Forensic DNA: Use, Abuse, Forensic DNA: Use, Abuse, Promise, and PerilPromise, and Peril

William M. Shields

DNA IdentificationDNA Identification

• Where does DNA come from?Where does DNA come from?1/2 from mom1/2 from mom1/2 from dad1/2 from dad

What is it?What is it? ““Blue print” of lifeBlue print” of life

How is DNA different among us?How is DNA different among us?Common vs DifferentCommon vs Different

What does “DNA” mean?What does “DNA” mean?Deoxyribonucleic AcidDeoxyribonucleic Acid

Cell Types

Where can DNA be found?Where can DNA be found?Where can DNA be found?Where can DNA be found?

Cell Blood

Sweat

Hair Roots Saliva

Various Tissue Semen

SAME

Nucleus

Where is DNA in the body?Where is DNA in the body?Where is DNA in the body?Where is DNA in the body?

Cell

Nuclear DNA

Where are the types of DNA Where are the types of DNA found in a cell?found in a cell?

Where are the types of DNA Where are the types of DNA found in a cell?found in a cell?

Mitochondrial DNACellCell

MaternalChromosome

PaternalChromosome

Nucleus

Where is DNA in the body?Where is DNA in the body?Where is DNA in the body?Where is DNA in the body?

Where is DNA packaged in the Where is DNA packaged in the body?body?

Where is DNA packaged in the Where is DNA packaged in the body?body?

Chromosome

DNA

A =Adenine

T =Thymine

G =Guanine

C =Cytosine

UnitsDouble Helix

GATC

DNA- What does it look like?DNA- What does it look like?DNA- What does it look like?DNA- What does it look like?

Sources of Biological EvidenceSources of Biological Evidence

BloodBlood SemenSemen SalivaSaliva UrineUrine HairHair TeethTeeth BoneBone TissueTissue

Types of objects where DNA may Types of objects where DNA may be foundbe found

Types of objects where DNA may Types of objects where DNA may be foundbe found

Blood Stains

Semen Stains

Chewing Gum

Stamps & Envelopes

Penile Swabs

Plant Material

Sweaty Clothing

Bone

Hair

Fingernail Scraping

Saliva

Animal Material

Where DNA Evidence is Found

BloodHair RootsSalivaSweatTissue

Chemical

DNA

Isolation of DNAIsolation of DNAIsolation of DNAIsolation of DNA

Semen stainChemical

RemoveEpithelial

DNA

Differential Isolation of DNADifferential Isolation of DNADifferential Isolation of DNADifferential Isolation of DNA

Different Chemical

Sperm DNA

Semen stain

Epithelial DNA

Sperm DNA

DNA

Solution

AmplificationAmplificationAmplificationAmplification(making copies)(making copies)(making copies)(making copies)

TG C

ATTA

G C

A T

A

G

TA

GA

A T

C

AT

CT

Heat

Step one of a single cycle

DENATUREDENATURE

TAC TA TTCTT AT C

AA TA G G

Step two of a single cycle

ANNEALANNEAL

Step three of a single cycle

TAAATA G G

T ACTT AT C TAC TT

CA

AA

G

GG

GT

TT

T

A G

EXTENDEXTEND

1 Cycle2 Cycles

3 Cycles

4 Cycles

5 Cycles

28 Cycles

AmplificationAmplificationAmplificationAmplification

DNA

PCR (Polymerase Chain Reaction)PCR (Polymerase Chain Reaction)PCR (Polymerase Chain Reaction)PCR (Polymerase Chain Reaction)

Analysis of amplified DNAAnalysis of amplified DNA Analysis of amplified DNAAnalysis of amplified DNA

AmplifiedDNA

DNAProfile

Brief History of Forensic DNA TypingBrief History of Forensic DNA Typing

1980 - Ray White describes first 1980 - Ray White describes first polymorphic RFLP markerpolymorphic RFLP marker

1985 - Alec Jeffreys discovers multilocus 1985 - Alec Jeffreys discovers multilocus VNTR probesVNTR probes

1985 - first paper on PCR1985 - first paper on PCR 1988 - FBI starts DNA casework1988 - FBI starts DNA casework 1991 - first STR paper1991 - first STR paper 1995 - FSS starts UK DNA database1995 - FSS starts UK DNA database 1996 – First mtDNA case1996 – First mtDNA case 1998 - FBI launches CODIS database1998 - FBI launches CODIS database

DNA Use in Forensic CasesDNA Use in Forensic Cases

Most are rape cases or murders Most are rape cases or murders Looking for match between Looking for match between

evidence and suspectevidence and suspectMust compare victim’s DNA profileMust compare victim’s DNA profile

•Mixtures must be resolved

•DNA is often degraded

•Inhibitors to PCR are often present

Challenges

Human Identity TestingHuman Identity Testing Forensic cases -- Forensic cases -- matching suspect with matching suspect with

evidenceevidence Paternity testing -- Paternity testing -- identifying fatheridentifying father

Historical investigations-Czar Nicholas, Historical investigations-Czar Nicholas, Jesse JamesJesse James

Missing persons investigationsMissing persons investigations Mass disasters -- Mass disasters -- putting pieces back togetherputting pieces back together

Military DNA “dog tag”Military DNA “dog tag” Convicted felon DNA databasesConvicted felon DNA databases

Sample Obtained from Crime Scene or

Paternity Investigation Biology

DNAExtraction

DNAExtraction

DNAQuantitation

DNAQuantitation

PCR Amplificationof Multiple STR

markers

PCR Amplificationof Multiple STR

markers

TechnologySeparation and Detection of

PCR Products(STR Alleles)

Sample Genotype

Determination

GeneticsComparison of Sample

Genotype to Other Sample Results

Comparison of Sample Genotype to Other

Sample Results

If match occurs, comparison of DNA profile to population databases

If match occurs, comparison of DNA profile to population databases

Generation of Case Report with Probability

of Random Match

Generation of Case Report with Probability

of Random Match

Steps in DNA Sample Processing

BloodHair RootsSalivaSweatTissue

Chemical

DNA

Extraction of DNAExtraction of DNAExtraction of DNAExtraction of DNA

Short Tandem Repeat

AGAT AGATAGAT

AGAT

AGAT

AGAT

AGAT

AGAT

AGAT

AGAT

6

4

DNA Profile =4,6

TCTA TCTA TCTA

TCTA

TCTA

TCTA

TCTA

TCTA

TCTA

TCTA

7

5

DNA Profile =5,7

TCTA

TCTA

STRSTR

Multiplex PCR Over 10 Markers Can Be Over 10 Markers Can Be

Copied at OnceCopied at Once Sensitivities to levels less Sensitivities to levels less

than 1 ng of DNAthan 1 ng of DNA Ability to Handle Mixtures Ability to Handle Mixtures

and Degraded Samplesand Degraded Samples Different Fluorescent Dyes Different Fluorescent Dyes

Used to Distinguish STR Used to Distinguish STR Alleles with Overlapping Alleles with Overlapping Size RangesSize Ranges

An Example Forensic STR Multiplex KitAn Example Forensic STR Multiplex Kit

D3 FGAvWA 5-FAM (blue)

D13D5 D7 NED (yellow)

A D8 D21 D18 JOE (green)

GS500-internal lane standard

ROX (red)

AmpFlSTR® Profiler Plus™Kit available from PE Biosystems (Foster City, CA)

9 STRs amplified along with sex-typing marker amelogenin in a single PCR reaction

100 bp 400 bp300 bp200 bpSize Separation

Col

or S

epar

atio

n

DNA Quantitation using Slot Blot

AMEL

D3

TH01 TPOX

Penta D

Penta EFGAD21 D18

CSF

D16D7

D13D5VWA D8

PCR Amplification with Fluorescent STR Kits and Separation with Capillary Electrophoresis

Blood Stain

Overview of Steps Involved in DNA Overview of Steps Involved in DNA TypingTyping

Genotyping by Comparison to Allelic Ladder

Calculation of DNA Quantities Calculation of DNA Quantities in Genomic DNAin Genomic DNA

Important values for calculations:1 bp = 618 g/mol A: 313 g/mol; T: 304 g/mol; A-T base pairs = 617 g/mol

G: 329 g/mol; C: 289 g/mol; G-C base pairs = 618 g/mol 1 genome copy = ~3 x 109 bp = 23 chromosomes (one member of each pair) 1 mole = 6.02 x 1023 molecules Standard DNA typing protocols with PCR amplification of STR markers typically ask for 1 ng of DNA template. How many actual copies of each STR locus exist in 1 ng?  1 genome copy = (~3 x 109 bp) x (618 g/mol/bp) = 1.85 x 1012 g/mol 

= (1.85 x 1012 g/mol) x (1 mole/6.02 x 1023 molecules) 

= 3.08 x 10-12 g = 3.08 picograms (pg)  Since a diploid human cell contains two copies of each chromosome, then  each diploid human cell contains ~6 pg genomic DNA  1 ng genomic DNA (1000 pg) = ~333 copies of each locus (2 per 167 diploid genomes)

Short Tandem Repeats Short Tandem Repeats (STRs)(STRs)

the repeat region is variable between samples while the flanking regions where PCR primers bind are constant

AATG

7 repeats

8 repeats

AATG AATG

Primer positions define PCR product size

Fluorescent dye label

ABI Prism 310 Genetic Analyzer

capillary

Syringe with polymer solution

Autosampler tray

Outlet buffer

Injection electrode

Inlet buffer

Chemistry InvolvedChemistry Involved InjectionInjection

electrokinetic injection processelectrokinetic injection process importance of sample preparation importance of sample preparation

(formamide)(formamide)SeparationSeparation

capillarycapillaryPOP-4 polymerPOP-4 polymerbufferbuffer

DetectionDetection fluorescent dyes with excitation and emission fluorescent dyes with excitation and emission

traits traits virtual filters (hardware/software issues)virtual filters (hardware/software issues)

Sample Tube

DNA-

-

Electrokinetic Injection Electrokinetic Injection ProcessProcess

Electrode

CapillaryD

NA

-

-

Q is the amount of sample injected

r is the radius of the capillary

cs is the sample concentration

E is the electric field applied

t is the injection time

s is the sample conductivity

b is the buffer conductivity

ep is the mobility of the sample molecules

eo is the electroosmotic mobilityRose et al (1988) Anal. Chem. 60: 642-648

Q =s

r2cs(ep + eo)Etb

Separation IssuesSeparation Issues

Run temperatureRun temperature -- 60 -- 60 ooC helps reduce C helps reduce secondary structure on DNA and secondary structure on DNA and improves precisionimproves precision

Electrophoresis bufferElectrophoresis buffer -- urea in running -- urea in running buffer helps keep DNA strands denaturedbuffer helps keep DNA strands denatured

Capillary wall coatingCapillary wall coating -- dynamic coating -- dynamic coating with polymerwith polymer

Polymer solutionPolymer solution -- POP-4 -- POP-4

DNA Separation MechanismDNA Separation Mechanism

+-DNA-

DNA-

DNA-DNA- DNA-

• Size based separation due to interaction of DNA molecules with entangled polymer strands

• Polymers are not cross-linked (as in slab gels)• “Gel” is not attached to the capillary wall• Pumpable -- can be replaced after each run• Polymer length and concentration determine the separation

characteristics

ABI 310 Filter Set FABI 310 Filter Set F

520 540 560 580 600 620 640WAVELENGTH (nm)

100

80

60

40

20

0

5-FAM JOE NED ROX

Laser excitation(488, 514.5 nm)Laser excitation(488, 514.5 nm)

Normalized Fluorescent Intensity

Fluorescent Emission Spectra for ABI Fluorescent Emission Spectra for ABI DyesDyes

Sample Detection

CCD Panel

ColorSeparation

Ar+ LASER (488 nm)

Fluorescence ABI Prism spectrograph

Capillary or Gel Lane

Size Separation

Labeled DNA fragments (PCR products)

Detection region

Principles of Sample Principles of Sample Separation and Separation and

DetectionDetection

15,16 16,17 20,23 12,14 30,30X,Y 13.2,15Evidence

Area 1 Area 2 Area 3 Area 4 Area 5

AREAS OF DNA

SAMPLE Sex Area 6

Ref.Std.2

Ref.Std.1

15,16 16,17 20,23 12,14 30,30X,Y 13.2,15

14,15 17,18 23,24 13,13 30,30X,X 15,1914,15 17,18 23,24 13,13 30,30X,X 15,19

amelogenin

D19

D3

D8

TH01

VWA D21FGA

D16D18 D2

amelogeninD19

D3D8 TH01

VWA D21

FGA

D16D18 D2

Tw

o di

ffer

ent i

ndiv

idua

ls

DNA Size (base pairs)

Results obtained in less than 5 hours with a spot of blood the size of a pinhead

probability of a random match: ~1 in 3 trillion

Human Identity Testing with Multiplex STRs

Simultaneous Analysis of 10 STRs and Gender ID

AmpFlSTR® SGM Plus™ kit

PERKIN-ELMER’S PERKIN-ELMER’S PROFILER+ AND COFILERPROFILER+ AND COFILER

STATE OF TENNESSEE STATE OF TENNESSEE VERSUS VERSUS

TAYLOR LEE SMITHTAYLOR LEE SMITH

JUST THE FACTS:JUST THE FACTS:NOT A MIXTURE?NOT A MIXTURE?

1. Sperm Fraction: Eight of thirteen loci have a total 1. Sperm Fraction: Eight of thirteen loci have a total of nine alleles not found in either the victim or the of nine alleles not found in either the victim or the suspect.suspect.

2. Suspect Known: Eight of thirteen loci have a 2. Suspect Known: Eight of thirteen loci have a total of 12 different alleles not found in the sperm total of 12 different alleles not found in the sperm fraction “mixture”.fraction “mixture”.

3. Victim Known: Ten of thirteen loci have a total of 3. Victim Known: Ten of thirteen loci have a total of 11 different alleles not found in the sperm fraction 11 different alleles not found in the sperm fraction “mixture”.“mixture”.

COINCIDENCE OR EVIDENCE?COINCIDENCE OR EVIDENCE?The likelihood ratios for producing homozygous genotypes at The likelihood ratios for producing homozygous genotypes at four of thirteen STR loci* with DNA from a single individual four of thirteen STR loci* with DNA from a single individual versus a mixture of DNA from two individuals.versus a mixture of DNA from two individuals.

Theta = 0.03Theta = 0.03 Theta = 0.05 Theta = 0.05

African AmericanAfrican American 1 in1 in 278,000,000278,000,000 1 in1 in 43,000,00043,000,000

Likelihood RatioLikelihood Ratio 16,60016,600 6,5006,500

CaucasianCaucasian 1 in1 in 183,000,000183,000,000 1 in1 in 27,500,00027,500,000

Likelihood RatioLikelihood Ratio 13,50013,500 5,2005,200

HispanicHispanic 1 in1 in 15,000,000 1 in 3,700,000 15,000,000 1 in 3,700,000 Likelihood Ratio Likelihood Ratio 3,9003,900 1,9901,990

*Observed Sperm fraction genotypes: vWA=16, TPOX=8, D5S818=12, and D16S539=10).

Why the Y Chromosome?Why the Y Chromosome? ApplicationsApplications

forensic investigations forensic investigations (98% of violent crime by (98% of violent crime by men)men)

genealogical purposesgenealogical purposes evolutionary studiesevolutionary studies

Advantages to Human Identity TestingAdvantages to Human Identity Testing male component isolated without differential male component isolated without differential

extractionextraction paternal lineagespaternal lineages

NeedsNeeds population studies to evaluate diversity of population studies to evaluate diversity of

haplotypeshaplotypes robust assay for accurate characterization of Y robust assay for accurate characterization of Y

markersmarkers

Y STR Multiplex AssayY STR Multiplex Assay

100 bp 400 bp300 bp200 bp

DYS19 389II389I

390Primer Amounts Dye

Y19 0.25 M JOE

Y389 0.125 M FAM

Y390 0.25 M JOE

Primer Amounts Dye

Y19 0.25 M JOE

Y389 0.125 M FAM

Y390 0.25 M JOE

Prinz et al. 1997 (Forensic Sci Int, vol. 85, pp. 209-218)

“Quadruplex I”

Mitochondrial DNAMitochondrial DNA

What is mtDNA What is mtDNA Typing?Typing?

Database and Database and statistical issuesstatistical issues

A Mitochondrial Exclusion

A Mitochondrial Inclusion

Mitochondrial Inconclusive?

The Future of Forensic The Future of Forensic DNADNA

CODISCODIS

SNP’s & ChipsSNP’s & Chips

FBI’s CODIS DNA DatabaseFBI’s CODIS DNA Database

CoCombined mbined DDNA NA IIndex ndex SSystemystem Used for linking serial crimes and Used for linking serial crimes and

unsolved cases with repeat offendersunsolved cases with repeat offenders Launched October 1998Launched October 1998 Links all 50 statesLinks all 50 states Requires >4 RFLP markersRequires >4 RFLP markers

and/or 13 core STR markersand/or 13 core STR markers Current backlog of >600,000 samplesCurrent backlog of >600,000 samples

13 CODIS Core STR Loci with Chromosomal Positions

CSF1PO

D5S818

D21S11

TH01

TPOX

D13S317

D7S820

D16S539 D18S51

D8S1179

D3S1358

FGA

VWA

AMEL

AMEL

On August 25, 1979, an 8-year old girl was brutally raped and murdered inSan Pablo, CA. Semen was collected from the body and placed in anevidence room, where it sat for 22 years. Through this program, a DNA profilewas made and submitted to the state and federal databases. This resulted ina “cold hit” identifying Joseph Cordova Jr. as the suspect. Cordova was ahabitual child molester who at the time of the DNA analysis was incarceratedin a Colorado prison. Cordova was subsequently charged with molesting,raping and murdering the 8-year old girl.

On November 8, 2000, a 12 year old girl, was kidnapped off of the street inRancho Cordova, CA, and driven to Feather River in Sutter County where shewas sexually assaulted and then killed. Nine months later, Justin Weinbergerwas stopped for a traffic violation in New Mexico. A check by police revealedthat Weinberger was wanted on a federal warrant for child pornography. Hewas detained and voluntarily provided a DNA sample. Analysis of that DNAsample resulted in a match with evidence identifying Weinberger as the suspect in this case. Weinberger was subsequently extradited to California where he was tried and convicted of the murder of the 12-year old girl.

Cold Hits and Solved Cases

STR Analysis by Hybridization on Microchips