Transcript No Slide Title

Forensic DNA Typing
or
Did you kill (rape…) that person?
How DNA can “definitively” say.
Adapted from:
National Institutes of Science & Technology
http://www.cstl.nist.gov/div831/strbase/intro.htm
Brief History of Forensic DNA Typing
• 1980 - Ray White describes first polymorphic RFLP marker (Restriction
Fragment Length Polymorphism [alleles]).
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Different RFLP for different people
1985 - Alec Jeffreys discovers multilocus VNTR (variable number of
tandem repeats) probes.
1985 - first paper on PCR
1988 - FBI starts DNA casework
1991 - first STR paper (renaming of VNTR– could be larger repeats, STR 4-6 bp’s.
now using mostly 4 bases )
• 1995 - FSS (Forensic Science Service-UK) starts UK DNA database
• 1998 - FBI launches CODIS (Combined DNA Information Service)
Now FBI use 13 loci: PCR identifies it: in the quadrillions – except
for identical. Except for police mistakes, it’s done deal.
RFLP’s: Sickle Cell hemoglobin
Case 1: Screening for the sickle-cell gene
Sickle cell disease is a genetic disorder in which both genes in the patient encode
the amino acid valine (Val) in the sixth position of the beta chain (betaS) of the
hemoglobin molecule. "Normal" beta chains (betaA) have glutamic acid at this
position.
The only difference between the two genes is the substitution of a T for an A in
the middle position of codon 6.
This
•converts a GAG codon (for Glu) to a GTG codon for Val and
•abolishes a sequence (CTGAGG, which spans codons 5, 6, and 7) recognized
and cut by one of the restriction enzymes.
Brief History of Forensic DNA Typing
• 1980 - Ray White describes first polymorphic RFLP (Restriction Fragment Length
Polymorphism) marker—detect to transferring to membrane. Probe w
southern blot (radiological). Diff. RFLP for dif. People. Single rflp
• 1985 - Alec Jeffreys discovers multilocus VNTR (variable number
of tandem repeats) probes (stat. very impressive identical 4-6 bp
that are spec. 7 and 9 repeat, one from mom and dad, on chrom. 1nowadays use pcr- but flanking sequence that is unique to
chromo1)). Jeffreys almost ident. Typing. Now use PCR.
• 1985 - first paper on PCR (Kerry Mullis)
• 1988 - FBI starts DNA casework
• 1991 - first STR paper ( renaming of VNTR– could be larger
repeats, STR 4-6 bp’s. now using mostly 4 bases )
• 1995 - FSS (Forensic Science Service-UK) starts UK DNA
database
• 1998 - FBI launches CODIS (Combined DNA Information
Service) database. Now FBI use 13 loci: PCR identifies it: in the
quadrilians – except for identical.
DNA Use in Forensic Cases
• Most are rape cases (>2 out of 3)
• Looking for match between evidence
and suspect
• Must compare victim’s DNA profile
Challenges
•Mixtures must be resolved
•DNA is often degraded (stored wet- have mold, nuclease)
•Inhibitors to PCR are often present
Human Identity Testing
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Forensic cases -- matching suspect with evidence
Paternity testing -- identifying father
Historical investigations
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
Quantization
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
Sources of Biological Evidence
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Blood
Semen
Saliva
Urine
Hair
Teeth (useful in fires).
Bone (there are cells. Decalcify it.
100,000 year old- has DNA. Has Dinosaur!)
• Tissue
All felony arrests- cheek swab.
DNA in the Cell
chromosome
cell nucleus
Double stranded
DNA molecule
Target Region for PCR
Individual
nucleotides
DNA Amplification with the
Polymerase Chain Reaction (PCR)
5’
3’
5’
3’
3’
3’
5’
5’
Starting DNA
Template
Separate
strands
(denature)
Forward primer
5’
3’
5’
3’
Make copies
Add
primers
(extend
primers)
5’
(anneal)
3’
3’
5’
Reverse primer
PCR (Polymerase Chain Reaction) Copies DNA
Exponentially through Multiple Thermal Cycles
Original DNA
target region
1 copy
Heat
Oligo’s
DNA Poly.
dUTP
Cool
Heat
Cool
2 copies
Heat
4 copies
In 32 cycles at 100% efficiency, 1.07 billion
copies of targeted DNA region are created
…
Short Tandem Repeats (STRs)
(say chromo 3)
Identical in all people
AATG
7 repeats
Identical in all people
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
Diff. PCR primers sets, can amplify the same region. Different companies sell different kits.
195 bp
170 bp
TCAT repeat unit
Different primer sets produce different PCR product sizes for the
same STR allele
Variation Among STRs
Choosing which STRs:
Significant statistical variation – but not too
many. Freq. that are measured in pop. : Loc 1 10%. Loc 2 – 10%; locus 1+2 -1/100. Random
match with 13 primers 1/1013.
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
Most rxns: require 2 PCR (tubes) 7 or 8
primer pairs in one tube– need total of
about 2 tubes for 13 different STRs.
$20-$25 per rxn in lab. $150 incl
labor. Cost for forensic up to $1000.
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
Available Kits for STR Analysis
• Kits make it easy for labs to just add DNA
samples to a pre-made mix
• 13 CODIS core loci
– Profiler Plus and COfiler (PE Applied Biosystems)
– PowerPlex 1.1 and 2.1 (Promega Corporation)
• Increased power of discrimination
– CTT (1994): 1 in 410
– SGM Plus™ (1999): 1 in 3 trillion
– PowerPlex ™ 16 (2000): 1 in 2 x 1017
ABI Prism 310 Genetic Analyzer
5 min from inj.
to output.
capillary
Syringe with
polymer solution
Injection
electrode
Outlet
buffer
Autosampler
tray
Inlet
buffer
Close-up of ABI Prism 310 Sample Loading Area
Electrode
Capillary
Sample Vials
Autosampler Tray
See Technology section for more information on CE
D tells chromosome 21—happens to be down’s syndrome. 2 peaks cause heterozygotic
Human Identity Testing with Multiplex STRs
Amelogenin amel protein that happens to be on sex chromosome, tooth
enamel– top: 2 peaks: x and y. (universal that two diff. people.) Bottom
only 1 peak cause they have two X chromosomes.
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
STR Allele Frequencies
45
40
TH01 Marker
Frequency
35
30
Caucasians (N=427)
Blacks (N=414)
Hispanics (N=414)
25
20
15
10
*Proc. Int. Sym. Hum. ID
5
(Promega) 1997, p. 34
0
6
7
8
9
9.3
Number of repeats
10
FBI’s CODIS DNA Database
Combined DNA Index System --all 50 states can upload
their convicted felony and seq. of unsolved cases…. In Florida to convicted felon.
• 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
The End