DNA and Biometrics (Mitretek Seminar) May 12, 2004

Examples of DNA in the News

• Saddam Hussein Identification
• Source of Cow with “Mad cow” Disease
Forensic DNA Typing and • Scot Peterson Murder Trial

Prospects for Biometrics

• Identification of WTC Victims
• “Thomas Jefferson fathered slave’s children”

Dr. John M. Butler

NIST Biotechnology Division

May 12, 2004

“A Forensic Paternity Test”

December 14, 2003 Captured December 13, 2003

Y-chromosome tests were also

“We got him!” used to verify male lineage…

Portion of DNA Profile

DNA Profile Suspected

Saddam

Saddam Hussein’s From Son #1

capture verified with
DNA testing
Source: www.cnn.com; The Scientist Dec 19, 2003
Killed
July 22, 2003 From Son #2
DNA Used as the Ultimate Biometric
for Confirming Saddam’s Identity
Source: www.cnn.com Uday and Qusay Hussein

Our DNA Comes from our Parents Genetic Inheritance Pattern of

DNA Profiles
Father’s
Sperm
Mother’s DAD
Egg

CHILD

MOM
Child’s Cell

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DNA and Biometrics (Mitretek Seminar) May 12, 2004

PATERNITY TESTING PATERNITY TESTING

Family Inheritance of STR Alleles (D13S317) Family Inheritance of STR Alleles (D13S317)
PCR product size (bp) PCR product size (bp)

11 14 11 14
Results Father
Me Father
Me
Resultsof
ofDNA
DNATests
Tests
Impact
ImpactFamilies
Families
12 14 12 14
Child #1 Amanda
Child #1

8 14 8 14
Child #2 Child #2
Marshall

11 12 11 12
Child #3 Child
Katy#3

8 12 8 12
Mother Mother
My Wife

Historical Investigation DNA Study Methods for Human Identification

(Matching Relatives to Remains or Relatives to Relatives)
Thomas Jefferson II Failure
Failure to
to Exclude
Exclude
Jefferson
Jefferson as
as Eston
Eston
Hemings’
Hemings’ Father
Father
Field Jefferson Peter Jefferson

President Thomas Woodson

Thomas Jefferson
?
Eston Hemings

Genetic Genealogy Companies

Different Y
Haplotype
Same Y
Jefferson Haplotype
Y Haplotype
Jefferson Nature article (Nov 1998)
Y Haplotype
Fingerprints have been used since 1901 DNA since 1986
Butler, J.M. (2001) Forensic DNA Typing, Figure 17.4, ©Academic Press

Forensic DNA Testing Law Judicial

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

Probe subsets of genetic variation in order to

differentiate between individuals

DNA typing must be done efficiently and reproducibly

(information must hold up in court)

Typically, we are not looking at genes – little/no

information about race, predisposal to disease, or
phenotypical information (eye color, height, hair color) is
obtained Laboratory

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DNA and Biometrics (Mitretek Seminar) May 12, 2004

Applications for Human Identity Testing Armed Forces DNA Repository

>4.5 million blood cards on

Forensic cases - matching suspect with file from members of U.S.
evidence military
Paternity testing - identifying father Are being used to identify
Historical investigations remains in case of combat
casualties in Iraq
Missing persons investigations
Mass disasters - putting pieces back together
Military DNA “dog tag”
Convicted felon DNA databases
As
AsDNA
DNAanalysis
analysishas
hasshown
shownits
itsusefulness,
usefulness,the
thenumber
numberof
ofsamples
samples Located in Gaithersburg,
gathered
gatheredfor
fortesting
testingpurposes
purposeshas
hasgone
goneupupdramatically…
dramatically… Maryland

Tomb of Unknown Soldier Roles of Biological Evidence in

Armed Forces DNA
Identification Laboratory Criminal Investigation
(Rockville, MD)

Identification of Skeletal Remains from Identify a person

Previous Military Conflicts Exclude a suspect
Identification of Link suspect, victim and crime scene
Michael J. Blassie - Vietnam Tomb of the
Unknown Link weapon to victim
Link witness to scene
>2100 Unaccounted for
from Southeast Asia Prove or disprove an alibi Post-conviction DNA testing is
used to exonerate falsely
>8000 Unaccounted for incarcerated individuals
from Korea Reconstruct the scene (143 so far)
>75,000 Unaccounted for
from WWII
Provide investigative leads

Sources of Biological Evidence Steps in DNA Analysis

Slot Blot

• Blood 1 ng
0.3 ng

• Semen Collection No DNA

0.5 ng
0.5 ng
• Saliva Specimen Storage 0.7 ng
1 ng
• Urine Blood Stain Buccal swab 1 ng

Extraction Sample Collection DNA DNA

• Hair & Storage Extraction Quantitation
• Teeth Quantitation
• Bone
• Tissue Genotyping Multiplex PCR Amplification
Blood stain
Interpretation
Only a very small of Results
amount of blood is STR Typing
needed to obtain a
DNA profile Database Male: 13,14-15,16-12,13-10,13-15,16
Storage & Searching
Interpretation of Results DNA Database

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DNA and Biometrics (Mitretek Seminar) May 12, 2004

DNA in the Cell What Type of Genetic Variation?

chromosome 22 pairs + XX or XY •Length Variation

short tandem repeats (STRs)
cell nucleus
CTAGTCGT(GATA)(GATA)(GATA)GCGATCGT

Double stranded
DNA molecule Target Region for PCR •Sequence Variation
single nucleotide polymorphisms (SNPs)
~3 billion total base pairs
insertions/deletions
GCTAGTCGATGCTC(G/A)GCGTATGCTGTAGC
Individual
nucleotides

PCR Process
Basic Concepts 5’ 3’ Starting DNA Template
80-500 bases
3’ 5’
PCR polymerase chain reaction – method of
amplifying a specific region of the genome – go from 1 to Separate
over a billion copies in about 2 hours Forward Primer strands
5’ 3’ (denature) 5’ 3’

Locus region of the genome being examined 3’ 5’

Add primers
(anneal) 3’ 5’
Reverse Primer
Allele the state of the genetic variation being examined
Make copies
(STRs = number of repeat units) (extend primers)
(SNPs = base sequence at the site)

Chromosomes are paired so… Repeat Cycle,

Copying DNA
Homozygous – Alleles are identical on each chromosome Exponentially

Heterozygous - Alleles differ on each on each chromosome

Position of Forensic STR Markers

Short Tandem Repeats (STRs)
on Human Chromosomes
Fluorescent TPOX 13 CODIS Core STR Loci
Fluorescent Fluorescentdye
dyecreates
creates
dye label
aalabeled
labeledPCR
PCRproduct
product
AATG AATG D3S1358
primer1
TH01
D8S1179
D5S818 VWA
primer2
primer1 7 repeats FGA D7S820
CSF1PO

8 repeats primer2

the repeat region is variable between samples while the AMEL

flanking regions where PCR primers bind are constant Sex-typing
Homozygote = both alleles are the same length D13S317
Heterozygote = alleles differ and can be resolved from one another D16S539 D18S51 D21S11 AMEL

Primer positions define PCR product size

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DNA and Biometrics (Mitretek Seminar) May 12, 2004

Labeled DNA fragments

Capillary Electrophoresis System (PCR products) Principles of Sample
Capillary or Separation and Detection
Laser Gel Lane
36 cm
Sample Detection
Capillary filled with
Detection
polymer solution CCD Panel
window Size Ar+
Separation LASER
- 5-20 + (488 nm)

(cathode) (anode)
kV

Outlet
Inlet
Buffer Detection
Color
Buffer
region Separation

Fluorescence ABI Prism

Data
Sample tray moves spectrograph
automatically beneath the Acquisition
Sample cathode end of the capillary
tray to deliver each sample in
succession
Butler, J.M. (2001) Forensic DNA Typing, Figure 9.3, ©Academic Press Butler, J.M. (2001) Forensic DNA Typing, Figure 10.8, ©Academic Press

Scanned
Capillary Electrophoresis Instrumentation Gel
ABI 310 ABI 3100 Image
single capillary 16-capillary array

8 repeats

9 repeats

Capillary Electropherogram

PCR Product Size (bp) PCR Product Size (bp)

loci loci
Allelic 8 11 14 Allelic 8 11 14
Ladders Ladders

Sample Sample alleles

#1 #1

All heterozygous All heterozygous

Sample Sample
#2 #2

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DNA and Biometrics (Mitretek Seminar) May 12, 2004

Crime Scene - Two Suspects Methods for Parallel Sample Processing

Multiplex
Multiplexby
bySize
Size Multiplex
Multiplexby
byDye
Dye Color
Color
Suspect 1 Blue

Green
D3 vWA FGA
Yellow
S1 14,15 17,18 23,24
S2 15,18 17,19 23.2,24
Suspect 2 Internal sizing standard in red Combined
E 15,18 17,19 23.2,24

Multiplex
Multiplexby
byNumber
Number of
ofCapillaries
Capillaries
Evidence

Information is tied together with multiplex PCR and data analysis High-Throughput STR Typing on the ABI 3100 (16-capillary array)

AmpFlSTR® Identifiler™ (Applied Biosystems)

D8S1179 D21S11 D7S820 CSF1PO

TH01 D13S317
256 data points in 45 minutes with
STR 16plex and 16 capillaries
D3S1358 D16S539 D2S1338

D19S433 VWA TPOX D18S51

AMEL D5S818 FGA

11 integrated
integrated analysis
analysis
vs.
vs. 16
16 separate
separate runs
runs

DNA Profile Frequency with all 13 CODIS STR loci

DNA Statistics AmpFlSTR® Identifiler™
TH01 TPOX D7 CSF
D13
For heterozygous loci What
would
(Applied Biosystems) D19 be
D3 entered into a D21
D8 DNA database forD16
searching:
D18 D2
AMEL D5 VWA
16,17-17,18-21,22-12,14-28,30-14,16-12,13-11,14-9,9-11,13-6,6-8,8-10,10
FGA
P = 2pq
P = probability; p and q are frequencies of allele Locus allele value allele value frequency, 1 in

in a given population D3S1358 16 0.2315 17 0.2118 10.20

VWA 17 0.2628 18 0.2219 8.57
FGA 21 0.1735 22 0.1888 15.26
Example: For the locus D3S1358 and individual D8S1179 12 0.1454 14 0.2015 17.07
is 16,17 with frequencies of 0.2315 and 0.2118 D21S11 28 0.1658 30 0.2321 12.99
respectively D18S51 14 0.1735 16 0.1071 26.91
D5S818 12 0.3539 13 0.1462 9.66
D13S317 11 0.3189 14 0.0357 43.92
P = 2(0.2315)(0.2118) = 0.0981 or 1 in 10.2
D7S820 9 0.1478 43.28
D16S539 11 0.2723 13 0.1634 11.24
For independent loci, the genotype frequencies can THO1 6 0.2266 18.83
be combined through multiplication… TPOX 8 0.5443 3.35
Profile Probability = (P1)(P2)…(Pn) CSF1PO 10 0.2537 15.09
= 1 in a very large number… The Random Match Probability for this profile in the FBI Caucasian population
is 1 in 1.56 quadrillion (1015)

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DNA and Biometrics (Mitretek Seminar) May 12, 2004

All 50 states now require convicted offenders

CODIS DNA Database to submit a sample for DNA testing purposes

16,160 Investigations Aided

Combined DNA Index System as of March 2004
Used for linking serial crimes and unsolved cases
with repeat offenders
Convicted offender and forensic case samples As of March 2004 the profile composition of the National DNA Index
Launched October 1998 and links all 50 states System (NDIS) is as follows:

Requires 13 core STR markers Total number of profiles: 1,719,551

Total Forensic profiles: 78,475
Current backlog of >750,000 samples (millions of Total Convicted Offender Profiles: 1,641,076
dollars are spent each year to reduce backlog)

http://www.fbi.gov/hq/lab/codis/clickmap.htm

1,641,076 offenders National DNA Database

78,475 forensic
102 missing persons
175 CODIS labs in 50 states, FBI, US Army Crime Lab & Puerto Rico
16,160 investigations aided
Results from Virginia
Our Human Identification Project Team
Statistical Information Total • Prepares Standard Reference Materials (SRMs)
– SRM 2391b PCR-based DNA Profiling Standard
Offender Profiles 185,387
– SRM 2395 Human Y-Chromosome DNA Profiling Standard
Forensic Samples 3,068 • Creates databases with useful information
Number of CODIS Labs 4 – STRBase (http://www.cstl.nist.gov/biotech/strbase)
NDIS Participating Labs 4 • Evaluates and develops new technologies
Investigations Aided 1,773 • Conducts interlaboratory testing
• Performs quality control testing for labs & companies
http://www.fbi.gov/hq/lab/codis/va.htm

Ensuring Accurate Forensic DNA Results

STRBase
ASCLD-LAB Proficiency Short Tandem Repeat DNA
Accreditation Testing of Internet Database
Analysts General Information Forensic Interest Data Supplemental Info
•Intro to STRs •FBI CODIS Core Loci •Reference List
(downloadable PowerPoint)
•DAB Standards •Technology Review
Inspections/ •STR Fact Sheets
•NIST SRM 2391 •Addresses for Scientists

DAB Audits •Sequence Information

•Published PCR Primers •Links to Other Web Sites

Standards- NIST •Multiplex STR Kits

•Variant Allele Reports
•Y-Chromosome STRs
Standardized
Standardized
•Population Data information
informationformats
SWGDAM Standard •Validation Studies
formats

Guidelines (SRMs) http://www.cstl.nist.gov/biotech/strbase

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DNA and Biometrics (Mitretek Seminar) May 12, 2004

New DNA Test for Cats Developed in Our Lab Telling Cats Apart…
(the “MeowPlex”)
“Kitty Biometrics”

male

Different peak patterns between samples

female

Disclaimer DNA within the Biometric Model

Deny Entry
Creating the reference sample…
“Exonerated”
• Our laboratory performs research and
development in human identification techniques
involving DNA testing
Testing the “evidence”…

• I am not an “expert” in biometrics and therefore

do not understand all of the issues involved
• The following ideas are my own and in no way
represent the official position of the National
Institute of Standards and Technology (NIST)
“Implicated”
http://www.itl.nist.gov/div893/biometrics/Biometricsfromthemovies.pdf
Permit Entry
Match of 13 points (each with 2 variable alleles) within DNA
String of 26 numbers (order of listing DNA results would have to be standardized)
16,17-17,18-21,22-12,14-28,30-14,16-12,13-11,14-9,9-11,13-6,6-8,8-10,10

Issues with DNA as a Biometric Tool Collection of Test Sample

Individualization of everyone except identical twins
• Invasive collection of test sample (swab of cheek cells or finger • Buccal (cheek cell)
prick for blood)
• Concern over genetic privacy if sample is stored (would or could swab is not as
sample ever be used for other purposes?) invasive as a finger
• Expense for testing (>$20 per sample; forensic testing is >$500)
• Time required for testing (minimum 4-5 hours with forensic markers)
prick to draw blood

• Database search/storage of DNA profile (who controls access?; would

it ever be used for criminal investigations?) • DNA can be
• Development of reference databases for terrorists, etc. (how would a reliably extracted
match be used?)
• Chance for falsifying your sample (e.g., movie GATTACA)
Used routinely in Virginia for off of the paper or
felon arrestee DNA collection Q-tip swab
with 99.5% success (only 23
failures in first 6 months of use)

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DNA and Biometrics (Mitretek Seminar) May 12, 2004

Time Required for Testing

Genetic Privacy Concerns Collection
Now typically a minimum of 4-5 hours

• The DNA profile itself is neutral (and uninformative)— Extraction Could be <5 minutes
just a string of numbers like a Social Security Number Not necessary if samples are
Quantitation uniform in amount Biggest problem is
• DNA markers used in forensics were selected to be Rapid thermal cycling to-date done with length of time for PCR
neutral and are located away from or between genes Amplification singleplexes; typically 2-3 hours (with multiplex amplification)

rather than being part of gene products and therefore DNA separations (STR analysis) of <5 minutes
are not generally thought to be associated with any Genotyping have been demonstrated; typically ~30 minutes
genetic disease Currently performed manually in most labs;
Interpretation expert systems are under development to
of Results enable rapid interpretation
• Concern is really with the DNA sample collected—
would it be retained and used for any other type of Database Search could be similar to fingerprint
testing? (e.g., Armed Forces DNA Repository can Storage & Searching search in terms of speed
only be used for identifying combat casualties)
Comparison a DNA profile to a reference or database
Male: 13,14-15,16-12,13-10,13-15,16-…..

Possible Ways DNA Could Be Used Now How close are we to GATTACA?
Gattaca Corp. is an aerospace firm in the future. During this
As part of check before issuing visa for foreign visitors wanting to visit U.S. time society analyzes your DNA and determines where you
belong in life.
– DNA samples could be shipped from each embassy to U.S.-based lab for
testing Ethan Hawke's character was born with a congenital heart
condition which would cast him out of getting a chance to
– Visa would not be issued until DNA profile was generated and searched travel in space. So in turn he assumes the identity of an
against a national/international database athlete named Jerome who has genes that would allow him
– DNA profile would be included in biometric passport for future confirmation to achieve his dream of space travel.
of identity as needed Hawke’s character Vincent constantly passes genetic tests
by diligently using samples of Jerome's hair, skin, blood and
Confirming identity upon entry into the U.S. urine.
while the plane is in the air
– Would require DNA labs operating in Entry to secure locations controlled by
foreign airports rapid genetic profiling
– 200+ DNA profiles would be generated
in ~8 hours (before a plane coming from Ability to predict future health risks
Europe landed) based on genetic testing
http://www.imdb.com/title/tt0119177/

In FY2003, 7,300,667 U.S. passports were issued

http://travel.state.gov/passport_statistics.html
1997 movie with a futuristic story of a
genetically imperfect man and his seemingly NOT THERE YET!
unobtainable goal to travel in space.

Lagally et al., Lab-on-a-Chip, 1, 102 (2001)

15 minutes for PCR amplification and detection

From Richard Mathies presentation at 14th International Symposium on Human Identification, Oct 2003 From Richard Mathies presentation at 14th International Symposium on Human Identification, Oct 2003

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DNA and Biometrics (Mitretek Seminar) May 12, 2004

Virginia DNA Testing of Felon Arrestees If you want to know more…

As of January 1, 2003, any individual arrested for a violent felony crime (Code of Virginia §
19.2-310.2:1) must provide a buccal sample for DNA analysis, with the resultant profile • Forensic DNA Typing: Biology and Technology behind
incorporated into the Virginia DNA Data Bank (Code of Virginia § 19.2-310.5).
STR Markers
Since January 2003 • NIST website: http://www.cstl.nist.gov/biotech/strbase
• Buccal swab collected upon arrest • John Butler email: john.butler@nist.gov
• DNA sample processed within 72 hours
• DNA profile searched against state database STRBase
(national database does not currently allow searches
for individuals prior to conviction)
• If a match results, then arrestee is detained and later
prosecuted
• From Jan 2003 – Dec 2003, VA processed 7,836
arrestee samples (not all analyzed) and scored 63
hits against their state database (Profiles in DNA, 2004, 7(1):3-5)

Thank you for your attention…

NIST Project Team:

John Butler (leader)
Margaret Kline
Jan Redman
Pete Vallone
Dave Duewer
Jill Appleby
Amy Decker
Mike Coble

National Institute of Justice

Funding through NIST Office of
Law Enforcement Standards

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