Fundamentals of Forensic
DNA Typing
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.
John M. Butler (2009) Fundamentals of Forensic DNA Typing, Figure 1.1
Interactions between the Three Components
of the Criminal Justice System
Law Enforcement
Police Agencies
Scientific Analysis
Legal Proceedings
Forensic Laboratory
Court System
Validated scientific tests
Legal framework
and precedent
(local, state, federal)
Laws and police training
Investigators/
Detectives
Other
Forensic
Disciplines
CSI
DNA Unit
DNA Analysts
Evidence
submitted
Evidence
returned
Judge
Prosecution
Defense
Scientific
report(s)
completed
References
submitted
Research
(introduces new methods)
Trial
Conviction
or
exoneration
Historical Perspective on DNA Typing
2009: DNA is an important part
www.dna.gov
of the criminal justice system
President’s DNA Initiative
(>$600M from 2004-2008)
NDIS launched
(October 13, 1998)
Identifiler 5-dye kit
and ABI 3100
2002
UK National
Database launched
CODIS loci
(April 10, 1995)
defined
Gill et al. (1985) Forensic
application of DNA 'fingerprints‘.
Nature 318:577-9
FSS
1994
1990
1985
1992
PCR developed
RFLP
2006
miniSTRs
2004
Y-STRs
PowerPlex® 16
(16 loci in single amp)
1998
2000
STR typing with
CE is fairly routine
Quadruplex
First STRs
developed
2009
1996
First commercial
fluorescent STR
multiplexes
mtDNA
Capillary electrophoresis
of STRs first described
DQA1 & PM
(dot blot)
Multiplex STRs
Stages of Forensic DNA Progression
Stages
Time Frame Description
Exploration
1985-1995
Beginnings, different methods
tried (RFLP and early PCR)
Stabilization
1995-2005
Growth
2005-2009
Standardization to STRs,
selection of core loci,
implementation of Quality
Assurance Standards
Rapid growth of DNA
databases, extended
applications pursued
Expanding tools available,
confronting privacy concerns
Sophistication The Future
From John M. Butler (Feb 2009) Presentation at AAFS session on “Envisioning the Future”
John M. Butler (2009) Fundamentals of Forensic DNA Typing, D.N.A. Box 1.2
Lessons from the First Case Involving DNA Testing
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.
John M. Butler (2009) Fundamentals of Forensic DNA Typing, D.N.A. Box 1.1
The Innocence Project
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
Steps in DNA Sample
Processing
John M. Butler (2009) Fundamentals of Forensic DNA Typing, Figure 1.2
Sample Obtained from
Crime Scene or Paternity
Investigation
DNA
Extraction
Biology
DNA
Quantitation
PCR Amplification
of Multiple STR markers
Technology
Separation and Detection of
PCR Products
(STR Alleles)
Sample Genotype
Determination
Genetics
Comparison of Sample
Genotype to Other Sample
Results
Generation of Case Report
with Probability of Random
Match
If match occurs, comparison of
DNA profile to population
databases
Suspect developed
Crime committed
Biological material transferred
May match another
Evidence (Question)
sample “Q”
Database Search
Steps Involved
Serology
Collection
Sample Storage
Characterization
Biology
Extraction
Quantitation
Amplification
Genetics
Technology
STR Markers
Separation/
Detection
Data
Interpretation
Statistical
Interpretation
Profile put on database
Reference (Known)
sample “K”
(K’)
Q
U
A
L
I
T
Y
A
S
S
U
R
A
N
C
E
Exclusion (no match)
Q≠K
DNA Profile
Comparison
Q
May be
Inconclusive
due to
Steps Involved
May be
Inconclusive
Collection
due to
Sample Storage
Lack of
Available
Reference
Extraction
K
Q=K
Inclusion (match)
Forensic Issues
Quantitation
Amplification
STR Markers
Separation/
Detection
(degradation,
mixtures, low levels)
Report
(with statistical weight)
Plea
Data
Interpretation
Court
John M. Butler (2009) Fundamentals of Forensic DNA Typing, Figure 1.3
Profile put on database
Q
U
A
L
I
T
Y
A
S
S
U
R
A
N
C
E
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)
Victim’s Remains compared to Biological Relative (Mass Disaster ID)
Soldier’s Remains compared to Direct Reference Sample (Armed Forces ID)
The Three Possible Outcomes
of Evidence Examination
“Suspect”
Known (K) Sample
• Exclusion (no match)
• Non-exclusion
13
11 12
11 12
11 12
– “Match” or “inclusion”
11 12
• Inconclusive result
“Evidence”
Question (Q) Sample
No result
(or a complex mixture)
Steps Involved
Steps in Forensic DNA Analysis
Usually 1-2 day process (a minimum of ~5 hours)
Collection
Slot Blot
1 ng
Specimen Storage
0.3 ng
No DNA
0.5 ng
0.5 ng
0.7 ng
1 ng
Extraction
Blood Stain Buccal swab
STR Typing
Interpretation
of Results
Genetics
If a match occurs, comparison of
DNA profile to population allele
frequencies to generate a case
report with probability of a random
match to an unrelated individual
Database
Storage & Searching
Calculation of
Match Probability
DNA
Database
Search
Biology
Multiplex PCR
1 ng
DNA
DNA
Extraction Quantitation
Multiplex PCR Amplification
DNA separation and sizing
Technology
Sample Collection
& Storage
Quantitation
STR Typing
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.
John M. Butler (2009) Fundamentals of Forensic DNA Typing, D.N.A. Box 1.3
Example Laboratory Report
from a DNA Examination
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
Q2
K1
K2
Swab from broken, bloodstained glass in window frame (Item #2)
Swab from keyboard of laptop computer (Item #7)
Blood sample from SUSPECT 1
Buccal swab from SUSPECT 2
This report contains the results of the serological and nuclear DNA analyses.
John M. Butler (2009) Fundamentals of Forensic DNA Typing, D.N.A. Box 1.3
Example Laboratory Report
from a DNA Examination
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
John M. Butler (2009) Fundamentals of Forensic DNA Typing, Table 1.1
Compared to Timeline for Microsoft Corporation
Year
Forensic DNA Science &
Application
Parallel Developments in
Biotechnology
Microsoft Corporation
Chronology
1985
Alec Jeffreys develops multi-locus
RFLP probes
PCR process first described
First version of Windows
shipped
1986
DNA testing goes public with
Cellmark and Lifecodes in
United States
automated DNA sequencing with 4colors first described
Microsoft goes public
1988
FBI begins DNA casework with
single locus RFLP probes
1989
TWGDAM established; NY v. Castro
case raises issues over quality
assurance of laboratories
DNA detection by gel silver-staining,
slot blot, and reverse dot blots
first described
1990
Population statistics used with RFLP
methods are questioned; PCR
methods start with DQA1
Human Genome Project begins with
goal to map all human genes
1991
fluorescent STR markers first
described; Chelex extraction
Windows 3.0 released
(quality problems);
exceeds $1 billion in
sales
Windows 3.1 released
Major Historical Events in Forensic DNA
John M. Butler (2009) Fundamentals of Forensic DNA Typing, Table 1.1
Compared to Timeline for Microsoft Corporation
Year
Forensic DNA Science &
Application
Parallel Developments in
Biotechnology
1992
NRC I Report; FBI starts casework
with PCR-DQA1
capillary arrays first described
1993
first STR kit available; sex-typing
(amelogenin) developed
first STR results with CE
1994
Congress authorizes money for
upgrading state forensic labs;
“DNA wars” declared over; FBI
starts casework with PCR-PM
Hitachi FMBIO and Molecular
Dynamics gel scanners; first
DNA results on microchip CE
1995
O.J. Simpson saga makes public
more aware of DNA; DNA
Advisory Board setup; UK DNA
Database established; FBI
starts using D1S80/amelogenin
ABI 310 Genetic Analyzer and
TaqGold DNA polymerase
introduced
1996
NRC II Report; FBI starts mtDNA
testing; first multiplex STR kits
become available
STR results with MALDI-TOF and
GeneChip mtDNA results
demonstrated
1997
13 core STR loci defined;
Y-chromosome STRs described
Microsoft Corporation
Chronology
Windows 95 released
Internet Explorer begins
overtaking Netscape
Major Historical Events in Forensic DNA
John M. Butler (2009) Fundamentals of Forensic DNA Typing, Table 1.1
Compared to Timeline for Microsoft Corporation
Year
Forensic DNA Science &
Application
Parallel Developments in
Biotechnology
Microsoft Corporation
Chronology
1998
FBI launches national Combined DNA
Index System; Thomas Jefferson
and Bill Clinton implicated with
DNA
2000 SNP hybridization chip described
Windows 98 released; anti-trust
trial with U.S. Justice
Department begins
1999
Multiplex STR kits are validated in
numerous labs; FBI stops testing
DQA1/PM/D1S80
ABI 3700 96-capillary array for highthroughput DNA analysis;
chromosome 22 fully sequenced
2000
FBI and other labs stop running RFLP
cases and convert to multiplex
STRs; PowerPlex 16 kit enables
first single amplification of CODIS
STRs
First copy of human genome completed
Bill Gates steps down as
Microsoft CEO; Windows
2000 released
2001
Identifiler STR kit released with 5-dye
chemistry; first Y-STR kit becomes
available
ABI 3100 Genetic Analyzer introduced
Windows XP released
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
Windows XP Tablet PC Edition
released
Human Genome Project completed with
the “final” sequence coinciding
with 50th anniversary of WatsonCrick DNA discovery
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