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1
Aspects of Forensic DNA Typingusing John M Butler’s slides
http://www.cstl.nist.gov/biotech/strbase/FDT2e.htm
Statistic 246 Week 2, Lecture 2 Spring 2006
Copyright symbols have been placed at the bottom of figures used directly fromthe book. The publisher requests that these copyright symbols are retained byslide users in their presentations.This has been retained, but material has been added or removed to suit thepurposes of the class. Please check the originals to see exactly what changeshas been made. Most other slides are from John Butler’s collection too.
2
Some generalities
3http://www.ojp.usdoj.gov/nij/pubs-sum/183697.htm
•Report published in Nov 2000
•Asked to estimate where DNAtesting would be 2, 5, and 10 yearsinto the future
ConclusionsSTR typing is here tostay for a few yearsbecause of DNAdatabases that havegrown to containmillions of profiles
The Future of Forensic DNA Testing
4
Forensic DNA Typing, 2nd Edition:Biology, Technology, and Genetics of STR Markers
Chapter 1 Overview & History of DNA TypingChapter 2 DNA Biology Review Chapter 3 Sample Collection, Extraction, QuantitationChapter 4 PCR AmplificationChapter 5 Common STRs and Commercial KitsChapter 6 Biology of STRsChapter 7 Forensic IssuesChapter 8 Single Nucleotide PolymorphismsChapter 9 Y-Chromosome DNA TestsChapter 10 Mitochondrial DNA AnalysisChapter 11 Non-Human DNA and Microbial ForensicsChapter 12 DNA Separation MethodsChapter 13 DNA Detection Methods Chapter 14 Instrumentation for STR Typing: ABI 310, ABI 3100, FMBIO Chapter 15 STR Genotyping IssuesChapter 16 Lab ValidationChapter 17 New Technologies, Automation, and Expert SystemsChapter 18 CODIS and DNA DatabasesChapter 19 Basic Genetic Principles and StatisticsChapter 20 STR Database AnalysesChapter 21 Profile Frequency EstimatesChapter 22 Statistical Analysis of Mixtures and Degraded DNAChapter 23 Kinship and Paternity TestingChapter 24 Mass Disaster DNA Victim IdentificationAppendix I Reported STR AllelesAppendix II U.S. Population Data-STR Allele FrequenciesAppendix III Suppliers of DNA Analysis EquipmentAppendix IV DAB QA StandardsAppendix V DAB Recommendations on StatisticsAppendix VI Application of NRC II to STR TypingAppendix VII Example DNA Cases
John Butler
5
Human Identity Testing
• Forensic cases -- matching suspect withevidence
• Paternity testing -- identifying father• Mass disasters -- putting pieces back together• Historical investigations• Missing persons investigations• Military DNA “dog tag”• Convicted felon DNA databases
Involves generation of DNA profiles usually withthe same core STR (short tandem repeat) markers
6
Basis of DNA Profiling The genome of each individual is unique (with theexception of identical twins) and is inherited from parents
Probe subsets of genetic variation in order to differentiatebetween individuals (statistical probabilities of a randommatch are used)
DNA typing must be performed efficiently andreproducibly (information must hold up in court)
Current standard DNA tests DO NOT look at genes –little/no information about race, predisposal to disease, orphenotypical information (eye color, height, hair color) isobtained
7
Speed of Analysis(Technology)
Power ofDiscrimination
(Genetics)
Low
High
Slow Fast
Markers Used(Biology)
RFLPSingle Locus Probes
RFLPMulti-Locus Probes
ABOblood groups
Multiplex STRs
DQαsingle STR
D1S80mtDNA
PolyMarker
Figure 1.1, J.M. Butler (2005) Forensic DNA Typing, 2nd Edition © 2005 Elsevier Academic Press
Some DNA marker types & technologies)
8
DNAExtraction
DNAQuantitation
PCR Amplificationof Multiple STR markers
Biology
Separation and Detection ofPCR Products(STR Alleles)
Technology
Sample GenotypeDetermination
Genetics
Comparison of SampleGenotype to Other Sample
Results
If match occurs, comparison ofDNA profile to population
databases
Generation of Case Reportwith Probability of Random
Match
Figure 1.2, J.M. Butler (2005) Forensic DNA Typing, 2nd Edition © 2005 Elsevier Academic Press
Steps in DNA sample analysis and interpretation
9
genderID
A
B
C
D
E FG
H IJ
genderID A
B C D EF
G
HI
J
Figure 1.3, J.M. Butler (2005) Forensic DNA Typing, 2nd Edition © 2005 Elsevier Academic Press
Comparison of two electropherograms
10
Quick review of some biology
11
http
://w
ww
.ncb
i.nlm
.nih
.gov
/gen
ome/
guid
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
Human Genome23 Pairs of Chromosomes + mtDNA
Sex-chromosomes
mtDNA
16,569 bp
Autosomes
MitochondrialDNA
Nuclear DNA
3.2 billion bp
Located in cell nucleus
Located inmitochondria
(multiple copiesin cell cytoplasm)
2 copiesper cell
100s of copiesper cell
Figure 2.3, J.M. Butler (2005) Forensic DNA Typing, 2nd Edition © 2005 Elsevier Academic Press
12
p(short arm)
centromere
telomere
q(long arm)
telomere
Band 5
Band 3
Chromosome 12
12p3
12q5
Figure 2.4, J.M. Butler (2005) Forensic DNA Typing, 2nd Edition © 2005 Elsevier Academic Press
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2 repeats
3 repeats
--------AGACTAGACATT---------------AGATTAGGCATT-------
---------(AATG)(AATG)(AATG)----------
---------(AATG)(AATG)----------
(B) Short tandem repeat (STR) polymorphism
(A) Single nucleotide polymorphism (SNP)
Based on Figure 2.5, J.M. Butler (2005) Forensic DNA Typing, 2nd Edition © 2005 Elsevier Academic Press
Two important polymorphic marker types
14
63
4 5
Homologous pair ofchromosomes
Homologous pair ofchromosomes
Locus A
Locus B
Allele 1 Allele 2
Allele 2Allele 1
Figure 2.6, J.M. Butler (2005) Forensic DNA Typing, 2nd Edition © 2005 Elsevier Academic Press
15
Overview of the technology
16
Sources of Biological Evidence• Blood• Semen• Saliva• Urine• Hair• Teeth• Bone• Tissue
Blood stain
Only a very smallamount of blood isneeded to obtain a
DNA profile
17
ORGANIC FTA PaperCHELEX
Bloodstain
PUNCH
WASH Multiple Timeswith extraction buffer
PERFORM PCR
PCRReagents
SDS, DTT, EDTAand
proteinase K
INCUBATE (56 oC)
Phenol,chloroform,
isoamyl alcohol
QUANTITATEDNA
Apply blood topaper and allow
stain to dryBloodstain
VORTEX
(NO DNA QUANTITATIONTYPICALLY PERFORMED WITH
UNIFORM SAMPLES)
Water
INCUBATE (ambient)
5%Chelex
INCUBATE (100 oC)
REMOVE supernatant
INCUBATE (56 oC)
QUANTITATEDNA
PERFORM PCR
PERFORM PCR
Centrifuge
Centrifuge
Centrifuge
Centrifuge
REMOVE supernatantTRANSFER aqueous (upper) phaseto new tube
CONCENTRATE sample(Centricon/Microcon-100 or ethanol
precipitation)
Centrifuge
TE buffer
Figure 3.1, J.M. Butler (2005) Forensic DNA Typing, 2nd Edition © 2005 Elsevier Academic Press
Common DNA extraction methods
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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
Calibrationstandards
Calibrationstandards
Unknown Samples
~2.5 ng
Figure 3.3, J.M. Butler (2005) Forensic DNA Typing, 2nd Edition © 2005 Elsevier Academic Press
Slot blot for human DNA quantitation
Uses a 40 bp primate-specific DNA probe (for details see book)
19
Importance of DNA Quantitation(prior to multiplex PCR)
DNA amount(log scale)
0.5 ng
-A
+AToo much DNA
Off-scale peaks Split peaks (+/-A) Locus-to-locus imbalance
100 ng
10 ng
1 ng
0.1 ng
0.01 ng
2.0 ng
Too little DNA Heterozygote peak imbalance Allele drop-out Locus-to-locus imbalance
Stochastic effect when amplifying lowlevels of DNA produces allele dropout
STR Kits Work Best in This Range
High levels of DNA create interpretationchallenges (more artifacts to review)
Well-balanced STR multiplex
20
Calculation of the quantity of DNA in a cell
1. Molecular Weight of a DNA Basepair = 618g/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
2. Molecular weight of DNA = 1.85 x1012 g/mol There are 3 billion base pairs in a haploid cell ~3 x 109 bp
(~3 x 109 bp) x (618 g/mol/bp) = 1.85 x 1012 g/mol
3. Quantity of DNA in a haploid cell = 3 picograms1 mole = 6.02 x 1023 molecules
(1.85 x 1012 g/mol) x (1 mole/6.02 x 1023 molecules) = 3.08 x 10-12 g = 3.08 picograms (pg)
A diploid human cell contains ~6 pg genomic DNA
4. One ng of DNA contains the DNA from 167 diploid cells
1 ng genomic DNA (1000 pg)/6pg/cell = ~333 copies of each locus (2 per 167 diploid genomes)
21
Brief review of thePolymerase Chain Reaction
22
Separatestrands
(denature)
Add primers(anneal)
Make copies(extend primers)
Repeat Cycle,Copying DNAExponentially
Starting DNATemplate
5’
5’
3’
3’
5’
5’
5’3’ 3’
3’3’5’
Forward primer
Reverse primer
Figure 4.2, J.M. Butler (2005) Forensic DNA Typing, 2nd Edition © 2005 Elsevier Academic Press
Schematic: the precisedetails are different
23
94 oC
60 oC
72 oC
Time
Tem
pera
ture
Single Cycle
Typically 25-35 cyclesperformed during PCR
94 oC 94 oC 94 oC
60 oC60 oC
72 oC72 oC
The denaturation time in the firstcycle is lengthened to ~10 minuteswhen using AmpliTaq Gold toperform a “hot-start” PCR
Figure 4.1, J.M. Butler (2005) Forensic DNA Typing, 2nd Edition © 2005 Elsevier Academic Press
Thermal cycling: temperature profile
24
(A) Simultaneous amplification of three locations on a DNA template
Locus A Locus CLocus B
(B) Resolution of PCR products with a single size-based separation method
AC
B
small large
Figure 4.3, J.M. Butler (2005) Forensic DNA Typing, 2nd Edition © 2005 Elsevier Academic Press
Schematic for multiplex PCR
25
Commonly used Short TandemRepeat (STR) markers
26
Minisatellite Marker (D1S80)
GAGGACCACCAGGAAG
Repeat region
Flanking regions
16 bp repeat unit
STR Marker (TH01)
TCAT
Repeat region
Flanking regions
4 bp repeat unit
Figure 5.1, J.M. Butler (2005) Forensic DNA Typing, 2nd Edition © 2005 Elsevier Academic Press
27
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
Figure 5.2, J.M. Butler (2005) Forensic DNA Typing, 2nd Edition © 2005 Elsevier Academic Press
Example of DNA sequencein an STR repeat region
Note different repeat motifs and starting positions on different strands. By convention, this is a TCAT repeat, read 5’ to 3’.
28
AmpFlSTR® Identifiler™ kit (Applied Biosystems)
6-FAM (Blue)
VIC (Green)
NED (Yellow)
PET (Red)
D8S1179 D21S11 D7S820 CSF1POD3S1358 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)
Figure 5.4, J.M. Butler (2005) Forensic DNA Typing, 2nd Edition © 2005 Elsevier Academic Press
Commercially available STR kits for the 13 CODIS loci
29
D3S1358 TH01 D21S11D18S51 Penta E
D5S818D13S317
D7S820D16S539
Penta DCSF1PO
Amelogenin(sex-typing)
VWA D8S1179
TPOXFGA
blue panel
Overlay of all 4 colors(including internal size
standard)
yellow panel
green panel
red panelILS600 DNA sizing standard
200 bp 300 bp100 bp 400 bp 500 bp325 350 375 425 450 475225 250 275120 140 160 180
Figure 5.5, J.M. Butler (2005) Forensic DNA Typing, 2nd Edition © 2005 Elsevier Academic Press
30
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 standard
100 bp
Red panel
D19S433(15 alleles)
D5S818(10 alleles)
TPOX(8 alleles)
D13S317(8 alleles)
D7S820(10 alleles)
CSF1PO(10 alleles)
AMEL(2 alleles)
Figure 5.6, J.M. Butler (2005) Forensic DNA Typing, 2nd Edition © 2005 Elsevier Academic Press
31
X
Y
6 bpdeletion
Female: X, X
Male: X, Y
1:1 Mixture: 3X + 1Y
X = 212 bpY = 218 bp
X = 106 bpY = 112 bp
AmpFlSTR kitsand PowerPlex 16
PowerPlex 1.1
Figure 5.11, J.M. Butler (2005) Forensic DNA Typing, 2nd Edition © 2005 Elsevier Academic Press
Amelogenin sex-typing assay
32
Short Tandem Repeat DNAInternet DataBase
Created by John M. Butler and Dennis J. Reeder (NIST Biotechnology Division), with invaluable helpfrom Jan Redman, Christian Ruitberg and Michael Tung
*Partial support for the design and maintenance of this website is being provided by The NationalInstitute of Justice through the NIST Office of Law Enforcement Standards.*
STRs101: Brief Introduction to STRsSTR Fact Sheets (observed alleles and PCR product sizes)Sequence Information (annotated)Multiplex STR setsSTR Training MaterialsNon-published Variant Allele ReportsThree-Banded PatternsFBI CODIS Core STR LociDNA Advisory Board Quality Assurance StandardsNIST Standard Reference Material for PCR-Based Testing
Chromosomal LocationsMutation Rates for Common LociPublished PCR primersValidation studiesPopulation dataY-chromosome STRsSex-typing markersTechnology for resolving STR allelesReference List Now 2059 referencesAddresses for scientists working with STRs
Links to other web sitesGlossary of commonly used terms
Publications and Presentations from NIST Human Identity Project Team
These data are intended to benefit research and application of short tandem repeat DNA markers tohuman identity testing. The authors are solely responsible for the information herein.
Figure 5.12, J.M. Butler (2005) Forensic DNA Typing, 2nd Edition © 2005 Elsevier Academic Press
33
D21S11 D18S51
D8S1179
DNA Size (bp)R
elat
ive
Fluo
resc
ence
Uni
ts
StutterProduct
6.3% 6.2% 5.4%
Allele
Figure 6.1, J.M. Butler (2005) Forensic DNA Typing, 2nd Edition © 2005 Elsevier Academic Press
STR alleles with stutter products
34
GATA GATA GATA
CTAT CTAT CTAT CTAT CTAT CTAT3’ 5’
5’
1 2 3 4 5 6
1 2 3
(A) Normal replication
GATA GATA
CTAT CTAT CTAT CTAT CTAT CTAT3’ 5’
5’
1 2 3 4 5 6
1
2’
2
(B) Insertion caused by backward slippage
GATA GATA GATA
CTAT CTAT CTAT3’ 5’
5’
1 2 3CTAT CTAT
5 6
1 2 3(C) Deletion caused by forward slippage
GATA
5
4
CT AT
Primary mechanism forstutter product formation
Figure 6.2, J.M. Butler (2005) Forensic DNA Typing, 2nd Edition © 2005 Elsevier Academic Press
Conjectured mechanisms
35
0
2.5
5
7.5
10
12.5
15
Stut
ter P
erce
ntag
e TH01 TPOX CSF1PO D7S820
0
2.5
5
7.5
10
12.5
15
Stu
tter
Per
cent
ages D5S818
VWAD3S1358
D13S317D8S1179
0
2.5
5
7.5
10
12.5
15
Stu
tter
Per
cent
age FGA D16S539 D18S51 D21S11
Alleles
Alleles
Alleles
LOCI
Figure 6.3, J.M. Butler (2005) Forensic DNA Typing, 2nd Edition © 2005 Elsevier Academic Press
Stutter percentages for CODIS loci
36
5’5’3’3’
Polymerase extension
ReversePrimer
ForwardPrimer
Polymerase extension
5’5’
5’5’AA
OR
(-A form) (+A form)
Shoulder peak
-A
+A
Split peak
+A-A
+A-A
(A)
(B)
Full-length allele(n)
allele + 1 base(n+1)
Measurement Result with dye labeled DNA strand
Incompleteadenylation
D8S1179
-A
+A
-A
+A
Figure 6.4, J.M. Butler (2005) Forensic DNA Typing, 2nd Edition © 2005 Elsevier Academic Press
Non-template nucleotide addition
37
D3S1358 VWA FGA
-A
+A 10 ngtemplate
(overloaded)
2 ng template(suggested level)
DNA Size (bp)
Rel
ativ
e Fl
uore
scen
ce (R
FUs) off-scale
Figure 6.5, J.M. Butler (2005) Forensic DNA Typing, 2nd Edition © 2005 Elsevier Academic Press
Incomplete non-template additionwith high levels of DNA template
38
28.1
δ1 = S25-L25 = 244.34 - 244.46 = -0.12 bp
δ2 = SOL - L28 = 257.51-256.64 = +0.87 bp
c = |δ1 -δ2| = |-0.12-0.87| = 0.99 bp
Figure 6.6, J.M. Butler (2005) Forensic DNA Typing, 2nd Edition © 2005 Elsevier Academic Press
Detection of a microvariant allele at the locus FGA
39
AMELD8S1179 D21S11 D18S51
(B)
TPOX(A)
Figure 6.7, J.M. Butler (2005) Forensic DNA Typing, 2nd Edition © 2005 Elsevier Academic Press
Tri-allelic patterns atTPOX and D18S51
40
*
*
*A)
B)
C)
Example:TH01 9.3 allele(-A in 7th repeat)
Example:D18S51 13.2 allele(+AG in 3’-flanking region)
Example:Rare VWA allele amplified withAmpFlSTR primers(A-to-T in 2nd base from 3’end offorward primer)
Repeat regionReverse primerbinding region
3’flankingregion
5’flankingregionForward primer
binding regionSTR
Figure 6.8, J.M. Butler (2005) Forensic DNA Typing, 2nd Edition © 2005 Elsevier Academic Press
Possible sequence variation (*) andimpact on PCR amplification
Amplicon size may be nonstandard
Amplicon size may be nonstandard
PCR may fail
41
*
*8
86
6 8
Allele 6 ampliconhas “dropped out”
Imbalance in allelepeak heights
Heterozygous allelesare well balanced
Figure 6.9, J.M. Butler (2005) Forensic DNA Typing, 2nd Edition © 2005 Elsevier Academic Press
Impact of sequence polymorphismin the primer binding site
42
14,18
(a) (b)
15,18
15,17 14,18
13,17
15,17
Figure 6.10, J.M. Butler (2005) Forensic DNA Typing, 2nd Edition © 2005 Elsevier Academic Press
Mutational events observed in family trees
43
Mutation Rates for Common STR Loci
0.64%330/51,940Nonereported
330/51,610 (0.64)0/330 (<0.30)SE33(ACTBP2)
0.11%187/173,4907178/103,489 (0.075)38/70,001 (0.05)D19S4330.12%262/225,14090157/152,310 (0.10)15/72,830 (0.021)D2S13380.16%163/100,0305975/55,719 (0.135)29/44,311 (0.065)Penta E0.14%57/41,2022421/22,501 (0.09)12/18,701 (0.06)Penta D0.19%1,816/962,096580772/526,708 (0.15)464/435,388 (0.11)D21S110.22%1,746/790,3424661,094/494,098 (0.22)186/296,244 (0.06)D18S510.11%1,041/962,239372540/494,465 (0.11)129/467,774 (0.03)D16S5390.14%1,558/1,103,282485881/621,146 (0.14)192/482,136 (0.04)D13S3170.14%1,239/899,837364779/489,968 (0.16)96/409,869 (0.02)D8S11790.10%1,089/1,085,305285745/644,743 (0.12)59/440,562 (0.013)D7S8200.11%1,259/1,107,339385763/655,603 (0.12)111/451,736 (0.025)D5S8180.12%1,152/964,288379713/558,836 (0.13)60/405,452 (0.015)D3S13580.17%2,480/1,437,9458141,482/873,547 (0.17)184/564,398 (0.03)VWA0.01%100/857,4812854/457,420 (0.012)18/400,061 (0.004)TPOX0.01%100/779,5542841/452,382 (0.009)31/327,172 (0.009)TH010.28%3,125/1,101,0067102,210/692,776 (0.32)205/408,230 (0.05)FGA0.16%1,487/947,425410982/643,118 (0.15)95/304,307 (0.03)CSF1PO
MutationRate
Total Number ofMutations
Number fromeither
Paternal Meioses(%)
Maternal Meioses (%)STR Systemhttp://www.aabb.org/About_the_AABB/Stds_and_Accred/ptannrpt03.pdf, Appendix 2
J.M. Butler (2005) J. Forensic Sci., in press
44
Some special issues arisingwith forensic DNA typing
45
Degraded DNA sample
D5S818 D13S317D7S820
D16S539CSF1PO Penta D
Agarose yield gel results
Smear of degradedDNA fragmentsHigh molecular
weight DNA ina tight band
(A)
(B)
Good qualityDNA
DegradedDNA
Figure 7.1, J.M. Butler (2005) Forensic DNA Typing, 2nd Edition © 2005 Elsevier Academic Press
Impact of degraded DNA
46
<15%Stutter region
>70%
100%
Heterozygouspeak region
85%
MIXTUREREGION
9%
Higher than typicalstutter product (>15%)
100%
<15%
>70%60%
10%
25%
Wrong side of allele to betypical stutter product
Smaller peak area than normally seenwith heterozygote partner alleles(<70%)
(a)
(b)
Figure 7.3, J.M. Butler (2005) Forensic DNA Typing, 2nd Edition © 2005 Elsevier Academic Press
Illustration of typical single source vs mixed sample
47
Some of the DNA technology
48
-
Voltage
Gel
Loading well
+anode cathode
Side view Top view
Gel lanes
DNAbands
Buffer
+
-
Figure 12.1, J.M. Butler (2005) Forensic DNA Typing, 2nd Edition © 2005 Elsevier Academic Press
Schematic of gel electropheresis
49
Laser
InletBuffer
Capillary filled withpolymer solution
5-20 kV- +
OutletBuffer
Sample tray
Detectionwindow
(cathode) (anode)
DataAcquisition
Sample tray moves automatically beneath thecathode end of the capillary to deliver eachsample in succession
Figure 12.3, J.M. Butler (2005) Forensic DNA Typing, 2nd Edition © 2005 Elsevier Academic Press
Schematic of capillary electropheresis
50
(a)
Larger DNA molecules interactmore frequently with the gel and arethus retarded in their migrationthrough the gel
Gel
(b)
Ogston Sieving Reptation
Small DNAmolecules
Long DNAmolecules
Gel
Figure 12.4, J.M. Butler (2005) Forensic DNA Typing, 2nd Edition © 2005 Elsevier Academic Press
Illustration of DNAseparation modes
51
hνex hνem1
2
3
So
S’1S1energy
(a)
Excitation Emission
Wavelength (nm)
1 3
λex max λem max
Fluo
resc
ence
(b)
Stokesshift
Figure 13.1, J.M. Butler (2005) Forensic DNA Typing, 2nd Edition © 2005 Elsevier Academic Press
Illustration offluorescence &
excitation/emissionspectra
52
FAM(blue)
JOE(green)
TAMRA(yellow)
ROX(red)
Figure 13.3, J.M. Butler (2005) Forensic DNA Typing, 2nd Edition © 2005 Elsevier Academic Press
Dyes used by ABI in 4-color detection
53
520 540 560 580 600 620 640WAVELENGTH (nm)
100
80
60
40
20
0
310 Filter Set Fwith color contributions
5-FAM JOE NED ROX
Laser excitation(488, 514.5 nm)
Nor
mal
ized
Flu
ores
cent
Inte
nsity
Figure 13.4, J.M. Butler (2005) Forensic DNA Typing, 2nd Edition © 2005 Elsevier Academic Press
Emission spectra of ABI dyes and wavelengthbands used for detection
54
Figure 13.6, J.M. Butler (2005) Forensic DNA Typing, 2nd Edition © 2005 Elsevier Academic Press
Example of a matrix used for color separation
55
Scan number
Rel
ativ
e Fl
uore
scen
ce U
nits
DNA size in base pairs
Rel
ativ
e Fl
uore
scen
ce U
nits
Region shown below
(a)
(b)
Figure 13.7, J.M. Butler (2005) Forensic DNA Typing, 2nd Edition © 2005 Elsevier Academic Press
Traces before and after color separation(and perhaps other processsing)
56
Mixture of dye-labeledPCR products from
multiplex PCRreaction
SampleSeparation
Sample DetectionCCD Panel (with virtual filters)
Argon ionLASER(488 nm)
ColorSeparationFluorescence
ABI Prismspectrograph
Capillary
SampleInjection
SizeSeparation
Processing with GeneScan/Genotyper software
Sample Interpretation
Figure 13.8, J.M. Butler (2005) Forensic DNA Typing, 2nd Edition © 2005 Elsevier Academic Press
Schematic of the separationand detection process
57
Profiler Plus™ multiplex STR resultDNA size (bp)
DNA size (bp)COfiler™ multiplex STR result
FGAD21S11
D18S51
D8S1179
VWA D13S317
D5S818
Amel
D3S1358 D7S820
TH01
AmelD16S539
D7S820CSF1POTPOX
D3S1358
Figure 13.9, J.M. Butler (2005) Forensic DNA Typing, 2nd Edition © 2005 Elsevier Academic Press
Traces from ABI 310 capillary electrophoresis system
58
Figure 13.11, J.M. Butler (2005) Forensic DNA Typing, 2nd Edition © 2005 Elsevier Academic Press
Image of data fromFig 14.9 below
59
Fluorescein Scan JOE Scan Rhodamine Red-X Scan Texas Red-X Scan
200
100
225
250
275
180
160
140
120
300325350375400425450
TPOX
D8S1179
VWA
FGA
Amelogenin
Penta D
CSF1PO
D7S820
D13S317
D5S818
D16S539
Penta E
D18S51
D21S11
TH01
D3S1358
Figure 14.9, J.M. Butler (2005) Forensic DNA Typing, 2nd Edition © 2005 Elsevier Academic Press
60
Dye blob
STR alleles
stutter
Pull-up(bleed-through)
spike
Blue channel
Green channel
Yellow channel
Red channel
Figure 15.4, J.M. Butler (2005) Forensic DNA Typing, 2nd Edition © 2005 Elsevier Academic Press
D3S1358
Stutter products
6.0% 7.8%
Incompleteadenylation
D8S1179
-A
+A
-A
+A
Biological (PCR)artifacts
Deciphering Artifacts from the True Alleles
61
STR population databasesand statistical calculations
This material which follows ispresented without comment as
illustrative of the approach taken bylaw enforcement agencies.
62
Decide on Number of Samplesand Ethnic/Racial Grouping
Gather Samples Get IRB approval
Analyze Samples atDesired Genetic Loci
Summarize DNA types
Ethnic/ RacialGroup 1
Ethnic/ RacialGroup 2
Determine Allele Frequenciesfor Each Locus
Perform StatisticalTests on Data
Hardy-Weinberg equilibrium for allele independenceLinkage equilibrium for locus independence
Usually >100 per group (see Table 20.1)
Use Database(s) to Estimate anObserved DNA Profile Frequency See Chapter 21
Often anonymous samples from a blood bank
See Table 20.2 and Appendix II
See Chapter 5 (STR kits available) andChapter 15 (STR typing/interpretation)
Examination of genetic distance between populations
Figure 20.1, J.M. Butler (2005) Forensic DNA Typing, 2nd Edition © 2005 Elsevier Academic Press
63
U.S. Population Samples(Appendix II)
0.000
0.050
0.100
0.150
0.200
0.250
0.300
0.350
0.400
0.450
8 9 10 11 12 13 14 15
D13S317 Allele
Fre
qu
en
cy
African Americans (N=258) Caucasians (N=302) Hispanics (N=140)
**
Figure 20.3, J.M. Butler (2005) Forensic DNA Typing, 2nd Edition © 2005 Elsevier Academic Press
64
How Statistical Calculations are Made
• Generate data with set(s) of samples from desiredpopulation group(s)– Generally only 100-150 samples are needed to obtain
reliable allele frequency estimates
• Determine allele frequencies at each locus– Count number of each allele seen
• Allele frequency information is used to estimate therarity of a particular DNA profile– Homozygotes (p2), Heterozygotes (2pq)– Product rule used (multiply locus frequency estimates)
For more information, see Chapters 20 and 21 in Forensic DNA Typing, 2nd Edition
65
Assumptions with Hardy-Weinberg Equilibrium
None of these assumptions are really true…
Table 20.6, J.M. Butler (2005) Forensic DNA Typing, 2nd Edition © 2005 Elsevier Academic Press
66
Individual Genotypes Are Summarizedand Converted into Allele Frequencies
The 11,12 genotype was seen54 times in 302 samples(604 examined chromosomes)
Table 20.2, J.M. Butler (2005) Forensic DNA Typing, 2nd Edition © 2005 Elsevier Academic Press
67
Allele Frequency Tables
CaucasianN= 302
0.0017*
--0.10270.2616
--
0.25330.2152
0.152320.01160
AfricanAmerican
N=258
--
0.0019*0.08920.3023
0.0019*0.33530.20540.06010.0039*
20 0.0017* 0.0001*
D3S1358
Butler et al. (2003)JFS 48(4):908-911
Allele frequencies denoted withan asterisk (*) are below the5/2N minimum allele thresholdrecommended by the NationalResearch Council report (NRCII)The Evaluation of Forensic DNAEvidence published in 1996.
Mostcommonallele
CaucasianN= 7,636
0.0009
0.12400.2690
--
0.24300.20000.14600.0125
Einum et al. (2004)JFS 49(6)
Allele
11
131415
15.216171819
12 0.0017* --0.0007
0.0031
AfricanAmericanN= 7,602
0.0003*
0.00770.09050.2920
0.00100.33000.20700.06300.0048
0.0045
20
Allele
11
131415
15.216171819
12
68
Illustrative calculations
69
DNA Profile Frequency with all 13 CODIS STR loci
21.283.50
18.6213.8
31.8530.69
9.2526.1811.31
16.29
12.35
8.87
9.171 in
8.37 x 10140.216910CSF1PO
3.94 x 10130.53488TPOX
1.13 x 10130.23186THO1
6.05 x 10110.3212110.11269D16S539
4.38 x 10100.17729D7S820
1.38 x 1090.0480140.339411D13S317
44,818,2590.1407130.384112D5S818
4,845,2170.1391160.137414D18S51
185,0730.2782300.158928D21S11
16,3640.1656140.185412D8S1179
10050.2185220.185421FGA
810.2003180.281517VWA
9.170.2152170.253316D3S1358
Combined value allele value alleleLocus
The Random Match Probability for this profile in the U.S. Caucasian populationis 1 in 837 trillion (1012)
AmpFlSTR® Identifiler™(Applied Biosystems)
AMELD3
TH01 TPOX
D2D19FGA
D21 D18
CSFD16
D7D13
D5 VWAD8
What wouldbe enteredinto a DNA
database forsearching:
16,17-17,18-21,22-12,14-28,30-14,16-12,13-11,14-9,9-
9,11-6,6-8,8-
10,10
PRODUCT
RULE
70
The Same 13 Locus STR Profilein Different Populations
1 in 0.84 quadrillion (1015) in U.S. Caucasian population (NIST)1 in 2.46 quadrillion (1015) in U.S. Caucasian population (FBI)*1 in 1.86 quadrillion (1015) in Canadian Caucasian population*
1 in 16.6 quadrillion (1015) in African American population (NIST)1 in 17.6 quadrillion (1015) in African American population (FBI)*
1 in 18.0 quadrillion (1015) in U.S. Hispanic population (NIST)
*http://www.csfs.ca/pplus/profiler.htm
1 in 837 trillion
These values are for unrelated individualsassuming no population substructure (using only p2 and 2 pq)
NIST study: Butler, J.M., et al. (2003) Allele frequencies for 15 autosomal STR loci on U.S.Caucasian, African American, and Hispanic populations. J. Forensic Sci. 48(4):908-911.(http://www.cstl.nist.gov/biotech/strbase/NISTpop.htm)
71
STR Cumulative Profile Frequency with Multiple Population Databases
1014 to 1021
D.N.A. Box 21.1, J.M. Butler (2005) Forensic DNA Typing, 2nd Edition © 2005 Elsevier Academic Press
72
Example Calculations with PopulationSubstructure Adjustments
73
Example Calculations with Corrections for Relatives