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Contribution of the National Institute of Standards and Technology, 2010
Law Enforcement Scientific Analysis Legal Proceedings
Police Agencies(local, state, federal)
Forensic Laboratory
Investigators/Detectives
CSI
DNA UnitDNA Analysts
Court System
Prosecution
Defense
Judge
Trial
Legal framework and precedent
Evidence submitted
Scientific report(s)
completed
References submitted
Validated scientific testsLaws and police training
Conviction or
exoneration
Other Forensic
Disciplines
Research (introduces new methods)
Evidence returned
Contribution of the National Institute of Standards and Technology, 2010
Sample Obtained from Crime Scene or
Paternity Investigation
DNAExtraction
DNAExtraction
DNAQuantitation
DNAQuantitation
PCR Amplificationof Multiple STR markers
PCR Amplificationof Multiple STR markers
Biology
Separation and Detection of PCR Products
(STR Alleles)
Technology
Sample Genotype Determination
Genetics
Comparison of Sample Genotype to Other
Sample Results
Comparison of Sample Genotype to Other
Sample Results
If match occurs, comparison of DNA profile to population databases
If match occurs, comparison of DNA profile to population databases
Generation of Case Report with Probability
of Random Match
Generation of Case Report with Probability
of Random Match
Contribution of the National Institute of Standards and Technology, 2010
DNA Profile ComparisonQ K
Court
Database Search
Exclusion (no match)
Inclusion (match)
May match another (K’)
Evidence (Question) sample “Q”
Profile put on database
Plea
Report(with statistical weight)
Q = K
Q ≠ K
Crime committedBiological material transferred
Collection
Extraction
Quantitation
STR Markers
DataInterpretation
Sample Storage
Amplification
Separation/Detection
Reference (Known) sample “K”
Profile put on database
Steps Involved
Suspect developed
Collection
Extraction
Quantitation
STR Markers
DataInterpretation
Sample Storage
Amplification
Statistical Interpretation
Characterization
Separation/Detection
Bio
log
yT
ech
no
log
yG
en
etic
sS
ero
log
y
Steps Involved
QUALITY
ASSURANCE
QUALITY
ASSURANCE
May match another (K’)
Contribution of the National Institute of Standards and Technology, 2010
O
Base(A, T, C, or G)
HO
1’
3’ 2’
4’
5’
CH2OP
H
HH
H
O
O-
HO
O
Base(A, T, C, or G)
HOH
1’
3’ 2’
4’
5’
CH2OP
H
HH
H
O-
HO
5’end|
Phosphate|
Sugar—Base…|
Phosphate|
Sugar—Base…|
3’end
(a) (b)
5’
3’
Contribution of the National Institute of Standards and Technology, 2010
A = TG C
T = A
A = T
C G
T
CCA
GG
TA
G C
T = A
T = A
C G
A = T
A = TG C
5’
3’
5’
3’
5’ 3’
3’ 5’denatured
strands
hybridizedstrands
Hydrogen bonds
C G C
G
G C
Phosphate-sugar backbone
Contribution of the National Institute of Standards and Technology, 2010
The Human Genomeh
ttp
://w
ww
.ncb
i.nlm
.nih
.go
v/g
en
om
e/g
uid
e/
1 2 3 4 5 6 7 8 9 10 11 12
13 14 15 16 17 18 19 20 21 22 X Y
Sex-chromosomes
Autosomes
3.2 billion bp
Nuclear DNA - Located in cell nucleus
2 copies per cell
mtDNA
16,569 bp
mtDNA
Located in mitochondria
(multiple copies in cell cytoplasm)
100s of copies per cell
Onl
y si
ngle
cop
y of
ea
ch a
utos
ome
show
n
Contribution of the National Institute of Standards and Technology, 2010
p(short arm)
centromere
telomere
q(long arm)
telomere
Band 5
Band 3
Chromosome 12
12p312p3
12q512q5
Contribution of the National Institute of Standards and Technology, 2010
2 repeats
3 repeats
--------AGACTAGACATT-------
--------AGATTAGGCATT-------
---------(AATG)(AATG)(AATG)----------
---------(AATG)(AATG)----------
(B) Length polymorphism
(A) Sequence polymorphism
Contribution of the National Institute of Standards and Technology, 2010
63
4 5
Homologous pair of chromosomes
Homologous pair of chromosomes
Locus A
Locus B
Allele 1 Allele 2
Allele 2Allele 1
Contribution of the National Institute of Standards and Technology, 2010
1 2 3 4 5 6 7 8 9 1110 12 13 14 15 16 17 18 19 20 21 22
X mtDNA
Y1 2 3 4 5 6 7 8 9 1110 12 13 14 15 16 17 18 19 20 21 22
Maternal Contribution
Paternal Contribution
1 2 3 4 5 6 7 8 9 1110 12 13 14 15 16 17 18 19 20 21 22X Y
mtDNAMale Child’s Full Genome
Nuclear DNAMitochondrial DNA
AutosomesSex
chromosomes
X
or
Sex chromosome
Sex chromosome
Zyg
ote
(d
iplo
id)
Sp
erm
(h
aplo
id)
Eg
g
(hap
loid
)
Contribution of the National Institute of Standards and Technology, 2010
AA
A
A
a
a Aa
aA
aa
Freq (A) = p
Freq (a) = q (p + q)2 = p2 + 2pq + q2
Punnett square
p2 qp
pq q2
p
q
p q
Fat
her
gam
etes
(sp
erm
)Mother gametes (egg)
p + q = 1
Resulting genotype combinations and frequencies
AA
Aa
p2
2pq
aa
q2
Contribution of the National Institute of Standards and Technology, 2010
AA aa
Aa
1.0
Frequency of a allele (q)
Frequency of A allele (p)
1.0 0.8 0.6 0.4 0.2 0.0
0.0 0.2 0.4 0.6 0.8
Fre
quen
cy o
f ge
noty
pe in
pop
ulat
ion
0.2
0.4
0.6
0.8 p2
2pq
q2
Contribution of the National Institute of Standards and Technology, 2010
1 2
3 4 56 7 8
9 10 11
20,22 23.2,25
20,25 20,25
20,22
20,22
22,25
20,22
22,25 20,22 20,25
14 15 16
22,2422,
23.222,
23.2
12 13
22,23.2
22,22
17
(a)
(b) (c)
22,2522,23.2
20,2520,23.220
22
23.2 25
Fat
her
’s a
llele
s
Mother’s alleles
#3
#5#4#1
#2
22,2520,22
20,2520,2020
22
20 25
Fat
her
’s a
llele
s
Mother’s alleles
#13
#14
#12
#7
#4
Contribution of the National Institute of Standards and Technology, 2010
Speed of Analysis (Technology)
Power of Discrimination
(Genetics)
Low
High
Slow Fast
Markers Used (Biology)
Markers Used (Biology)
RFLPSingle Locus Probes
RFLPMulti-Locus Probes
ABO blood groups
Multiplex STRs
DQsingle STR
D1S80mtDNA
PolyMarker
Contribution of the National Institute of Standards and Technology, 2010
A B AB O
A A or OA,B,AB,
or OA,B, or
ABA or O
BA,B,AB,
or OB or O
A,B, or AB
B or O
ABA,B, or
ABA,B, or
ABA,B, or
ABA or B
O A or O B or O A or B OFat
her
’s B
loo
d T
ype
Mother’s Blood Type
Child’s Blood Type
Contribution of the National Institute of Standards and Technology, 2010
Multi-Locus Probe
Probe 1 Probe 2 Probe 3
D1S7 D2S44 D4S139
Single-Locus Probe
Probe 33.6
Contribution of the National Institute of Standards and Technology, 2010
probe
Restriction site
Restriction site
VNTR
Small allele
Large allele
Small allele
Large allele
Sizing ladder
13 repeats
7 repeats
Bands seen on autoradiogram of probed membrane
probe
Contribution of the National Institute of Standards and Technology, 2010
TGCAGG CCTAACGACGTCC GGATTGC
TGCACTGCA GTAACGACGTG ACGTCATTGC
TGCAG ANTCTAACGACGTCTNA GATTGC
HaeIII
HinfI
PstI
Contribution of the National Institute of Standards and Technology, 2010
GTCCAGTCG PCR product (denatured)Biotin
Strepavidin
HRP
TMB (colorless)
Colored precipitate
CAGGTCAGC
Nylon membrane
Immobilized SSO probe5’
3’
hybridization
match no match
View from above nylon membrane
Allele 1 Allele 2
Contribution of the National Institute of Standards and Technology, 2010
Nominal allele specific dots
Control dot
Subtype allele specific dots
4.24.3
1.21.3 4
1 2 3 4 CAll but1.3
1.3 4.11.1 DQA1
1.21.3 4
1 2 3 4 CAll but1.3
1.31.1 AMPLITYPETM
DQ-Alpha
S A B
LDLR
A B
GYPA
A B
HBGG
A B
D7S8
A B
GC
C C
“S” dot
(A)
(B)
1.2/3
AB AB BB AB BC
Contribution of the National Institute of Standards and Technology, 2010
AllelicLadder
AllelicLadder
Pos
itive
Con
trol
Sam
ple
1
Sam
ple
2
14
18
24
34
31
1617
1920212223
41
252627282930
14
18
24
34
31
1617
1920212223
41
252627282930
Contribution of the National Institute of Standards and Technology, 2010
Minisatellite Marker (D1S80)
GAGGACCACCAGGAAGGAGGACCACCAGGAAG
Repeat region
Flanking regions
16 bp repeat unit
STR Marker (TH01)
TCATTCAT
Repeat region
Flanking regions
4 bp repeat unit
Contribution of the National Institute of Standards and Technology, 2010
AllelicLadders
Sam
ple
1
Sam
ple
2
5
8
67
91011
CSF1PO
TPOX
TH01
89
101112
AllelicLadders
6
14
Contribution of the National Institute of Standards and Technology, 2010
3’-TAAATGATTCC-5’
ATT
ATTTACTAA
ATTTACT ATTTAC
ATTTATTTA
AT
ATTTACTA
ATTTACTAAGATTTACTAAGG
A
DNA template5’ 3’
Primer anneals Extension produces a series of
ddNTP terminated products each one base different in length
Each ddNTP is labeled with a different color fluorescent dye
Sequence is read by noting peak color in electropherogram (possessing single base resolution)
Contribution of the National Institute of Standards and Technology, 2010
Contribution of the National Institute of Standards and Technology, 2010
Contribution of the National Institute of Standards and Technology, 2010
Contribution of the National Institute of Standards and Technology, 2010
ORGANIC FTA PaperCHELEX
Blood stain
PUNCH
WASH Multiple Times with extraction buffer
PERFORM PCR
PCR Reagent
s
SDS, DTT, EDTA and
proteinase K
INCUBATE (56 oC)
Phenol,chloroform,
isoamyl alcohol
QUANTITATE DNA
Apply blood to paper and
allow stain to dry
Blood stain
VORTEX
(NO DNA QUANTITATION TYPICALLY PERFORMED
WITH UNIFORM SAMPLES)
Water
INCUBATE (ambient)
5% Chelex
INCUBATE (100 oC)
REMOVE supernatant
INCUBATE (56 oC)
QUANTITATE DNA
PERFORM PCR
PERFORM PCR
Centrifuge
Centrifuge
Centrifuge
Centrifuge
REMOVE supernatantTRANSFER aqueous (upper) phase to new tube
CONCENTRATE sample (Centricon/Microcon-100 or
ethanol precipitation)
Centrifuge
TE buffer
Contribution of the National Institute of Standards and Technology, 2010
Perpetrator’s sperm mixed with victim’s
epithelial cells
Centrifuge
REMOVE supernatant
SDS, EDTA and proteinase K
(cell lysis buffer)
Remove a portion of the mixed stain
SDS, EDTA and proteinase K + DTT
Incubate at 37 oC
sperm pellet
DTT lyses sperm heads
“Male Fraction” “Female Fraction”sperm pellet
Differential Extraction
Contribution of the National Institute of Standards and Technology, 2010
Too much DNA amplified
(a) (b)
Too little DNA amplified
(c)
Within optimal range
Contribution of the National Institute of Standards and Technology, 2010
20 ng
10 ng
5 ng
2.5 ng
1.25 ng
0.63 ng20 ng
10 ng
5 ng
2.5 ng
1.25 ng
0.63 ng
Calibration standards
Calibration standards
Unknown Samples
≈2.5 ng
Contribution of the National Institute of Standards and Technology, 2010
Polymerization and Strand Displacement
RQ
Forward primer
Reverse primer
3’
5’ 3’
5’3’5’
5’
5’
Forward primer
Reverse primer
3’
5’ 3’
5’5’
5’
Q
R
3’ Probe Cleavage (release of reporter dye)
Forward primer
Reverse primer
3’
5’ 3’
5’5’
5’
QR
Completion of Polymerization
TaqMan probe
Fluorescence occurs when reporter dye and quencher dye are no longer in close proximity
Contribution of the National Institute of Standards and Technology, 2010
Contribution of the National Institute of Standards and Technology, 2010
Cycle Number
Nor
mal
ized
Flu
ores
cenc
e
threshold
CT
Exponential product growth
Linear product growth
Plateau
ΔRn
Negative control
a b c d e
Standard curve
CT
Log[DNA]
a
b
c
d
e
Nc = No (1 + E)c
If efficiency is close to 100% (E = 1), then the product copy number (Nc) doubles the target copy number (No) with each cycle (c).
Contribution of the National Institute of Standards and Technology, 2010
94 oC
60 oC
72 oC
TimeTemperature
Single Cycle
Typically 25-35 cycles performed during PCR
94 oC 94 oC 94 oC
60 oC60 oC
72 oC72 oC
The denaturation time in the first cycle is lengthened to ~10 minutes when using AmpliTaq Gold to perform a “hot-start” PCR
Contribution of the National Institute of Standards and Technology, 2010
Separate strands
(denature)
Add primers (anneal)
Make copies (extend primers)
Repeat Cycle, Copying DNA Exponentially
Starting DNA
Template
5’
5’
3’
3’
5’
5’
5’3’ 3’
3’3’5’
Forward primer
Reverse primer
Contribution of the National Institute of Standards and Technology, 2010
Contribution of the National Institute of Standards and Technology, 2010
(a)
(b)
Contribution of the National Institute of Standards and Technology, 2010
(a) Simultaneous amplification of three locations on a DNA template
Locus A Locus CLocus B
(b) Resolution of PCR products with size-based separation method
A
CB
small large
Contribution of the National Institute of Standards and Technology, 2010
STR repeat region
GATA GATAGATAGATA
PCR product size generated
DNA template containing STR marker
Reverse PCR primer
Forward PCR primer
Fluorescent dye
(a)
(b)
Flanking regions
4 repeat units
5 repeat units
6 repeat units
Contribution of the National Institute of Standards and Technology, 2010
1 2 3 4 5 65’-TTTCCC TCAT TCAT TCAT TCAT TCAT TCAT TCACCATGGA-3’3’-AAAGGG AGTA AGTA AGTA AGTA AGTA AGTA AGTGGTACCT-5’
6 5 4 3 2 1
Contribution of the National Institute of Standards and Technology, 2010
Combine and re-amplify
Allelic Ladder
Individual Samples
6 8
7 9
8
9 10 6 7 8 9 10
Contribution of the National Institute of Standards and Technology, 2010
AmpFlSTR Identifiler kit (Applied Biosystems)
6-FAM (Blue)
VIC (Green)
NED (Yellow)
PET (Red)
D8S1179 D21S11 D7S820 CSF1PO
D3S1358 TH01 D13S317 D16S539 D2S1338
D19S433 D18S51TPOXVWA
AMEL D5S818 FGA
GS500 LIZ size standardLIZ (Orange)
D3S1358 TH01 D21S11 D18S51 Penta E
D5S818 D13S317 D7S820 D16S539 CSF1PO Penta D
AMEL VWA D8S1179 TPOX FGA
PowerPlex 16 kit (Promega Corporation)
ILS600 CXR size standardCXR (Red)
FL (Blue)
JOE (Green)
TMR (Yellow)
PCR product size (bp)
Contribution of the National Institute of Standards and Technology, 2010
D3S1358 TH01 D21S11D18S5
1
Penta E
D5S818
D13S317D7S820
D16S539Penta D
CSF1PO
Amelogenin(sex-typing)
VWA D8S1179
TPOXFGA
blue panel
Overlay of all 4 colors
(including internal size standard)
yellow panel
green panel
red panel
ILS600 DNA sizing standard
200 bp 300 bp100 bp 400 bp 500 bp325 350 375 425 450 475225 250 275
120 140 160 180
Contribution of the National Institute of Standards and Technology, 2010
D3S1358(8 alleles)
VWA(14 alleles)
D16S539(9 alleles)
D2S1338(14 alleles)
Blue panel
Green panel
Yellow panel
Orange panel
D21S11(24 alleles)
D8S1179(12 alleles)
D18S51(23 alleles)
TH01(10 alleles)
FGA low(19 alleles)
FGA high(9 alleles)
250 bp*139bp 200 bp160 bp 300 bp 340 bp 350 bp150 bp LIZ-labeled GS500 DNA sizing
standard100 bp
Red panel
D19S433(15 alleles)
D5S818(10 alleles)
TPOX(8 alleles)
D13S317(8 alleles)
D7S820(10 alleles)
CSF1PO(10 alleles)
AMEL(2 alleles)
Contribution of the National Institute of Standards and Technology, 2010
Fluorescent dye at 5’end
Non-nucleotide linkers (mobility modifiers)
Primer sequence
PCR amplification generates a labeled PCR product containing the mobility modifiers
5’-end
3’-end
For each linker unit added, there is an apparent
migration shift of ≈2.5 bp
Contribution of the National Institute of Standards and Technology, 2010
D7S820
CSF1PO
6 15
6 15
NED-labeled (yellow)
JOE-labeled (green)
D7S820 CSF1PO6 15 6 15
6FAM-labeled (blue) 6FAM-labeled (blue)
(a) COfiler kit allele relative size ranges
(b) Identifiler kit allele relative size ranges
256.01 bp 292.62 bp
279.65 bp 317.67 bp
255.15 bp 291.58 bp 304.69 bp 341.84 bp
Size overlap
10 non-nucleotide linkers
= ≈ +25 bp shift
Contribution of the National Institute of Standards and Technology, 2010
CSF1PO forward primer
CSF1PO reverse primer
(AGAT)6-15
(a) PowerPlex 1.1 Kit
91 bp 128 bp
TMR-labeled
PCR product sizes = 291-327 bp
CSF1PO forward primer
CSF1PO reverse primer
(b) PowerPlex 16 Kit
13 bp238 bpJOE-labeled
(AGAT)6-15
PCR product sizes = 221-357 bp +30 bp shift in size+30 bp shift in size
Contribution of the National Institute of Standards and Technology, 2010
X
Y
6 bp deletion
Female: X, X
Male: X, Y
1:1 Mixture: 3X + 1Y
X = 212 bpY = 218 bp
X = 106 bpY = 112 bp
AmpFlSTR kits and PowerPlex 16
PowerPlex 1.1
Contribution of the National Institute of Standards and Technology, 2010
Contribution of the National Institute of Standards and Technology, 2010
-
Voltage
Gel
Loading well
+anode cathode
Side view Top view
Gel lanes
DNA bands
Buffer
+
-
Gel stand
Contribution of the National Institute of Standards and Technology, 2010
Laser
InletBuffer
Capillary filled with polymer solution
5-20 kV- +
OutletBuffer
Sample tray
Detection window
(cathode) (anode)
Data Acquisition
Sample tray moves automatically beneath the cathode end of the capillary to deliver each sample in succession
Contribution of the National Institute of Standards and Technology, 2010
Mixture of dye-labeled PCR products from multiplex PCR
reaction
CCD Panel (with virtual filters)
Argon ion
LASER (488 nm)
ColorSeparationFluorescenc
e
ABI Prism spectrograph
SizeSeparation
Processing with GeneScan/Genotyper software
Sample Interpretation
Sample Injection
Sample Separation
Sample Detection
Sample Preparatio
n
Capillary(filled with
polymer solution)
Contribution of the National Institute of Standards and Technology, 2010
(a)
Larger DNA molecules interact more frequently with the gel and are thus retarded in their migration through the gel
Gel
(b)
Ogston Sieving Reptation
Small DNA molecules
Long DNA molecules
Gel
Contribution of the National Institute of Standards and Technology, 2010
hex hem1
2
3
So
S’1S1energy
(a)
Excitation Emission
Wavelength (nm)
1 3
ex max em max
Fluorescence
(b)
Stokes shift
Contribution of the National Institute of Standards and Technology, 2010
Fluorescent dNTPs are incorporated into both strands of PCR product
Ethidium bromide
DNA labeled with intercalating dye
Unlabeled DNA
SYBR Green
Intercalator inserts between base pairs on double-stranded DNA
One strand of PCR product is labeled with fluorescent dyeFluorescent dye
labeled primer
(a)
(b)
(c)
Contribution of the National Institute of Standards and Technology, 2010
FAM(blue)
JOE(green)
TAMRA(yellow)
ROX(red)
Contribution of the National Institute of Standards and Technology, 2010
520 540 560 580 600 620 640
WAVELENGTH (nm)
100
80
60
40
20
0
310 Filter Set F with color contributions
5-FAM JOE NED ROX
Laser excitation(488 nm, 514.5 nm)
Laser excitation(488 nm, 514.5 nm)
Normalized Fluorescent Intensity
Contribution of the National Institute of Standards and Technology, 2010
Scan number
Relative Fluorescence Units
DNA size in base pairs
Relative Fluorescence UnitsRegion shown below
(a)
(b)
Contribution of the National Institute of Standards and Technology, 2010
Capillary
Heat plate
Detection
window
electrode
Autosampler
Gel block
Syringe (with
polymer)
Outlet buffer
reservoir
Inlet buffer reservoir
Sample tray
Samples
Contribution of the National Institute of Standards and Technology, 2010
Mechanical pump(with polymer)
Capillaryarray Oven
Detection
window
electrodes
Autosampler
Lower gel
block
Polymer bottle Outlet
buffer reservoir
Inlet buffer reservoir
Sample tray
Fan
Contribution of the National Institute of Standards and Technology, 2010
Capillaries
Electrodes for Injection
Contribution of the National Institute of Standards and Technology, 2010
Data Collection
Peak Identification
Data Review by Analyst/Examiner
Color Separation
Peak Sizing
Comparison to Allelic Ladder
Confirmation of Results by Second Analyst/Examiner
Genotype Assignment to
Alleles
GeneScan software
Genotyper software
Internal size standard
Matrix file (spectral
calibration)
Allelic ladder sample
GeneMapperIDsoftware
Expert Systems (e.g., FSS-i3, TrueAllele)
Peak Editing to Remove Artifact
Calls
User-defined thresholds
Contribution of the National Institute of Standards and Technology, 2010
50 RFUs
150 RFUs
Analytical Threshold
Interpretation Threshold
Baseline Noise
Peak reliable, but only used for exclusions
Peak reliable, can be used for inclusions
Peak not considered
reliable
Val
ues
show
n fo
r ex
ampl
e pu
rpos
es o
nly
(sho
uld
be
base
d em
piric
ally
on
a la
b’s
inte
rnal
val
idat
ion)
Contribution of the National Institute of Standards and Technology, 2010
DNA fragment peaks in sample
DNA Size
Data Point
147.32 bp147.32 bp
165.05 bp
165.05 bp
100
139
150160
200
250
DNA fragment peaks are sized based on the sizing curve produced from the points on the internal size standard
3550
75 100139 160 200 250
300 340350
400 450 490500150
(a)
(b)
Time (minutes)
Contribution of the National Institute of Standards and Technology, 2010
Allelic ladder PCR-amplified sample
Internal size standard Internal size standard
Co
lor-
sep
arat
ed
an
d s
ize
d
alle
le p
eaks
fo
r e
ach
lo
cu
s
10 11 12 13 14 15
Data from CE instrument (prior to color separation
and peak sizing)
Genotyping performed by comparing allelic
ladder to sample results
Color separation and peak
sizing
Color separation and peak
sizingLocus 1
Genotype = 12, 14All ladder
alleles sized using internal size standard
All sample alleles sized
using internal size standard
Genotyping allele bins (+/-0.5 bp around ladder allele)
Alleles (# repeats)
Contribution of the National Institute of Standards and Technology, 2010
Dye blob
STR alleles
stutter
Pull-up (bleed-through)
spike
Blue channel
Green channel
Yellow channel
Red channel
D3S1358
Stutter products
6.0% 7.8%
Incomplete adenylation
D8S1179
-A
+A
-A
+A
Biological (PCR) artifacts
Contribution of the National Institute of Standards and Technology, 2010
(a) (b) (c)
Allelic ladder Allelic ladder Allelic ladder
Contribution of the National Institute of Standards and Technology, 2010
28.128.1
1 = S25-L25 = 244.34 - 244.46 = -0.12 bp
2 = SOL - L28 = 257.51-256.64 = +0.87 bp
c = |1 -2| = |-0.12-0.87| = 0.99 bp
Contribution of the National Institute of Standards and Technology, 2010
(a) (b)
12
31 2 3
Type 1 Type 2
Contribution of the National Institute of Standards and Technology, 2010
*
*8
86
6 8
Allele 6 amplicon has ‘dropped out’
Imbalance in allele peak heights
Heterozygous alleles are well balanced
No mutation
Mutation at 3’-end of primer binding site (allele dropout)
Mutation in middle of primer
binding site
(a)
(b)
(c)
Contribution of the National Institute of Standards and Technology, 2010
DNA Profile(with specific alleles)
Rarity estimate of DNA profile(e.g., RMP or LR)
Genetic formulas
Population allele frequencies
Contribution of the National Institute of Standards and Technology, 2010
Decide on Number of Samples and Ethnic/Racial Grouping
Gather Samples
Analyze Samples at Desired Genetic Loci
Summarize DNA types
Ethnic/ Racial Group 1
Ethnic/ Racial Group 2
Determine Allele Frequencies for Each Locus
Perform Statistical Tests on Data
Hardy-Weinberg equilibrium for allele independenceLinkage equilibrium for locus independence
Usually >100 per group
Use Database(s) to Estimate an Observed DNA Profile Frequency
Often anonymous samples from a blood bank
See Table 11.1
Examination of genetic distance between populations
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Paternal Allele
Maternal Allele
Genotype
Locus 1
DNA Profile
Paternal Allele
Maternal Allele
Genotype
Locus 2
Paternal Allele
Maternal Allele
Genotype
Locus 3
HWE
Linkage Equilibrium (product rule)
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National LevelNDIS
(FBI Laboratory)
SDIS(Richmond, Virginia)
SDIS(Tallahassee, Florida)
LDIS(Tampa)
LDIS(Orlando)
LDIS(Broward County)
LDIS(Roanoke)
LDIS(Norfolk)
LDIS(Fairfax)
State Level
Local Level
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Convicted Offender Index
Offenders (N) Crime Samples (C)
Forensic Index Arrestee Index
Arrestees (A)
1
2
3
‘Offender Hit’
‘Forensic Hit’
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Degraded DNA sample
D5S818D13S317
D7S820D16S539
CSF1PO Penta D
Agarose yield gel results
Smear of degraded DNA fragments
High relative molecular mass DNA in a tight band
(a)
(b)
Good quality DNA
Degraded DNA
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Full Profile (Good Quality)
Partial Profile (Poor Quality)
(a)
(b)
DNA size (bp) relative to an internal size standard (not shown)R
ela
tiv
e fl
uo
resc
enc
e u
nit
s (
RF
Us)
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STR repeat regionminiSTR primer
miniSTR primer
Conventional PCR primer
Conventional PCR primer
(a)
(b)
Conventional STR test (COfiler kit)
MiniSTR assay (using Butler et al. 2003 primers)
150 bp smaller
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(a) Single SourceD3S1358 TH01 D13S317 D16S539 D2S1338
16,16 9,9.3 8,12 9,9 17,19
(b) Mixed Source
D3S1358 TH01 D13S317 D16S539 D2S1338
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Type A Type B Type C
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>2 alleles at a
locus, except tri-allelics?
Single Source DNA Sample
NO
Mixed DNA Sample
YES
Differentiate a
Major/Minor Component?
Determine STR profile and compute RMP
YES
Is the sample a mixture?
TYPE B
NO
YES
Stochastic Effects ? Possible
Low Level DNA) ?
YES
Assume # Contributor
s?
TYPE C
TYPE ANO
A biostatistical analysis must be performed
Probability of Exclusion [CPE]
“RMNE”
Likelihood Ratio [LR]
YES
NO
Are # of contributors
defined?
A biostatistical analysis should not be performed
Determine component profile(s) and compute RMP for
major
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Identify the Presence of a Mixture
Consider All Possible Genotype Combinations
Estimate the Relative Ratio of the Individuals Contributing to the Mixture
Identify the Number of Potential Contributors
Designate Allele Peaks
Compare Reference Samples
Step #1
Step #2
Step #3
Step #4
Step #5
Step #6
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100 pg
50 pg
10 pg
Allele dropout
Severe imbalance
Good heterozygote peak balance
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Two possible allelesTwo possible alleles
Human Alu Repeat(~300 bp)
AluI
400 bp
100 bp
“long” (+) allele
“short” (-) allele
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Autosomal (passed on in part, from all ancestors)
Y-Chromosome(passed on complete,
but only by sons)
Mitochondrial (passed on complete, but only by daughters)
Lineage Markers
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Female-Male Mixture Performance with Autosomal vs. Y-Chromosome DNA Markers
Female Victim DNA Profile
Male Perpetrator DNA Profile
DNA Profile from Crime Scene
Autosomal STR Profile
Y-Chromosome STR Profile
No signal observed
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?uncle 3rd cousin
(paternal)
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Control region (D-loop)
1/16,569
cyt b
HV1 HV2
16024 16365 73 340
16024 576
mtGenome‘16,569’ bp
1
Heavy (H) strand
Light (L) strand
Coding region
HV3
438 574
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1 2
3 54
1211109
6 7 8
181715 16
13 14
MtDNA Haplotype Groups:1
2,3,6,8,11,13,15,164,9,10
57
1214,17,18
MtDNA Haplotype Groups:1
2,3,6,8,11,13,15,164,9,10
57
1214,17,18
A B
B C
C C
D B
B
B
B
B B
E
F
G
G
G
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Compare with database to determine haplotype frequency
Extract mtDNA from evidence
(Q) sample
PCR Amplify HV1 and HV2 Regions
Sequence HV1 and HV2 Amplicons
(both strands)
Confirm sequence with forward and reverse strands
Note differences from Anderson (reference) sequence
Compare Q and K sequences
Performed separately and preferably after
evidence is completed
Extract mtDNA from reference
(K) sample
PCR Amplify HV1 and HV2 Regions
Sequence HV1 and HV2 Amplicons
(both strands)
Confirm sequence with forward and reverse strands
Note differences from Anderson (reference) sequence
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Sample Q16093C16129A
Sample K16093C16129A
ACCGCTATGT ATTTCGTACA TTACTGCCAG CCACCATGAA TATTGTACGG TACCATAAAT 16090 16100 16110 16120 16130 16140
rCRS
ACCGCTATGT ATCTCGTACA TTACTGCCAG CCACCATGAA TATTGTACAG TACCATAAAT Q
K ACCGCTATGT ATCTCGTACA TTACTGCCAG CCACCATGAA TATTGTACAG TACCATAAAT
(a) mtDNA Sequences Aligned with rCRS (positions 16071-16140)
(b) Reporting Format with Differences from rCRS
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Thomas Jefferson II
Field Jefferson Peter Jefferson
President Thomas Jefferson
Eston Hemings
Same Y Haplotype
Jefferson Y Haplotype
Jefferson Y Haplotype
?
Randolph Jefferson
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TsarinaAlexandra
Tsar Nicholas II
Xenia Cheremeteff-Sfiri
Xenia Cheremeteff-Sfiri
Prince PhilipDuke of Edinburgh
Prince PhilipDuke of Edinburgh
GeorgijRomanov
GeorgijRomanov
Mitotype16111T16357C263G
315.1C
Mitotype16126C16169T16294T16296T
73G263G
315.1C
16169T/C
16169T/C Louise of Hesse-Cassel
Louise of Hesse-Cassel
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11,14 8,12
12,14 11,128,14 12,14 8,11
Obligate paternal allele 14 14 11 14 11
C,DA,B
B,C
mother father
child
(b) Example
(a) Mendelian Inheritance
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child
Mother (known parent)
Alleged father
?Rules of Inheritance
1) Child has two alleles for each autosomal marker (one from mother and one from biological father)
2) Child will have mother’s mitochondrial DNA haplotype (barring mutation)
3) Child, if a son, will have father’s Y-chromosome haplotype (barring mutation)
Random man
Missing child
Alleged mother
Alleged father
?
Parentage (Paternity) Testing
Reverse Parentage Testing (Missing Persons Investigation)
(a)
(b)
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DNA profile from mass disaster victim
DNA profile from direct reference
(toothbrush believed to have belonged to the victim)
(a) Direct comparison
(b) Kinship analysis
D5S818 D13S317D7S820
D16S539CSF1PO
Penta D
?
son
wife
victim D5S818 D13S317 D7S820 D16S539 CSF1PO Penta D
10,10 9,109,138,98,1411,13 son
wife10,12 8,108,98,128,1211,13
10,10 9,1211,139,911,1412,13
?,10 9,??,139,??,1411,? or ?,13
victim (father)
actual profile
Predicted victim profile
mass disaster victim profile
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14,18
(a) (b)
15,18
15,17 14,18
13,17
15,17
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Core set of markers(e.g., CODIS 13 STRs)
Past and Present
Future
(a)
(b)
(c)
(d)
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Loci Described
Use in Casework
Court Presentation/ Acceptance
Internal Validation
ResearchGovernment
Funded or Private
Development
Typically by Commercial Manufacturer
ForensicApplicationForensic Labs
Assay Constructed
Population Study
Information GatheredReleased to Community
Kit Developed
Kit Tested
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Lower amounts of DNA being
tested
Challengingkinship search
questionsStandard
STR Typing (DNA Profile)
Core Competency
Sufficient DNA quantity (ng)
Direct Matching (or Parentage)
Solution: Replicate Testing
Solution: Additional Markers (Y-chromosome, more STRs) and
Multiple Reference Samples
Touch DNA Attempts (poor quality, mixtures, low-level
stochastic effects)
Familial Searching Attempts (fishing for brothers or other relatives)
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8,128,13
12,138,9
9,129,13
Q(query)
K(database)
(b)
9,139,12
8,138,128
9
12 13
Fat
her’s
alle
les
Mother’s alleles
(a)
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Set up two hypotheses (H0 and H1)
Collect data and calculate the test
statistic (S)
Look up the critical value (C) and define
the region of rejection for the test statistic
Is S ≤ C?
yes
Accept H0 (Reject H1)
Accept H1 (Reject H0)
no
Select appropriate statistical model
Specify the level of significance and its
critical value (C)
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Truth about the population
Decision based on sample examined
Correct decision
Type II error
Type I error Correct decision
H0 True H0 False
Accept H0
Reject H0 (Accept H1)
Correct decision
Wrongfully acquitted
Wrongfully accused
Correct decision
(b) Example
Not Guilty
Guilty
Courtroom VerdictSaint Sinner
Defendant
(a) Hypothesis Testing Decisions