Advances in the Biochemistry of DNA Fingerprinting Analysis

J.M. Butler talk at ACS San Diego March 14, 2005

Methods for Human Identification Advances in the Biochemistry of DNA Fingerprinting Analysis

Dr. John M. Butler National Institute of Standards and Technology Biotechnology Division

March 14, 2005 American Chemical Society Meeting Fingerprints have been used since 1901 DNA since 1986

Justice for All Act of 2004 www.dna.gov

$1 billion into forensic DNA over the next 5 years Unfortunately, current DNA testing cannot be performed as quickly as a commercial break…

Lessons from the First Case Involving DNA Testing

Presentation Outline Describes the first use of DNA (in 1986) to solve a double rape-homicide case in England; about 5,000 men asked to give • Where has the field come in the past 20 years blood or saliva to compare to crime stains

• Connection of two crimes (1983 and 1986) • Current DNA typing methods and chemistry • Use of DNA database to screen for perpetrator (DNA only done on 10% with • Comments on the future of forensic DNA 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) STR Typing 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. http://www.cstl.nist.gov/biotech/strbase/NISTpub.htm 1 J.M. Butler talk at ACS San Diego March 14, 2005

http://www.innocenceproject.org Uses for DNA Typing

• 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… • Inheritance claims – who gets the money… DNA EXONERATIONS BY YEAR IN THE U.S.

AllAll usesuses involveinvolve accurateaccurate measurementmeasurement ofof DNADNA profilesprofiles andand PATTERNPATTERN MATCHINGMATCHING

Forensic DNA Testing Historical Perspective on DNA Typing 2005: DNA is an important Justice for All Act 2005 The genome of each individual is unique (with the part of the criminal ($1B over 5 years) exception of identical twins) justice system Identifiler 5-dye kit and ABI 3100 2004 UK National 2002 Y-STRs Probe subsets of genetic variation in order to Database launched CODIS loci differentiate between individuals (statistical probabilities (April 10, 1995) defined PowerPlex® 16 (16 loci in single amp) of a random match are used) 1998 2000 FSS STR typing with CE is fairly routine DNA typing must be performed efficiently and Quadruplex 1996 First commercial First STRs 1994 reproducibly (information must hold up in court) fluorescent STR developed mtDNA multiplexes 1990 1992 Current standard DNA tests DO NOT look at genes – Capillary electrophoresis little/no information about race, predisposal to disease, of STRs first described or phenotypical information (eye color, height, hair color) 1985 DQA1 & PM PCR developed Multiplex STRs is obtained RFLP (dot blot)

Sources of Biological Evidence DNA in the Cell • Blood WeWe examine examine length length variations variations or or sequence sequence • Semen variationsvariations in in the the DNA DNA molecules. molecules. • Saliva chromosome 22 pairs + XX or XY • Urine cell nucleus • Hair • Teeth • Bone Double stranded DNA molecule Target Region for PCR • Tissue Blood stain ~3 billion total base pairs

Only a very small amount of blood is needed to obtain a Individual Short oligonucleotides used nucleotides DNA profile Short oligonucleotides used asas primersprimers toto targettarget regionregion http://www.cstl.nist.gov/biotech/strbase/NISTpub.htm 2 J.M. Butler talk at ACS San Diego March 14, 2005

Polymerase Chain Reaction (PCR) Process 5’ 3’ Starting DNA Template 80-500 bases What Type of Genetic Variation? 3’ 5’ More alleles are possible Separate •Length Variation Forward Primer strands 5’ 3’ (denature) 5’ 3’ short tandem repeats (STRs)

Add primers CTAGTCGT(GATA)(GATA)(GATA)GCGATCGT 5’ 3’ 5’ (anneal) 3’ Reverse Primer •Sequence Variation Make copies (extend primers) single nucleotide polymorphisms (SNPs) insertions/deletions

Repeat Cycle, GCTAGTCGATGCTC(G/A)GCGTATGCTGTAGC Copying DNA Exponentially InIn 3232 cyclescycles atat 100%100% efficiency,efficiency, 1.071.07 billionbillion copiescopies ofof targetedtargeted DNADNA regionregion areare createdcreated

Short Tandem Repeats (STRs) Variation of STRs Among Individuals An allelic ladder, which is a mixture of common alleles, is used to convert DNA size to STR repeat number Fluorescent dye creates Fluorescent Fluorescent dye creates Familial Relationships Can Be Tracked aa labeled labeled PCR PCR product product dye label AATG AATG

Father 7 repeats Unrelated Individuals

8 repeats Mother the repeat region is variable between samples while the flanking regions where PCR primers bind are constant Daughter Homozygote = both alleles are the same length Heterozygote = alleles differ and can be resolved from one another

Son Primer positions define PCR product size

Position of Forensic STR Markers Scanned Gel on Human Chromosomes Image TPOX 13 CODIS Core STR Loci

D3S1358 8 repeats PCR 9 repeats TH01 D8S1179 D5S818 VWA PCR product FGA D7S820 DNA size (bp) CSF1PO

AMEL Sex-typing D13S317 D16S539 D18S51 D21S11 AMEL

Capillary Electropherogram http://www.cstl.nist.gov/biotech/strbase/NISTpub.htm 3 J.M. Butler talk at ACS San Diego March 14, 2005

Argon ion Steps in STR Typing LASER STR Typing Technologies (488 nm) Size http://www.cstl.nist.gov/biotech/strbase/tech.htm ABI Prism

Separation spectrograph Gels Capillary Electrophoresis Capillary Arrays

i B

Color

Separation s e

Fluorescence t

r u

Sample o

a t

Separation a D

J. Forensic Sci. (1998) 43: 1168-1180 Electrophoresis (1998) 19: 86-93 Nucleic Acids Res. (1999) 27: e36 Capillary CCD Panel (with virtual filters) Sample Detection Microchip CE Mass Spectrometry Hybridization Arrays

Sample m

u h

Injection Processing with GeneScan/Genotyper software c

f o

Mixture of dye-labeled

s e

PCR products from t

u o www.sequenom.com

multiplex PCR c

a t

reaction a Sample Interpretation D

PNAS (1997) 94: 10273-10278 Int. J. Legal Med. (1998) 112: 45-49 Nucleic Acids Res. (2000) 28: e17 Butler, J.M. (2005) Forensic DNA Typing, 2nd Edition, Figure 13.8, © Elsevier Science/Academic Press

Capillary Electrophoresis Instrumentation Steps in DNA Analysis ABI 310 ABI 3100 Usually 1-2 day process (a minimum of ~5 hours) Slot Blot single capillary 16-capillary array 1 ng 0.3 ng Collection No DNA 0.5 ng 0.5 ng Specimen Storage 0.7 ng 1 ng Blood Stain Buccal swab 1 ng Extraction Sample Collection DNA DNA & Storage Extraction Quantitation Quantitation

Genotyping Multiplex PCR Amplification

Interpretation of Results STR Typing

Database Male: 13,14-15,16-12,13-10,13-15,16 Storage & Searching Interpretation of Results DNA Database

DNA Profile Frequency with all 13 CODIS STR loci

Commercial STR 16plex Kits TH01 TPOX D7 CSF AmpFlSTR® Identifiler™ SRM 2391b component 1 D13 D16 (Applied Biosystems) D3 D19 D8 D21 D18 D2 AMEL VWA D5 FGA What would D8 Identifiler™ kit (Applied Biosystems) be entered Locus allele value allele value frequency, 1 in multiplex STR result into a DNA TH01 database for D3S1358 16.0 0.2315 17.0 0.2118 10.20 D13 VWA VWA 17.0 0.2628 18.0 0.2219 8.57 D19 D5 TPOX D16 1313 core core STR STR loci loci + + searching: AMEL D21 CSF 16,17- D3 FGA 2 additional loci + FGA 21.0 0.1735 22.0 0.1888 15.26 D18 D2 2 additional loci + 17,18- D7 D8S1179 12.0 0.1454 14.0 0.2015 17.07 AMELAMEL sex-typing sex-typing 21,22- 12,14- D21S11 28.0 0.1658 30.0 0.2321 12.99 28,30- D18S51 14.0 0.1735 16.0 0.1071 26.91 14,16- ® D5S818 12.0 0.3539 13.0 0.1462 9.66 PowerPlex 16 kit (Promega Corporation) 12,13- D13S317 11.0 0.3189 14.0 0.0357 43.92 multiplex STR result 11,14- D8 9,9- D7S820 9.0 0.1478 43.28 D18 Penta E D5 D21 CSF 11,13- D16S539 11.0 0.2723 13.0 0.1634 11.24 AMEL D7 VWA 6,6- THO1 6.0 0.2266 18.83 D3 D13 Penta D 8,8- TH01 D16 FGA TPOX 8.0 0.5443 3.35 TPOX 10,10 CSF1PO 10.0 0.2537 15.09 The Random Match Probability for this profile in the FBI Caucasian population From Butler, J.M. (2005) Constructing STR multiplex assays. Methods in Molecular Biology: Forensic DNA Typing Protocols is 1 in 1.56 quadrillion (1015) (Carracedo, A., ed.), Humana Press: Totowa, New Jersey, in press. http://www.csfs.ca/pplus/profiler.htm

http://www.cstl.nist.gov/biotech/strbase/NISTpub.htm 4 J.M. Butler talk at ACS San Diego March 14, 2005

Databank vs Casework Common Casework Challenges Data Challenges DEGRADED DNA • Databank (single source samples) D5S818 D7S820 – Too much DNA may be added to the PCR reaction resulting D13S317 Loss of signal at D16S539 CSF1PO Penta D larger size loci in pull-up between dye colors – Lots of data to review – often produced by contractors

• Casework (mixtures or low level samples) MIXTURES – Often limited DNA material to work with More than two alleles – Low copy number samples can result in allele dropout at multiple loci – Can produce complicated STR profiles to interpret

Improved computer software for rapid data interpretation D3S1358 TH01 D13S317 D16S539 D2S1338

is really the biggest need currently From Butler, J.M. (2004) Short tandem repeat analysis for human identity testing. Current Protocols in Human Genetics, John Wiley & Sons, Hoboken, NJ, Unit 14.8, (Supplement 41), pp. 14.8.1-14.8.22

Remove a portion of Differential Extraction the mixed Advantages for STR Markers stain SDS, EDTA and proteinase K • Small product sizes are generally compatible with (cell lysis buffer) degraded DNA and PCR enables recovery of Incubate information from small amounts of material at 37 oC

Centrifuge • Multiplex amplification with fluorescence detection Perpetrator’s sperm mixed with victim’s sperm enables high power of discrimination in a single test epithelial cells pellet REMOVE SDS, EDTA and supernatant • Commercially available in an easy to use kit format proteinase K + DTT DTT lyses sperm • Uniform set of core STR loci provide capability for heads national and international sharing of criminal DNA profiles “Male Fraction” sperm “Female Fraction” pellet

Differential extraction used to separate sperm (male fraction) from Male-Specific Amplification in Presence of Excess Female DNA vaginal epithelial cells (female fraction) PowerPlex Y kit results 2 ng male female Evidence (female fraction)

2 ng male: 15 ng female Evidence (male fraction) male

Suspect 500 pg male: 408 ng female male

Victim female 800X female DNA 1 ng male: 816 ng female

USACIL case 01-1738 The four samples typically associated with a forensic DNA case…

http://www.cstl.nist.gov/biotech/strbase/NISTpub.htm 5 J.M. Butler talk at ACS San Diego March 14, 2005

Different Inheritance Patterns http://www.fbi.gov

Lineage Markers CODIS STR Loci

Selection of U.S. Core Loci:: DYS19, DYS385 a/b, Autosomal Y-Chromosome Mitochondrial DYS389I/II, DYS390, (passed on in part, (passed on complete, (passed on complete, DYS391, from all ancestors) but only by sons) but only by daughters) DYS392, DYS393, DYS438, DYS439

Mitochondrial DNA miniSTRs: new tool for degraded DNA Smaller PCR products work better with low High copy number is the primary advantage for degraded DNA samples Conventional miniSTR PCR primer primer copy number or fragmented DNA templates mtDNA Inheritance Patterns

12A B STR repeat region miniSTR Conventional primer PCR primer

354 678 B C D B E B Conventional STR test 9 10 11 12 13 14 (COfiler™ kit) C C B F B G

15 16 17 18 B B G G MiniSTR assay (using 150 bp smaller Butler et al. 2003 primers) Maternal relatives have the same mtDNA Isenberg et al. (1999) Forensic Sci. Comm. (barring mutation)

Ensuring Accurate Forensic DNA Results Future Methods Used in DNA Analysis ASCLD-LAB Proficiency • Improved capabilities for multiplex analysis Accreditation Testing of (parallel processing of genotypes) Analysts • More rapid separation/detection technology (higher throughputs) • More automated sample processing and data Inspections/ analysis DAB Audits • Improved sensitivities and resolution NIST • Less expensive sample analysis Standards- SWGDAM Standard We must maintain accurate and robust methods Guidelines (SRMs)

http://www.cstl.nist.gov/biotech/strbase/NISTpub.htm 6 J.M. Butler talk at ACS San Diego March 14, 2005

Law Judicial Enforcement •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

Laboratory http://www.ojp.usdoj.gov/nij/pubs-sum/183697.htm

If you want to know more… Thank you for your attention…

• Forensic DNA Typing: Biology and Technology behind Our publications and presentations are available at: http://www.cstl.nist.gov/biotech/strbase/NISTpub.htm STR Markers • NIST website: http://www.cstl.nist.gov/biotech/strbase NIST Project Team: Collaborators (past & present): • John Butler email: [email protected] John Butler (leader) Tom Parsons (AFDIL) Margaret Kline Bruce McCord (OhioU/FIU) Jan Redman Alan Redd (U Arizona) Pete Vallone Mike Hammer (U Arizona) STRBase Dave Duewer Sandy Calloway (Roche) Amy Decker Jim Girard (American U) Mike Coble Marilyn Raymond (NCI) Mecki Prinz (NYC OCME) Peter Gill (UK FSS)

National Institute of Justice Funding through NIST Office of Law Enforcement Standards

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