Chapter 1

Overview
Fundamentals of Forensic
DNA Typing

Slides prepared by John M. Butler

June 2009
 Chapter 1 - Overview
Chapter Summary
Since its introduction in the mid-1980s, forensic DNA
testing techniques have enabled crime scene evidence
to be matched to perpetrators with increasing sensitivity
and speed. An example is used to illustrate how DNA
analysis aided the investigation of a sexual assault
committed in Charlottesville, Virginia in 1999. The role of
forensic science and DNA testing are considered in the
context of the criminal justice system. The steps in DNA
sample processing are briefly reviewed and
improvements to DNA testing are compared to advances
in computer technology.
News Story on Montaret Davis DNA Database
Match to University of Virginia Student Rape
 Overview of the Criminal Justice System

The criminal justice system consists of three broad areas:

(1) law enforcement, (2) scientific analysis, and (3) legal proceedings

Detectives or investigators serving in police agencies submit

evidence collected from crime scenes to forensic laboratories. This
evidence is then compared to suspect reference samples (when
available) or in the case of DNA or fingerprints -- searched against
a database of previous offenders as performed in the Virginia case
just described.

A scientific report of the analysis of the evidence and comparison to

the reference samples is then produced. This report is used by law
enforcement and the legal community (prosecutors or defense
attorneys) to make further decisions that may result in the evidence
being presented in a court of law.
 Interactions between the Three Components
of the Criminal Justice System
John M. Butler (2009) Fundamentals of Forensic DNA Typing, Figure 1.1

Law Enforcement Scientific Analysis Legal Proceedings

Police Agencies Forensic Laboratory Court System
(local, state, federal)
Laws and police training Validated scientific tests Legal framework
and precedent
Other Judge
Investigators/ CSI Forensic
Detectives Disciplines Prosecution
DNA Unit Defense
DNA Analysts
Scientific Conviction
Evidence Trial
report(s) or
submitted
completed exoneration
Evidence
returned
References
submitted

Research
(introduces new methods)
 Historical Perspective on DNA Typing
2009: DNA is an important part www.dna.gov
of the criminal justice system Presidents DNA Initiative 2009
(>$600M from 2004-2008)
NDIS launched
2006
Identifiler 5-dye kit miniSTRs
(October 13, 1998) and ABI 3100
UK National 2002 2004 Y-STRs
Database launched
(April 10, 1995) CODIS loci
PowerPlex 16
defined (16 loci in single amp)
Gill et al. (1985) Forensic
application of DNA 'fingerprints. 1998 2000
Nature 318:577-9 FSS STR typing with
Quadruplex CE is fairly routine

1994 1996 First commercial

First STRs
fluorescent STR mtDNA
developed
multiplexes
1990 1992 Capillary electrophoresis
of STRs first described

1985 PCR developed DQA1 & PM

(dot blot) Multiplex STRs
RFLP
 Stages of Forensic DNA Progression
Stages Time Frame Description

Exploration 1985-1995 Beginnings, different methods

tried (RFLP and early PCR)
Stabilization 1995-2005 Standardization to STRs,
selection of core loci,
implementation of Quality
Assurance Standards
Growth 2005-2009 Rapid growth of DNA
databases, extended
applications pursued
Sophistication The Future Expanding tools available,
confronting privacy concerns
From John M. Butler (Feb 2009) Presentation at AAFS session on Envisioning the Future
 Lessons from the First Case Involving DNA Testing
John M. Butler (2009) Fundamentals of Forensic DNA Typing, D.N.A. Box 1.2

Describes the first use of DNA (in 1986) to

solve a double rape-homicide case in
England; about 5,000 men asked to give
blood or saliva to compare to crime stains

Connection of two crimes (1983 and 1986)

Use of DNA database to screen for

perpetrator (DNA only done on 10% with
same blood type as perpetrator)

Exoneration of an innocent suspect

DNA was an investigative tool did not

solve the case by itself (confession of
accomplice)
A local baker, Colin Pitchfork, was arrested and his DNA profile matched with the
semen from both murders. In 1988 he was sentenced to life for the two murders.
 The Innocence Project
John M. Butler (2009) Fundamentals of Forensic DNA Typing, D.N.A. Box 1.1

http://www.innocenceproject.org

Defense attorneys Barry Scheck and Peter Neufeld launched the

Innocence Project in 1992 at the Benjamin N. Cardozo School of Law
in New York City.

The Innocence Project promotes cases where evidence is available

for post-conviction DNA testing and can help demonstrate innocence.
The fact that truly innocent people have been behind bars for a
decade or more has promoted legislation in a number of states
and also at the federal level to fund post-conviction DNA testing.
 Basis of DNA Profiling
The genome of each individual is unique (with the
exception of identical twins) and is inherited from parents

Probe subsets of genetic variation in order to differentiate

between individuals (statistical probabilities of a random
match are used)

DNA typing must be performed efficiently and

reproducibly (information must hold up in court)

Current standard DNA tests DO NOT look at genes

little/no information about race, predisposal to disease, or
phenotypical information (eye color, height, hair color) is
obtained
 Human Identity Testing
Forensic cases -- matching suspect with
evidence
Paternity testing -- identifying father
Mass disasters -- putting pieces back together
Historical investigations
Missing persons investigations
Military DNA dog tag
Convicted felon DNA databases

Involves generation of DNA profiles usually with

the same core STR (short tandem repeat) markers
 Sample Obtained from
Crime Scene or Paternity
Steps in DNA Sample
Investigation Processing
Biology
John M. Butler (2009) Fundamentals of Forensic DNA Typing, Figure 1.2

DNA DNA PCR Amplification

Extraction Quantitation of Multiple STR markers

Technology
Separation and Detection of Sample Genotype
PCR Products Determination
(STR Alleles)

Genetics
Comparison of Sample Generation of Case Report
Genotype to Other Sample with Probability of Random
Results Match

If match occurs, comparison of

DNA profile to population
databases
 Crime committed Suspect developed
Biological material transferred

May match another Reference (Known)

Evidence (Question)
sample Q (K) sample K
Database Search
Steps Involved Steps Involved
May be
Collection Collection
Inconclusive
Q Q
Serology

due to Sample Storage

Sample Storage
U Exclusion (no match) Lack of U
Characterization Available
A A
Reference
L QK Extraction
L
Extraction I I
Biology

DNA Profile
Quantitation

Amplification
T
Y Q Comparison K Quantitation

Amplification
T
Y

A Q=K A
May be
S S
STR Markers Inconclusive STR Markers
S due to Inclusion (match) S
U Forensic Issues U
Separation/ Separation/
R (degradation, R
Technology

Detection mixtures, low levels) Detection

A A
N N
Data C Report Data C
Interpretation E (with statistical weight) Interpretation E
Genetics

Statistical
Interpretation
Plea Court
Profile put on database John M. Butler (2009) Fundamentals of Forensic DNA Typing, Figure 1.3 Profile put on database
 DNA Testing Requires a Reference Sample

A DNA profile by itself is

fairly useless because it
has no context

DNA analysis for identity

only works by comparison
you need a reference
sample

Crime Scene Evidence compared to Suspect(s) (Forensic Case)

Child compared to Alleged Father (Paternity Case)
Victims Remains compared to Biological Relative (Mass Disaster ID)
Soldiers Remains compared to Direct Reference Sample (Armed Forces ID)
 The Three Possible Outcomes
of Evidence Examination
Suspect Evidence
Known (K) Sample Question (Q) Sample
13
Exclusion (no match) 11 12

11 12
Non-exclusion 11 12
Match or inclusion

11 12
No result
Inconclusive result (or a complex mixture)
Steps Involved Steps in Forensic DNA Analysis
Collection Usually 1-2 day process (a minimum of ~5 hours)
Slot Blot
1 ng
Specimen Storage 0.3 ng
No DNA
0.5 ng
0.5 ng
Extraction 0.7 ng
1 ng
Blood Stain Buccal swab
1 ng
Quantitation Sample Collection DNA DNA

Biology
& Storage
Extraction Quantitation
Multiplex PCR

STR Typing
Genetics

If a match occurs, comparison of

DNA profile to population allele Multiplex PCR Amplification
Interpretation frequencies to generate a case
report with probability of a random
of Results match to an unrelated individual DNA separation and sizing

Technology
Database
Storage & Searching DNA
Database STR Typing
Calculation of Search
Match Probability Male: 13,14-15,16-12,13-10,13-15,16
Interpretation of Results
 The Laboratory Report
The end result of a forensic examination is a laboratory report,
which represents a brief summary of work conducted by a forensic
examiner (i.e., DNA analyst).

The work represented in a laboratory report is based on following

standard operating procedures. Prior to release of a lab report, data
and conclusions are vetted through an internal review process
culminating with a second reviewer and/or the DNA technical leader
approving the work.

A lab report is typically submitted to police investigators to

describe DNA typing results obtained from evidence and
reference samples submitted. Depending on the results, this report
may also be used by a prosecuting attorney during court proceedings
to illustrate that a defendant matches (or cannot be eliminated as a
possible contributor to) DNA evidence from a crime scene.
 Example Laboratory Report
from a DNA Examination
John M. Butler (2009) Fundamentals of Forensic DNA Typing, D.N.A. Box 1.3

ABC Laboratory
Hometown, U.S.A.
Report of Examination

Date: December 8, 2008

Examiner Name: Sherlock Holmes
Unit: Forensic Biology
Case File Number: 08-3101-042

The specimens listed below were received in the Forensic Biology unit under
cover of communication dated April 1, 2008 (080412001) and April 15, 2008
(080412312):

Q1 Swab from broken, bloodstained glass in window frame (Item #2)

Q2 Swab from keyboard of laptop computer (Item #7)
K1 Blood sample from SUSPECT 1
K2 Buccal swab from SUSPECT 2

This report contains the results of the serological and nuclear DNA analyses.
 Example Laboratory Report
from a DNA Examination
John M. Butler (2009) Fundamentals of Forensic DNA Typing, D.N.A. Box 1.3

Results of Examinations:

Blood was identified on specimen Q1. Specimen Q2 was examined for the presence of blood; however, no evidence of
blood was found.

Deoxyribonucleic acid (DNA) was isolated from specimens Q1, Q2, K1 (SUSPECT 1), and K2 (SUSPECT 2) and subjected
to DNA typing by the polymerase chain reaction (PCR) at the amelogenin sex typing locus and fifteen (15) short tandem
repeat (STR) loci of the AmpFlSTR Identifiler PCR Amplification Kit. The DNA typing results are detailed below:

Specimen D8 D21 D7 CSF D3 TH01 D13 D16 D2 D19 VWA TPOX D18 AMEL D5 FGA

Q1 12,14 28,30 9,9 10,10 16,17 6,6 11,14 9,11 22,23 12,14 17,18 8,8 14,16 X,Y 12,13 21,22

Q2 12,14 28,30 9,9 10,10 16,17 6,6 11,14 9,11 22,23 12,14 17,18 8,8 14,16 X,Y 12,13 21,22

K1 12,14 28,30 9,9 10,10 16,17 6,6 11,14 9,11 22,23 12,14 17,18 8,8 14,16 X,Y 12,13 21,22

K2 13,14 30.2,32 8,12 10,12 17,17 6,9 8,12 7,8 23,25 14,14 17,20 8,10 14,17 X,X 11,13 21,25

Based on the typing results from the amelogenin locus (for sex determination), male DNA is present in the DNA obtained
from specimens Q1, Q2, and K1 (SUSPECT 1). Based on the STR typing results and to a reasonable degree of
scientific certainty, the contributor of specimen K1 (SUSPECT 1) is the source of the DNA obtained from
specimens Q1 and Q2. The probability of selecting an unrelated individual at random having an STR profile matching the
DNA obtained from the questioned specimens is approximately 1 in 840 trillion from the Caucasian population, 1 in 16
quadrillion from the African American population, and 1 in 18 quadrillion from the Hispanic population.

The STR typing results for specimen Q1 will be entered into the Combined DNA Index System (CODIS) and maintained by
the ABC Laboratory for future comparisons.

No further serological or nuclear DNA examinations were conducted.

 Applications for DNA Testing
Crime solving matching suspect with evidence
Accident victims after airplane crashes
Soldiers in war who is the unknown soldier
Paternity testing who is the father
Immigration testing are two people related
Missing persons investigations whose remains
Convicted felons databases cases solved

Involves generation of DNA profiles usually with

the same core STR (short tandem repeat) markers
and then MATCHING TO REFERENCE SAMPLE
 Advantages for STR Markers
Small product sizes are generally compatible with
degraded DNA and PCR enables recovery of information
from small amounts of material

Numerous alleles per locus aid mixture interpretation

Multiplex amplification with fluorescence detection enables

high power of discrimination in a single test

Commercially available in an easy to use kit format

Uniform set of core STR loci provide capability for national

(and international) sharing of criminal DNA profiles
 The Future of Forensic DNA Testing

Report published in Nov 2000

Asked to estimate where DNA

testing would be 2, 5, and 10 years
into the future

Conclusions
STR typing is here to
stay for a few years
because of DNA
databases that have
grown to contain
millions of profiles
http://www.ojp.usdoj.gov/nij/pubs-sum/183697.htm
 Major Historical Events in Forensic DNA
Compared to Timeline for Microsoft Corporation
John M. Butler (2009) Fundamentals of Forensic DNA Typing, Table 1.1

Year Forensic DNA Science & Parallel Developments in Microsoft Corporation

Application Biotechnology Chronology

1985 Alec Jeffreys develops multi-locus PCR process first described First version of Windows
RFLP probes shipped

1986 DNA testing goes public with automated DNA sequencing with 4- Microsoft goes public
Cellmark and Lifecodes in colors first described
United States

1988 FBI begins DNA casework with

single locus RFLP probes

1989 TWGDAM established; NY v. Castro DNA detection by gel silver-staining,

case raises issues over quality slot blot, and reverse dot blots
assurance of laboratories first described

1990 Population statistics used with RFLP Human Genome Project begins with Windows 3.0 released
methods are questioned; PCR goal to map all human genes (quality problems);
methods start with DQA1 exceeds $1 billion in
sales

1991 fluorescent STR markers first Windows 3.1 released

described; Chelex extraction
 Major Historical Events in Forensic DNA
Compared to Timeline for Microsoft Corporation
John M. Butler (2009) Fundamentals of Forensic DNA Typing, Table 1.1
Year Forensic DNA Science &
Application
1992 NRC I Report; FBI starts casework
with PCR-DQA1
1993 first STR kit available; sex-typing
(amelogenin) developed
1994 Congress authorizes money for
upgrading state forensic labs;
DNA wars declared over; FBI
starts casework with PCR-PM
1995 O.J. Simpson saga makes public
more aware of DNA; DNA
Advisory Board setup; UK DNA
Database established; FBI
starts using D1S80/amelogenin
1996 NRC II Report; FBI starts mtDNA
testing; first multiplex STR kits
become available
1997 13 core STR loci defined;
Y-chromosome STRs described
Parallel Developments in Microsoft Corporation
Biotechnology Chronology
capillary arrays first described
first STR results with CE
Hitachi FMBIO and Molecular
Dynamics gel scanners; first
DNA results on microchip CE
ABI 310 Genetic Analyzer and Windows 95 released
TaqGold DNA polymerase
introduced
STR results with MALDI-TOF and
GeneChip mtDNA results
demonstrated
Internet Explorer begins
overtaking Netscape
 Major Historical Events in Forensic DNA
Compared to Timeline for Microsoft Corporation
John M. Butler (2009) Fundamentals of Forensic DNA Typing, Table 1.1
Year Forensic DNA Science &
Application
1998 FBI launches national Combined DNA
Index System; Thomas Jefferson
and Bill Clinton implicated with
DNA
1999 Multiplex STR kits are validated in
numerous labs; FBI stops testing
DQA1/PM/D1S80
2000 FBI and other labs stop running RFLP
cases and convert to multiplex
STRs; PowerPlex 16 kit enables
first single amplification of CODIS
STRs
2001 Identifiler STR kit released with 5-dye
chemistry; first Y-STR kit becomes
available
2002 FBI mtDNA population database
released; Y-STR 20plex published
2003 U.S. DNA database (NDIS) exceeds 1
million convicted offender profiles;
the U.K. National DNA Database
passes the 2 million sample mark
Parallel Developments in
Biotechnology
2000 SNP hybridization chip described
ABI 3700 96-capillary array for high-
throughput DNA analysis;
chromosome 22 fully sequenced
First copy of human genome completed
ABI 3100 Genetic Analyzer introduced
Human Genome Project completed with
the final sequence coinciding
with 50th anniversary of Watson-
Crick DNA discovery
Microsoft Corporation
Chronology
Windows 98 released; anti-trust
trial with U.S. Justice
Department begins
Bill Gates steps down as
Microsoft CEO; Windows
2000 released
Windows XP released
Windows XP Tablet PC Edition
released
Windows Server 2003 released;
64-Bit Operating Systems
expand capabilities of
software
 Chapter 1 Points for Discussion

What role does a forensic laboratory play in the

criminal justice system?

What are some ways that DNA testing has

impacted forensic science and the criminal
justice system?

Discuss some communication skills that might

be beneficial for a forensic DNA scientist to have
in interacting with law enforcement and the legal
community