Human DNA forensics

1. DNA Fingerprinting & Its
Applications
Dr. Abdul Hameed, Ph.D
Chief Scientific Officer
Institute of Biomedical & Genetic Engineering,
Islamabad, Pakistan
ahameed0786@hotmail.com

2. Also known as,
DNAFingerprinting
 DNA profiling
 DNA testing
 DNA typing
 Genetic fingerprinting

3. What is DNA
Fingerprinting?
 A technique used by scientists
to distinguish between
individuals of the same
species using only samples of
their DNA

4. Who Invented it?
 The process of DNA
fingerprinting was
invented by Alec
Jeffreys at the
University of Leicester
in 1985.

5. DNA
Our bodies are composed of cells.
We have approximately 70 trillion
cells in our bodies, of different
shapes and sizes according to their
function.
All of them have something in
common: they contain genetic
material called DNA
(deoxyribonucleic acid).
Islamabad Police- June 2018

6. DNA
The cells have central
circumscribed zone called
nucleus that contains
most of our DNA. This
DNA is organized in
discrete units called
chromosomes.
We inherit half of them
from our father and the
other half from our
mother.
Except for the Y-
chromosome: only in
males.
Outside of the nucleus there is the
smaller mitochondrial DNA. It is
inherited ONLY from our mothers.

8. Genetic Variation Types
 Mini-satellites - repeated sequences, 10–100 base pairs
 Short Tandem Repeats (STRs) – repeated sequences, 2–9 base pairs
 Single Nucleotide Polymorphisms (SNPs) - Single Nucleotide — A, T, C or G —
in the genome differs between members of a biological species or paired
chromosomes.
...CCTGACTTAGGATTGCCA...

9. Inheritance of autosomal,
Y-Chromosomal and mtDNA
Variations

10. Mode of inheritance: biparental
Applied to
Paternity
Kinship
Family search
Criminal databases

11. Uniparental inheritance:
Y chromosome
Y-chromosome: ONLY IN MALES
Transmitted as a single block of
genetic information from fathers
to sons.
Applications:
Pedigree, clans (paternal lineage).
Paternity
Familial search
Rape cases

12. Uniparental inheritance:
mitochondrial DNA
mtDNA: transmitted as a single block of genetic
information from mothers to all their children.

13. Genetic profiling
DNA markers
Our genome
Markers
(DNA fragments defined by a
criteria for their utility,: e.g.
how diverse they are, etc)
Application
Autosomal STRs (Short Tandem Repeats)
Autosomal SNPs (Single Nucleotide Polymorphisms)
Autosomal indels (insertion deletion polymorphisms)
Y- STRs (Short Tandem Repeats)
Y- SNPs (Single Nucleotide Polymorphisms)
Individual ID ( kinship)
Control region
sequence information
mtDNA SNPs
Individual ID (sex. assault,
. genealogy studies)
Lineage ID (ancestry studies)
Individual ID ( kinship, mass
disasters)
Lineage ID
(mass disasters, ancestry studies)

14. The Forensic DNA process:
collection of evidence
Source: The DNA project SA Classification and storage
Submission to relevant labs/
institutions for analysis
Storage policies.
Genetic profiling
Reporting
Crime scene
Reference material
Suspect
Islamabad Police- June 2018

15. We’ve got DNA.
Now what ?
• Identify a clear question
• Choose DNA markers according to the question.
• Criteria: type of inheritance, resistance to degradation
etc.
Not all DNA in the genome is equally informative.
Some DNA markers are more efficient for
o kinship and paternity investigations,
o degraded DNA situations e.g. mass disasters, and other
o for inference of ethnic ancestry, or geographic origin.
Islamabad Police- June 2018

16. Blood
Semen
Saliva
Teeth
Bone
Urine
Muscle
Hair (w/Root &Shaft)
Common Biological Evidence
DNA Profiling always starts with a biological sample

17. DNA extraction
Organic DNA extraction
Chelex-100 DNA extraction
Two step DNA digestion

18. Differential DNA Extractions
• Used when sperm is present in a sample
• Designed to separate sperm cells from
non-sperm cells
• One sample becomes split into two
different fractions (sperm and non-sperm)
• Involves additional washing steps
• After washing, fractions receive the same
purification and concentration steps

19. Perpetrator’s sperm
mixed with victim’s
epithelial cells
Centrifuge
REMOVE
supernatant
SDS, EDTA and
proteinase K
(cell lysis buffer)
Remove a
portion of
the mixed
stain
SDS, EDTA and
proteinase K + DTT
Incubate
at 37 oC
sperm
pellet
DTT lyses
sperm
heads
“Sperm
Fraction”
“Epithelial/Non-
sperm Fraction”
sperm
pellet
Figure 3.2, J.M. Butler (2005) Forensic DNA Typing, 2nd Edition © 2005 Elsevier Academic Press
Differential DNA Extraction

20. The cell and nuclear membrane
of sperm contain abundant
disulfide bond, to destory them
need a special chemical
substance named DTT .

21. Forensic Science: Fundamentals & Investigations, 2e
Chapter 7
All rights Reserved
Cengage/NGL/South-Western © 2016
Early DNA Fingerprinting
Using Gel Electrophoresis
o In DNA fingerprinting, DNA is isolated and
cut using restriction enzymes, creating
fragments of DNA called restriction
fragments.
o Each person's length and number of DNA
restriction fragments differs.
21

22. RFLP Based DNA Fingerprinting

23. Modern DNA Fingerprinting
• Gel electrophoresis has been replaced by the
use of STR analysis, which analyzes shorter
pieces of DNA.

24. How do we study biparental DNA
variation?
Islamabad Police- June 2018
• The most common DNA fragments (markers) used in individual
ID are called microsatellites or STRs (Short Tandem Repeats)
GATA GATA GATA GATA
GATA GATA GATA GATA
GATA GATA GATA
GATA GATA GATA GATA GATA
Individual 1
Profile = 4-4
Individual 2
Profile = 3-4
Polymerase Chain Reaction (PCR)
A molecular “ photocopy machine”

25. STR
• Short tandem repeat
• Describes a type of DNA polymorphism in which:
– a DNA sequence repeats
– over and over again
– and has a short (usually 4 base pair) repeat unit
• A length polymorphism -- alleles differ in their length
5 repeats: AATG AATG AATG AATG AATG
6 repeats: AATG AATG AATG AATG AATG AATG
4 repeats: AATG AATG AATG AATG
3 repeats: AATG AATG AATG

26. Analyze Raw Data
Conclusion:
Providing the
authentication
report
1 2 3
4
5
6
DNA Extraction
In the laboratory
Multiplex
PCR Amplification
Capillary
Electrophoresis
Collect Biological
Evidence at the
crime scene
The whole Process of Modern DNA
Fingerprinting

27. Extract and Purify DNA

28. Definition of PCR
Polymerase chain reaction (PCR) enables
researchers to produce millions of copies
of a specific DNA sequence in vitro in
approximately two hours.

29. Set up PCR Reaction-What do you need?
5’
3’
5
’
3
’
3
’
5
’
3
’
5
’
Forward
primer
Reverse
primer
Target
Buffer containing salts (ions) Magnesium chloride (enzyme cofactor)
Nucleotides(dNTPs)
Primers(Sequence-specific
primers flanking the target
sequence)
DNA
Polymerase
Template DNA(containing
the STR gene locus you
want to amplify for the
study

30. Denaturation
Annealing
Extension
The PCR programe-How does it work?
(denature double strands DNA)
（anneal primers to template）
（activate Taq DNA polymerase, which
extends primers and replicates DNA）

31. Make copies
(extend primers)
Starting DNA
Template
5’
5’
3’
3’
5’
5’
3’
3’
Add primers
(anneal)
5’
3’
3’
5’
Forward primer
Reverse primer
DNA Amplification with the
Polymerase Chain Reaction (PCR)
Separate
strands
(denature)
5’
5’
3’
3’

32. In 32 cycles at 100% efficiency, 1.07 billion
copies of targeted DNA region are created
PCR Copies DNA Exponentially through
Multiple Thermal Cycles
Original DNA target region
Thermal cycle
Thermal cycle
Thermal cycle

33. Multiplex PCR
• 15 STR Markers Can Be
Amplified in 1 reaction.
• Sensitivity = less than 250
pg of DNA.
• Different Fluorescent Dyes
Used to Distinguish STR
Alleles with Overlapping
Size Ranges.

34. The principle of capillary
electrophoresis

35. If the PCR products have the same
length , primers are labeled with
different fluorescent dyes
Different length of PCR products,
primers are labeled with same
fluorescent dye
The principle of
separation

36. Running PCR fragments on
Genetic Analyzer
• We mix 1ul of PCR product, 1ul of size
standard in 18 ul of Hi-Di formamide.
• Heat denature at 95oC for 5 minutes. (Samples
and allelic ladder)
• Chill quickly on ice and load on to the genetic
analyzer.

37. The ABI 310 Genetic Analyzer

38. ABI 310 Genetic Analyzer: Capillary Electrophoresis
•Amplified STR DNA injected
onto column
•Electric current applied
•DNA separated out by size:
– Large STRs travel slower
– Small STRs travel faster
•DNA pulled towards the
positive electrode
•Color of STR detected and
recorded as it passes the
detector
Detector
Window

39. Analysis of Raw Data
• The Raw data files generated by the genetic
analyzer to generate DNA profiles are further
analyzed using software, such as:
• GeneMapper version 3
• GeneMapper version 5

40. Profiler Plus: Raw data

41. D3 vWA FGA
D8 D21 D18
D5 D13 D7
Am
RAW DATA
PROCESSED DATA
•GENESCAN divides the raw data into a
separate electropherogram for each
color:
•Blue
•Green
•Yellow
•Red
•GENOTYPER identifies the different
loci and makes the allele calls
•The type of this sample is:
–D3: 16, 17
–vWA: 15, 15
–FGA: 21,23
–Amelogenin: X, Y
–D8: 16, 16
–D21: 28, 29
–D18: 14, 19
–D5: 8, 12
–D13: 11, 13
–D7: 10 10

42. Short Tandem Repeats (STRs)
the repeat region is variable among samples while the flanking
regions where PCR primers bind are constant
7 repeats
8 repeats
AATG
Homozygote = both alleles are the same length
7 8
Heterozygote = alleles differ and can be resolved from one another
8 repeats
8 repeats 8

43. Reading an electropherogram
Peaks correspond to alleles
Electropherogram
D3 vWA FGA
D8 D21 D18
D5 D13 D7
BLUE
GREEN
YELLOW
RED
Amelogenin
Amelogenin
XX = female
XY = male
75 100 139
150
160
200 245 300 bps
Red = ROX size standard

44. Reading an electropherogram
L
A
R
G
E
S
M
A
LL
NUMBER OF PEAKS
– 1 peak = homozygous
– 2 peaks = heterozygous
– 3 or more peaks = mixed sample
(?)
POSITION OF PEAK
– Smaller alleles on left
– Larger alleles on right
HEIGHT OF PEAK
– Proportional to amount of allele
(approx)

45. Statistical estimates: the product rule
0.222 x 0.222 x 2
= 0.1

46. Statistical estimates: the product rule
= 0.1
1 in 79,531,528,960,000,000
1 in 80 quadrillion
1 in 10 1 in 111 1 in 20
1 in 22,200
x x
1 in 100 1 in 14 1 in 81
1 in 113,400
x x
1 in 116 1 in 17 1 in 16
1 in 31,552
x x

47. “The chance of a coincidental
match is one in 80 quadrillion?”

48. 48

49. Familial Relationships and DNA
Profiles
• Paternity Testing
– Analyze samples from child and adults involved
– Is this man the father of the child?
Mother
Child
Father

50.  Crime scene
 Human relatedness
Paternity
 Animal relatedness
 Anthropology studies
 Disease-Causing
 Food identification
 Human remains
 Monitoring transplants
DNA Fingerprinting
Applications

51.  Crime scene
 Human relatedness
Paternity
 Animal relatedness
 Anthropology studies
 Disease-Causing
 Food identification
 Human remains
 Monitoring transplants
DNA Fingerprinting Real
World Applications

52. 2. Brief introduction to DNA forensics.
DNA markers. Crime scenes.
DNA forensics is the specialized field of forensics that
used DNA evidence in criminal investigations.
These include paternity disputes, kinship analysis, rape,
identification of missing people, mass graves, mass
disasters, crime scene genotyping, cold cases, etc, and the
novel non-traditional developments for “profiling without
a suspect”.
Many of these applications are regulated by law, e.g.
“phenotyping” is not allowed in most countries.
Islamabad Police- June 2018

53. © 2013 Pearson Education, Inc.
Putting DNA to Use

54. Food Identification

55. The detection of pork in
processed food
L 1 2 3 4 5 6 7 8 9 10 11 12 13
14
BseD1 restriction profile of cyt b PCR products amplifiedfrom meat and food
samples. L: 100 bp DNA ladder; 1: chicken, 2: beef; 3: mutton; 4: pork;
5, 6, 7: beef burgers; 8, 9, 10: chicken burgers; 11, 12, 13: sausages; 14: pork cocktail