Forensic DNA: Use, Abuse,
Promise, and Peril
William M. Shields
DNA Identification
• Where does DNA come from?
1/2 from mom
 1/2 from dad
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What is it?
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How is DNA different among us?
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“Blue print” of life
Common vs Different
What does “DNA” mean?
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Deoxyribonucleic Acid
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
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Blood
Semen
Saliva
Urine
Hair
Teeth
Bone
Tissue
Types of objects where DNA may
be found
Blood Stains
Sweaty Clothing
Semen Stains
Bone
Chewing Gum
Hair
Stamps & Envelopes Fingernail Scraping
Penile Swabs
Saliva
Plant Material
Animal Material
Where DNA Evidence is Found
Isolation of DNA
Chemical
DNA
Blood
Hair Roots
Saliva
Sweat
Tissue
Differential Isolation of DNA
Semen stain
Semen
stain
Remove
Epithelial
DNA
EpithelialChemical
DNA
Different
ChemicalDNA
Sperm
Sperm
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
T
EXTEND
Step three of a single cycle
T
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
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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 victim’s DNA profile
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Challenges
•Mixtures must be resolved
•DNA is often degraded
•Inhibitors to PCR are often present
Human Identity Testing
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Forensic cases -- matching suspect with
evidence
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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
DNA
Extraction
PCR Amplification
of Multiple STR markers
Technology
Separation and Detection of
PCR Products
(STR Alleles)
Comparison of Sample
Genotype to Other
Sample Results
Sample Genotype
Determination
Genetics
If match occurs, comparison
of DNA profile to population
databases
Generation of Case
Report with Probability
of Random Match
Progression of DNA Typing
Markers
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RFLP
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multilocus VNTR probes
single locus VNTR probes (P32 and
chemiluminescence)
PCR
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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
Extraction of DNA
Chemical
DNA
Blood
Hair Roots
Saliva
Sweat
Tissue
RFLP Analysis
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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
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PCR-based
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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
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
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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
AGAT
AGAT
AGAT
AGAT
4
AGAT
AGAT
6
DNA Profile =4,6
TCTA
TCTA
TCTA
TCTA
TCTA
TCTA
TCTA
TCTA
TCTA
TCTA
DNA Profile =5,7
5
TCTA
TCTA
7
Multiplex PCR
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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
Color Separation
100 bp
Size Separation
D3
A
vWA
D8
D5
FGA
300 bp
400 bp
5-FAM (blue)
D21
D18
JOE (green)
D13
D7
NED (yellow)
ROX (red)
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
TPOX
TH01
D3
AMEL
D5
VWA
D7
D13
D21 D8
CSF
D16
D18
FGA
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
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Injection
electrokinetic injection process
 importance of sample preparation
(formamide)
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Separation
capillary
 POP-4 polymer
 buffer
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Detection
fluorescent dyes with excitation and emission
traits
 virtual filters (hardware/software issues)
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Electrokinetic Injection
Process
Capillary
Electrode
Q=
r2cs(ep + eo)Etb
s
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
DNA-
ep is the mobility of the sample molecules
Sample
Tube
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
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DNA Separation Mechanism
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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
ROX
100
80
60
40
20
0
520
540
560
580 600
620
640
WAVELENGTH (nm)
Laser excitation
(488, 514.5 nm)
ABI 310 Filter Set F
Principles of Sample
Separation and Detection
Labeled DNA fragments
(PCR products)
Capillary or
Gel Lane
Sample Detection
Size
Separation
Ar+
LASER
CCD Panel
(488 nm)
Color
Separation
Detection
region
Fluorescence
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
12,14
30,30 13.2,15
Ref.Std.1 14,15 17,18 23,24 X,X
13,13
12,14
30,30 15,19
30,30 13.2,15
Ref.Std.2 15,16 16,17 20,23 X,Y
Human Identity Testing with Multiplex STRs
AmpFlSTR® SGM Plus™ kit
Two different individuals
DNA Size (base pairs)
amelogenin
D19
D3
D8 TH01
VWA D21
D16
D18
D2
FGA
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
D16
D21
FGA
D18
Simultaneous Analysis of 10 STRs and Gender ID
D2
PERKIN-ELMER’S
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.
African American
Likelihood Ratio
Caucasian
Likelihood Ratio
Hispanic
Likelihood Ratio
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
*Observed Sperm fraction genotypes: vWA=16, TPOX=8, D5S818=12, and D16S539=10).
The Future of Forensic
DNA
CODIS
SNP’s & Chips
FBI’s 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
D5S818
FGA
CSF1PO
TH01
VWA
D7S820
AMEL
D13S317
D16S539
D18S51
D21S11
AMEL
STR Analysis by Hybridization
on Microchips