Aspects of Forensic DNA Typing using John M Butler's slides

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


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


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


12

p(short arm)

centromere

telomere

q(long arm)

telomere

Band 5

Band 3

Chromosome 12

12p3

12q5


13

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


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


Common DNA extraction methods

18

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


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


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


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


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


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


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)


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


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)


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


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.


33

D21S11 D18S51

D8S1179

DNA Size (bp)R

elat

ive

Fluo

resc

ence

Uni

ts

StutterProduct

6.3% 6.2% 5.4%

Allele


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


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


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


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


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


Detection of a microvariant allele at the locus FGA

39

AMELD8S1179 D21S11 D18S51

(B)

TPOX(A)


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


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


Impact of sequence polymorphismin the primer binding site

42

14,18

(a) (b)

15,18

15,17 14,18

13,17

15,17


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


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)


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

+

-


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


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


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


Illustration offluorescence &

excitation/emissionspectra

52

FAM(blue)

JOE(green)

TAMRA(yellow)

ROX(red)


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


Emission spectra of ABI dyes and wavelengthbands used for detection

54


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)


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


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


Traces from ABI 310 capillary electrophoresis system

58


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


60

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%

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


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)

**


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

Documents

Aspects of Forensic DNA Typing using John M Butler's slides