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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