Forensic DNA: Use, Abuse, Promise, and Peril

DNA Identification Where does DNA come from?

1/2 from mom 1/2 from dad

What is it?

Blue print of life Common vs Different Deoxyribonucleic Acid

How is DNA different among us?

What does DNA mean?

Where can DNA be found?

Cell Cell Types Blood Hair Roots Saliva SAME Sweat Semen Various Tissue

Where is DNA in the body?

Cell

Nucleus

Where are the types of DNA found in a cell?

Cell
Nuclear DNA Mitochondrial DNA

Where is DNA in the body?

Nucleus

Maternal Chromosome

Paternal Chromosome

Where is DNA packaged in the body?

Chromosome

DNA

DNA- What does it look like?

Double Helix Units A =Adenine AT GC

T =Thymine
G =Guanine

C =Cytosine

Sources of Biological Evidence

Blood Semen Saliva Urine Hair Teeth Bone Tissue

Types of objects where DNA may be found

Blood Stains Sweaty Clothing

Semen Stains
Chewing Gum Penile Swabs

Bone
Hair Saliva

Stamps & Envelopes Fingernail Scraping Plant Material Animal Material

Where DNA Evidence is Found

Isolation of DNA
Chemical DNA

Blood Hair Roots Saliva Sweat Tissue

Differential Isolation of DNA

EpithelialChemical DNA
Different Chemical Sperm DNA Sperm DNA

Semen stain

Semen stain
Remove Epithelial DNA

Amplification
(making copies)
Solution

DNA

DENATURE
Step one of a single cycle A G A T A G
Heat

T
C T A T C

ANNEAL
Step two of a single cycle

EXTEND
Step three of a single cycle

Amplification
PCR (Polymerase Chain Reaction)

28 Cycles 1 Cycle

2 Cycles

DNA

3 Cycles 4 Cycles

5 Cycles

Analysis of amplified DNA

DNA Profile

Amplified DNA

Brief History of Forensic DNA Typing

1980 - Ray White describes first polymorphic RFLP marker 1985 - Alec Jeffreys discovers multilocus VNTR probes 1985 - first paper on PCR 1988 - FBI starts DNA casework 1991 - first STR paper 1995 - FSS starts UK DNA database 1996 First mtDNA case 1998 - FBI launches CODIS database

DNA Use in Forensic Cases

Most are rape cases or murders Looking for match between evidence and suspect Must compare victims DNA profile

Challenges

Mixtures must be resolved DNA is often degraded Inhibitors to PCR are often present

Human Identity Testing

Forensic cases -- matching suspect with

evidence

Paternity testing -- identifying father Historical investigations-Czar Nicholas, Jesse James Missing persons investigations Mass disasters -- putting pieces back together Military DNA dog tag Convicted felon DNA databases

Steps in DNA Sample Processing

Sample Obtained from Crime Scene or Paternity Investigation

Biology
DNA Quantitation PCR Amplification of Multiple STR markers

DNA Extraction

Technology
Separation and Detection of PCR Products (STR Alleles)
Sample Genotype Determination

Comparison of Sample Genotype to Other Sample Results

Genetics

Generation of Case Report with Probability of Random Match

If match occurs, comparison of DNA profile to population databases

Progression of DNA Typing Markers

RFLP

multilocus VNTR probes single locus VNTR probes (P32 and chemiluminescence) DQ-alpha (reverse dot blot) PolyMarker (6 plex PCR; dots for SNPs) D1S80 (AMP-FLPs) singleplex STRs with silver staining multiplex STRs with fluorescent dyes Mitochondrial DNA sequencing Multiplex Y-STR with fluorescent dyes

PCR

Extraction of DNA
Chemical DNA

Blood Hair Roots Saliva Sweat Tissue

RFLP Analysis

Enzymes break DNA into restriction fragments Measurements taken of fragments that vary in length across people (length polymorphism) because they contain VNTRs can produce extremely low random match probabilities requires relatively large fresh samples (>50 ng DNA) slow and expensive

Which Suspect, A or B, cannot be excluded from the class of potential perpetrators of this assault?

PM+DQA1 Test

PCR-based

Possible Problems:

Extremely sensitive(1ng DNA) degraded samples faster and cheaper than RFLP

interpretation is subjective and can be difficult

mixtures difficult to interpret statistical characterization of mixed samples is tricky

Statistics less impressive, particularly with mixed samples

DNA in the Cell

chromosome cell nucleus

Double stranded DNA molecule

Target Region for PCR

Individual nucleotides

Short Tandem Repeats (STRs)

1. CTTA with silverstained gel PCR-based 3 loci for identification plus sex-typing Easier interpretation of mixtures

Short Tandem Repeats (STRs)

2. Gel-based systems with Fluorescent Detection

Short Tandem Repeats (STRs)

3. Capillary Electrophoresis AmpFlstr Profiler Plus

Groups of amplified STR products are labeled with different colored dyes (blue, green, yellow) Electrophoresis and detection occur in computer-controlled capillary device (ABI Prism 310 Genetic Analyzer)

Short Tandem Repeats (STRs)

AATG

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

STR
Short Tandem Repeat
AGAT AGAT AGAT AGAT

4
AGAT AGAT

AGAT

AGAT

AGAT

AGAT

DNA Profile =4,6

TCTA TCTA TCTA TCTA TCTA

5
TCTA TCTA

TCTA

TCTA

TCTA

TCTA

TCTA

DNA Profile =5,7

Multiplex PCR

Over 10 Markers Can Be Copied at Once Sensitivities to levels less than 1 ng of DNA Ability to Handle Mixtures and Degraded Samples Different Fluorescent Dyes Used to Distinguish STR Alleles with Overlapping Size Ranges

An Example Forensic STR Multiplex Kit

AmpFlSTR Profiler Plus
Kit available from PE Biosystems (Foster City, CA)
200 bp 300 bp 400 bp 100 bp

Size Separation
FGA D21 D18

Color Separation

D3 A D8 D5

vWA

5-FAM (blue) JOE (green) NED (yellow) ROX (red)

D13

D7

GS500-internal lane standard

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

Overview of Steps Involved in DNA Typing

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

Penta D Penta E

Blood Stain

PCR Amplification with Fluorescent STR Kits and Separation with Capillary Electrophoresis

DNA Quantitation using Slot Blot

Genotyping by Comparison to Allelic Ladder

Calculation of DNA Quantities in 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 (STRs)

Fluorescent dye label
AATG AATG AATG

7 repeats 8 repeats the repeat region is variable between samples while the flanking regions where PCR primers bind are constant Primer positions define PCR product size

ABI Prism 310 Genetic Analyzer

capillary

Syringe with polymer solution Injection electrode Outlet buffer Autosampler tray Inlet buffer

Chemistry Involved

Injection
electrokinetic injection process importance of sample preparation (formamide)

Separation
capillary POP-4 polymer buffer

Detection
fluorescent dyes with excitation and emission traits virtual filters (hardware/software issues)

Electrokinetic Injection Process

Capillary
Electrode

Q=

r2cs(ep + eo)Etb

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
Sample Tube

DNA-

eo is the electroosmotic mobility

Rose et al (1988) Anal. Chem. 60: 642-648

Separation Issues
Run temperature -- 60 oC helps reduce secondary structure on DNA and improves precision Electrophoresis buffer -- urea in running buffer helps keep DNA strands denatured Capillary wall coating -- dynamic coating with polymer Polymer solution -- POP-4

DNA 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

Fluorescent Emission Spectra for ABI Dyes

5-FAM JOE NED
100 80 60 40 20

ROX

520

540

560

580 600

620

640

WAVELENGTH (nm)

Laser excitation (488, 514.5 nm)

ABI 310 Filter Set F

Labeled DNA fragments (PCR products)

Capillary or Gel Lane

Principles of Sample Separation and Detection

Sample Detection
CCD Panel

Size Separation

Ar+ LASER
(488 nm)

Detection region
Fluorescence

Color Separation
ABI Prism spectrograph

AREAS OF DNA SAMPLE Area 1 Area 2 Area 3 Sex Area 4 Area 5 Area 6

Evidence 15,16 16,17 20,23 X,Y Ref.Std.1 14,15 17,18 23,24 X,X Ref.Std.2 15,16 16,17 20,23 X,Y

12,14 13,13 12,14

30,30 13.2,15 30,30 15,19 30,30 13.2,15

Human Identity Testing with Multiplex STRs

AmpFlSTR SGM Plus kit DNA Size (base pairs)

Two different individuals

amelogenin D19

D3

D8 TH01 VWA D21 FGA

D16 D18 D2

probability of a random match: ~1 in 3 trillion

amelogenin D3 D19 D8 VWA TH01

Results obtained in less than 5 hours with a spot of blood the size of a pinhead
D21 FGA D16 D18 D2

Simultaneous Analysis of 10 STRs and Gender ID

PERKIN-ELMERS PROFILER+ AND COFILER STATE OF TENNESSEE VERSUS TAYLOR LEE SMITH

JUST THE FACTS: NOT A MIXTURE?

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

2. Suspect Known: Eight of thirteen loci have a total of 12 different alleles not found in the sperm fraction mixture.
3. Victim Known: Ten of thirteen loci have a total of 11 different alleles not found in the sperm fraction mixture.

COINCIDENCE OR EVIDENCE?
The likelihood ratios for producing homozygous genotypes at four of thirteen STR loci* with DNA from a single individual versus a mixture of DNA from two individuals. Theta = 0.03 1 in 278,000,000 1 in 16,600 1 in 183,000,000 1 in 13,500 1 in 15,000,000 1 in 3,900 Theta = 0.05 43,000,000 6,500 27,500,000 5,200 3,700,000 1,990

African American Likelihood Ratio Caucasian Likelihood Ratio Hispanic Likelihood Ratio

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

Why the Y Chromosome?

Applications

forensic investigations (98% of violent crime by men) genealogical purposes evolutionary studies

Advantages to Human Identity Testing

male component isolated without differential extraction paternal lineages

population studies to evaluate diversity of haplotypes robust assay for accurate characterization of Y markers

Needs

Y STR Multiplex Assay

Prinz et al. 1997
(Forensic Sci Int, vol. 85, pp. 209-218)
100 bp 200 bp 300 bp 400 bp

DYS19 390

389I

389II

Primer Amounts Dye

Y19 Y389 Y390 0.25 M 0.125 M 0.25 M JOE FAM JOE

Quadruplex I

Mitochondrial DNA
What is mtDNA Typing? Database and statistical issues

A Mitochondrial Exclusion

A Mitochondrial Inclusion

Mitochondrial Inconclusive?

The Future of Forensic DNA

CODIS SNPs & Chips

FBIs CODIS DNA Database

Combined DNA Index System Used for linking serial crimes and unsolved cases with repeat offenders Launched October 1998 Links all 50 states Requires >4 RFLP markers and/or 13 core STR markers Current backlog of >600,000 samples

13 CODIS Core STR Loci with Chromosomal Positions

TPOX D3S1358 D8S1179 D7S820 TH01 VWA

D5S818 FGA CSF1PO

AMEL D13S317 D16S539 D18S51 D21S11

AMEL

Cold Hits and Solved Cases

On August 25, 1979, an 8-year old girl was brutally raped and murdered in San Pablo, CA. Semen was collected from the body and placed in an evidence room, where it sat for 22 years. Through this program, a DNA profile was made and submitted to the state and federal databases. This resulted in a cold hit identifying Joseph Cordova Jr. as the suspect. Cordova was a habitual child molester who at the time of the DNA analysis was incarcerated in 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 in Rancho Cordova, CA, and driven to Feather River in Sutter County where she was sexually assaulted and then killed. Nine months later, Justin Weinberger was stopped for a traffic violation in New Mexico. A check by police revealed that Weinberger was wanted on a federal warrant for child pornography. He was detained and voluntarily provided a DNA sample. Analysis of that DNA sample 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.

STR Analysis by Hybridization on Microchips