Forensic DNA: Use, Abuse, Promise, and Peril

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Forensic DNA Use, Abuse, Promise, and Peril
William M. Shields
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DNA Identification

• Where does DNA come from?
• 1/2 from mom
• 1/2 from dad
• What is it?
• Blue print of life
• How is DNA different among us?
• Common vs Different
• What does DNA mean?
• Deoxyribonucleic Acid

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Where can DNA be found?
Cell Types
Blood
Hair Roots
Saliva
SAME
Sweat
Semen
Various Tissue
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Where is DNA in the body?
Cell
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Where are the types of DNA found in a cell?
Mitochondrial DNA
Nuclear DNA
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Where is DNA in the body?
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Where is DNA packaged in the body?
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DNA- What does it look like?
Units
A Adenine
T Thymine
G Guanine
C Cytosine
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Sources of Biological Evidence

• Blood
• Semen
• Saliva
• Urine
• Hair
• Teeth
• Bone
• Tissue

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Types of objects where DNA may be found

• Blood Stains
• Semen Stains
• Chewing Gum
• Stamps Envelopes
• Penile Swabs
• Plant Material

• Sweaty Clothing
• Bone
• Hair
• Fingernail Scraping
• Saliva
• Animal Material

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Where DNA Evidence is Found
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Isolation of DNA
Chemical
DNA
Blood Hair Roots Saliva Sweat Tissue
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Differential Isolation of DNA
Semen stain
Remove Epithelial DNA
Sperm DNA
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Amplification
(making copies)
Solution
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DENATURE
Step one of a single cycle
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ANNEAL
Step two of a single cycle
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EXTEND
Step three of a single cycle
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Amplification
PCR (Polymerase Chain Reaction)
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Analysis of amplified DNA
DNA Profile
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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

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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

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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

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Steps in DNA Sample Processing
Sample Obtained from Crime Scene or Paternity
Investigation
Biology
Technology
Genetics
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Progression of DNA Typing Markers

• RFLP
• multilocus VNTR probes
• single locus VNTR probes (P32 and
  chemiluminescence)
• PCR
• 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

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Extraction of DNA
Chemical
DNA
Blood Hair Roots Saliva Sweat Tissue
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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 (gt50 ng
  DNA)
• slow and expensive

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Which Suspect, A or B, cannot be excluded from
the class of potential perpetrators of this
assault?
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PMDQA1 Test

• PCR-based
• Extremely sensitive(1ng DNA)
• degraded samples
• faster and cheaper than RFLP
• Statistics less impressive, particularly with
  mixed samples

• Possible Problems
• interpretation is subjective and can be difficult
• mixtures difficult to interpret
• statistical characterization of mixed samples is
  tricky

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DNA in the Cell
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Short Tandem Repeats (STRs)

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

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Short Tandem Repeats (STRs)

• 2. Gel-based systems with Fluorescent Detection

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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)

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Short Tandem Repeats (STRs)
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
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STR
Short Tandem Repeat
AGAT
DNA Profile 4,6
TCTA
DNA Profile 5,7
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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

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An Example Forensic STR Multiplex Kit
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Overview of Steps Involved in DNA Typing
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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)
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Short Tandem Repeats (STRs)
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
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ABI Prism 310 Genetic Analyzer
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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)

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Electrokinetic Injection Process
Capillary
Electrode
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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

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DNA Separation Mechanism

• 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

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Fluorescent Emission Spectra for ABI Dyes
JOE
NED
ROX
5-FAM
100
80
60
Normalized Fluorescent Intensity
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20
0
600
520
540
560
580
620
640
WAVELENGTH (nm)
Laser excitation (488, 514.5 nm)
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Principles of Sample Separation and Detection
Labeled DNA fragments (PCR products)
Capillary or Gel Lane
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15,16
16,17
20,23
12,14
30,30
X,Y
13.2,15
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PERKIN-ELMERS PROFILER AND COFILERSTATE OF
TENNESSEE VERSUS TAYLOR LEE SMITH
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JUST THE FACTSNOT 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.

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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 Theta 0.05
• African American 1 in 278,000,000 1 in 43,000,000
• Likelihood Ratio 16,600 6,500
• Caucasian 1 in 183,000,000 1 in 27,500,000
• Likelihood Ratio 13,500 5,200
• Hispanic 1 in 15,000,000 1 in
  3,700,000 Likelihood Ratio 3,900 1,990

Observed Sperm fraction genotypes vWA16,
TPOX8, D5S81812, and D16S53910).
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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
• Needs
• population studies to evaluate diversity of
  haplotypes
• robust assay for accurate characterization of Y
  markers

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Y STR Multiplex Assay
Prinz et al. 1997 (Forensic Sci Int,
vol. 85, pp. 209-218)
DYS19
389II
389I
390
Primer Amounts Dye Y19 0.25 ?M JOE Y389 0.125
?M FAM Y390 0.25 ?M JOE
Quadruplex I
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Mitochondrial DNA

• What is mtDNA Typing?
• Database and statistical issues

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A Mitochondrial Exclusion
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A Mitochondrial Inclusion
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Mitochondrial Inconclusive?
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The Future of Forensic DNA

• CODIS
• SNPs Chips

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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 gt4 RFLP markers
• and/or 13 core STR markers
• Current backlog of gt600,000 samples

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13 CODIS Core STR Loci with Chromosomal Positions
TPOX
D3S1358
TH01
D8S1179
D5S818
VWA
FGA
D7S820
CSF1PO
AMEL
D13S317
AMEL
D21S11
D16S539
D18S51
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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.
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STR Analysis by Hybridization on Microchips