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Forensic Science DNA Testing

Biologia Molecular

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Albino Velasquez
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© © All Rights Reserved
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67 views

Forensic Science DNA Testing

Biologia Molecular

Uploaded by

Albino Velasquez
Copyright
© © All Rights Reserved
Available Formats
Download as PPT, PDF, TXT or read online on Scribd
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Forensic Sciences: DNA testing

The application of a broad spectrum of sciences to


answer questions of interest to the legal system.

Latent Print analysis


Toxicology
Crime Scene
Photography
Document analysis
Blood Spatter analysis
Ballistics

Fiber analysis
Arson
Explosives
DNA
Computer
Other

Fingerprints
Palm prints
Footwear and tire impressions
Other ears, lips, etc.

Blood alcohol, urinalysis, poisons


Blood, urine, organs, tissue, vitreous humor

Accurate and complete documentation of scene and evidence


Establish spatial locations, conditions, scale

Analysis of inks using Raman Spectroscopy.

Comparison of inks, paper, printers, copiers, and handwriting

Physics of flight, trigonometry used to determine origin point of blood


Size and orientation of spatters can determine method by which stains are
created

Polymer analysis on a pyrolysis gas


chromatograph

Fibers have distinct color, diameter, shape, and chemical composition


Microscopic and chemical analysis to compare

Direct comparison of known samples and unknowns from crime


Striations or firing pin impressions
Also used to do tool-mark comparisons (screwdrivers, etc.)

Search for chemical signs of accelerants (gasoline, etc.)


Test burn scenarios

Search for unique chemical traces or bomb-making materials


Look for evidence from makers of bomb (DNA, fingerprints)

Serology (body fluids)


Drug analysis (marijuana, cocaine, meth)
Anthropology
Pathology (medical examiner)

Used Since?

Identification Method

Accuracy?

1800

Measurement of height

1 in 4

(Qutelets method)
Comparison of Pubic hair

1 in 800

Late 1800s Early 1900s

Comparison of Scalp hair

1 in 4500

Late 1800s early 1900s

Anthropometry

1 in 268 million

(Bertillons method)
Forensic odontology
Teeth bite marks

1 in 2.5 billion

Evidence in Early Egypt


documented forensic use 1800s
-1900s

Dactylography

Late 1900s

DNA Fingerprinting

1 in 2 x 1022

Late 1900s early 2000s

Facial recognition

(Fingerprints)

http://lifeloom.com/I2Aggrawal.htm and

1980 - Ray White describes first polymorphic


RFLP marker
1985 - Alec Jeffreys discovers multi-locus
VNTR probes
1985 - first paper on PCR
1988 - FBI starts DNA casework
1991 - first STR paper
1995 - FSS starts UK DNA database
1998 - FBI launches CODIS database

Unique identifying
characteristics

DNA
RNA
Protein

What regions of DNA would you expect to use for identification?


- Regions from genes expressing proteins?
- Other regions?
- Why

Restriction Fragment Length


Polymorphism (RFLP)
PCR Analysis
STR Analysis
Mitochondrial DNA Analysis
Y-Chromosome Analysis

Less than 2% codes for Proteins


50% of the genome contains repeated
sequences
No apparent function
Recombination?
Formation of new genes?
Types of repeated DNA
Tandomly repeated
Telomeres
Satellite (VNTRs)
Minisatellite (STRs)
Interspersed repetitive DNA
SINES (Alu sequences)
LINES
Transposable elements

DNA in the Cell

Chromosome
cell nucleus

double-stranded
DNA molecule

Target Region for PCR

Individual
nucleotides

Short Tandem Repeats (STR) are repetitive


sequences:

Tetranucleotide:
Trinucleotide:
Dinucleotide:

AAAG AAAG AAAG AAAG


CTT CTT CTT CTT CTT
AG AG AG AG AG AG

Tetranucleotides are favored in human identity

Good balance of ease of interpretation and variability


found in nature

1 aattgagcnc aggagtttaa gaccagcctg ggtaacacag tgagacccct gtctctaca


61 aaaaatacaa aaatnagttg ggcatggtgg cacgtgcctg tagtctcagc
tacttgcagg 121 gctgaggcag gaggagttct tgagcccaga aggttaaggc
tgcagtgagc catgttcatg 181 ccactgcact tcactctgag tgacaaattg
agaccttgtc tcagaaagaa agaaagaaag 241 aaagaaagaa agaaagaaag
aaagaaagaa agaaagaaag aaaaagagag ggaaagaaag 301 agaaanagna
aanaaatagt agcaactgtt attgtaagac atctccacac accagagaag 361
ttaattttaa ttttaacatg ttaagaacag agagaagcca acatgtccac cttaggctga
421 cggtttgttt atttgtgttg ttgctggtag tcgggtttgt tatttttaaa
gtagcttatc 481 caatacttca ttaacaattt cagtaagtta tttcatcttt
caacataaat acgnacaagg 541 atttcttctg gtcaagacca aactaatatt
agtccatagt aggagctaat actatcacat 601 ttactaagta ttctatttgc
aatttgactg tagcccatag ccttttgtcg gctaaagtga 661 gcttaatgct
gatcgactct agag

The repeat sequence is aaga this particular individual has 14 repeats

Locusthe physical
position of an STR and its
associated flanking
sequence

Both chromosomes of a
homologous pair contain
this locus

The allele contained on either chromosome can be the same


or different lengths (homozygous or heterozygous)

TPOX

The PowerPlex 16
System amplifies
16 loci.

D3S1358

D5S818

FGA

CSF1PO

D7S820

D13S317

D8S1179

Penta E

THO1

D16S539

D21S11
Penta D

vWA

Amelogenin

D18S51

Amelogenin

Short Tandem Repeats (STRs)


AATG

~
7 repeats

~
8 repeats

Repeat region is variable (polymorphic)


Each variant is referred to as an allele

Flanking region is constant


KEY: Alleles are distinguished by length
Homozygote = both alleles are the same length
Heterozygote = alleles differ and can be resolved from one another

DNA Amplification with PCR


5

3
3

Forward primer

5
5

Starting DNA
Template

Separate strands
(denature)

3
Add primers
(anneal)

Reverse primer

Make copies (extend


primers)
5

Exponential Amplification with PCR


Original DNA target region

Thermal cycle

In 32 cycles at 100% efficiency, 1.07


billion copies of amplicon are made.
http://www.cstl.nist.gov/div831/strbase/

Multiplex PCR

16 Loci Are Copied at


Once
Sensitivities to levels less
than 0.5 ng of DNA
Ability to Handle Mixtures
and Degraded Samples
Different Fluorescent Dyes
Used to Distinguish STR
Alleles with Overlapping
Size Ranges

Separating and Seeing STRs

Electrophoresis

Separates amplification products based on size

Fluorescent detection

Amplification products have a fluorescent label


attached to the primer
Label is seen through excitation via a laser and
corresponding emission captured with a camera
377

310

3130

100 bp

200 bp

D3

TH01

D5
A

100

300 bp

D21

D13

vWA

D7
D8

200

400 bp

D18
D16
TPOX

300

CSF

500 bp

600 bp

500

600

Penta E
Penta D
FGA

400

ILS600 Size Standard

100

200

300

400

500

A sizing standard is used in all samples and allelic ladders


The known standard is used to determine the size of the allelic ladders
and the unknown samples

600

Allelic Ladder

Allele Calls

Allele
possibilities

Hypothetical
likelihood of
occurrence
1 locus: 1 in 18
2 loci: 1 in 360
3 loci: 1 in 18000
4 loci: 1 in 792000
9 loci: ~1 in 1010
16 loci: ~1 in

1017

Current World Pop:


~6.3 billion

Sample
Genotype

1 in 1 in 1 in
18 X 20 X 50

1 in
44

Controls

STR Allelic Ladders

Negative control devoid of amplification products


Compare positive control 9947a with locus-specific
ladder
Comparison with samples allows precise assignment of
alleles

Fluorescent Ladder (CXR)

Internal Size Standard

Forensic cases: matching suspect with


evidence
Paternity testing: identifying father
Convicted felon DNA databases
Missing persons investigations
Mass disasters -- putting pieces back together
Historical investigations
Military DNA dog tag

Complete STR Profile

DNA from Small Stains/challenging samples

0.1l blood stain on denim

1/5 of eluted material used for amplification

Most are rape cases (>2 out of 3)


Looking for matches between evidence,
victim, and suspect
Must compare DNA profiles

Challenges

Mixtures must be resolved if present


DNA is often degraded
Inhibitors to PCR and sample
contamination are often present

Steps in DNA Sample Processing


Sample Obtained
from Crime Scene or
Paternity Investigation

Biology
DNA
Extraction

DNA
Quantitation

PCR Amplification
of Multiple STR markers

Technology
Separation and Detection of PCR
Products
(STR Alleles)

Comparison of Sample
Genotype to Other Sample
Results

Genetics
If match occurs, comparison of
DNA profile to population
databases

Sample Genotype
Determination

Generation of Case Report


with Probability of Random
Match

Sources of Biological Evidence


Blood
Semen
Saliva
Urine
Hair
Teeth
Bone
Tissue

Samples can have extremely small amounts of DNA


Available Technologies for DNA Isolation
Phenol:Chloroform Extraction (Homebrew)
Chelex (ReadyAmp)
FTA Paper
Qiagen
DNA IQ System
DNA IQ Reference Sample Kit for Maxwell 16

Forensic labs in the US are required by law to


quantitate the amount of Human DNA in crime
scene samples
How might this be done?

Forensic

FBI
Standards for Combined DNA Index System (CODIS) labs
http://www.fbi.gov/hq/lab/codis/index1.htm

The Scientific Working Group for DNA Analysis


Methods(SWGDAM) publishes guidelines

Paternity

American Association of Blood Banks (AABB)


http://www.aabb.org

Analysis based on
population statistics and
data
Probability that the
evidence matches the
suspect

http://www.cstl.nist.gov/biotech/strbase/intro.htm
(some information in this presentation is from this
ppt)
http://www.promega.com/applications/hmnid/
(Promega Human identity testing products)
http://www.promega.com/profiles/ (Profiles in DNA)
http://journalsip.astm.org/JOURNALS/FORENSIC/jof
s_home.html (Journal of Forensic Science)
http://appliedbiosystems.com Supplier of Human
identification systems

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