reference Sequence for HVRII

73: ATGCACGCGA TAGCATTGCG AGACGCTGGA

GCCGGAGCAC
113: CCTATGTCGC AGTATCTGTC TTTGATTCCT
GCCTCATCCT
153: ATTATTTATC GCACCTACGT TCAATATTAC
AGGCGAACAT
193: ACTTACTAAA GTGTGTTAAT TAATTAATGC
TTGTAGGACA
233: TAATAATAAC AATTGAATGT CTGCACAGCC
ACTTTCCACA
273: CAGACATCAT AACAAAAAAT TTCCACCAAA
CCCCCCCTCC
313: CCCGCTTCTG GCCACAGCAC TTAAACAC-340(end)

8 Precautions
l Wear gloves and goggles during isolation of
mitochondrial DNA.
l Always use disposable plasticware which are pre-sterilized and
DNase free.
l Use filter tips to avoid cross-sample contamination.
l Do not store the reagents under a light source.
l Always include both positive and negative controls throughout
the process.
l As some of the chemicals are hazardous, they should be handled
carefully.
l Always prepare amplification mixture for 1–2 extra reactions to
avoid any chance of possible pipetting error.
 Troubleshooting 291

l Always store the reagents in appropriate storage conditions.

l Never use expired reagents.
l In between runs do not close the data collection software and
open it. It may hamper the life span of the laser present in the
instrument.
l Never mix the working area for nuclear DNA and
mitochondrial DNA.

9 Troubleshooting

Problem Tentative cause Possible solutions

Mixed sequences RNA contamination Run the samples on
(no mixed sequence agarose gel; check
in positive control the quality of sample;
and no sequences in if RNA is present at
negative control) higher amount use
RNase treatment or
re-isolate the samples
Check A260/A280 of
the extracted DNA;
if the observed value
is greater than 2.0 go
for RNase treatment
or re-isolate the
samples
DNA contamination Run on agarose gel to
check the presence of
two different bands;
take extra precaution
and re-isolate the
sample
Primer not optimized Use recommended
primer
concentration; check
the specificity of the
primer before
proceeding for
sequencing
Carryover Replace septa with a
contamination from fresh one and rerun
septa the samples
Leftover primers present Remove leftover PCR
in the product reagents using post-
PCR purification kit

(continued)
292 Sequencing Control Region of Human Mitochondrial Genome to Assess Matrilineal Lineage

Problem Tentative cause Possible solutions

Little or “no” sequence Template quantity not Always recommended
signals adequate amount of DNA
template
Primer quantity not Use recommended
adequate concentration of
primer during
sequencing PCR
Broken or blocked Check and replace the
capillaries defunct capillaries
PCR process not Check expiry of the
complete PCR reagents and
re-PCR the samples
Issue with data analysis Threshold settings for Correct the settings
quality and mixed base and reanalyze the
settings are not result
defined properly
Heterozygous Recheck the sequence
insertion/deletion on the reverse strand
 Chapter 31

Application of Next-Generation Sequencing (NGS) in

Forensic DNA Analysis and DNA Phenotyping

Objective: To analyze forensic samples by using next-generation

sequencing (NGS).

1 Introduction

Use of DNA fingerprinting to exploit the variation of fragment

length of STR markers has become an integral part of the conven-
tional forensic DNA analysis. Mostly capillary electrophoresis tech-
nique is applied in such cases to determine the fragment length
variation of STR markers present on X, Y, and autosomal chromo-
somes. In certain instances, certain regions on mitochondrial DNA
(mtDNA) are also analyzed by conventional Sanger sequencing
technology mostly to analyze the matrilineal inheritance. However,
none of the currently used technique allows any additional lead to
the investigation purpose without a reference sample or a hit in the
database. In this context, after the completion of the Human
Genome Project in 2003, the next-generation sequencing tech-
nique has become widely useful in conventional as well as forensic
applications with the advanced features of massively parallel
sequencing (MPS), high throughput, fastness, depth coverage,
low cost per nucleotide, and reduction in error. With the increase
in load of casework samples and requirement of establishment of
criminal intelligence database throughout the globe, the NGS
technique is widely useful to meet the current demand of forensic
DNA typing.
In the conventional capillary electrophoresis technique, the
difference in sizes of the individual markers is being assessed,
whereas in targeted MPS both length and sequence variation can
be measured for higher number of markers in a single run. Use of

H. R. Dash et al., Principles and Practices of DNA Analysis: A Laboratory Manual for Forensic DNA Typing, Springer Protocols
Handbooks, https://doi.org/10.1007/978-1-0716-0274-4_31, © Springer Science+Business Media, LLC, part of Springer Nature 2020

293
294 Application of Next-Generation Sequencing (NGS)

different dye sets limits the number of STR markers analyzed in a

single multiplex reaction; however, each molecule is read indepen-
dently enabling more number of markers to be analyzed simulta-
neously in a single MPS reaction. Thus, in contrary to the current
capacity of CE technique to analyze 15–27 STR markers, a single
MPS reaction can analyze ~400 markers of autosomal, Y, and X
STRs as well as various single-nucleotide polymorphism (SNP)
markers. Additionally, mtDNA can also be analyzed in this tech-
nique. Another advantage of the NGS technique over the conven-
tional CE technique is the high sensitivity of the process.
Many NGS platforms are currently available from Roche, Ion
torrent (Thermo Fisher Scientific), Illumina, Solid, PacBio, Heli-
cose, Oxford Nanopore, Qiagen, and BGI (Sobiah et al. 2018).
However, for forensic application two most common platforms
being used include the MiSeq® FGx™ Forensic Genomics System
(Illumina) and the HID-Ion Personal Genome Machine (PGM)™
(Thermo Fisher Scientific). Though the workflow varies a little
between these two systems, both of them are capable of providing
an enhanced solution for the smallest, most compromised, as well
as mixed samples. The MiSeq® FGx™ Forensic Genomics System
(Illumina) has been described in details in this chapter.

2 Principle

Amplicon-based sequencing is the basic principle behind an NGS

workflow to be used for forensic application. The first step involves
the library preparation where the library is prepared using a
two-step amplification process. In the first amplification process
the target sequence is amplified using sequence-specific primer
pairs and the second amplification step involves addition of indexes
and adaptors to give a unique identity to each amplicon. After
preparation of library, they are pooled in one tube and linearized.
Further, the library is injected into the flow cell where oligos
complementary to the adaptors are present and the fragments are
amplified by bridge amplification to generate clonal clusters.
Finally, the sequencing by synthesis (SBS) technique is employed
for sequencing which involves the detection of single bases during
their incorporation into the DNA strands. Finally, the obtained
sequence data are analyzed using the ForenSeq™ Universal Analy-
sis Software to generate the result.
In the primary step, library is prepared using ForenSeq™ DNA
Signature Prep Kit for simultaneous amplification of 231 markers
(27 autosomal, 24 Y, 7 X, 95 identity SNPs, 22 phenotypic SNPs,
and 56 ancestry SNPs) useful for forensic casework and database
purpose. Besides autosomal, Y-, and X-STR markers, the kit
 Reagents/Materials Required and Their Role 295

contains identity informative single-nucleotide polymorphism

(SNP) markers that are useful for source designation in case of
degraded samples or samples with potential PCR inhibitors. Addi-
tionally, some phenotype SNPs informative for tentative eye and
hair color of the individual as well as ancestry SNPs informative for
biogeographical origin of the individual are also included in the kit.
The analysis software automatically analyzes the obtained result
leading to generation of report. Additionally, mitochondrial DNA
can also be analyzed either using D-loop amplification protocol or
with a whole mitochondrial genome protocol using Nextera® XT
DNA Library Prep Kit for library preparation.

3 Reagents/Materials Required and Their Role

3.1 ForenSeq™ DNA This kit contains all the chemicals and reagents required for target-
Signature Prep Kit specific library preparation from the DNA samples simultaneously
for global autosomal STRs, Y-STRs, X-STRs, and different varieties
of SNPs. The kit includes DNA primer mix A which harbors
primers for 58 STRs and 94 identity SNPs. However, DNA primer
mix B contains primer sets for all the markers present in primer mix
A and additional 56 ancestry SNPs and 22 phenotypic SNPs.
Besides, the kit contains control DNA, reaction mix, enzyme mix,
storage buffer, i5 and i7 indexed adaptors, other reagents, and
sample purification beads.

3.2 ForenSeq™ During cluster amplification to retain the integrity of each ampli-
Index Plate Fixture Kit con, indexes are added to each of them. In this process two index
(i7 and i5) adapters are used to tag the DNA with a unique
combination of indices. Tagging the samples is useful in later step
of data analysis to distinguish among the amplified products. Index
adapters and PCR2 reaction mixture are the major components of
this kit which should be stored at
25 to
15  C.

3.3 MiSeq® FGx™ It is the validated next-generation sequencing system to be used

Forensic Genomics exclusively for forensic genomics applications. Due to its high
System accuracy and increased resolution, the system can analyze the
degraded, low DNA samples commonly observed in forensic
setup. The advantage of using this system is the simultaneous
analysis of >200 genetic markers with low level of DNA input,
thus increasing the efficiency of the system in comparison to the
currently used CE system. The system is capable of increasing the
speed and accuracy of generating sequencing results to serve as a
boon to the criminal justice system.

3.4 ForenSeq™ It is a complete DNA to data software solution which analyzes the
Universal Analysis results originated from MiSeq FGx™ Forensic Genomics System
Software when the samples are prepared using ForenSeq DNA Signature
296 Application of Next-Generation Sequencing (NGS)

Prep Kit. The software interface provides solutions for population

statistics, sample comparison, as well as predicting phenotype of an
individual through its ancestry, hair, and eye color estimation.

3.5 Template DNA As sequencing is carried out during NGS, the template DNA
should be prepared with utmost care to minimize the level of
contamination. The optimum concentration of template DNA to
be used for target-based NGS reaction is 0.2–1.0 ng/μl. Always use
recommended amount of template DNA for a better result. Due to
high sensitivity and specificity of the NGS reaction, prior estimation
of DNA concentration by qRT-PCR is always useful. Additionally,
most of the NGS library preparation kits can also use stains on FTA
card directly.

4 Procedure

1. Prepare template DNA containing the recommended concen-

tration of DNA required for a NGS reaction.
2. Use ForenSeq™ DNA Signature Prep Kit for preparing the
DNA libraries using the manufacturer’s guidelines, briefly as
follows:
(a) Prepare a master mixture by mixing 4.7 μl of PCR 1, 0.3 μl
of FEM, and 5.0 μl of DPMA or DPMB per reaction.
(b) Transfer 10 μl of the master mixture to each well of a
96-well plate and add 5 μl of DNA samples to each well.
Additionally, add positive control and Milli-Q water as
negative control to the designated wells.
(c) Centrifuge the plate at 1000  g for 30 s and run on the
PCR machine at 98  C for 3 min; followed by 8 cycles of
96  C for 45 s, 80  C for 30 s, 54  C for 2 min, and 68  C
for 2 min; followed by 10 cycles of 96  C for 30 s, 68  C
for 3 min, and finally 68  C for 10 and 10  C for 1.
(d) After completion of the PCR program, you can either
store the plate at 2–8  C for 2 days after proper sealing
or proceed immediately for the next step.
3. Use ForenSeq™ Index Plate Fixture Kit for adding of adapters
to the amplicons for cluster amplification using the manufac-
turer’s protocol which is as follows:
(a) Centrifuge the 96-well plate containing the PCR products
from the previous step at 1000  g for 30 s.
(b) Arrange i7 and i5 adaptors at columns (1–12) and rows
(A–H) besides the plate, respectively.
(c) Add 4 μl of i7 and i5 adaptors to each column and row,
respectively, followed by addition of 27 μl of PCR2 master
mixture to each well.
 Procedure 297

(d) Centrifuge the plate at 1000  g for 30 s and run on the

PCR machine programmed at 98  C for 30 s followed by
15 cycles of 98  C for 20 s, 66  C for 30 s, and 68  C for
90 s followed by 68  C for 10 min and 10  C for 1.
(e) After PCR program is over, you may proceed with the
next step or store the plate at 2–8  C for 7 days after
proper sealing.
4. Purify the libraries from other unused/leftover primers,
dNTPs, and adaptors using sample purification beads (SPB)
following the manufacturer’s protocol which is as follows:
(a) Add 45 μl of sample purification beads (SPB) to each well
of the 96-well plate and centrifuge the plate at 1000  g
for 30 s.
(b) Add 45 μl of tagged libraries to the corresponding wells
containing SPB in the 96-well plate.
(c) Mix the contents of the plate at 1800 rpm for 2 min after
proper sealing of the plate, followed by incubation at
room temperature for 5 min.
(d) Place the plate on the magnetic stand and wait for ~2 min
or till the liquid becomes transparent; decant the whole
supernatant from each well.
(e) Wash the magnetic beads present on each well using
200 μl of 80% ethanol followed by incubating on magnetic
stand for 30 s and removal of the supernatant.
(f) Centrifuge the plate at 1000  g for 30 s and place it on
the magnetic stand.
(g) Remove the excess ethanol from each well using a
micropipette.
(h) After removing the plate from the magnetic stand add
52.5 μl of resuspension buffer (RSB) and mix the plate
contents at 1800 rpm for 2 min after proper sealing.
(i) Incubate the plate first at room temperature followed by
on magnetic stand for 2 min each.
(j) Transfer 50 μl of the purified libraries to a fresh 96-well
plate and centrifuge the plate at 1000  g for 30 s for
settling down of the contents.
(k) Immediately proceed for the next step or occasionally
store the plate at
25  C till 1 year.
5. Optionally, normalize the libraries to achieve consistent cluster
density.
6. Pool libraries by adding 5 μl of each library in a 1.5 ml micro-
centrifuge tube, vortex, and centrifuge briefly.
7. Denature and dilute the libraries as follows:
298 Application of Next-Generation Sequencing (NGS)

(a) Add 36 μl of nuclease-free water, 2 μl of human sequenc-

ing control (HSC), and 2 μl of HP3 (2 N NaOH) in a
microcentrifuge tube (MCT); vortex; and centrifuge
briefly.
(b) Incubate the mixture at room temperature for 5 min.
(c) Mix 591 μl of hybridization buffer (HT1), 7 μl of the
pooled normalized libraries, and 2 μl of HSC mixture in
a 1.5 ml MCT.
(d) Vortex and centrifuge the tube briefly.
(e) Place the tube at 96  C on a dry bath for 2 min.
(f) Mix the contents by inverting the tube multiple times and
immediately snap cool the tube at
25  C for 5 min.
8. Load the entire content on the reagent cartridge/flow cell.
9. Load flow cell by opening the “Load Flow Cell” log on the
MiSeq FGx Control Software of MiSeq FGx™ instrument.
10. Load SBS solution at the designated place and make sure that
the waste bottle is completely empty.
11. Load the reagent cartridge at designated place of the instru-
ment and select “Start Run.”
12. During run, monitor the progress of the run, intensities, and
quality scores.
13. After completion of the run, click on the “Next” button to
proceed for a post-run wash of the instrument.
14. Analyze the run results using ForenSeq™ Universal Analysis
Software as follows:
(a) Log in to the software, create a new run, import samples
for analysis, and enter sample information.
(b) Review the positive control samples (P), negative control
samples (N), and quality matrices for the run (Q).
(c) Select “Create New Analysis” button and “Generate New
Analysis.”
(d) Select STR sample details table to see the allele present on
the selected marker.
(e) Select SNP sample details to see the allele present on each
marker.
(f) Select analysis and population statistics to generate ran-
dom match probability, Hardy-Weinberg expectations,
and linkage equilibrium.
(g) From analysis you may select “Start Comparison” to gen-
erate the Table of Discordance.
(h) Select “Actions” and “Phenotype estimation” from the
drop-down menu.
 Precautions 299

5 Observation and Result

1. Before proceeding for result analysis, always check the positive

control, negative control, and quality matrix (Fig. 31.1). In this
case of amplification of positive control the HSC region can be
assessed by “P,” whereas reagent blank can be assessed by
negative control “N” and “Q” representing the overall quality
matrices for the current NGS run.
2. Outcome of the controls and quality standards can be assessed
by the color indicators. Green color indicates that the sample
has been sequenced with sufficient coverage and the calculated
genotype is concordant. However, when green color appears,
sufficient coverage may not be present for the sample and hence
the genotypes are not concordant.
3. By selecting a sample for STR sample details table the following
icon will appear (Fig. 31.2).
4. By clicking on a box containing the alleles, the signal intensity
and nucleotide sequence can be visualized (Fig. 31.3).
5. Similarly, the SNP sample details can be visualized as follows
(Fig. 31.4).

6 Precautions
l Wear gloves and goggles during the whole process of NGS.
l Always use disposable plasticware which are pre-sterilized and
DNase free.
l Use filter tips to avoid cross-sample contamination.

Fig. 31.1 Results of controls and quality standards of an NGS run

300 Application of Next-Generation Sequencing (NGS)

Fig. 31.2 Table showing STR details of a sample

l Do not store the reagents under a light source.

l Always store the reagents in appropriate storage conditions.
l Never use expired reagents.
l Assess the quality and quantity of template DNA prior to setting
up of an NGS reaction.
l Always prepare the master mixture for extra 10% of the samples.
l Always change tips in between samples.
l Set up PCR-1 in a pre-PCR environment.
l Avoid repeated freeze-thaw of the reagents.
l Check and remove bubbles (if any) in the PCR tubes.
l Do not forget to bring the sample purification beads to room
temperature before use.
l Always change tips when adding adapters and primers for each
column and row.
 Precautions 301

Fig. 31.3 Icon showing the signal intensity and nucleotide sequence

l Handle the reagents with utmost care as you are dealing with the
hazardous known chemicals.
l Use dedicated separate waste disposal containers for solid and
liquid wastes.
l Ensure that reagents in the cartridge are thawed thoroughly to
enable proper sequencing.
l Never pierce any reagents on the reagent cartridge as they will be
pierced automatically during the run.
l Never touch the MiSeq FGx instrument after commencement of
a run as it is highly sensitive to vibration.
l Do not open the flow cell compartment during a run.
l Always perform a post-sequencing instrument wash.
l Regularly perform the maintenance wash of the sequencer ide-
ally once in every month.
302 Application of Next-Generation Sequencing (NGS)

Fig. 31.4 Table showing SNP details of a sample

7 Troubleshooting

Problem Tentative cause Possible solutions

Low sample Less quality or Reprocess the sample after
intensity quantity of samples re-isolating DNA
Try with few samples for
sequencing, at least eight samples
in one run

(continued)
 Troubleshooting 303

Problem Tentative cause Possible solutions

High cluster Too much library Rerun the sample with sufficiently
density added in the diluted libraries
mixture
More amount of Normalize the libraries and
magnetic beads re-sequence the samples
added
Too much adapter Check the amount of dimers in a
dimer in library Bioanalyzer; if the amount
preparation exceeds 5%, repeat library
purification process
Low cluster Low/degraded DNA Repeat library preparation step with
density input higher input DNA
DNA did not denature Ensure that denaturation step is
properly performed properly
Blockage of Perform a maintenance wash
instrument
Run HSC not added Add HSC and repeat the
incomplete sequencing step
Reagents not Contact technical support
performing up to personnel of your instrument
the mark
Error during Analysis software is Reload the software and look for the
analysis not running possible solution in the “help”
icon corresponding to the error
command
Any other error Look for the possible solution in the
command “help” icon corresponding to the
error command
 Chapter 32

Forensic Trace and Touch DNA Analysis

1 Introduction

The earlier version of DNA fingerprinting analysis, i.e., restriction

fragment length polymorphism (RFLP), was dependent on a huge
quantity of input DNA for its successful operation. However, with
the advent of polymerase chain reaction (PCR)-based short tandem
repeat (STR) typing, a trace amount of DNA as low as 1 ng is
required to generate a DNA profile successfully. In this regard,
traces of DNA transferred to any object at the crime scene by the
personnel present can be analyzed and the suspects can be linked. A
healthy human sheds around tens of thousands of epithelial cells
every day (Williamson 2012), which is sufficient enough to yield a
DNA accounting for a successful generation of DNA profile. Addi-
tionally, many genotyping kits sensitive for DNA typing are in use
nowadays favoring the chances of trace DNA analysis (Einot et al.
2017). In general, touch DNA refers to the transfer of trace
amount of epithelial cells by the user/perpetrator on various
objects present at the crime scene, i.e., murder weapon, vehicles,
or questioned documents.
The amount of cells shed by an individual varies from person to
person which is approximately 400,000 cells per day (Wickenheiser
2002). Various physiological factors also affect the shedding of cells
as the nervous suspects perspire more to leave more number of cells
in the crime scene (Alketbi 2018). Touching of own face or head
while committing a crime increases the chance of adding more
number of cells of the suspect to the crime scene. Similarly, touch-
ing a rough surface increases the chance of transfer of more number
of cells than a smooth surface. Additionally, contact pressure also
affects the amount of cells shed by an individual on the object
which increases the probability of yielding the suspect’s DNA
profile.

H. R. Dash et al., Principles and Practices of DNA Analysis: A Laboratory Manual for Forensic DNA Typing, Springer Protocols
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306 Forensic Trace and Touch DNA Analysis

There are different terminologies associated with the DNA

analysis with its lesser quantity of availability, i.e., trace DNA,
touch DNA, low-copy DNA, and low-template DNA. However,
these terminologies cannot be generalized for their use in a singlet
form. Trace or touch DNA can be used for the analysis of a tiny
amount of biological samples collected from the crime scene. This
can be used for any sample which does not meet the recommended
threshold of various stages of DNA analysis, i.e., detection, DNA
extraction, PCR, and result interpretation. Low-template DNA is
used to describe the process of amplification which will generate
stochastic effects, whereas low copy number is generally used to
describe the process of increased cycle number of PCR conditions
rather than the template concentrations (van Oorschot et al. 2010).
Thus, in general all touch DNA samples are trace DNA but all trace
DNA samples are not touch DNA.
Thresholds of DNA amount for biological samples vary from
laboratory to laboratory, i.e., 100 pg (Gill 2001) or 200 pg
(Budowle et al. 2009). In addition to template threshold, all the
steps involving DNA typing should be monitored properly during
trace DNA analysis. It is not necessary that a trace DNA sample will
remain as trace sample during all the processing steps. It is also
possible that the sample may meet the threshold level during PCR
process to generate a good DNA profile. Thus, all the steps of DNA
analysis such as sample collection, DNA extraction, PCR, and
interpretation should be carried out carefully to avoid possible
contaminations.

2 Targets of Trace DNA Samples

Trace/touch DNA samples become of evidentiary value only when

the possibility of innocent transfer is ruled out. The primary target
surfaces carrying touch/trace DNA should be understood properly
by the investigator or sample collector. In cases of murder/homi-
cide, touch DNA samples may be targeted from the surfaces of the
murder weapons such as knife, revolver, or pistol used by the
perpetrator. Similarly, in sexual assault cases clothing of the victim
may be used as a potential source of culprit’s touch DNA samples.
Additionally, certain suspected skin surfaces can be checked for the
presence of any trace amount of touch DNA samples of the suspect
(s). Touch DNA analysis also plays a useful role in cases of burglary
where the recovered items can be analyzed for the presence of
touch DNA samples of the culprits. Similarly, criminal detection
of the forgerer’s DNA on the forged documents can be analyzed to
reach the culprit. Additionally, a wider range of exhibits can be
targeted for forensic touch/trace DNA analysis, i.e., tools, cloth-
ing, vehicles, food, bedding, condoms, lip cosmetics, wallets,
 Collection Strategies for Trace DNA Samples 307

jewellery, glass, whole body surface, cables, windows and doors,

and many others.
Beyond doubt, touch DNA plays a useful role in solving
property-related issues. 8% of the success has been achieved for
touch DNA analysis during 2009–2015 by the Harris County
Institute of Forensic Sciences (HCIFS). A study on 252 trace
DNA samples constituting 201 burglary, robbery, and drug cases
shows a success rate in 14% of the cases, whereas in 21% of cases, a
mixed genetic profile was obtained (Raymond et al. 2009). Despite
low success rate the awareness regarding this technology is being
increased among the investigators, mostly in the cases associated
with burglaries and thefts. In violent criminal cases, where no blood
or seminal stains are visible, the suspected articles can be submitted
for touch DNA analysis generating investigative leads. Additionally,
the cold cases where visible stains are not found or the samples are
highly degraded can be re-examined for touch DNA analysis.

3 Collection Strategies for Trace DNA Samples

The most common technique of collecting trace/touch DNA sam-

ple is by rubbing the surface using a moistened swab. Swabbing of
the target surface should be carried out perfectly by simultaneous
application of pressure and rotation along the full surface area.
Currently double-swabbing method is also in practice for the
same purpose by the use of a moisten swab followed by a dry
swab (Bartlett and Short 2017). During the process of double-
swab technique, the use of moisten swab dislodges the cells from
the surface, whereas the second dry swab picks the cellular material
from the surface. Though water is the most commonly used moist-
ening agent, 0.01% sodium dodecyl sulfate (SDS) and/or isopro-
panol is also recommended by certain studies (Prinz et al. 2006).
After collection of samples in the swab it can be processed immedi-
ately or air-dried; however, air-drying reduces the DNA yield from
the swab. For enhanced recovery of DNA, the swab should be kept
in refrigerated conditions immediately after its collection (van
Oorschot et al. 2003).
Besides the traditional methods of swabbing, tape lifting is also
recommended by various workers. It can be used for lifting sus-
pected samples from the fabrics as well as from the ridged surfaces.
A study has also revealed the effectiveness of tape lifting technique
in comparison to double-swabbing technique for the recovery of
higher quantity of DNA (Williams et al. 2013). Besides, FTA®
paper can also be used in place of common swabs. In addition to
that, cutting method can be used for soft items such as clothing;
however it adds unnecessary substrates into the processing tubes
that increases the cost of processing per sample. Though scrapping
method is also used by some laboratories using a piece of sterile
308 Forensic Trace and Touch DNA Analysis

scalpel blade, it is not recommended for outdoor collection of

samples.
Due to the presence of small amount of DNA in trace DNA
samples, they are highly prone to degradation when destructive
methods are used for its detection. Hence, nondestructive methods
such as use of alternative light sources and fingerprint techniques
can be used for recognition of the source DNA (Sowmyya 2016).
Additionally, personal protective equipment (PPE) such as gloves,
masks, head, and shoe cover, and in certain instances complete
body cover, can be used during collection of touch DNA samples
from the crime scene.

4 Analysis of Trace DNA Samples

Trace DNA samples can be processed like other samples with extra
precaution to avoid possible contaminations. The earlier DNA
extraction techniques like Chelex method or manual method
using phenol-chloroform reagents cannot recover around 75% of
the total available DNA in a sample (Côté et al. 2008). As most of
the genotyping methodologies require DNA samples in the range
of 0.1–1 ng of DNA and trace DNA samples itself contain very little
amount of DNA in them, any loss of DNA samples during the
extraction steps can only give rise to a partial or no DNA profile. In
this regard, development of a new robust DNA extraction method-
ology using silica-coated magnetic beads is highly useful in
recovering trace amount of DNA from the substrate. Additionally,
complete automation with suitable robotic systems minimizes the
chance of contamination and is useful for the processing of higher
number of samples in small time which is the biggest concern in
processing trace DNA samples. When large volume of trace DNA
sample is available it is always recommended to use the concentra-
tion devices, i.e., Microcon (Millipore), MinElute (Qiagen), or
NucleoSpin (Clontech). To minimize sample loss during concen-
tration step addition of poly A RNA or salmon sperm DNA is
recommended by many users (Lever et al. 2015). Though many
techniques are available for the extraction of DNA from trace DNA
samples, the selection of suitable methodology is always dependent
on sample, technique, and analyst’s preference.
Touch DNA samples do not contain trace amount of DNA
always. Hence, it is recommended to quantify these samples prior
to their further processing which gives an indication of the tentative
DNA profile to be generated. Out of many techniques available for
DNA quantitation, probe-based qRT-PCR technique is considered
to be the gold standard for quantification of touch DNA extract.
The commercially available kits for DNA quantification have been
listed in Appendix B. The next step for analysis involves the multi-
plex PCR by various genotyping kits. As the sensitivity of most of
 Concerns and Limitations of Trace DNA Analysis 309

the genotyping kits falls in the range of 0.1–1 ng, the presence of
DNA in such quantity generates a complete DNA profile. Other-
wise a partial DNA profile is obtained.
Technological advancement has revolutionized the analysis of
touch DNA samples where importance has been given to avoid
allele drop in/out in DNA profiles. The new generation kits such
as AmpFlSTR®, Identifiler® Direct, GlobalFiler™ Express, and
PowerPlex® 18D have the capability of direct PCR from the sam-
ples minimizing allele dropout rate. Additionally, the mini-STR
analysis can also be performed to generate a complete analyzable
profile from the low-template touch DNA samples (Nieuwerburgh
et al. 2014). Currently, massive parallel sequencing of SNP markers
for ancestry and phenotype information is being used which
requires a little amount of DNA compared to the STR-based
capillary electrophoresis, and thus it becomes highly useful for
touch DNA analysis (Bruijns et al. 2018). In this regard, some
researchers have also tried with the addition of extra Taq Polymer-
ase (Kloosterman and Kersbergen 2003) and increase in the PCR
cycle conditions (Whitaker et al. 2001) to get an improved DNA
profile from touch DNA samples.

5 Concerns and Limitations of Trace DNA Analysis

The most controversial issue pertaining to trace DNA analysis is the

result interpretation. Though detailed guidelines are in existence
for the interpretation of trace DNA result, its implementation
throughout the global laboratories is in question till now which
raises a huge concern for its application in the medicolegal cases.
Preferential amplification leading to allele dropout is a huge prob-
lem for low DNA samples. Additionally, confirming the zygosity
of a trace DNA sample becomes extremely difficult due to high
stochastic effect and peak height imbalance. Drop-in of alleles
also creates a huge problem in such types of samples creating
confusion regarding the number of contributors in a STR profile
(Hong 2014).
Interpretation of mixed profile is still in debate and it becomes
extremely difficult for the profiles from low DNA samples due to a
higher chance of allele drop-in, dropout, and stutter percentage.
Many software systems are available nowadays to deal with this
problem and separate the contributor’s profiles in a mixed profile
depending on the quantitative models of peak heights and likeli-
hood ratios. Some of the software programs include TrueAllele,
STRmix, DNA VIEW Mixture Solution™, LiRaHT, DNAmix-
tures, EuroForMix, likeLTD, CEESIt, and Kongoh (Manabe
et al. 2017). These mixed DNA profiles in touch DNA samples
may arise from the background contaminations and leftover DNA
present at the crime scene. These left over DNA samples do not
310 Forensic Trace and Touch DNA Analysis

create a significant problem in normal cases as these minute peaks

are masked in the large quantity of target DNA due to preferential
amplification. These contaminant DNA may be deposited either
before the crime, during committing the crime, during crime
investigation, or during experimentation procedure. Though con-
tamination generated prior to the crime and during the crime
cannot be controlled, it can be minimized during sample collection
and their processing steps by adopting a standard operating proce-
dure (Llamas et al. 2017).
Though many studies have been conducted so far to increase
the efficacy of touch DNA analysis, the technique remains unavail-
able till date to distinguish among the origin of DNA due to
primary, secondary, or tertiary transfer. The sequence of events
during primary, secondary, and tertiary transfer of DNA is illu-
strated in Fig. 32.1. In primary transfer, the DNA deposits directly
on an object when an individual touches it. Transfer of DNA on an
object through another individual giving rise to a mixed DNA
profile is called as secondary transfer (Edwards et al. 1991). Simi-
larly, tertiary transfer of DNA can also occur with the involvement
of third party leading to its DNA deposition on the object. In such
cases, a mixed DNA profile from three sources is observed on the
object. In this regard, it becomes imperative to distinguish between
these three types of DNA transfers and unfold the chain of

Fig. 32.1 Mechanism of (a) primary, (b) secondary, and (c) tertiary touch DNA transfers
 Conclusion 311

incidences that lead to the generation of mixed DNA profiles on an

object when touch DNA samples are analyzed (Goray et al. 2010).

6 Conclusion

Forensic trace or touch DNA analysis is of huge importance in

criminal justice system. However, till date no gold standard tech-
nique is available to deal with such extremely low-template DNA
samples. Over the years trace DNA analysis has been improved with
the introduction of magnetic bead-based DNA extraction and
robust, highly sensitive kits for STR amplification. However,
other problems associated with trace DNA analysis are still persist-
ing such as contamination issues, mixture interpretation, and
resolving among primary, secondary, and tertiary transfer of
DNA. Thus, proper training, methodology development, policy
making, and proficiency testing will increase the usefulness of
trace DNA typing in forensic investigations.
 Chapter 33

RAPID DNA Technology: A Boon to Forensic DNA Typing

1 Introduction

With the emergence of forensic DNA typing technology, criminal

justice system has considered it to be the gold standard among
other evidences. During the processing of forensic samples, there
are many labor-intensive processes such as DNA isolation, quantifi-
cation, amplification of short tandem repeat (STR) markers by
multiplex PCR, genotyping by capillary electrophoresis, and data
interpretation. Traditional forensic DNA typing technology
requires a number of expensive instruments and trained manpower.
Routine samples may take at least a day per sample for analysis.
Considering the backlogs and waiting period, which is a common
fact for most of the DNA typing laboratories, quite a long time is
required for obtaining a DNA fingerprinting result. With the
increase in demand from the law enforcement agencies, time-
bound result from the demanded samples has become the need of
the hour. In this context, the recently discovered completely auto-
mated platforms are on demand for the forensic DNA typing
laboratories.
The RAPID DNA instruments provide completely automatic
solutions from sample to results without any human intervention.
These commercially available robotic systems have integrated all the
requisite steps of forensic DNA typing in a single machine to
minimize the processing time as well as the chance of contamina-
tion during manual handling of samples. Besides shortening the
processing time, ease of operating this instrument has also advo-
cated its use in the field conditions.

H. R. Dash et al., Principles and Practices of DNA Analysis: A Laboratory Manual for Forensic DNA Typing, Springer Protocols
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313
314 RAPID DNA Technology: A Boon to Forensic DNA Typing

2 The Technology

The technology uses a prefilled cartridge containing all the chemi-

cals required for the routine processes of DNA fingerprinting. After
insertion of the sample to the instrument, the operation com-
mences with automatic calibration. In this regard, it is useful for
the semiskilled personnel in the field to generate a DNA profile.
Due to low processing time, the machine allows identification of
criminal(s) within 75–90 min and is helpful in exonerating the
suspect(s) in a time-bound manner. The principle behind the oper-
ation of this instrument lies with the micro-fluidic processing tech-
nique. Micro-fluidic processing technique utilizes smaller amounts
of chemicals, hence minimizing the operation cost, reaction time,
as well as size of the instrument and the cartridges containing
chemicals. All the steps including extraction, amplification, electro-
phoresis, and data interpretation take place in one micro-
fluidic chip.
A typical RAPID DNA instrument consists of three parts, i.e.,
the swab, a chip, and the instrument. The swab is required for the
input of desired amount of biological samples to the instrument for
analysis. The chip contains all the reagents, materials, and waste
containments necessary for the process which include reagents for
DNA extraction, amplification by PCR, and separation of amplified
fragments by capillary electrophoresis. The samples present in the
swab are taken by pneumatic pressure and at no stage there is the
direct contact between the instrument and the sample or reagents.
This limits the chance of possible cross contamination. The addi-
tional advantage of this instrument is that the generated DNA
profiles are compatible to the CODIS, Expanded European Stan-
dard Set, Australia’s National Criminal Investigation DNA Data-
base, Canada’s National DNA Data Bank, China’s National DNA
Database, Germany’s DNA-Analyze-Datei, New Zealand’s
National DNA profile databank, and United Kingdom’s National
DNA Database (Carney et al. 2019). The various steps performed
by a RAPID DNA system are described in Fig. 33.1.
Cases of various natures are continuously being solved
promptly by using RAPID DNA technology such as reuniting of
families separated during mass migration or natural calamities,
prevention of human trafficking, and identification of mass casualty
victims. Though the RAPID DNA technology has received huge
applause throughout the globe, it faces many challenges courtesy
variant nature of the samples pertaining to forensic cases. Some of
the challenges of RAPID DNA technology are as follows:
1. All sample types mostly the tough samples, i.e., bone and teeth,
cannot be processed using the currently available RAPID DNA
technology.
 Currently Available Technologies 315

Fig. 33.1 Various steps performed by the RAPID DNA system

2. Some samples also require pre-processing limiting the sample

to result nature of the technology.
3. The technology is yet to reach a ~100% result in all the samples
tested.
4. Non-cost-effectiveness in comparison to the traditional DNA
technology for varied nature of the forensic case samples.
5. Pendency and backlog of cases may result in degraded samples,
where the current technology is of limited applications.

3 Currently Available Technologies

The first fully automatic “swab in-profile out” RAPID DNA analy-
sis approved by FBI is the NetBio (http://netbio.com/) DNAscan
Rapid DNA Analysis™ (Network Biosystems). The two systems of
NetBio, i.e., ANDE™ and DNAscan™, use a common cotton
swab for input of reference samples to be processed within 90 min
by the instruments. The currently used DNAscan 6C has the capa-
bility to analyze four samples at 27 loci within 90 min.
Another RAPID DNA instrument in the market is Rapid-
HIT™ 200 system (IntergenX) which is slightly smaller in size in
comparison to NetBio instruments and is capable of processing
up to eight samples at the same time. The second-generation
316 RAPID DNA Technology: A Boon to Forensic DNA Typing

IntergenX (https://integenx.com/) instrument RapidHIT ID has

reduced its cost to half of the first-generation instrument and is
capable of using both GlobalFiler Express and NGMSelect 17 kit.
Another fully automatic portable DNA Analyzer has been
manufactured by NEC (http://in.nec.com/en_IN/products/pub
lic-safetysecurity/product/portable-dna-analyzer.html). This
instrument is highly popular in the Asian subcontinent and is
capable of performing the entire DNA analysis within 25 min. As
this instrument is portable enough to be carried to the crime scene,
it is highly useful in speeding up of the crime investigation and
prevention processes. Recently LGC (https://www.lgcgroup.com/
products/paradnatechnology/#.WJ4-KcLHxD9) has manufac-
tured ParaDNA® instruments capable of providing high success
rate, fastness, and reliable profile generation in the quickest
possible time.
Currently, National DNA Index System (NDIS) is the respon-
sible agency to provide approval of the RAPID DNA systems for
forensic applications. As the process has limitation for analyzing the
low-DNA-content (LDC) samples, many developmental valida-
tions are in progress for improving the associated methodologies.
Incorporation of single-use BioChipSet (BCS) has increased the
performance of the technology to generate a readable DNA profile
from as low as 5 pg/μl of DNA samples (Turingan et al. 2016;
Buscaino et al. 2018).

4 Conclusion

RAPID DNA technology possesses a definitive advantage for its use

in definitive scenario such as at the crime scene. Due to ease in
operation and complete automation, processing of the samples at
crime scene or police stations may not require highly skilled per-
sonnel besides interpretation of the obtained results. Regarding
cost of the instrument and its associated consumables, if we include
the operational as well as infrastructure cost, the RAPID DNA
technology is highly useful for processing fewer samples with the
requirement of urgent results for investigation purpose. Though
the technology has improved a lot since its inception, it is still not so
useful in challenging forensic samples. Hence, the technology
works fine for database generation and analysis of reference samples
in a short period of time.
Appendix A: Composition of Reagents, Buffers,
and Solutions

Lysis Buffer I

Tris: 30 mM
EDTA: 5 mM
NaCl: 50 mM

Lysis Buffer II

NaCl: 75 mM
EDTA: 2 mM

Forensic Buffer (pH 8.2)

Tris: 100 mM
EDTA: 5 mM
NaCl: 50 mM

Hair Digestion Buffer

Mix the following stock solution as per the following:

1 M Tris–HCl (pH 7.5): 1 ml
0.5 M EDTA: 2 ml
5 M NaCl: 10 ml
20% SDS: 10 ml

H. R. Dash et al., Principles and Practices of DNA Analysis: A Laboratory Manual for Forensic DNA Typing, Springer Protocols
Handbooks, https://doi.org/10.1007/978-1-0716-0274-4, © Springer Science+Business Media, LLC, part of Springer Nature 2020

317
318 Appendix A: Composition of Reagents, Buffers, and Solutions

Deionized water: 86 ml
Note: Do not autoclave, and store at 4
C till further use.

Proteinase-K (20 mg/ml)

Add 20 mg of proteinase-K in 500 μl of TE buffer first and then add

500 μl of 100% glycerol. Mix well with slow pipetting, prepare
aliquots, and store at 20
C.
Note: As the protein is soluble in water, the solvent (water/
buffer) to be used depends on the condition of the supplied pow-
der. If proteinase-K powder is supplied in a lyophilized form using a
buffer, it can be dissolved in deionized water. However, if it is
lyophilized from water, it should be dissolved in TE buffer. Thus,
check the manufacturer’s guidelines before dissolving proteinase-K.

1 M DTT

Weigh out 3.86 g of DL-dithiothreitol (DTT) (C4H10O2S2) of

molecular weight 154.25 in a 50 ml disposable flask. Add 20 ml of
deionized water to it and mix well to dissolve the DTT completely.
Make up the volume to 25 ml with deionized water. Sterilize the
solution by passing through 0.22 μm syringe filter under aseptic
conditions. Store the solution at 20
C till further use.
Caution: Do not autoclave the DTT solution; autoclaving
destroys the DTT.

20% (w/v) SDS

Sodium dodecyl sulfate (SDS) has the molecular formula of

CH3(CH2)11OSO3Na. To prepare 100 ml of 20% SDS solution,
take 20 g of SDS in a 250 ml of conical flask and add 80 ml of
deionized water to it. Mix gently using a magnetic stirrer. Option-
ally you may heat the solution at 68
C using a water bath for
thorough dissolving. Adjust the volume to 100 ml using deionized
water. Make small aliquots and store at room temperature.
Note: Autoclaving is not required for SDS solution. Wear mask
while preparing SDS solution to avoid inhalation of the fine
powder.

Tris-Saturated Phenol (TSP)

Open 100 g of phenol in a fume hood and pour in a 100 ml of

50 mM Tris-Cl pH 8.0. Close the lid of the bottle and mix gently.
 Appendix A: Composition of Reagents, Buffers, and Solutions 319

Allow it to stand for 1–2 h till phenol liquefies and two phases get
separated. Remove the supernatant using a glass pipette and further
add 100 ml of 50 mM Tris-Cl pH 8.0. Close the bottle and mix
gently. Allow it to stand for 1–2 h till phenol liquefies and two
phases get separated. Repeat the saturation step 4–5 times till pH of
the supernatant reaches 7–8. Dispense in aliquots and add 10 ml of
50 mM Tris-Cl (pH 8.0) to each aliquot. Store at 20
C till
further use.
Alternatively you may try using the commercially available Tris-
saturated phenol (TSP). Some manufacturers add stabilizers in the
solution. As pure phenol is colorless only oxidation in phenol
changes its color to pale yellow to pink. Storing the solution at
20
C minimizes the risk of oxidation of the solution.

Chloroform: Isoamyl Alcohol (24:1)

Mix 96 ml of chloroform with 4 ml of isoamyl alcohol (3-methyl-1-

butanol) in a glass measuring cylinder. Make small aliquots and
store at room temperature.

3 M Sodium Acetate

Weigh 24.61 g of sodium acetate (CH3COONa) and transfer in a

250 ml of conical flask. Add 80 ml of deionized water and mix until
sodium acetate is completely dissolved. Adjust the pH to 5.2 by
using glacial acetic acid. Finally, make up the volume to 100 ml with
deionized water. Sterilize the solution by autoclaving at 121
C and
15 pounds per square inch pressure for 15 min. Allow the solution
to cool. Dispense in aliquots and use.

70% Ethanol

Use the formula of N1  V1 ¼ N2  V2 to prepare the desired

quantity of 70% ethanol solution from the absolute ethanol (95%
solution). For example, to prepare 200 ml of 70% ethanol solution,
dissolve 147.4 ml of 95% ethanol with 52.6 ml of deionized water.
Mix well before use.

Tris-EDTA (TE) Buffer (pH 8.0)

The constituents of TE buffer are 10 mM Tris [Tris (hydroxyl-

methyl) aminomethane] and 0.5 M EDTA [diaminoethane tetra-
acetic acid] (pH 8.0). Prepare 1 M stock of Tris by dissolving
60.57 g of Tris in 500 ml of deionized water after adjusting the
320 Appendix A: Composition of Reagents, Buffers, and Solutions

pH to 8.0. Prepare 0.5 M EDTA (pH 8.0) solution by dissolving

18.6 g of EDTA in 100 ml of deionized water and adjusting the pH
to 8.0. To prepare 500 ml of TE buffer, mix 5 ml of 1 M Tris
(pH 8.0) and 1 ml of 0.5 M EDTA (pH 8.0). Make up the volume
to 500 ml by adding 496 ml of deionized water. Sterilize the
solution by autoclaving at 121
C and 15 pounds per square inch
pressure for 15 min.
Caution: Disodium EDTA salt will not dissolve in the solution
until pH 8.0 is achieved.

Normal Saline Solution (0.85%)

Dissolve 8.5 g of NaCl in deionized water. Sterilize the solution by

autoclaving at 121
C and 15 pounds per square inch pressure for
15 min. Allow the solution to cool. Dispense in aliquots and use.

0.5 M EDTA (pH 8.0)

Add 186.1 g of disodium EDTA.2H2O salt to 800 ml of deionized

water. Mix vigorously on a magnetic stirrer. Add NaOH pellets to
adjust the pH to 8.0. Make up the volume to 1000 ml using
deionized water. Sterilize the solution by autoclaving at 121
C
and 15 pounds per square inch pressure for 15 min. Allow the
solution to cool. Dispense in aliquots and use.
Caution: Disodium EDTA salt will not dissolve in the solution
until pH 8.0 is achieved.
Appendix B: List of Commercially Available Kits and
Instruments for Forensic DNA Analysis

(a) List of Commercially Available DNA Extraction Kits/Systems

Sl. Manufacturer’s name and

no. Name of kits address
1 QIAamp Micro DNA kit Qiagen, Valencia, CA
2 QIAamp DNA Investigator Kit
3 EZ1 DNA Investigator Kit
4 QIAsymphony DNA Investigator Kit
5 DNA IQ™ System and DNA IQ Promega Corporation,
Reference Sample Kit for Maxwell® Madison, WI
16
6 DNA IQ Casework Sample Kit for
Maxwell 16
7 PrepFiler™ Forensic DNA Extraction Thermo Fisher, South San
Kit Francisco, CA
8 PrepFiler Express™ DNA Extraction
Kit
9 Forensic GEM Kits ZyGEM, Hamilton,
New Zealand
10 MagPurix Forensic DNA Extraction Zinexts, New Taipei City,
Kit Taiwan
11 MPure™ Forensic DNA Extraction Kit MP Biomedicals, Santa Ana,
CA
12 E.Z.N.A. Forensic DNA Kit Omega Bio-tek, Inc.,
NorCross, GA

(continued)

H. R. Dash et al., Principles and Practices of DNA Analysis: A Laboratory Manual for Forensic DNA Typing, Springer Protocols
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321
322 Appendix B: List of Commercially Available Kits and Instruments for Forensic DNA Analysis

Sl. Manufacturer’s name and

no. Name of kits address
13 Forensic DNA Extraction Kit GeneON GmbH,
Deutschland/Germany
14 GF-1 Forensic DNA Extraction Kit Vivantis Technologies,
Selangor Darul Ehsan,
Malaysia
15 MagListo™ 5M Forensic Sample DNA Vivantis Technologies,
Extraction Kit Selangor Darul Ehsan,
Malaysia
16 AGOWA® mag Maxi DNA Isolation AGOWA, Germany
Kit
17 InviMag Forensic Kit Invitek mbH, Germany
18 UltraClean™ Forensic DNA Isolation Mo Bio Laboratories Inc.,
Kit Carlsbad, CA
19 HiPurA™ Forensic Sample Genomic HiMedia Laboratories,
DNA Purification Kit Mumbai, India
20 HiPurA™ Quick Bone DNA HiMedia Laboratories,
purification kit Mumbai, India

(b) List of Commercially Available DNA Quantitation Kits/Systems

Sl. Manufacturer’s name

no. Name of kits and address
1 Quantifiler Trio Thermo Fisher, South
San Francisco, CA
2 Quantifiler HP
3 Quantifiler Duo
4 Quantifiler Human
Quantifiler® Y human male DNA
quantification kit
5 Investigator® Quantiplex HYres Kit Qiagen, Valencia, CA
®
Investigator Quantiplex Pro Kit
Investigator® Quantiplex Kit
6 PowerQuant® System Promega Corporation,
® Madison, WI
Plexor HY System
7 InnoQuant™ Human DNA Quantification InnoGenomics
& Degradation Assessment Kit Technologies, LLC
Appendix B: List of Commercially Available Kits and Instruments for Forensic DNA Analysis 323

(c) List of Commercially Available Genotyping Kits

Sl. Manufacturer’s name

no. Name of the kit List of STR markers and address
1 AmpFlSTR® D8S1179, D21S11, Thermo Fisher
Identifiler® kit D7S820, CSF1PO, Scientific, Foster
D3S1358, TH01, City, CA, USA
D13S317, D16S539,
D2S1338, D19S433,
vWA, TPOX, D18S51,
amelogenin, D5S818,
FGA
2 AmpFlSTR® CSF1PO, D2S1338, Thermo Fisher
Identifiler® D3S1358, D5S818, Scientific, Foster
Plus kit D7S820, D8S1179, City, CA, USA
D13S317, D16S539,
D18S51, D19S433,
D21S11, FGA, TH01,
TPOX, vWA,
amelogenin
3 AmpFlSTR® D8S1179, D21S11, Thermo Fisher
Identifiler® D7S820, CSF1PO, Scientific, Foster
Direct kit D3S1358, TH01, City, CA, USA
D13S317, D16S539,
D2S1338, D19S433,
vWA, TPOX, D18S51,
D5S818, FGA,
amelogenin
4 AmpFlSTR® CSF1PO, FGA, Thermo Fisher
MiniFiler™ D16S539, D18S51, Scientific, Foster
kit amelogenin, D2S1338, City, CA, USA
D21S11, D7S820
5 AmpFℓSTR® D3S1358, vWA, Thermo Fisher
NGM™ kit D16S539, D2S1338, Scientific, Foster
D8S1179, D21S11, City, CA, USA
D18S51, D19S433,
TH01, FGA, D1S1656,
D12S391, D10S1248,
D22S1045, D2S441,
amelogenin
6 AmpFℓSTR® D3S1358, vWA, Thermo Fisher
NGM D16S539, D2S1338, Scientific, Foster
SElect™ kit D8S1179, D21S11, City, CA, USA
D18S51, D19S433,
TH01, FGA,
amelogenin,
D10S1248,

(continued)
324 Appendix B: List of Commercially Available Kits and Instruments for Forensic DNA Analysis

Sl. Manufacturer’s name

no. Name of the kit List of STR markers and address
D22S1045, D2S441,
D1S1656, D12S391,
SE33
7 AmpFℓSTR® D3S1358, vWA, Thermo Fisher
NGM D16S539, D2S1338, Scientific, Foster
SElect™ D8S1179, D21S11, City, CA, USA
Express D18S51, D19S433,
TH01, FGA,
amelogenin,
D10S1248,
D22S1045, D2S441,
D1S1656, D12S391,
SE33
8 AmpFℓSTR® FGA, TH01, vWA, Thermo Fisher
NGM D3S1358, D8S1179, Scientific, Foster
Detect™ kit D18S51, D21S11, City, CA, USA
D12S391, D1S1656,
D2S441, D10S1248,
D22S1045, D16S539,
D2S1338, D19S433,
SE33, IQCS, IQCL, Y
indel, amelogenin
9 GlobalFiler™ kit D3S1358, vWA, Thermo Fisher
D16S539, CSF1PO, Scientific, Foster
TPOX, D8S1179, City, CA, USA
D21S11, D18S51,
D2S441, D19S433,
TH01, FGA,
D22S1045, D5S818,
D13S317, D7S820,
SE33, D10S1248,
D1S1656, D12S391,
D2S1338, amelogenin,
DYS391, Y indel
10 GlobalFiler™ D3S1358, vWA, Thermo Fisher
Express kit D16S539, CSF1PO, Scientific, Foster
TPOX, D8S1179, City, CA, USA
D21S11, D18S51,
D2S441, D19S433,
TH01, FGA,
D22S1045, D5S818,
D13S317, D7S820,
SE33, D10S1248,
D1S1656, D12S391,
D2S1338, amelogenin,
DYS391, Y indel

(continued)
Appendix B: List of Commercially Available Kits and Instruments for Forensic DNA Analysis 325

Sl. Manufacturer’s name

no. Name of the kit List of STR markers and address
11 VeriFiler™ D3S1358, vWA, Thermo Fisher
Express kit D16S539, CSF1PO, Scientific, Foster
TPOX, D8S1179, City, CA, USA
D21S11, D18S51,
D2S441, D19S433,
TH01, FGA,
D22S1045, D5S818,
D13S317, D7S820,
D10S1248, D1S1656,
D12S391, D2S1338,
D6S1043, Penta D,
Penta E, Y indel,
amelogenin
12 AmpFlSTR® DYS19, DYS385a/b, Thermo Fisher
Yfiler® kit DYS389I/II, DYS390, Scientific, Foster
DYS391, DYS392, City, CA, USA
DYS393, DYS438,
DYS439, DYS437,
DYS448, DYS456,
DYS458, DYS635
(YGATAC4),
YGATAH4
13 AmpFlSTR® DYS19, DYS385a/b, Thermo Fisher
Yfiler® Direct DYS389I/II, DYS390, Scientific, Foster
kit DYS391, DYS392, City, CA, USA
DYS393, DYS438,
DYS439, DYS437,
DYS448, DYS456,
DYS458, DYS635
(YGATAC4),
YGATAH4
14 Yfiler™ Plus kit DYS576, DYS389I, Thermo Fisher
DYS635, DYS389II, Scientific, Foster
DYS627, DYS460, City, CA, USA
DYS458, DYS19,
YGATAH4, DYS448,
DYS391, DYS456,
DYS390, DYS438,
DYS392, DYS518,
DYS570, DYS437,
DYS385a/b, DYS449,
DYS393, DYS439,
DYS481, DYS387S1,
DYS533
15 PowerPlex® D18S51, D21S11, TH01, Promega Corporation,
18D System D3S1358, FGA, Madison, WI
TPOX, D8S1179,

(continued)
326 Appendix B: List of Commercially Available Kits and Instruments for Forensic DNA Analysis

Sl. Manufacturer’s name

no. Name of the kit List of STR markers and address
vWA, CSF1PO,
D16S539, D7S820,
D13S317, D5S818,
amelogenin, Penta E,
Penta D, D2S1338,
D19S433
16 PowerPlex® Amelogenin, D1S1656, Promega Corporation,
21 System D2S1338, D3S1358, Madison, WI
D5S818, D6S1043,
D7S820, D8S1179,
D12S391, D13S317,
D16S539, D18S51,
D19S433, D21S11,
amelogenin, CSF1PO,
FGA, Penta D, Penta E,
TH01, TPOX, vWA
17 PowerPlex® CS7 LPL, F13B, FESFPS, Promega Corporation,
System F13A01, Penta D, Madison, WI
Penta C, Penta E
18 PowerPlex® ESX D18S51, D21S11, TH01, Promega Corporation,
16 and ESI D3S1358, amelogenin, Madison, WI
16 Fast D16S539, D2S1338,
Systems D1S1656, D10S1248,
FGA, D8S1179, vWA,
D22S1045, D19S433,
D12S391, D2S441
19 PowerPlex® ESX D3S1358, D8S1179, Promega Corporation,
17 and ESI D18S51, D21S11, Madison, WI
17 Fast FGA, TH01, vWA,
Systems D2S441, D10S1248,
D22S1045, D1S1656,
D12S391, D2S1338,
D16S539, D19S433,
SE33, amelogenin
20 PowerPlex® CSF1PO, FGA, TH01, Promega Corporation,
Fusion 6C vWA, D1S1656, Madison, WI
System D2S1338, D2S441,
D3S1358, D5S818,
D7S820, D8S1179,
D10S1248, D12S391,
D13S317, D16S539,
D18S51, D19S433,
D21S11, amelogenin,
DYS391, Penta D,
Penta E, D22S1045,
TPOX, SE33, DYS570,
DYS576

(continued)
Appendix B: List of Commercially Available Kits and Instruments for Forensic DNA Analysis 327

Sl. Manufacturer’s name

no. Name of the kit List of STR markers and address
21 PowerPlex® CSF1PO, FGA, TH01, Promega Corporation,
Fusion System TPOX, vWA, Madison, WI
D3S1358, D5S818,
D7S820, D8S1179,
D13S317, D16S539,
D18S51, D21S11,
D10S1248,
D22S1045, D2S441,
D1S1656, D12S391,
amelogenin, DYS391,
Penta D, Penta E,
D2S1338, D19S433
22 PowerPlex® Y23 DYS576, DYS389I/II, Promega Corporation,
System DYS448, DYS19, Madison, WI
DYS391, DYS481,
DYS549, DYS533,
DYS438 (penta),
DYS437, DYS570,
DYS635, DYS390,
DYS439, DYS392,
DYS643 (penta),
DYS393, DYS458,
DYS385a/b, DYS456,
Y-GATA-H4
23 Investigator Amelogenin, TH01, QIAGEN, Hilden,
24plex QS Kit D3S1358, vWA, Germany
D21S11, TPOX,
DYS391, D1S1656,
D12S391, SE33,
D10S1248,
D22S1045, D19S433,
D8S1179, D2S1338,
D2S441, D18S51,
FGA, QS1, D16S539,
CSF1PO, D13S317,
D5S818, D7S820, QS2
24 Investigator Amelogenin, TH01, QIAGEN, Hilden,
24plex GO! D3S1358, vWA, Germany
Kit D21S11, TPOX,
DYS391, D1S1656,
D12S391, SE33,
D10S1248,
D22S1045, D19S433,
D8S1179, D2S1338,
D2S441, D18S51,
FGA, QS1, D16S539,
CSF1PO, D13S317,
D5S818, D7S820, QS2

(continued)
328 Appendix B: List of Commercially Available Kits and Instruments for Forensic DNA Analysis

Sl. Manufacturer’s name

no. Name of the kit List of STR markers and address
25 Investigator QS1, amelogenin, TH01, QIAGEN, Hilden,
ESSplex SE D3S1358, vWA, Germany
QS Kit D21S11, QS2,
D163539, D1S1656,
D19S433, SE33,
D10S1248,
D22S1045, D12S391,
D8S1179, D2S1338,
D2S441, D18S51,
FGA
26 Investigator Amelogenin, TH01, QIAGEN, Hilden,
ESSplex SE D3S1358, vWA, Germany
GO! Kit D21S11, D16S539,
D1S1656, D19S433,
SE33, D10S1248,
D22S1045, D12S391,
D8S1179, FGA,
D2S1338, D2S441,
D18S51
27 Investigator Amelogenin, TH01, QIAGEN, Hilden,
ESSplex SE D3S1358, vWA, Germany
Plus Kit D21S11, D16S539,
D1S1656, D19S433,
D8S1179, D2S1338,
D10S1248,
D22S1045, D12S391,
FGA, D2S441,
D18S51
28 Investigator Amelogenin, TH01, QIAGEN, Hilden,
ESSplex Plus D3S1358, vWA, Germany
Kit D21S11, D16S539,
D1S1656, D19S433,
D8S1179, D2S1338,
D10S1248,
D22S1045, D12S391,
FGA, D2S441,
D18S51
29 Investigator Amelogenin, TH01, QIAGEN, Hilden,
IDplex Plus D3S1358, vWA, Germany
Kit D21S11, TPOX,
D7S820, D19S433,
D5S818, D2S1338,
D16S539, CSF1PO,
D13S317, FGA,
D18S51, D8S1179

(continued)
Appendix B: List of Commercially Available Kits and Instruments for Forensic DNA Analysis 329

Sl. Manufacturer’s name

no. Name of the kit List of STR markers and address
30 Investigator Amelogenin, TH01, QIAGEN, Hilden,
IDplex GO! D3S1358, vWA, Germany
Kit D21S11, TPOX,
D7S820, D19S433,
D5S818, D2S1338,
D16S539, CSF1PO,
D13S317, FGA,
D18S51, D8S1179
31 Investigator QS1, amelogenin, QIAGEN, Hilden,
Argus X-12 DXS10103, DXS8378, Germany
QS Kit DXS10101,
DXS10134,
DXS10074, DXS7132,
DXS10135, DXS7423,
DXS10146,
DXS10079,
DXSHPRTB,
DXS10148, D21S11
32 Investigator Amelogenin, D7S1517, QIAGEN, Hilden,
HDplex Kit D3S1744, D12S391, Germany
D2S1360, D6S474,
D4S2366, D8S1132,
D5S2500, D18S51,
D21S2055,
D10S2325, SE33
33 Investigator DYS439, DYS437, QIAGEN, Hilden,
Argus Y-12 DYS390, DYS385, Germany
QS Kit DYS391, DYS389-I,
DYS19, DYS389-II,
DYS393, DYS438,
DYS392
34 COrDIS Plus kit D3S1358, D5S818, Gordiz, Moskva, Russia
D7S820, D8S1179,
D13S317, D16S539,
D18S51, D21S11,
CSF1PO, FGA, TH01,
TPOX, VWA,
D1S1656, D2S441,
D10S1248, D12S391,
D22S1045 and SE33,
amelogenin
35 COrDYS-Y kit DYS19, DYS389I/II, Gordiz, Moskva, Russia
DYS390, DYS391,
DYS392, DYS393,
DYS385a/b, DYS438
and DYS439, DYS437,
DYS456, DYS635,
DYS448, DYS576,
DYS481, DYS449

(continued)
330 Appendix B: List of Commercially Available Kits and Instruments for Forensic DNA Analysis

Sl. Manufacturer’s name

no. Name of the kit List of STR markers and address
36 COrDX kit DXS10148, DXS10135, Gordiz, Moskva, Russia
DXS8378, DXS7132,
DXS10079,
DXS10075,
DXS10074,
DXS10103, HPRTB,
DXS10101,
DXS10146, DXS8377,
DXS10134, DXS7423
37 iPLEX-STR™ Amelogenin, D3S1358, Independent Forensics,
Kit TH01, D12S391, 500 Waters Edge
D1S1656, D10S1248, Lane, Lombard
D2S441, D7S820,
D13S317, FGA,
TPOX, D18S51,
D16S539, D8S1179,
CSF1PO, D5S818,
vWA, D21S11, SE33
38 iPLEX-STR™ Y DYS19, DYS389I, Independent Forensics,
Kit DYS389II, DYS390, 500 Waters Edge
DYS391, DYS392, Lane, Lombard
DYS393, DYS385a/b,
DYS438, DYS439,
DYS437, DYS456,
DYS635, DYS448,
DYS576, DYS481,
DYS449
39 iPLEX X Kit DXS10148, DXS10135, Independent Forensics,
DXS8378, DXS7132, 500 Waters Edge
DXS10079, Lane, Lombard
DXS10075,
DXS10074,
DXS10103, HPRTB,
DXS10101,
DXS10146, DXS8377,
DXS10134, DXS7423
40 SureID® Amelogenin, D18S1364, HEALTH Gene
23comp D1S1656, D13S325, Technologies
Human DNA D9S1122, D4S2366, Co. Ltd., China
Identification D3S1744, D12S391,
Kit D11S2368,
D21S2055, D20S482,
D8S1132, D7S3048,
D2S441, D19S253,
D10S1248,
D17S1301,
D22-GATA198B05,
D16S539, D6S474,
D14S1434, D15S659

(continued)
Appendix B: List of Commercially Available Kits and Instruments for Forensic DNA Analysis 331

Sl. Manufacturer’s name

no. Name of the kit List of STR markers and address
41 SureID® 21G CSF1PO, FGA, TH01, HEALTH Gene
Human TPOX, vWA, Technologies
STR D3S1358, D5S818, Co. Ltd., China
Identification D7S820, D8S1179,
Kit D13S317, D16S539,
D18S51, D21S11,
D12S391, D19S433,
D1S1656, D2S1338,
D6S1043, Penta E,
Penta D, Amelogenin
42 SureID® 27Y DYS456, DYS576, HEALTH Gene
Human DYS570, DYS481, Technologies
STR DYF387S1, DYS627, Co. Ltd., China
Identification DYS458, DYS460,
Kit DYS437, DYS439,
DYS392, DYS385,
DYS393, DYS391,
DYS390, DYS456,
DYS635, DYS449,
DYS533, DYS438,
DYS389I, DYS448,
DYS389II, DYS19,
GATA_H4, DYS518
43 DNATyper™15 D6S1043, D21S11, Institute of Forensic
PCR D7S820, CSF1PO, Science, Ministry of
Genotyping D2S1338, D3S1358, Public Security,
System D13S317, D8S1179, Beijing, China
D16S539, Penta E,
D5S818, vWA,
D18S51, FGA,
amelogenin

(d) List of Commercially Available Capillary Electrophoresis Instruments

Sl. no. Systems Manufacturer Capillary no.

1 ABI 310 Thermo Scientific 1
2 ABI 3130 Thermo Scientific 4
3 ABI 3130xl Thermo Scientific 16
4 ABI 3500 Thermo Scientific 8
5 ABI 3500xL Thermo Scientific 24

(continued)
332 Appendix B: List of Commercially Available Kits and Instruments for Forensic DNA Analysis

Sl. no. Systems Manufacturer Capillary no.

6 ABI 3730 Thermo Scientific 48
7 ABI 3730xl Thermo Scientific 96
8 MegaBACE 500 Amersham Biosciences 48
9 MegaBACE 1000 Amersham Biosciences 96
10 MegaBACE 4000 Amersham Biosciences 384
11 SCE 2410 SpectruMedix Corporation 24
12 SCE 9610 SpectruMedix Corporation 96
13 SCE 19210 SpectruMedix Corporation 192
14 CEQ 8800 Beckman Coulter 8

(e) List of Data Analysis Software

Sl.
no. Software Manufacturer
1 GeneMapperID and ID-X Thermo Scientific
2 TrueAllele (Pittsburgh, PA)
3 STRess (STR Expert System Suite) and Forensic Science Service
FSS i-cubed (FSS)
4 Open Source Independent Review and National Center for
Interpretation System (OSIRIS) Biotechnology Information
(NCBI)
5 GenoProof 2 and GenoProof Mixture Qualitype AG
6 GeneMarker Soft Genetics, LLC (State
College, PA)
Appendix C: Good Laboratory Practices

General Molecular Biology Laboratories

1. Always wear lab coats and gloves inside the laboratory.

2. Always practice a good housekeeping policy in the laboratory.
3. Gloves should be changed frequently.
4. Use paper, pens, lab coats, and other accessories dedicatedly in
one lab.
5. Always maintain the unidirectional workflow.
6. Never drink or eat anything inside the laboratory.
7. Avoid mouth pipetting in the laboratory.
8. While opening/closing the centrifuge tubes, avoid touching
the inner surface of the lids.
9. Never use any reagent from the stock itself; first prepare small
aliquots and use from the aliquots.
10. Pulse centrifuge each reagent prior to use.
11. All the workers should have proper lab induction and training.
12. Always maintain the logbook.
13. Expose the reagents for minimal freeze-thaw cycle.
14. Reagents containing fluorophores should be stored away from
light to prevent photo-leaching.
15. Always use controls during experiments.
16. Decontaminate the working area using UV irradiation, sodium
hypochlorite, or 1 M HCl.

General Forensic DNA Fingerprinting Laboratories

1. The DNA fingerprinting laboratory should be designed in such

a way to have a separate pre-PCR and post-PCR lab.

H. R. Dash et al., Principles and Practices of DNA Analysis: A Laboratory Manual for Forensic DNA Typing, Springer Protocols
Handbooks, https://doi.org/10.1007/978-1-0716-0274-4, © Springer Science+Business Media, LLC, part of Springer Nature 2020

333
334 Appendix C: Good Laboratory Practices

2. The pre-PCR lab comprises evidence handling, sample prepa-

ration, DNA isolation, preparation of DNA samples for quan-
tification, and PCR amplification.
3. The laboratory should use validated instruments.
4. The laboratory should use validated laboratory protocols
(SOPs).
5. Suitable allelic ladders, positive and negative amplification con-
trols, and reagent blanks should be used for each dataset.
6. Internal size standard should be used in each individual sample.
7. For correct interpretation, a qualified analyst or supervisor
should review the result.
8. Quality assurance audits of the laboratory should be carried out
from time to time.
9. All the forensic DNA analysis should be performed as per
SWGDAM guidelines.
10. The laboratory should be accredited by the proper agencies to
carry out DNA typing work.
11. Individual DNA analysts should undergo training, proficiency
tests, and continuing education.
12. Statistical evaluation for the significance of match should be
conducted for each match report.
13. Inconclusive result should be reported in situations where two
analysts remain in disagreement on the data and if it is felt that
insufficient information exists to draw any conclusion.
14. All the analysis should be validated for their reproducibility,
robustness, and reliability.
15. The DNA laboratory must maintain documentation of all
aspects of DNA analysis procedure, related documents, and
interpretation of results.
16. Prior to analysis of a reagent, kit, or instrument, internal labo-
ratory validation should be performed each time.

For Equipment, Materials, and Facilities

1. The inventory of instruments present in the laboratory should

be maintained for their manufacturers, model numbers, serial
numbers, agency inventory numbers, and acquisition dates.
2. Operating manual of each instrument should be available
readily.
3. Calibration, maintenance procedure, and logs should be main-
tained for each instrument.
 Appendix C: Good Laboratory Practices 335

4. Dedicated instruments should be used for dedicated

experiments.
5. Logs should be maintained for kits and other reagents having
an expiration period.
6. Written validated procedures must be present for preparation
of all chemicals, reagents, standards, as well as controls.
7. All the reagents should contain labels mentioning the name of
the reagent, concentration, date of preparation, initial of the
individual preparing the reagent, storage requirements, and its
expiry date.
8. Data regarding the supplier, catalog number, lot number,
receiving data, and storage location should be maintained.
9. Specific procedure should be maintained for cleaning, prepara-
tion, and sterilization in the laboratory.

For Evidence Handling

1. Each case and their exhibits should be labeled with the unique
identifier that may be a number or barcode.
2. A clear, well-documented chain of custody should be main-
tained from time to time for all the evidences and the biological
samples.
3. A written policy should be present to ensure proper storage of
the exhibits in their appropriate storage conditions.
4. Care should be taken to protect the samples from loss, contam-
ination, and degradation over time during storage in the
laboratory.
5. Specific law and regulations of the agency/agencies involved
should be maintained during evidence handling.
Appendix D: Allele Frequency Data for Calculation of
Paternity Index

H. R. Dash et al., Principles and Practices of DNA Analysis: A Laboratory Manual for Forensic DNA Typing, Springer Protocols
Handbooks, https://doi.org/10.1007/978-1-0716-0274-4, © Springer Science+Business Media, LLC, part of Springer Nature 2020

337
Table D.1
338

Allele frequency data and paternity and forensic parameters of the 21 genetic markers tested during the current study (n ¼ 551)

Genetic markers/loci tested

Alleles D3S1358 vWA D16S539 CSF1PO TPOX D8S1179 D21S11 D18S51 D2S441 D19S433 TH01 FGA D22S1045 D5S818 D13S317 D7S820 SE33 D10S1248 D1S1656 D12S391 D2S1338

6 0.001 0.239 0.001

7 0.002 0.003 0.004 0.001 0.155 0.001 0.004 0.028

8 0.073 0.004 0.339 0.007 0.142 0.002 0.232 0.219 0.012 0.040

9 0.002 0.154 0.028 0.150 0.008 0.005 0.005 0.304 0.027 0.105 0.063 0.001 0.012

9.3 0.141

10 0.002 0.092 0.188 0.094 0.203 0.008 0.351 0.001 0.016 0.005 0.108 0.087 0.240 0.006

11 0.290 0.288 0.384 0.065 0.018 0.360 0.006 0.002 0.299 0.389 0.214 0.256 0.005 0.013 0.151

11.3 0.068

12 0.002 0.235 0.393 0.025 0.086 0.095 0.063 0.082 0.005 0.295 0.274 0.165 0.009 0.016 0.098

12.2 0.007

13 0.003 0.008 0.124 0.088 0.003 0.145 0.125 0.020 0.295 0.170 0.059 0.024 0.012 0.111 0.136 0.001

13.2 0.021 0.001

14 0.035 0.141 0.029 0.007 0.002 0.162 0.002 0.293 0.108 0.246 0.065 0.008 0.024 0.015 0.262 0.108

14.2 0.057 0.001

15 0.277 0.062 0.002 0.001 0.001 0.199 0.001 0.164 0.020 0.128 0.002 0.364 0.002 0.018 0.303 0.138 0.004 0.001

15.2 0.046 0.005

15.3 0.004 0.016

16 0.315 0.227 0.106 0.119 0.005 0.061 0.174 0.037 0.203 0.146 0.012 0.005
Appendix D: Allele Frequency Data for Calculation of Paternity Index

16.2 0.025 0.006

16.3 0.002 0.039

17 0.252 0.268 0.013 0.001 0.078 0.012 0.077 0.065 0.085 0.058 0.135 0.064

17.2 0.005 0.001 0.001

17.3 0.022 0.005

18 0.107 0.205 0.001 0.035 0.005 0.010 0.001 0.083 0.007 0.009 0.246 0.192
18.2 0.002

18.3 0.012 0.011

19 0.010 0.076 0.027 0.055 0.001 0.001 0.087 0.001 0.001 0.168 0.171

19.2 0.001

20 0.001 0.006 0.013 0.110 0.085 0.096 0.125

20.2 0.001 0.004

20.3 0.001

21 0.011 0.001 0.115 0.041 0.095 0.058

21.1 0.001

21.2 0.011 0.015

22 0.007 0.001 0.159 0.011 0.001 0.108 0.055

22.1 0.001

22.2 0.006 0.019

23 0.005 0.001 0.180 0.007 0.001 0.069 0.142

23.1 0.001

23.2 0.006 0.030

24 0.001 0.166 0.002 0.028 0.087

24.2 0.004 0.031

25 0.001 0.001 0.116 0.014 0.082

25.2 0.036

26 0.001 0.049 0.001 0.004 0.015

26.2 0.001 0.037

27 0.008 0.005 0.002 0.001

27.2 0.001 0.051

28 0.145 0.001

28.2 0.001 0.054

Appendix D: Allele Frequency Data for Calculation of Paternity Index

29 0.205

29.2 0.006 0.001 0.059

339

(continued)
Table D.1
340

(continued)

Genetic markers/loci tested

Alleles D3S1358 vWA D16S539 CSF1PO TPOX D8S1179 D21S11 D18S51 D2S441 D19S433 TH01 FGA D22S1045 D5S818 D13S317 D7S820 SE33 D10S1248 D1S1656 D12S391 D2S1338

30 0.001 0.173

30.2 0.031 0.001 0.075

30.3 0.003

31 0.035

31.2 0.103 0.037

32 0.006 0.002

32.2 0.185 0.021

33.2 0.003 0.085 0.009

34 0.001

34.2 0.008 0.001 0.005

35 0.001
Appendix D: Allele Frequency Data for Calculation of Paternity Index
 References 341

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Further Reading
Butler JM (2012) Advanced topics in forensic Jamieson A, Bader S (2016) A guide to forensic
DNA typing: methodology. Academic Press, DNA profiling. Wiley, Boston, MA. ISBN:
Amsterdam. ISBN: 978-0-12-374513-2 978-1-118-75152-7
Dash HR, Shrovastava P, Mohapatra BK, Das S Taupin JM (2013) Introduction to forensic DNA
(2018) DNA fingerprinting: advancements and evidence for criminal justice professionals. CRC
future endeavors. Springer, Singapore. ISBN: Press, Hoboken, NJ. ISBN: 978-1-439-89909-0
978-981-13-1582-4