Fundamentals of Forensic DNA Typing 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 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)

Historical Perspective on DNA Typing 2009: DNA is an important part of the criminal justice system www.dna.gov President’s DNA Initiative (>$600M from 2004-2008) 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

Stages of Forensic DNA Progression Time Frame Description Beginnings, different methods tried (RFLP and early PCR) 1985-1995 Exploration Standardization to STRs, selection of core loci, implementation of Quality Assurance Standards 1995-2005 Stabilization Rapid growth of DNA databases, extended applications pursued 2005-2009 Growth Expanding tools available, confronting privacy concerns The Future Sophistication From John M. Butler (Feb 2009) Presentation at AAFS session on “Envisioning the Future”

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.

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.

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 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.

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

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 “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)

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

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 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. 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 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.

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 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

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

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 3700 96-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

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