FORENSIC

DNA
ANALYSIS
Zhaoshu ZENG, PhD, Vice Professor
Department of Forensic Medicine
Zhengzhou University

法医学教研室曾昭书博士
Tel: 63998705
E-mail: zzs@zzu.edu.cn
March, 2008
CONTENTS OF THIS LESSON
 Short introduction to forensic DNA analysis
 Famous cases involving forensic DNA analysis

 The basis of forensic DNA analysis: human

genome polymorphism
 STR introduction
 Sequence characteristics
 Nomenclature
 Technical analysis
 Automation
 Forensic usages
 DNA fingerprints
 Samples preparing for DNA testing
BRANCHES OF FORENSIC MEDICINE
 Forensic Pathology
 Forensic Biology

 Forensic Clinical Medicine

 Forensic Psychiatry

 Forensic Toxicology
 FORENSIC BIOLOGY
 It is a subject involving identifying the sources of
bloodstain, serum, semen, DNA, and so on.
 The identification of blood or semen on dried, stained
materials and subsequent individualization and
comparison to known persons for inclusion or
exclusion are the major functions of the Forensic
Biologists.
 One more important aspect is to identify the true
biological parents for a child.
 It can also be used to deal with the catastrophe such
as the persons died in plane crash and tsunami
 Many famous cases involved the DNA analysis, such
as Clinton-Lewinsky Sex Scandal
 Forensic DNA: A very specialized area

 The examination of DNA has become predominantly the province

of the pure forensic scientists.

 However, this field offers many ways in which the

young scientists can apply standard methods and
scientific disciplines. The field is open to new
concepts and offers unlimited potential for physical,
biological, and social scientists to participate in
service and educational and research activities in an
important and practical area of community service.
BRIEF HISTORY OF FORENSIC DNA
TYPING
 1980 - Ray White describes the first
polymorphic RFLP marker
 1985 - Alec Jeffreys discovers
multilocus 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

DNA: FOR HUMAN IDENTITY
TESTING
 Forensic cases -- matching suspect with
evidence
 Paternity testing – identifying the alleged
father
 Historicalinvestigations
 Missing persons investigations

 Mass disasters -- putting pieces back

together
 Military
DNA “dog tag”
 Convicted felon DNA databases
Part 2 : THE POWER OF DNA

Famous Cases
FAMOUS CASES 1: T. JEFFERSON AND
E. HEMINGS, A BRIEF REVIEW.

 Thomas Jefferson (1743-1826) , the 3rd President of U.S.

(1801-1809).
 Sally Hemings (1773-1835) was a slave at Monticello; she
lived in Paris with Thomas Jefferson and two of his
daughters from 1787 to 1789; and she had at least six
children.
 Sally Hemings' duties included being a nursemaid-
companion to Thomas Jefferson's daughter Maria (c. 1784-
1787), lady's maid to daughters Martha and Maria (1787-
1797), and chambermaid and seamstress (1790s-1827).
 According to contemporary accounts, some of Sally
Hemings' children strongly resembled Thomas Jefferson.
Male-line descendants: T. JEFFERSON AND E. HEMINGS

Randolph
Jefferson
 The study - which tested Y-chromosomal DNA samples from
male-line descendants of the Jefferson’s family and the Hemings’
family - indicated a genetic link between the Jefferson and
Hemings descendants.
 Shortly after the DNA test results were released in November
1998, the Thomas Jefferson Foundation formed a research
committee consisting of nine members of the foundation staff,
including four with Ph.D.s. In January 2000, the committee
reported its finding that the weight of all known evidence - from
the DNA study, original documents, written and oral historical
accounts, and statistical data - indicated a high probability that
Thomas Jefferson was the father of Eston Hemings, and that he
was perhaps the father of all six of Sally Hemings' children listed
in Monticello records - Harriet (born 1795; died in infancy);
Beverly (born 1798); an unnamed daughter (born 1799; died in
infancy); Harriet (born 1801); Madison (born 1805); and Eston
(born 1808).
DISPUTE EXISTS
 Since then, a committee commissioned by the
Thomas Jefferson Heritage Society, after reviewing
essentially the same material, reached different
conclusions, namely that Sally Hemings was only a
minor figure in Thomas Jefferson's life and that it is
very unlikely he fathered any of her children. This
committee also suggested in its report, issued in
April 2001, that Jefferson's younger brother
Randolph (1755-1815) was more likely the father of
at least some of Sally Hemings' children.
 Because the evidence is not definitive, DNA can’t
tell the father is either Thomas Jefferson or
Randolph Jefferson.
FAMOUS CASE 2: Y CHROMOSOME
MARKERS USED TO IDENTIFY SADAAM
HUSSEIN

House of Uday and Qusay in Mosul, Iraq destroyed by US forces

 Saddam Hussein’s capture was verified with DNA
testing conducted in Rockville, MD at the Armed
Forces DNA Identification Laboratory on
December 14, 2003.
Part 3: DNA IN THE CELL
 Chromosomes consist of A, T, C and G.
HUMAN GENOME
APPROX. 3.2 BILLION BASE PAIRS IN
GENOME
 The vast majority of DNA is the same from
person to person (99.9%);

 Only very small DNA sequence variations

exists from person to person (0.1%).
--This is called DNA polymorphism
POLYMORPHISM:
DEFINITION BY ENCYCLOPEDIA BRITANNICA

 In genetics, the existence of two or more forms that are

genetically distinct from one another but contained within
the same interbreeding population. The polymorphism may
be transient or it may persist over many generations, when
it is said to be balanced. Classical examples of
polymorphisms are the presence or absence of banding in
Cepaea snails, the number of spots on the wings of
ladybirds, and eye colour in humans. All these are visible
polymorphisms that can readily be seen in nature. Some
polymorphisms, however, are cryptic and require
biochemical techniques to identify phenotypic differences.
Such techniques include gel electrophoresis of enzymes and
other proteins, and the fragmentation of the DNA molecule
by restriction enzymes (which allows the sequencing of
nucleotides), both of which operate nearer to the level of the
genotype.
TWO TYPES OF POLYMORPHISM

~80%

~20%
STR POLYMORPHISM
 Short tandem repeats (STR) is a kind of Length polymorphism
THE NOMENCLATURE OF A STR
 D3S1358:
 D: Number of Chromosome;

 S: Number of unique sequence with repeats

found from the genomic DNA database.
 Allele is named after

its core repeat number.

 For example, DXS6800

has 5 alleles: 7, 8, 9, 10, 11
Part 4:

The Analysis of STR

ANALYSIS PROCEDURES
 Target Region For PCR
chromosome

cell nucleus

Double stranded
DNA molecule Target Region for PCR

Individual
nucleotides
 DNA Amplification with the
Polymerase Chain Reaction
(PCR)
5’ 3’
5’ 3’
Starting DNA
Template
3’ 5’
3’ 5’

Separate
strands
(denature)

Forward primer
Add primers 5’ 3’
5’ 3’
(anneal)
Make copies
(extend primers) 5’
3’ 5’ 3’
Reverse primer
DENATURE, ANNEAL AND EXTENSION
PCR SCHEME

Denaturation
PCR Copies DNA Exponentially
through Multiple Thermal
Cycles

Original DNA target region

Thermal cycle

In
In32
32cycles
cyclesatat100%
100%efficiency,
efficiency,1.07
1.07billion
billioncopies
copiesofof
targeted
targetedDNA
DNAregion
regionare
arecreated
created
 SHORT TANDEM REPEATS (STRS)
AATG

7 repeats

8 repeats

the repeat region is variable between samples while the flanking

regions where PCR primers bind are constant

Homozygote = both alleles are the same length

Heterozygote = alleles differ and can be resolved from one another
STR-PCR
 195 170
170bp
bp
195bp
bp

TCAT
TCATrepeat
repeatunit
unit

Different primer sets produce different PCR product sizes for

the same STR allele
CSF1PO
Polyacrylamide Gel Electrophoresis
(PAGE)
STR-PCR PAGE RESULTS, Silver staining

D3S1358 Allele 12-20

D12 VWA Allele 13-22

The Discrimination Power of D3S1358
and ABO
 It has 9 alleles from Allele 12 to 20;
 Everyone has 2 of these alleles, for example,
A has 15, 15;
 Totally there will be 9×9=81 genotypes exists
among the people;
 E.g., only D3S1358 is enough to divide people
into 81 subgroups;

 While ABO system can only divide people into

4 subgroups: A, B, AB and O;

 Thus a STR locus has much more higher DP

than ABO.
13 CODIS STR LOCI Combined: no 2 persons can be identical
 FBI’S CODIS DNA DATABASE
Combined DNA Index System
 Used for linking serial crimes and unsolved
cases with repeat offenders
 Launched October 1998
 Links all 50 states
 Requires >4 RFLP markers

and/or 13 core STR markers

 Current backlog of >600,000 samples
 Can reach a DP >0.9999 9999
 Can distinguish each person in this world
 Multiplex PCR
 Over 10 Markers Can Be
Copied at Once
 Sensitivities to levels less
than 1 ng of DNA
 Ability to Handle Mixtures
and Degraded Samples
 Different Fluorescent Dyes
Used to Distinguish STR
Alleles with Overlapping Size
Ranges
THE IMAGE OF A MULTIPLEX PCR
AUTOMATED FORENSIC DNA
ANALYSIS
 ABI Prism 310 Genetic Analyzer

capillary

Syringe with polymer

solution

Injection
electrode

Outlet Autosampler
buffer tray
Inlet
buffer
Close-up of ABI Prism 310 Sample Loading
Area

Electrode

Capillary

Sample Vials

Autosampler Tray

See Technology section for more information on CE

PROCEDURES
 AN EXAMPLE FORENSIC STR MULTIPLEX
KIT

AmpFlSTR® Profiler Plus™

Kit available from PE Biosystems (Foster City, CA)

100 bp 200 bp 300 bp 400 bp

Size Separation

D3 vWA FGA 5-FAM (blue)

Color Separation

A D8 D21 D18 JOE (green)

D5 D13 D7 NED (yellow)

ROX
(red)
GS500-internal lane standard
9 STRs amplified along with sex-typing marker amelogenin in a single PCR
reaction
PROFILER PLUS MULTIPLEX STR
RESULTS
ONE CASE OF PATERNITY TESTING
 D3S1358

Sample 1

Sample 2

Sample 3

Sample 4

Sample 5
STATISTICS
 STR ALLELE
FREQUENCIES
45
40
TH01 Marker
35
30
Frequency

25 Caucasians (N=427)
20 Blacks (N=414)
15 Hispanics (N=414)
10
5 *Proc. Int. Sym. Hum. ID
(Promega) 1997, p. 34
0
6 7 8 9 9.3 10
Number of repeats
THE USE OF STR IN FORENSIC CASES

 High Discrimination Power (DP) for

matching suspect with evidences

 High Parentage Exclusion (PE) value for

paternity testing
THE SAME 13 LOCUS STR PROFILE IN
DIFFERENT POPULATIONS
PARENTAGE TESTING
GENETIC BASIS
OUR PUBLICATIONS ON CODIS
 Zeng ZS, Zheng XD, Zhu YL, Wang ZQ, Xiang ZD,
Meng XS, Wang TP, Dong ZM. Population genetic
data of 15 STR loci in Han population of Henan
province (central China). Leg Med (Tokyo) 2007
Jan; 9(1): 30-2
 Zheng X, Zeng Z, Meng X, Xiang Z, Zhu Y, Yan H.
Genetic data of 9 STR loci from Henan Province
(central China). Forensic Sci Int. 2007 Jul 4;169(2-
3):244-6
 赵美乐，曾昭书，郑旭东等。中国河南汉族人群
D19S253 、 D12S391 和 D18S535 位点基因多态性检
测。郑州大学学报（医学版） 2007 ； 42 （ 2 ）：
289-91
PUBLICATIONS BY SCIENTISTS FROM PAKISTAN
 Rakha A, Yu B, Hadi S, Li S. Genetic analysis of Kashmiri Muslim
population living in Pakistan. Leg Med (Tokyo). 2008 Mar 5

 Firasat S, Khaliq S, Mohyuddin A, Papaioannou M, Tyler-Smith C,

Underhill PA, Ayub Q. Y-chromosomal evidence for a limited
Greek contribution to the Pathan population of Pakistan. Eur J
Hum Genet. 2007 Jan;15(1):121-6.
Biomedical and Genetic Engineering Division, Dr. AQ Khan Research
Laboratories, Islamabad, Pakistan

 Saqib Shahzad M, Abbas Bokhari SY, Rao AQ, Raza MH, Ullah O,
Zia-Ur-rahman, Shahid AA, Ahmad Z, Riazuddin S. Population
studies for STR loci (D3S1358, D5S818, D7S820, D18S51 and
FGA) in NWFP and Sindhi populations of Pakistan for forensic
use. Arch Med Sadowej Kryminol. 2004 Oct-Dec;54(4):215-22.
Centre of Excellence in Molecular Biology, University of the Punjab,
Lahore, Pakistan
PUBLICATIONS BY SCIENTISTS FROM INDIA
 Eaaswarkhanth M, Roy S, Haque I. Allele frequency distribution for
15 autosomal STR loci in two Muslim populations of Tamilnadu, India.
Leg Med (Tokyo). 2007 Nov;9(6):332-5.
 National DNA Analysis Center, Directorate of Forensic Science, Central
Forensic Science Laboratory, 30, Gorachand Road, Kolkata 700 014, India.
 Thangaraj K, Chaubey G, Singh VK, Reddy AG, Chauhan P, Malvee R,
Pavate PP, Singh L. Y-chromosomal STR haplotypes in two
endogamous tribal populations of Karnataka, India. J Forensic Sci.
2007 May;52(3):751-3.
 Centre for Cellular and Molecular Biology, Hyderabad 500 007, India
 Krithika S, Trivedi R, Kashyap VK, Vasulu TS. Allele frequency
distribution at 15 autosomal STR loci in Panggi, Komkar and Padam
sub tribes of Adi, a Tibeto-Burman speaking population of Arunachal
Pradesh, India. Leg Med (Tokyo). 2007 Jul;9(4):210-7.
 Biological Anthropology Unit, Indian Statistical Institute, Kolkata 700 108,
West Bengal, India
PUBLICATIONS ON NEPALESE
 Parkin EJ, Kraayenbrink T, Opgenort JR, van Driem GL, Tuladhar NM,
de Knijff P, Jobling MA. Diversity of 26-locus Y-STR haplotypes in a
Nepalese population sample: isolation and drift in the Himalayas.
Forensic Sci Int. 2007 Mar 2;166(2-3):176-81.
 Department of Genetics, University of Leicester, University Road, Leicester LE1
7RH, UK
 Ota M, Droma Y, Basnyat B, Katsuyama Y, Asamura H, Sakai H,
Fukuhsima H. Allele frequencies for 15 STR loci in Tibetan populations
from Nepal. Forensic Sci Int. 2007 Jul 4;169(2-3):234-8
 Department of Legal Medicine, Shinshu University School of Medicine, Asahi 3-
1-1, 390-8621 Matsumoto, Japan
 Kraaijenbrink T, van Driem GL, Opgenort JR, Tuladhar NM, de Knijff P.
Allele frequency distribution for 21 autosomal STR loci in Nepal.
Forensic Sci Int. 2007 May 24;168(2-3):227-31.
 MGC Department of Human and Clinical Genetics, Leiden University Medical
Centre, Wassenaarseweg 72, 2333 AL Leiden, The Netherlands.
 Kashyap VK, Guha S, Trived R. Concordance study on 15 STR loci in
three major populations of Himalayan State Sikkim. J Forensic Sci.
2002 Sep;47(5):1163-7.
Part 5:

Other DNA testing technique

-- DNA Fingerprints
OVERVIEW OF DNA “FINGERPRINT”
PROFILING
 Developed by Alec Jeffreys in 1985
 Was replaced by STR since 1990’s

 A kind of length polymorphism

 In its essence, DNA “Fingerprint” is a

kind of long repeats, while STR is a kind
of short repeats
 Instead of using primers, DNA
“Fingerprint” use isotopic 32P labeled
DNA sequences to detect the target
sequencs and to show the bands
PROCEDURES OF DNA Fingerprint

• Figure 1
 DNA

Cut

Separatio Visualization
n
THE DNA FINGERPRINT MAP OF 10
PERSONS

 Figure 12
USAGE: MATCHING SUSPECT WITH EVIDENCE

 F
FINGERPRINTS: ADVANTAGES AND
DISADVANTAGES
 Advantages
 Very high discrimination power (DP)

 Disadvantages
 Low specificity
 Results are hard to be repeated
 Results are hard to be digitalized

 Not used by current scientists!

Part 6:

Samples preparing
for DNA testing
SOURCE OF BIOLOGICAL EVIDENCE WITH
DNA
 Live tissue and blood are the best materials, but
autopsy samples can be used as long as marked
putrefactive changes have not occurred, sufficient to
destroy nuclear chromatin.
 In the living, a plain blood sample of at least 1 ml
and preferably 5 ml is taken. If there is to be any
delay in getting this to the laboratory, the sample (in
a plastic or glass tube) should be frozen solid at
-20oC.
 Multiple swabs are a second-best, but can still be
used. Again, if there is to be delay in transit, these
should all be deep-frozen to prevent autolytic
breakdown of nuclear DNA.
 Hairs, pulled out to secure cellular material in the
root bulbs, can also be used for data profiling.
STORAGE ADDITIVES
 Blood used for DNA analysis: anticoagulant
should be added, among which citric acid --
citric acid sodium solution is the best, EDTA-
Na2 is the second best.

 Tissues taken from the body should be kept

in the ethanol solution but avoiding the
formalin fixing.
DNA USE IN FORENSIC
CASES
 Most are rape cases (>2 out of 3)
 Looking for match between evidence and
suspect
 Must compare victim’s DNA profile

Challenges
•Mixtures must be resolved
•DNA is often degraded
•Inhibitors to PCR are often present
JOURNALS OF FORENSIC SCIENCES
OUR RESEARCH INTERESTS

 Focus on the polymorphism researches about

Chinese Hans and other populations including
Indians, Pakistani and Nepalese.

 Especially on the STR or SNP polymorphisms

between these populations.

 Seeking cooperation with Indian and Pakistani

colleagues to share DNA samples, to work on
the same research project and to find the
diversity among different populations.
My e-mail: zzs@zzu.edu.cn
The end

Thank you!