DNA since 1986

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

Justice for All Act of 2004

www.dna.gov

Unfortunately, current DNA testing cannot be performed as quickly as a commercial break

$1 billion into forensic DNA over the next 5 years

Lessons from the First Case Involving DNA Testing

Presentation Outline
Where has the field come in the past 20 years Current DNA typing methods and chemistry Comments on the future of forensic DNA
STR Typing

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.

http://www.cstl.nist.gov/biotech/strbase/NISTpub.htm

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.

All Alluses usesinvolve involveaccurate accuratemeasurement measurement of of DNA DNAprofiles profiles and andPATTERN PATTERN MATCHING MATCHING

Forensic DNA Testing

The genome of each individual is unique (with the exception of identical twins) 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

Historical Perspective on DNA Typing

2005: DNA is an important part of the criminal justice system
UK National Database launched (April 10, 1995) Justice for All Act ($1B over 5 years) Identifiler 5-dye kit and ABI 3100

2005

2002
CODIS loci defined

2004 Y-STRs

PowerPlex 16

1998
FSS Quadruplex First STRs developed

2000

(16 loci in single amp)

STR typing with CE is fairly routine mtDNA

1994 1992

1996

First commercial fluorescent STR multiplexes

1990

Capillary electrophoresis of STRs first described

1985

PCR developed

RFLP

DQA1 & PM (dot blot)

Multiplex STRs

Sources of Biological Evidence

Blood Semen Saliva Urine Hair Tissue Teeth Bone

DNA in the Cell

We variations or sequence Weexamine examinelength length variations or sequence variations variationsin inthe theDNA DNAmolecules. molecules.

chromosome cell nucleus

22 pairs + XX or XY

DNA profile

Double stranded DNA molecule

Target Region for PCR

Blood stain
Only a very small amount of blood is needed to obtain a

~3 billion total base pairs

Short Shortoligonucleotides oligonucleotides used used as asprimers primers to to target targetregion region

Individual nucleotides

http://www.cstl.nist.gov/biotech/strbase/NISTpub.htm

Sex-typing

J.M. Butler talk at ACS San Diego

Polymerase Chain Reaction (PCR) Process
5 3
Separate strands (denature) Add primers 5 (anneal)

March 14, 2005

3 Starting DNA Template 5

80-500 bases

What Type of Genetic Variation?

Length Variation
More alleles are possible short tandem repeats (STRs)

Forward Primer
5 3

3 3 5

CTAGTCGT(GATA)(GATA)(GATA)GCGATCGT

Reverse Primer

Make copies (extend primers)

Sequence Variation
single nucleotide polymorphisms (SNPs) insertions/deletions GCTAGTCGATGCTC(G/A)GCGTATGCTGTAGC

Repeat Cycle, Copying DNA Exponentially

In In 32 32cycles cycles at at 100% 100%efficiency, efficiency,1.07 1.07billion billion copies copiesof oftargeted targetedDNA DNAregion regionare arecreated created

Short Tandem Repeats (STRs)

Fluorescent dye label
AATG AATG

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 Fluorescentdye dyecreates creates a alabeled labeledPCR PCRproduct product

Familial Relationships Can Be Tracked

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

Unrelated Individuals

Father

Mother

Daughter

Primer positions define PCR product size

Son

Position of Forensic STR Markers on Human Chromosomes

TPOX D3S1358 D8S1179 D7S820 TH01 VWA

Scanned Gel Image

8 repeats

13 CODIS Core STR Loci PCR

DNA size (bp)
9 repeats

D5S818 FGA CSF1PO

PCR product

AMEL D13S317

D16S539

D18S51

D21S11

AMEL

Capillary Electropherogram

http://www.cstl.nist.gov/biotech/strbase/NISTpub.htm

J.M. Butler talk at ACS San Diego

Argon ion LASER
(488 nm)

March 14, 2005

STR Typing Technologies
http://www.cstl.nist.gov/biotech/strbase/tech.htm
Capillary Electrophoresis

Steps in STR Typing

ABI Prism spectrograph

Size Separation

Gels
w o l d u H l l i B f o y s e t r u o c a t a D

Capillary Arrays

Sample Separation

Fluorescence

Color Separation

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
m .co m o n e u www.seq

Hybridization Arrays
m m u h c S m i J f o y s e t r u o c a t a D

Sample Injection
Mixture of dye-labeled PCR products from multiplex PCR reaction

Processing with GeneScan/Genotyper software

Sample Interpretation
nd

Butler, J.M. (2005) Forensic DNA Typing, 2

Edition, Figure 13.8, Elsevier Science/Academic Press

PNAS (1997) 94: 10273-10278

Int. J. Legal Med. (1998) 112: 45-49

Nucleic Acids Res. (2000) 28: e17

Capillary Electrophoresis Instrumentation

ABI 310 single capillary ABI 3100 16-capillary array Collection
Specimen Storage

Steps in DNA Analysis

Usually 1-2 day process (a minimum of ~5 hours)
Slot Blot 1 ng 0.3 ng No DNA 0.5 ng 0.5 ng 0.7 ng

Blood Stain

Buccal swab

1 ng 1 ng

Extraction Quantitation Genotyping Interpretation of Results Database

Storage & Searching

Sample Collection & Storage

DNA Extraction

DNA Quantitation

Multiplex PCR Amplification

STR Typing
Male: 13,14-15,16-12,13-10,13-15,16

Interpretation of Results

DNA Database

Commercial STR 16plex Kits

SRM 2391b component 1
D8 TH01 D19 AMEL D3 D5 VWA D21 D13 TPOX FGA D7 D16 D18 CSF D2

DNA Profile Frequency with all 13 CODIS STR loci

AmpFlSTR Identifiler (Applied Biosystems) TH01 D19 D3 AMEL D8 D5 VWA D21 TPOX D13 FGA D7 D16 D18 CSF D2

Identifiler kit (Applied Biosystems) multiplex STR result

13 13core coreSTR STR loci loci + + 2 2additional additionalloci loci+ + AMEL sex-typing AMEL sex-typing

PowerPlex 16 kit (Promega Corporation) multiplex STR result

D8 D5 AMEL D3 VWA TH01 D13 TPOX D21 D7 D16 FGA Penta D D18 CSF Penta E

What would be entered into a DNA database for searching: 16,1717,1821,2212,1428,3014,1612,1311,149,911,136,68,810,10

Locus D3S1358 VWA FGA D8S1179 D21S11 D18S51 D5S818 D13S317 D7S820 D16S539 THO1 TPOX CSF1PO

allele 16.0 17.0 21.0 12.0 28.0 14.0 12.0 11.0 9.0 11.0 6.0 8.0 10.0

value 0.2315 0.2628 0.1735 0.1454 0.1658 0.1735 0.3539 0.3189 0.1478 0.2723 0.2266 0.5443 0.2537

allele 17.0 18.0 22.0 14.0 30.0 16.0 13.0 14.0 13.0

value 0.2118 0.2219 0.1888 0.2015 0.2321 0.1071 0.1462 0.0357 0.1634

frequency, 1 in 10.20 8.57 15.26 17.07 12.99 26.91 9.66 43.92 43.28 11.24 18.83 3.35 15.09

From Butler, J.M. (2005) Constructing STR multiplex assays. Methods in Molecular Biology: Forensic DNA Typing Protocols (Carracedo, A., ed.), Humana Press: Totowa, New Jersey, in press.

http://www.csfs.ca/pplus/profiler.htm

The Random Match Probability for this profile in the FBI Caucasian population is 1 in 1.56 quadrillion (10 15 )

http://www.cstl.nist.gov/biotech/strbase/NISTpub.htm

J.M. Butler talk at ACS San Diego

March 14, 2005

Databank vs Casework Data Challenges

Databank (single source samples)
Too much DNA may be added to the PCR reaction resulting in pull-up between dye colors Lots of data to review often produced by contractors

Common Casework Challenges

DEGRADED DNA
D5S818 D7S820 D16S539 CSF1PO Penta D

D13S317

Loss of signal at larger size loci

Casework (mixtures or low level samples)

Often limited DNA material to work with Low copy number samples can result in allele dropout Can produce complicated STR profiles to interpret

MIXTURES More than two alleles at multiple loci

Improved computer software for rapid data interpretation is really the biggest need currently

D3S1358

TH01

D13S317

D16S539

D2S1338

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

Advantages for STR Markers

Small product sizes are generally compatible with degraded DNA and PCR enables recovery of information from small amounts of material 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

Remove a portion of the mixed stain

Differential Extraction
SDS, EDTA and proteinase K (cell lysis buffer) Incubate at 37 oC Centrifuge

Perpetrators sperm mixed with victims epithelial cells

sperm pellet

SDS, EDTA and proteinase K + DTT

REMOVE supernatant

DTT lyses sperm heads

sperm pellet

Male Fraction

Female Fraction

Differential extraction used to separate sperm (male fraction) from vaginal epithelial cells (female fraction)
female

Male-Specific Amplification in Presence of Excess Female DNA

PowerPlex Y kit results

2 ng male

Evidence (female fraction)

The four samples typically associated with a forensic DNA case

male

Evidence (male fraction)

2 ng male: 15 ng female

male

Suspect

500 pg male: 408 ng female

female

Victim

800X female DNA

USACIL case 01-1738

1 ng male: 816 ng female

http://www.cstl.nist.gov/biotech/strbase/NISTpub.htm

J.M. Butler talk at ACS San Diego

Different Inheritance Patterns
CODIS STR Loci

March 14, 2005

http://www.fbi.gov

Lineage Markers

Autosomal
(passed on in part, from all ancestors)

Y-Chromosome
(passed on complete, but only by sons)

Mitochondrial
(passed on complete, but only by daughters)

Selection Selection of U. U.S. S. Core Core Loci Loci:: DYS19, DYS19, DYS385 DYS385 a/b, a/b, DYS389I/II, DYS389I/II, DYS390, DYS390, DYS391, DYS391, DYS392, DYS392, DYS393, DYS393, DYS438, DYS438, DYS439 DYS439

Butler, J.M. (2005) Forensic DNA Typing, 2

nd

Edition, Figure 9.1, Elsevier Science/Academic Press

Mitochondrial DNA
High copy number is the primary advantage for degraded DNA samples mtDNA Inheritance Patterns
1 A

miniSTRs: new tool for degraded DNA

Conventional PCR primer miniSTR primer

Smaller PCR products work better with low copy number or fragmented DNA templates

B 2

STR repeat region

miniSTR primer Conventional PCR primer

10

11

12

13

14

Conventional STR test (COfiler kit)

15

16

17

18

150 bp smaller

MiniSTR assay (using Butler et al. 2003 primers)

Isenberg et al. (1999) Forensic Sci. Comm.

Maternal relatives have the same mtDNA (barring mutation)

Future Methods Used in DNA Analysis

Improved capabilities for multiplex analysis (parallel processing of genotypes) More rapid separation/detection technology (higher throughputs) More automated sample processing and data analysis Improved sensitivities and resolution Less expensive sample analysis We must maintain accurate and robust methods

Ensuring Accurate Forensic DNA Results

ASCLD-LAB Accreditation

Proficiency Testing of Analysts

DAB

Inspections/ Audits

Standards-

NIST
(SRMs)

SWGDAM
Guidelines

Standard

http://www.cstl.nist.gov/biotech/strbase/NISTpub.htm

Judicial

J.M. Butler talk at ACS San Diego

March 14, 2005

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

http://www.cstl.nist.gov/biotech/strbase/NISTpub.htm

Law Enforcement

Laboratory

If you want to know more

Forensic DNA Typing: Biology and Technology behind STR Markers NIST website: http://www.cstl.nist.gov/biotech/strbase John Butler email: john.butler@nist.gov

Thank you for your attention

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

STRBase

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