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Three generations of DNA testing
RFLP
AUTORAD
Allele = BAND
DQ-alpha
TEST STRIP
Allele = BLUE DOT
Automated STR
ELECTROPHEROGRAM
Allele = PEAK
How do they fare?
DQ-alpha
RFLP
 Discriminating power □
□ Sensitivity

□ Technical artifacts

□ Speed

□ Mixtures
‫ ٱ‬Databasing
Discriminating power
Sensitivity
Technical artifacts
Speed
□ Mixtures
□ Databasing
STR
 Discriminating power
 Sensitivity
□ Technical artifacts
 Speed
□ Mixtures
 Databasing
RFLP technology
What we would
like for them to look like.
What they often
look like.
Incomplete digest
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
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
REPEATED DNA
• Satellite DNA
– Around chromosomal centromere
– Long repeats can be 100s to 1000s bp long
• Minisatellite DNA
– VNTR
– Medium repeats 10 to 100 bp long
• Microsatellites
– STR
– Short repeats 2 to 6 bp long
SHORT TANDEM REPEATS
• Easy to amplify
• Both heterozygote alleles amplify well
• Number of repeats highly variable
• Good for identification
• Many different sites
STR NOMENCLATURE
• Simple Repeats
– Identical length and sequence
• agat agat agat agat agat
• Compound Repeats
– Two or more adjacent simple repeats
• agat agat agat ttaa ttaa ttaa
• Complex Repeats
– Variable unit length & possible intervening seq
• agat agat aggat agat agat ttaacggccat agat agat
STR NOMENCLATURE
• Microvariants
– Alleles that contain incomplete units
• TH01 9.3
• aatg aatg aatg aatg aatg aatg aatg aatg aatg aatg - 10
• aatg aatg aatg aatg aatg aatg atg aatg aatg aatg - 9.3
STRs Used In Forensic Science
• Need lots of variation - polymorphic
• Overall short segments - 100-400 bp
– Can use degraded DNA samples
– Segment size usually limits preferential amplification of
smaller alleles
• Single base resolution
– TH01 9.3
STRs Used In Forensic Science
• TETRANUCLEOTIDE REPEATS
– Narrow allele size range - multiplexing
– Reduces allelic dropout (stochastic effects)
– Use with degraded DNA possible
– Reduced stutter rates - easier to interpret
mixtures
STR NOMENCLATURE
• Use the 5’ to 3’ (Top) strand
• Start with the first 5’ nucleotide in the
repeat
• Microvariants designated by decimal places
– Number of complete repeats
– Number of bases in incomplete repeat (9.3)
• Allelic ladders used as reference
– Contain all common alleles
ALLELIC LADDERS
• Artificial mixture of common alleles
• Reference standards
• Enable forensic scientists to compare results
– Different instruments
– Different detection methods
• Allele quantities balanced
• Produced with same primers as test samples
• Commercially available in kits
Allelic Ladder Formation
Separate PCR products from various
samples amplified with primers targeted
to a particular STR locus
Polyacrylamide Gel
Combine
Re-amplify
Find representative alleles
spanning population variation
Profiler Plus Allelic Ladders
VWA
D3S1358
AMEL
D8S1179
D5S818
FGA
D21S11
D13S317
D18S51
D7S820
ALLELIC LADDERS
THE 13 CODIS STR LOCI
• November 1997
• Average random match probability is
greater than 1in 1 trillion
• US slower than UK in selection of loci for
database
THE 13 CODIS STR LOCI
• Four categories
• Simple repeats
– TPOX, CSF1PO, D5S818, D13S317, D16S539
• Simple repeats w/ non-consensus alleles
– TH01, D18S51, D7S820
• Compound repeats w/ non-consensus alleles
– vWA, FGA, D3S1358, D8S1179
• Complex repeats
– D21S11
Commercial STR Kits
• Applied Biosystems
•
AmpFlSTR Profiler Plus
– 5-FAM (BLUE)
– Joe (GREEN)
– NED (YELLOW) Amelo
•
AmpFlSTR Cofiler
vWA
D3S1358
D8S1179
D5S818
FGA
D21S11
D13S317
D18S51
D7S820
– 5-FAM (BLUE)
– Joe (GREEN)
D16S539
D3S1358
– NED (YELLOW) Amelo
TH01
TPOX
CSF1PO
D7S820
Commercial STR Kits
• Promega Corporation
• PowerPlex 1.1
– Fluorescein (Blue)
D5S818
D13S317
– TMR (Yellow)
vWA
TH01
D7S820
TPOX
D16S539
CSF1PO
• PowerPlex 2.1
– Fluorescein (Blue)D3S1358
– TMR (Yellow)
Amelo
TH01
vWA
D21S11
D8S1179
D18S51
TPOX
Penta E
FGA
Same DNA Sample Run with
Each of the ABI STR Kits
PCR Product Size (bp)
D3S1358
vWA
FGA
Blue
TH01
Amel
Power of Discrimination
CSF1PO
TPOX
Green I
TH01
D13S317
D3S1358
Amel
D5S818 vWA TPOX FGA
D8S1179 vWA
D13S317
Amel
D3S1358 D5S818
D21S11 FGA
D3S1358
Amel
TH01
TPOX
D3S1358
vWA
Amel
D8S1179 TH01
D21S11
D19S433
1:5000
1:410
CSF1PO
D7S820
D7S820
D18S51
D7S820
CSF1PO
D16S539
D16S539 D18S51
D2S1338
FGA
Profiler
1:3.6 x 109
Profiler Plus
1:9.6 x 1010
COfiler
1:8.4 x 105
SGM Plus
1:3.3 x 1012
AmpFlSTR®
Identifiler™
D8S1179
6FAM
(blue)
D3S1358
D7S820
D21S11
TH01
D13S317
D16S539
VIC
(green)
TPOX
D19S433
D18S51
VWA
NED
(yellow)
AMEL
D5S818
FGA
PET
(red)
GS500 LIZ size standard
LIZ
(orange)
CSF1PO
D2S1338
Genotyping by Comparison to
Allelic Ladder
PCR Amplification with
Fluorescent STR Kits and
Separation with Capillary
Electrophoresis
TPOX
TH01
CSF
D7
D16
D3
D5
AMEL
VWA
D13
D21
D8
D18
FGA
Penta D
Penta E
Electropherogram
STR LOCI ALLELES
• CSF1PO
–
–
–
–
c-fms proto-oncogene
Chromosome 5
AGAT repeat
6 to 15 repeats
• FGA
–
–
–
–
alpha fibrinogen locus
Chromosome 4
CTTT repeat
15 to 51.2 repeats
STR LOCI ALLELES
• TH01
–
–
–
–
–
TYROSINE HYDROXYLASE
Chromosome 11
TCTA repeat (Bottom strand)
4 to 11 repeats
Common microvariant 9.3
• TPOX
–
–
–
–
THYROID PEROXIDASE
Chromosome 2
AATG repeat
6 to 13 repeats
STR LOCI ALLELES
• vWA
–
–
–
–
von Willebrand Factor
Chromosome 12
TCTA with TCTG repeat
10 to 22 repeats
• D3S1358
– Chromosome 3
– AGAT with AGAC repeat
– 12 to 20 repeats
STR LOCI ALLELES
• D5S818
– Chromosome 5
– AGAT repeat
– 7 to 16 repeats
• D7S820
–
–
–
–
Chromosome 7
GATA repeat
Some Microvariants
6 to 15 repeats
STR LOCI ALLELES
• D8S1179
– Chromosome 8
– TCTA repeat with TCTG in alleles >13
– 7 to 19 repeats
• D13S317
– Chromosome 13
– GATA repeat
– 7 to 15 repeats
STR LOCI ALLELES
• D16S539
– Chromosome 16
– AGAT repeat with TCTG in alleles >13
– 8 to 15 repeats
• D18S15
–
–
–
–
Chromosome 18
AGAA repeat
Some x.2 Microvariants
8 to 27 repeats
STR LOCI ALLELES
• D21S11
–
–
–
–
–
–
–
Chromosome 21
TCTA repeat with TCTG
Some x.2 Microvariants
24 to 38 repeats
Over 70 reported alleles
Fine differences must be sequenced
4 alleles are designated as 30 repeats
13 CODIS Core STR Loci
with Chromosomal Positions
TPOX
D3S1358
D8S1179
D5S818
FGA
CSF1PO
TH01
VWA
D7S820
AMEL
D13S317
D16S539
D18S51
D21S11
AMEL
Position of Forensic STR
Markers on
TPOX
Human
D3S1358
Chromosomes
D5S818
D2S1338
FGA
CSF1PO
D8S1179
D13S317
D16S539
VWA
D7S820
13 CODIS Core STR Loci
Penta E
TH01
AMEL
Sex-typing
D19S433
D18S51
D21S11
Penta D
AMEL
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
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
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
D2
Exclusions don’t require numbers
Matches do require statistics
Hardy - Weinberg
Equilibrium
A1A1 A1A2 A2A2
p12 2p1p2 p22
freq(A1) = p1
freq(A2) = p2
A1
p12
A1 A 1 A 1
p1p2
A2 A 1 A 2
A2
p1p2
A1A 2
p22
A2 A 2
(p1 + p2 )2 = p12 + 2p1p2 + p22
A Hardy-Weinberg Population
•
•
•
•
LARGE POPULATION
NO NATURAL SELECTION
NO MUTATION
NO IMMIGRATION / EMIGRATION
• RANDOM MATING
Estimate genotype frequency:
1. Frequency at each locus
2. Frequency across all loci
Product Rule
Product Rule
•
The frequency of a multi-locus STR
profile is the product of the genotype
frequencies at the individual loci
ƒ locus1 x ƒ locus2 x ƒ locusn = ƒcombined
Criteria for Use of Product Rule
Inheritance of alleles at one locus have no effect
on alleles inherited at other loci
Population database
• Look up how often each allele occurs at the
locus in a population (the “allele” frequency)
ProfIler Plus
Item
D3S1358 vWA
Q1
16,16
15,17
FGA
D8S1179 D21S11 D18S51 D5S818 D13S317 D7S820
21,22
13,13
29,30
16,20
8,12
12,12
8,11
CoFIler
Item
Q1
D3S1358
16,16
D16S539
10,12
TH01
8,9.3
TPOX
CSF1P0
9,10
12,12
D7S820
8,11
D3S1358 = 16, 16 (homozygote)
Frequency of 16 allele = ??
D3S1358 = 16, 16 (homozygote)
Frequency of 16 allele =
0.3071
When same allele:
Frequency = genotype frequency (p2)
(for now!)
Genotype freq = 0.3071 x 0.3071 = 0.0943
ProfIler Plus
Item
D3S1358 vWA
Q1
16,16
15,17
FGA
D8S1179 D21S11 D18S51 D5S818 D13S317 D7S820
21,22
13,13
29,30
16,20
8,12
12,12
8,11
CoFIler
Item
Q1
D3S1358
16,16
D16S539
10,12
TH01
8,9.3
TPOX
CSF1P0
9,10
12,12
D7S820
8,11
VWA = 15, 17 (heterozygote)
Frequency of 15 allele = ??
Frequency of 17 allele = ??
VWA = 15, 17 (heterozygote)
Frequency of 15 allele = ??
Frequency of 17 allele = ??
VWA = 15, 17 (heterozygote)
Frequency of 15 allele = 0.2361
Frequency of 17 allele = 0.1833
When heterozygous:
Frequency = 2 X allele 1 freq X allele 2 freq
(2pq)
Genotype freq = 2 x 0.2361 x 0.18331 = 0.0866
Overall profile frequency =
Frequency D3S1358 X Frequency vWA
0.0943 x 0.0866 = 0.00817
What if…
We encounter alleles not
represented in the population
database…
…or alleles that are
extremely rare in the
database???
Where the random match probability is the
sum of the squares of the observed
phenotype/genotype frequencies in a database,
The Power of Exclusion of a genetic locus is
based on the 1 – the sum of squares of all the
expected phenotypes/genotypes!
These measures tell us two things about our
markers and databases:
Power of Discrimination
– how powerful our loci are
at individualizing
Power of Exclusion
– how powerful our marker panel is
at excluding particular
genotypes
FBI’s CODIS DNA Database
Combined DNA Index System
• Launched October 1998
• Used for linking serial crimes and
unsolved cases with repeat offenders
• Links all 50 states
• Requires >4 RFLP markers and/or 13
core STR markers
• Current backlog of > 600,000 samples
As of June, 2004
• Total profiles = 1,857,093
• Total forensic profiles = 85,477
• Total convicted offender = 1,771,616