1. Fundamentals of Forensic DNA Typing
Chapter 1
Overview
Fundamentals of Forensic DNA Typing
Slides prepared by John M. Butler
June 2009

2. 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 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.

3. News Story on Montaret Davis DNA Database Match to University of Virginia Student Rape

4. 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.

5. Interactions between the Three Components of the Criminal Justice System
Law Enforcement
Scientific Analysis
Legal Proceedings
Police Agencies
(local, state, federal)
Forensic Laboratory
Court System
Validated scientific tests
Legal framework
and precedent
Laws and police training
Other Forensic Disciplines
Judge
Investigators/
Detectives
CSI
Prosecution
John M. Butler (2009) Fundamentals of Forensic DNA Typing, Figure 1.1
DNA Unit
DNA Analysts
Defense
Scientific report(s) completed
Conviction or exoneration
Evidence
submitted
Trial
Figure 1.1 Illustration of the interactions between the three components of the criminal justice system.
Evidence
returned
References
submitted
Research
(introduces new methods)

6. Historical Perspective on DNA Typing
2009: DNA is an important part of the criminal justice system
President’s DNA Initiative (>$600M from )
2009
2006
NDIS launched
(October 13, 1998)
Identifiler 5-dye kit and ABI 3100
miniSTRs
2002
2004
UK National Database launched (April 10, 1995)
Y-STRs
CODIS loci defined
PowerPlex® 16 (16 loci in single amp)
Gill et al. (1985) Forensic application of DNA 'fingerprints‘. Nature 318:577-9
1998
2000
STR typing with CE is fairly routine
FSS Quadruplex
DQA1 & PM (dot blot)
1996
1994
First commercial fluorescent STR multiplexes
First STRs developed
RFLP
mtDNA
1990
1992
Capillary electrophoresis of STRs first described
1985
PCR developed
Multiplex STRs

7. Stages of Forensic DNA Progression
Time Frame
Description
Beginnings, different methods tried (RFLP and early PCR)
Exploration
Standardization to STRs, selection of core loci, implementation of Quality Assurance Standards
Stabilization
Rapid growth of DNA databases, extended applications pursued
Growth
Expanding tools available, confronting privacy concerns
The Future
Sophistication
From John M. Butler (Feb 2009) Presentation at AAFS session on “Envisioning the Future”

8. 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)
John M. Butler (2009) Fundamentals of Forensic DNA Typing, D.N.A. Box 1.2
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.

9. The Innocence Project http://www.innocenceproject.org
John M. Butler (2009) Fundamentals of Forensic DNA Typing, D.N.A. Box 1.1
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.

10. 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

11. 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

12. Steps in DNA Sample Processing
Sample Obtained from Crime Scene or Paternity Investigation
DNA
Extraction
Quantitation
PCR Amplification
of Multiple STR markers
Biology
Separation and Detection of PCR Products
(STR Alleles)
Technology
Sample Genotype Determination
Genetics
Comparison of Sample Genotype to Other Sample Results
If match occurs, comparison of DNA profile to population databases
Generation of Case Report with Probability of Random Match
John M. Butler (2009) Fundamentals of Forensic DNA Typing, Figure 1.2
Figure 1.2 Overview of biology, technology, and genetic components of DNA typing using short tandem repeat (STR) markers.

13. Q K Exclusion (no match) Inclusion (match) Report Crime committed
Suspect developed
Biological material transferred
May match another
(K’)
Evidence (Question) sample “Q”
Reference (Known) sample “K”
Database Search
Steps Involved
Steps Involved
May be Inconclusive
due to
Lack of Available Reference
Collection
Collection Q U A L I T Y S R N C E Q U A L I T Y S R N C E
Sample Storage
Exclusion (no match)
Sample Storage
Serology
Characterization
Q ≠ K
Extraction
Extraction Q K
DNA Profile
Comparison
Biology
Quantitation
Quantitation
Amplification
Amplification
May be Inconclusive
due to
Forensic Issues (degradation, mixtures, low levels)
Q = K
STR Markers
STR Markers
Inclusion (match)
Figure 1.3 Overview of steps involved in DNA testing
Separation/
Detection
Separation/
Detection
Technology
Data
Interpretation
Report
(with statistical weight)
Data
Interpretation
Statistical Interpretation
Genetics
Plea
Court
Profile put on database
John M. Butler (2009) Fundamentals of Forensic DNA Typing, Figure 1.3
Profile put on database

14. 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)

15. The Three Possible Outcomes of Evidence Examination
“Suspect”
Known (K) Sample
“Evidence”
Question (Q) Sample
Exclusion (no match)
Non-exclusion
“Match” or “inclusion”
Inconclusive result 11 12 13 11 12 11 12 11 12
No result
(or a complex mixture)

16. Steps in Forensic DNA Analysis
Collection
Extraction
Quantitation
STR Typing
Interpretation of Results
Database Storage & Searching
Specimen Storage
Multiplex PCR
Calculation of Match Probability
Steps Involved
Usually 1-2 day process (a minimum of ~5 hours)
DNA Quantitation
Slot Blot
1 ng
0.3 ng
0.7 ng
0.5 ng
No DNA
Sample Collection & Storage
Buccal swab
Blood Stain
DNA Extraction
Biology
Multiplex PCR Amplification
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
Genetics
STR Typing
DNA separation and sizing
DNA Database Search
Technology
Male: 13,14-15,16-12,13-10,13-15,16
Interpretation of Results

17. 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.

18. 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:
The specimens listed below were received in the Forensic Biology unit under cover of communication dated April 1, 2008 ( ) and April 15, 2008 ( ):
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.
John M. Butler (2009) Fundamentals of Forensic DNA Typing, D.N.A. Box 1.3

19. 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
17,18
8,8
14,16
X,Y
12,13
21,22 Q2 K1 K2
13,14
30.2,32
8,12
10,12
17,17
6,9
7,8
23,25
14,14
17,20
8,10
14,17
X,X
11,13
21,25
John M. Butler (2009) Fundamentals of Forensic DNA Typing, D.N.A. Box 1.3
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.

20. 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

21. 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

22. 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

23. Major Historical Events in Forensic DNA 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 4-colors 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
Windows 3.0 released (quality problems); exceeds $1 billion in sales
1991
fluorescent STR markers first described; Chelex extraction
Windows 3.1 released
John M. Butler (2009) Fundamentals of Forensic DNA Typing, Table 1.1

24. Major Historical Events in Forensic DNA Compared to Timeline for Microsoft Corporation
Year
Forensic DNA Science & Application
Parallel Developments in Biotechnology
Microsoft Corporation Chronology
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
Windows 95 released
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
Internet Explorer begins overtaking Netscape
John M. Butler (2009) Fundamentals of Forensic DNA Typing, Table 1.1

25. Major Historical Events in Forensic DNA 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 capillary array for high-throughput 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
Windows XP Tablet PC Edition released
2003
U.S. DNA database (NDIS) exceeds 1 million convicted offender profiles; the U.K. National DNA Database passes the 2 million sample mark
Human Genome Project completed with the “final” sequence coinciding with 50th anniversary of Watson-Crick DNA discovery
Windows Server 2003 released; 64-Bit Operating Systems expand capabilities of software
John M. Butler (2009) Fundamentals of Forensic DNA Typing, Table 1.1

26. 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