FGB 5105; Molecular Genetics

- DNA Analysis

Dr. Md. Golam Sarower

Professor
Fisheries and Marine Resource Technology Discipline
Khulna University
DNA PROFILING
1980 - American researchers
discovered non-coding regions of
DNA
1984 - Professor Alec Jeffreys
developed the process of DNA
profiling at the University of
Leicester, UK

1987 - First conviction based

on DNA evidence
 Genetics Basics
• 100 trillion cells
• 23 pairs of chromosomes
• 99.9% identical between humans
• 0.1 % or 3 million bases accounts
for variation
• Est. 20,000 – 25,000 genes
• Up to hundreds of alleles per gene
(blue or brown eyes)
 Polymorphism = Variation
VNTR (minisatellites)
• Variable number of tandem repeats
• Repeat unit up to 25 bp (unit 20 kb)
ACAGGGTGTGGGGACAGGGTGTGGGG
vs
AGTAGTAGTAGTAGTAGT
STR (microsatellites)
• Short tandem repeats
• repeat unit less than 13 bp (unit <150 bp)
 Where do we get the DNA?

• Blood - extracted dry or wet (WBC)

• Buccal (cheek cells) – easy to obtain
• Semen – dried can be analyzed
years later
• Hair roots
• Saliva
• Skin cells
 DNA PROFILING
A process or technique of analysis
revealing unique patterns of an individual’s DNA
involving non-coding regions
NON-CODING REGIONS

(VNTR)

7
 STAGES INVOLVED
Cells broken down to
release DNA

DNA strands cut into

fragments

Fragments separated

Pattern of fragments
analysed
DNA is Tightly
Packaged into
Chromosomes
Which Reside
in the Nucleus
Model of
DNA

DNA is
Comprised
of Four
Base Pairs
Deoxyribonucleic Acid
(DNA)
 5’ phosphate
O
O P O
Base
O
CH2 O
Sugar

DNA O P O
O

Base
Schematic O
CH2 O
Sugar

OH
3’ hydroxyl
 DNA
Restriction
Enzymes
Evolved by bacteria
to protect against
viral DNA infection
Endonucleases =
cleave within DNA
strands
Over 3,000 known
enzymes
Enzyme Site
Recognition Restriction site

Palindrone

• Each enzyme digests

(cuts) DNA at a specific
sequence = restriction
site
• Enzymes recognize 4- or
6- base pair, palindromic
sequences
(eg GAATTC)

Fragment 1 Fragment 2
 Enzyme cuts
5 vs 3 Prime
Overhang

•Generates 5
prime overhang
 Common
Restriction EcoRI

Enzymes
– Eschericha coli
– 5 prime overhang

Pstl
– Providencia
stuartii
– 3 prime overhang
 Restriction enzymes

DNA molecules are very long

They may consist of millions of base pairs

In order to study the structure of DNA, the

molecules are broken up into smaller fragments
by enzymes called restriction enzymes

Restriction enzymes do not break up the DNA

molecule randomly but ‘cut’ it at particular sites
Restriction fragments

For example, a restriction enzyme called EcoR1*

‘recognises’ the base sequence CAATTC and cuts it
between the two As
recognised

--C-C-G-C-A-G-C-T-G-T-C-A-A-T-T-C-T-C-T-C-C-G-G-A-T-C-C-A

cut
--C-C-G-C-A-G-C-T-G-T-C-A A-T-T-C-T-C-T-C-C-G-G-A-T-C-C-A-

Other restriction enzymes cut the DNA in different places

and so produce fragments which are easier to analyse

--C-C-G-C-A-G-C-T-G-T-C-A A-T-T-C-T-C-T-C-C-G-G-A-T-C-C-C-A-

--C-C-G-C-A-G C-T-G-T-C-A A-T-T-C-T-C-C-G G-A-T-C-C-C-A-

Genetic fingerprinting
 90% or more of DNA does not carry nucleotide triplets that
code for proteins

 The non-coding DNA is often called ‘junk DNA’ but this only
means that its functions have not yet been discovered

 Some of the non-coding regions consist of repeated sequences

of nucleotides
For example -C-A-T-G-C-A-T-G-C-A-T-G-C-A-T-G- *

 The number of repeats in any one section of DNA varies from

one individual to the next

 Since these sections do not code for proteins (and, therefore

are not genes) there is no observable difference in these
individuals
Restriction enzymes
Particular repeat sequences can be ‘cut out’ by restriction
enzymes

For example
restriction enzyme cuts
here…………and…..….…..here

-CATCCACGACATGCATGCATGCATGCCACATCCA-
or

here…….…..…..………and…...….…………..here

-CCACGACATGCATGCATGCATGCATGCATGCCACAT-
The fragments cut by the restriction enzymes are called
restriction fragments

The fragments can be separated using gel electrophoresis

 Gel electrophoresis
 The different sized fragments are separated by a process
called gel electrophoresis

 The separation takes place in a sheet of a firm but jelly-like

substance (a ‘gel’)

 Samples of the DNA extracts are placed in shallow cavities

(‘wells’) cut into one end of the gel

 A voltage is applied to opposite ends of the gel

 DNA has a negative charge and moves slowly towards the

positive end

 The shorter fragments travel through the gel faster than the
longer fragments
 Agarose
Electrophoresis
Loading
• Electrical
current carries
negatively-
charged DNA
through gel
towards
positive (red)
Buffer
electrode
Dyes

Agarose gel

Power Supply
 Agarose
Electrophoresis
Running

• Agarose gel
sieves or moves
DNA fragments
according to size
– Small fragments
move farther
than large
fragments Gel running

Power Supply
Gel electrophoresis

DNA extract
added
well

gelatinous sheet

solution
Gel electrophoresis

DNA samples placed in

wells cut in gel
thin slab of
Voltage supply
gel
negative electrode

positive DNA fragments

electrode + Move from negative
To positive
A sample with the
shorter DNA fragments
travels through the gel
faster than a sample
with the larger fragments
Appearance of separated fragments on gel

These bands will

contain the shorter
DNA fragments

These bands will

contain the longer
DNA fragments

©©Prof.
Prof.E.J.Wood
E. Wood

starting positions
 Analysis of Stained Gel
Determine
restriction fragment
sizes

• Create standard curve

using DNA marker

• Measure distance
traveled by restriction
fragments

• Determine size of DNA

fragments

• Identify the related

samples
Molecular Weight
Fingerprinting Standard Curve: Semi-log
Determination 100,000

Size (bp) Distance

(mm)
23,000 11.0 10,000

Size, base pairs

9,400 13.0 B

6,500 15.0
1,000

4,400 18.0

2,300 23.0
100
A
2,000 24.0 0 5 10 15 20 25 30
Distance, mm
 • Restriction Buffer provides
The DNA optimal conditions
Digestion
• NaCl provides the correct
Reaction ionic strength

• Tris-HCI provides the

proper pH

• Mg2+ is an enzyme co-factor

 Why incubate at 37°C?
DNA • Body temperature is optimal for
Digestion these and most other enzymes

Temperature What happens if the temperature is

too hot or cool?

• Too hot = enzyme may be

denatured (killed)

• Too cool = enzyme activity

lowered, requiring
longer digestion time
 DNA TRANSFER
DNA split into single
strands using alkaline
solution
DNA fragments transferred
from gel to filter paper or
nylon membrane
(This is called Southern
blotting)
Gel, with filter paper
attached, is removed &
separated
 ANALYSIS

Radioactive probe in Revealing a pattern of

solution binds to DNA bands

X-ray film
HOW DNA PROBES WORK
 DNA PROFILE
Puppy (Off spring)

Sire (Father)
Dam (mother)
 SOME APPLICATIONS

Forensic Science

Family Relationships

Health Care
Genetic fingerprinting
 DNA analysis can be used for catching criminals, establishing
parentage, finding how closely organisms are related and
many other applications.

 The pattern of bands in a gel electrophoresis is known as a

genetic fingerprint or a ‘genetic profile’

 If a genetic fingerprint found in a sample of blood or other

tissue at the scene of a crime matches the genetic
fingerprint of a suspect, this can be used as evidence

 A DNA sample can be obtained from the suspect using

blood, cheek epithelial cells taken from the mouth lining or
even the cells clinging to the root of a hair
Chances of a match Suppose that…………

….there is a chance of 1 in 10 that this

fragment occurs in many individuals…

…and.there is a chance of 1 in 20 that this

fragment occurs in many individuals…

…and.there is a chance of 1 in 10 that this

fragment occurs in many individuals…

…and.there is a chance of 1 in 30 that this

fragment occurs in many individuals, but…
Probability of a match

…the probability of all 4 bands matching in any person other

than the suspect is

1 in 10 x 1 in 20 x 1 in 10 x 1 in 30

= 1 in 10 x 20 x 10 x 30 That is 1 in 60,000

When a larger number of bands is involved, the probability that

the suspect is not guilty becomes one in many thousands*
V S S1 S2 S3
DNA profiles
V Victim

S Sample from crime scene

S1 Suspect 1

S2 Suspect 2

S3 Suspect 3

More than 20 fragments

from Suspect 1 match those
taken from the crime scene
Evidence from genetic fingerprinting
 Genetic fingerprinting is powerful evidence in criminal trials but…

 Many restriction fragments may be crowded into a single band

 There may be variability in the speed with which a fragment travels

through the gel

 There is a chance of contamination with ‘foreign’ DNA e.g. from bacteria

 The jury may not understand the significance of genetic fingerprinting

and may be dependent on conflicting claims from ‘expert’ witnesses

 There may be arguments about the statistical significance of a match

between DNA profiles
Limitations of DNA evidence

 Even if there is agreement about a match between the suspect’s DNA

profile and forensic samples, it shows only that the suspect was
present at the scene of the crime and does not prove that he or she
committed the crime

 DNA evidence should be considered as conclusive proof of guilt only

if there is other supporting evidence

 In cases of paternity disputes, the genetic evidence can be conclusive

 Paternity can be decided on the basis of a single restriction site

Paternity test
mother father

position of part of DNA strand

restriction
fragment

child

Child will receive one copy of the restriction fragment from the
mother and one from the father. It could be any one of these
combinations
 Genetic fingerprint of …
1 mother
2 child

3 possible father A
4 possible father B

There is a match between one of

the child’s restriction fragments
and one of the mother’s.

There is also a match between the

child’s other fragment and one from
possible father A.

Neither of the child’s restriction

1 2 3 4 fragments match those of possible
father B
Starting position of sample
FAMILY RELATIONSHIPS

46
 21
The Human Genome Project

An organism’s genome is its entire genetic make-up

The genome includes …

all the chromosomes
all the genes on the chromosomes
and all the DNA of the chromosomes
The human genome project set out to …
identify the genes in the human genome (about 25,000*)
discover the sequence of the base pairs (about 2.8 billion)

99% of the gene-containing part of human DNA had been

analysed by 2003
Human genome project
 22
Mapping is the identification of genes and their positions in
the chromosome

Modern biochemical techniques are used to identify genes

and their positions in the chromosome

Special staining methods reveal bands in the chromosomes

These do not necessarily represent genes but help to identify
the position of genes

Chromosome 7

position of gene for cystic fibrosis

Chromosome 11

position of gene for sickle cell anaemia

Mapping
Giant chromosomes 23

By special staining techniques, bands appear in

the ‘giant chromosome’ of the fruit-fly (Drosophila)

© Biophoto Associates
The bands do not necessarily represent genes but if, in mutant
flies, some of the bands are missing, there is a corresponding
defect in the fly

If bands are missing from this If this band is missing there

region, the fly has no colour is an irregularity in the wing
in its eyes (normally red)

Loss of this band leads to a change One or more of the bands in

in the texture of the eye surface this region controls the normal
development of bristles
Sequencing
Sequencing aims to find out the sequence of nucleotides in a
stretch of DNA
The process can be automated to give results relatively quickly

Analysis of a small piece of DNA might give results something

like this
GCTTATCGATTCGGTGATACCATAGTGTAGTGTAGTCGCT
ATCCATCGCTTACGAGTCTGATGCGCATTAGCTAGCTAGCT
AGCTAGCCTCAGTTGATCATCGAGTGAGTACTGGACCATGC

Further analysis is needed to decide which sequences code for

proteins and represent genes
Applications of results from the Human Genome Project

It is hoped that a knowledge of the human genome will enable…

identification of defective genes and the chance of early treatment

identification of genes which could make a person susceptible to

certain diseases, and so lead to preventative measures

prediction of the proteins that genes produce, giving the opportunity

to enhance or inhibit these proteins by specially designed drugs

Among the possible drawbacks are the possibilities that …

insurance companies may refuse cover for people at risk of
developing a genetic disability or disease
prediction of a disease or disability could blight a person’s life
 Famous cases
• In 2002 Elizabeth
Hurley used DNA
profiling to prove that
Steve Bing was the
father of her child
Damien
 Famous Cases
• Colin Pitchfork was
the first criminal
caught based on DNA
fingerprinting
evidence.
• He was arrested in
1986 for the rape and
murder of two girls
and was sentenced in
1988.
 Famous Cases
• O.J. Simpson was
cleared of a double
murder charge in
1994 which relied
heavily on DNA
evidence.

• This case highlighted

lab difficulties.