Aspects of Forensic DNA Typing using John M Butlers 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 from the book. The publisher requests that these copyright symbols are retained by slide users in their presentations. This has been retained, but material has been added or removed to suit the purposes of the class. Please check the originals to see exactly what changes1 has been made. Most other slides are from John Butlers collection too.

Some generalities

The Future of Forensic DNA Testing

Report published in Nov 2000 Asked to estimate where DNA testing would be 2, 5, and 10 years into the future Conclusions

http://www.ojp.usdoj.gov/nij/pubs-sum/183697.htm

STR typing is here to stay for a few years because of DNA databases that have grown to contain millions of profiles 3

Forensic DNA Typing, 2nd Edition:

Biology, Technology, and Genetics of STR Markers
Chapter 1 Chapter 2 Chapter 3 Chapter 4 Chapter 5 Chapter 6 Chapter 7 Chapter 8 Chapter 9 Chapter 10 Chapter 11 Chapter 12 Chapter 13 Chapter 14 Chapter 15 Chapter 16 Chapter 17 Chapter 18 Chapter 19 Chapter 20 Chapter 21 Chapter 22 Chapter 23 Chapter 24 Appendix I Appendix II Appendix III Appendix IV Appendix V Appendix VI Appendix VII Overview & History of DNA Typing DNA Biology Review Sample Collection, Extraction, Quantitation PCR Amplification Common STRs and Commercial Kits Biology of STRs Forensic Issues Single Nucleotide Polymorphisms Y-Chromosome DNA Tests Mitochondrial DNA Analysis Non-Human DNA and Microbial Forensics DNA Separation Methods DNA Detection Methods Instrumentation for STR Typing: ABI 310, ABI 3100, FMBIO STR Genotyping Issues Lab Validation New Technologies, Automation, and Expert Systems CODIS and DNA Databases Basic Genetic Principles and Statistics STR Database Analyses Profile Frequency Estimates Statistical Analysis of Mixtures and Degraded DNA Kinship and Paternity Testing Mass Disaster DNA Victim Identification Reported STR Alleles U.S. Population Data-STR Allele Frequencies Suppliers of DNA Analysis Equipment DAB QA Standards DAB Recommendations on Statistics Application of NRC II to STR Typing Example DNA Cases

John Butler

Human Identity Testing

Forensic cases -- matching suspect with
evidence

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 with the same core STR (short tandem repeat) markers
5

Basis of DNA Profiling

The genome of each individual is unique (with the exception of identical twins) and is inherited from parents Probe subsets of genetic variation in order to differentiate between individuals (statistical probabilities of a random match are used) DNA typing must be performed efficiently and reproducibly (information must hold up in court) Current standard DNA tests DO NOT look at genes little/no information about race, predisposal to disease, or phenotypical information (eye color, height, hair color) is obtained

Some DNA marker types & technologies)

Markers Used (Biology) High RFLP Multi-Locus Probes RFLP Single Locus Probes PolyMarker D1S80 single STR DQ ABO blood groups Fast
7
Figure 1.1, J.M. Butler (2005) Forensic DNA Typing, 2nd Edition 2005 Elsevier Academic Press

Multiplex STRs

Power of Discrimination (Genetics)

mtDNA

Low Slow

Speed of Analysis (Technology)

Steps in DNA sample analysis and interpretation

Biology
DNA Extraction DNA Quantitation PCR Amplification of Multiple STR markers

Technology
Separation and Detection of PCR Products (STR Alleles) Sample Genotype Determination

Genetics
Comparison of Sample Genotype to Other Sample Results Generation of Case Report with Probability of Random Match If match occurs, comparison of DNA profile to population databases
Figure 1.2, J.M. Butler (2005) Forensic DNA Typing, 2nd Edition 2005 Elsevier Academic Press

Comparison of two electropherograms

gender ID A

E F G

gender B ID A C D E F H I

9
Figure 1.3, J.M. Butler (2005) Forensic DNA Typing, 2nd Edition 2005 Elsevier Academic Press

Quick review of some biology

10

Human Genome
23 Pairs of Chromosomes + mtDNA Located in cell nucleus

Autosomes
http://www.ncbi.nlm.nih.gov/genome/guide/

2 copies per cell

Located in mitochondria (multiple copies in cell cytoplasm)

mtDNA

10 11 12

16,569 bp

13 14 15 16 17 18 19 20 21 22 X Nuclear DNA

Mitochondrial DNA 100s of copies 11 per cell

3.2 billion bp

Sexchromosomes

Figure 2.3, J.M. Butler (2005) Forensic DNA Typing, 2nd Edition 2005 Elsevier Academic Press

Chromosome 12 telomere p (short arm) Band 3 12p3

centromere

q (long arm) Band 5 telomere

12
Figure 2.4, J.M. Butler (2005) Forensic DNA Typing, 2nd Edition 2005 Elsevier Academic Press

12q5

Two important polymorphic marker types

(A) Single nucleotide polymorphism (SNP)
--------AGACTAGACATT--------------AGATTAGGCATT-------

(B) Short tandem repeat (STR) polymorphism

---------(AATG)(AATG)(AATG)---------3 repeats

---------(AATG)(AATG)---------2 repeats

13
Based on Figure 2.5, J.M. Butler (2005) Forensic DNA Typing, 2nd Edition 2005 Elsevier Academic Press

Locus A
Allele 1 Allele 2 Homologous pair of chromosomes

5
Allele 1

Allele 2

Homologous pair of chromosomes

3 Locus B

6
14

Figure 2.6, J.M. Butler (2005) Forensic DNA Typing, 2nd Edition 2005 Elsevier Academic Press

Overview of the technology

15

Sources of Biological Evidence

Blood Semen Saliva Urine Hair Teeth Bone Tissue

Blood stain
Only a very small amount of blood is needed to obtain a DNA profile
16

ORGANIC

Common DNA extraction methods

CHELEX
SDS, DTT, EDTA and

FTA Paper
Apply blood to paper and allow stain to dry

Blood stain

proteinase K

Blood stain

Water PUNCH

INCUBATE (56 oC) Centrifuge

Phenol, chloroform, isoamyl alcohol

INCUBATE (ambient) Centrifuge REMOVE supernatant

VORTEX Centrifuge
TRANSFER aqueous (upper) phase to new tube

5% Chelex

WASH Multiple Times with extraction buffer REMOVE supernatant PCR Reagents

TE buffer CONCENTRATE sample

(Centricon/Microcon-100 or ethanol precipitation)

INCUBATE (56 oC) INCUBATE (100 oC) Centrifuge QUANTITATE DNA PERFORM PCR

Centrifuge QUANTITATE DNA PERFORM PCR

(NO DNA QUANTITATION TYPICALLY PERFORMED WITH UNIFORM SAMPLES)

PERFORM PCR 17

Figure 3.1, J.M. Butler (2005) Forensic DNA Typing, 2nd Edition 2005 Elsevier Academic Press

Slot blot for human DNA quantitation

Calibration standards 20 ng 10 ng 5 ng 2.5 ng 1.25 ng 0.63 ng Unknown Samples Calibration standards 0.63 ng 1.25 ng 2.5 ng 5 ng 10 ng 20 ng

~2.5 ng

Uses a 40 bp primate-specific DNA probe (for details see book)

18
Figure 3.3, J.M. Butler (2005) Forensic DNA Typing, 2nd Edition 2005 Elsevier Academic Press

Importance of DNA Quantitation

(prior to multiplex PCR)
DNA amount
(log scale) 100 ng

High levels of DNA create interpretation challenges (more artifacts to review)

-A +A

10 ng

Too much DNA Off-scale peaks Split peaks (+/-A) Locus-to-locus imbalance
2.0 ng Well-balanced STR multiplex

1 ng

STR Kits Work Best in This Range

0.5 ng

0.1 ng

0.01 ng

Too little DNA Heterozygote peak imbalance Allele drop-out Locus-to-locus imbalance Stochastic effect when amplifying low levels of DNA produces allele dropout
19

Calculation of the quantity of DNA in a cell

1. Molecular Weight of a DNA Basepair = 618g/mol
=: 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 A

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 picograms

1 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)
20

Brief review of the Polymerase Chain Reaction

21

5 3

3 5

Starting DNA Template

Schematic: the precise details are different

Forward primer

Separate strands (denature) Add primers 5 (anneal)

5 3

3 5

Reverse primer

Make copies (extend primers)

Repeat Cycle, Copying DNA Exponentially

22
Figure 4.2, J.M. Butler (2005) Forensic DNA Typing, 2nd Edition 2005 Elsevier Academic Press

Thermal cycling: temperature profile

94 oC 94 oC 72 oC 60 oC 72 oC 60 oC Single Cycle Time Typically 25-35 cycles performed during PCR 94 oC 72 oC 60 oC 94 oC

Temperature

The denaturation time in the first cycle is lengthened to ~10 minutes when using AmpliTaq Gold to perform a hot-start PCR
23
Figure 4.1, J.M. Butler (2005) Forensic DNA Typing, 2nd Edition 2005 Elsevier Academic Press

Schematic for multiplex PCR

(A) Simultaneous amplification of three locations on a DNA template

Locus A

Locus B

Locus C

(B) Resolution of PCR products with a single size-based separation method

A B C

small

large

24
Figure 4.3, J.M. Butler (2005) Forensic DNA Typing, 2nd Edition 2005 Elsevier Academic Press

Commonly used Short Tandem Repeat (STR) markers

25

Minisatellite Marker (D1S80)

Flanking regions

Repeat region
GAGGACCACCAGGAAG

16 bp repeat unit

STR Marker (TH01)

Flanking regions

Repeat region
TCAT

4 bp repeat unit
26
Figure 5.1, J.M. Butler (2005) Forensic DNA Typing, 2nd Edition 2005 Elsevier Academic Press

Example of DNA sequence in an STR repeat region

1 2 3 4 5 6 5-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

Note different repeat motifs and starting positions on different strands. By convention, this is a TCAT repeat, read 5 to 3.
27
Figure 5.2, J.M. Butler (2005) Forensic DNA Typing, 2nd Edition 2005 Elsevier Academic Press

PCR product size (bp)

PowerPlex 16 kit (Promega Corporation)

FL (Blue) JOE (Green) TMR (Yellow) CXR (Red)

D3S1358 D5S818 AMEL

TH01 D13S317

D21S11 D7S820

D18S51

Penta E Penta D

D16S539 CSF1PO TPOX FGA

VWA

D8S1179

ILS600 CXR size standard AmpFlSTR Identifiler kit (Applied Biosystems)

Commercially available STR kits for the 13 CODIS loci

6-FAM (Blue) VIC (Green) NED (Yellow) PET (Red) LIZ (Orange)

D8S1179 D3S1358 D19S433 AMEL

D21S11 TH01 VWA

D7S820 TPOX FGA

CSF1PO

D13S317 D16S539 D2S1338 D18S51

D5S818

GS500 LIZ size standard

28

Figure 5.4, J.M. Butler (2005) Forensic DNA Typing, 2nd Edition 2005 Elsevier Academic Press

Overlay of all 4 colors (including internal size standard)

D3S1358 TH01

D21S11

D18S51

Penta E

blue panel

D5S818

D7S820 D13S317

CSF1PO D16S539

green panel

Penta D

VWA
Amelogenin (sex-typing)

D8S1179 TPOX FGA

yellow panel

red panel ILS600 DNA sizing standard 100 bp

120 140 160

200 bp
180 225 250 275

300 bp

400 bp
325 350 375 425 450 475

500 bp

29

Figure 5.5, J.M. Butler (2005) Forensic DNA Typing, 2nd Edition 2005 Elsevier Academic Press

D8S1179
(12 alleles)

D21S11
(24 alleles)

D7S820
(10 alleles)

CSF1PO
(10 alleles)

Blue panel

D3S1358
(8 alleles)

TH01
(10 alleles)

D13S317
(8 alleles)

D16S539
(9 alleles)

D2S1338
(14 alleles)

Green panel

D19S433
(15 alleles)

VWA
(14 alleles)

TPOX
(8 alleles)

D18S51
(23 alleles)

Yellow panel

AMEL
(2 alleles)

D5S818
(10 alleles)

FGA low
(19 alleles)

Red panel

FGA high
(9 alleles)

100 bp

150 bp 139bp 160 bp

200 bp

250 bp*

300 bp

Orange panel 340 bp 350 bp

LIZ-labeled GS500 DNA sizing standard Figure 5.6, J.M. Butler (2005) Forensic DNA Typing, 2nd Edition 2005 Elsevier Academic Press

30

6 bp deletion

Amelogenin sex-typing assay

X Y

X = 106 bp Y = 112 bp AmpFlSTR kits and PowerPlex 16 Female: X, X

X = 212 bp Y = 218 bp PowerPlex 1.1

1:1 Mixture: 3X + 1Y Male: X, Y

31
Figure 5.11, J.M. Butler (2005) Forensic DNA Typing, 2nd Edition 2005 Elsevier Academic Press

Short Tandem Repeat DNA Internet DataBase

These data are intended to benefit research and application of short tandem repeat DNA markers to human identity testing. The authors are solely responsible for the information herein.
Created by John M. Butler and Dennis J. Reeder (NIST Biotechnology Division), with invaluable help from Jan Redman, Christian Ruitberg and Michael Tung *Partial support for the design and maintenance of this website is being provided by The National Institute of Justice through the NIST Office of Law Enforcement Standards.*

Publications and Presentations from NIST Human Identity Project Team STRs101: Brief Introduction to STRs STR Fact Sheets (observed alleles and PCR product sizes) Sequence Information (annotated) Multiplex STR sets STR Training Materials Non-published Variant Allele Reports Three-Banded Patterns FBI CODIS Core STR Loci DNA Advisory Board Quality Assurance Standards NIST Standard Reference Material for PCR-Based Testing Chromosomal Locations Mutation Rates for Common Loci Published PCR primers Validation studies Population data Y-chromosome STRs Sex-typing markers Technology for resolving STR alleles Reference List Now 2059 references Addresses for scientists working with STRs Links to other web sites Glossary of commonly used terms 32

Figure 5.12, J.M. Butler (2005) Forensic DNA Typing, 2nd Edition 2005 Elsevier Academic Press

STR alleles with stutter products

DNA Size (bp)

Relative Fluorescence Units

D8S1179 D21S11 D18S51

Allele Stutter Product 6.3% 6.2% 5.4%

33
Figure 6.1, J.M. Butler (2005) Forensic DNA Typing, 2nd Edition 2005 Elsevier Academic Press

(A) Normal replication

1 2
GATA CTAT

3
GATA CTAT

Conjectured mechanisms
CTAT CTAT CTAT

5 3

GATA CTAT

1 2 1

(B) Insertion caused by backward slippage

2
GATA CTAT CTAT CTAT CTAT CTAT

5 3

GATA CTAT

1 1

2 2
GATA CTAT

3 3
GATA CTAT

4 5
GATA CTAT C TA T

(C) Deletion caused by forward slippage 5 3

GATA CTAT

Primary mechanism for stutter product formation

CTAT

5
34

4
Figure 6.2, J.M. Butler (2005) Forensic DNA Typing, 2nd Edition 2005 Elsevier Academic Press

Stutter percentages for CODIS loci LOCI

15

Stutter Percentage

12.5 10 7.5 5 2.5 0

TH01

TPOX

CSF1P O

D7S820

Alleles

15

Stutter Percentages

12.5 10 7.5 5 2.5 0

D5S818 VWA

D3S1358 D13S317

D8S1179

Alleles

15

Stutter Percentage

12.5 10 7.5 5 2.5 0

FGA

D16S539

D18S51

D21S11

Alleles

35

Figure 6.3, J.M. Butler (2005) Forensic DNA Typing, 2nd Edition 2005 Elsevier Academic Press

(A)

Non-template nucleotide addition

Forward Primer 5 3
Polymerase extension Polymerase extension

3 5 Reverse Primer

OR
5 5

A
5

(-A form) (B)

Incomplete adenylation +A -A +A -A

(+A form)

Measurement Result with dye labeled DNA strand -A

Full-length allele (n)

+A
allele + 1 base (n+1)

+A -A Shoulder peak

-A +A

Split peak 36

D8S1179
Figure 6.4, J.M. Butler (2005) Forensic DNA Typing, 2nd Edition 2005 Elsevier Academic Press

Incomplete non-template addition with high levels of DNA template

DNA Size (bp)

Relative Fluorescence (RFUs)

-A +A

off-scale

10 ng template (overloaded)

D3S1358

VWA
2 ng template (suggested level)

FGA

37
Figure 6.5, J.M. Butler (2005) Forensic DNA Typing, 2nd Edition 2005 Elsevier Academic Press

Detection of a microvariant allele at the locus FGA

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
Figure 6.6, J.M. Butler (2005) Forensic DNA Typing, 2nd Edition 2005 Elsevier Academic Press

28.1
38

(A)

TPOX

Tri-allelic patterns at TPOX and D18S51

(B)

AMEL D8S1179 D21S11

D18S51

39
Figure 6.7, J.M. Butler (2005) Forensic DNA Typing, 2nd Edition 2005 Elsevier Academic Press

Possible sequence variation (*) and impact on PCR amplification

Forward primer binding region 5flanking region 3flanking region Reverse primer Repeat region binding region

STR

A)

* * *

Example: TH01 9.3 allele (-A in 7th repeat)

Amplicon size may be nonstandard

B)

Example: Amplicon size may D18S51 13.2 allele (+AG in 3-flanking region) be nonstandard

C)

Example: Rare VWA allele amplified with AmpFlSTR primers (A-to-T in 2nd base from 3end of forward primer)

PCR may fail

40
Figure 6.8, J.M. Butler (2005) Forensic DNA Typing, 2nd Edition 2005 Elsevier Academic Press

Impact of sequence polymorphism in the primer binding site

Heterozygous alleles are well balanced

6 8

Imbalance in allele peak heights

6 8

*
8

*
Allele 6 amplicon has dropped out

41
Figure 6.9, J.M. Butler (2005) Forensic DNA Typing, 2nd Edition 2005 Elsevier Academic Press

Mutational events observed in family trees

(a)
14,18 15,17

(b)
14,18 15,17

15,18

13,17

42
Figure 6.10, J.M. Butler (2005) Forensic DNA Typing, 2nd Edition 2005 Elsevier Academic Press

Mutation Rates for Common STR Loci http://www.aabb.org/About_the_AABB/Stds_and_Accred/ptannrpt03.pdf, Appendix 2

STR System CSF1PO FGA TH01 TPOX VWA D3S1358 D5S818 D7S820 D8S1179 D13S317 D16S539 D18S51 D21S11 Penta D Penta E D2S1338 D19S433 SE33 (ACTBP2) Maternal Meioses (%) 95/304,307 (0.03) 205/408,230 (0.05) 31/327,172 (0.009) 18/400,061 (0.004) 184/564,398 (0.03) 60/405,452 (0.015) 111/451,736 (0.025) 59/440,562 (0.013) 96/409,869 (0.02) 192/482,136 (0.04) 129/467,774 (0.03) 186/296,244 (0.06) 464/435,388 (0.11) 12/18,701 (0.06) 29/44,311 (0.065) 15/72,830 (0.021) 38/70,001 (0.05) 0/330 (<0.30) Paternal Meioses (%) 982/643,118 (0.15) 2,210/692,776 (0.32) 41/452,382 (0.009) 54/457,420 (0.012) 1,482/873,547 (0.17) 713/558,836 (0.13) 763/655,603 (0.12) 745/644,743 (0.12) 779/489,968 (0.16) 881/621,146 (0.14) 540/494,465 (0.11) 1,094/494,098 (0.22) 772/526,708 (0.15) 21/22,501 (0.09) 75/55,719 (0.135) 157/152,310 (0.10) 78/103,489 (0.075) 330/51,610 (0.64) Number from either 410 710 28 28 814 379 385 285 364 485 372 466 580 24 59 90 71 None reported

J.M. Butler (2005) J. Forensic Sci., in press

Total Number of Mutations 1,487/947,425 3,125/1,101,006 100/779,554 100/857,481 2,480/1,437,945 1,152/964,288 1,259/1,107,339 1,089/1,085,305 1,239/899,837 1,558/1,103,282 1,041/962,239 1,746/790,342 1,816/962,096 57/41,202 163/100,030 262/225,140 187/173,490 330/51,940

Mutation Rate 0.16% 0.28% 0.01% 0.01% 0.17% 0.12% 0.11% 0.10% 0.14% 0.14% 0.11% 0.22% 0.19% 0.14% 0.16% 0.12% 0.11% 43 0.64%

Some special issues arising with forensic DNA typing

44

(A)

Agarose yield gel results

Impact of degraded DNA

Smear of degraded DNA fragments

High molecular weight DNA in a tight band

(B)

Degraded DNA sample

Good quality Degraded DNA DNA

D5S818

D13S317 D7S820 D16S539 CSF1PO Penta D

45
Figure 7.1, J.M. Butler (2005) Forensic DNA Typing, 2nd Edition 2005 Elsevier Academic Press

Illustration of typical single source vs mixed sample

(a)
100%

Heterozygous peak region

MIXTURE REGION

85%

>70%

Stutter region

9%

<15%

(b)

100%

Higher than typical stutter product (>15%)

60% 25%

>70% Smaller peak area than normally seen with heterozygote partner alleles(<70%)
10%

<15%

Wrong side of allele to be typical stutter product 46

Figure 7.3, J.M. Butler (2005) Forensic DNA Typing, 2nd Edition 2005 Elsevier Academic Press

Some of the DNA technology

47

Schematic of gel electropheresis

anode Loading well

Gel Buffer

cathode

DNA bands

Voltage

Gel lanes

Side view

Top view

48
Figure 12.1, J.M. Butler (2005) Forensic DNA Typing, 2nd Edition 2005 Elsevier Academic Press

Schematic of capillary electropheresis

Laser Capillary filled with polymer solution
Detection window

(cathode)

5-20 kV

+
(anode)

Inlet Buffer

Outlet Buffer

Data Acquisition Sample tray

Sample tray moves automatically beneath the cathode end of the capillary to deliver each sample in succession Figure 12.3, J.M. Butler (2005) Forensic DNA Typing, 2nd Edition 2005 Elsevier Academic Press

49

(a)

Illustration of DNA separation modes

Gel

Larger DNA molecules interact more frequently with the gel and are thus retarded in their migration through the gel

(b)
Gel
Long DNA molecules Small DNA molecules

Ogston Sieving

Reptation

50

Figure 12.4, J.M. Butler (2005) Forensic DNA Typing, 2nd Edition 2005 Elsevier Academic Press

(a)

S1
energy

hex 1

S1
hem 3

So
(b)
Excitation Stokes shift

Illustration of fluorescence & excitation/ emission spectra

Emission

Fluorescence

ex max

em max
51

Wavelength (nm) Figure 13.1, J.M. Butler (2005) Forensic DNA Typing, 2nd Edition 2005 Elsevier Academic Press

Dyes used by ABI in 4-color detection

FAM
(blue)

JOE
(green)

TAMRA
(yellow)

ROX
(red)

52
Figure 13.3, J.M. Butler (2005) Forensic DNA Typing, 2
nd

Edition 2005 Elsevier Academic Press

Emission spectra of ABI dyes and wavelength bands used for detection
5-FAM
Normalized Fluorescent Intensity
100 80 60 40 20 0

JOE NED

ROX

520

540

560 580 600 620 WAVELENGTH (nm)

640

Laser excitation
(488, 514.5 nm)

310 Filter Set F with color contributions

53

Figure 13.4, J.M. Butler (2005) Forensic DNA Typing, 2nd Edition 2005 Elsevier Academic Press

Example of a matrix used for color separation

54
Figure 13.6, J.M. Butler (2005) Forensic DNA Typing, 2nd Edition 2005 Elsevier Academic Press

(a)

Traces before and after color Scan separation number (and perhaps other processsing)
Relative Fluorescence Units Region shown below

Relative Fluorescence Units

(b)

DNA size in base pairs

55

Figure 13.7, J.M. Butler (2005) Forensic DNA Typing, 2nd Edition 2005 Elsevier Academic Press

Argon ion LASER Size Separation

(488 nm)

Schematic of the separation and detection process

ABI Prism spectrograph

Sample Separation

Fluorescence

Color Separation

Capillary

CCD Panel (with virtual filters) Sample Detection

Sample Injection
Mixture of dye-labeled PCR products from multiplex PCR reaction

Processing with GeneScan/Genotyper software

Sample Interpretation
56
Figure 13.8, J.M. Butler (2005) Forensic DNA Typing, 2nd Edition 2005 Elsevier Academic Press

Traces from ABI 310 capillary electrophoresis system

Profiler Plus multiplex STR result
D8S1179 D3S1358 D5S818 Amel VWA DNA size (bp)

D21S11

FGA

D7S820 D18S51

D13S317

COfiler multiplex STR result

D3S1358 Amel TH01 TPOX

DNA size (bp)

D7S820 CSF1PO D16S539

57
Figure 13.9, J.M. Butler (2005) Forensic DNA Typing, 2nd Edition 2005 Elsevier Academic Press

Image of data from Fig 14.9 below

58
Figure 13.11, J.M. Butler (2005) Forensic DNA Typing, 2nd Edition 2005 Elsevier Academic Press

Fluorescein Scan
Penta E

JOE Scan
Penta D CSF1PO

Rhodamine Red-X Scan

FGA

Texas Red-X Scan

450 425 400 375 350 325 300 275

D18S51 D16S53 9 D21S11 D7S82 0 D13S31 7 TH01 VWA TPOX

D8S1179

250 225

200 180

160

D3S1358

D5S81 8

140

Amelogenin

120

100

59

Figure 14.9, J.M. Butler (2005) Forensic DNA Typing, 2nd Edition 2005 Elsevier Academic Press

Deciphering Artifacts from the True Alleles

Biological (PCR) artifacts
Stutter products

STR alleles

6.0%

7.8%

Dye blob

stutter

spike

D3S1358
Incomplete adenylation +A +A -A -A

Blue channel

Green channel

Pull-up (bleed-through)

Yellow channel

D8S1179

Red channel
60

Figure 15.4, J.M. Butler (2005) Forensic DNA Typing, 2nd Edition 2005 Elsevier Academic Press

STR population databases and statistical calculations

This material which follows is presented without comment as illustrative of the approach taken by law enforcement agencies.
61

Decide on Number of Samples and Ethnic/Racial Grouping Gather Samples Analyze Samples at Desired Genetic Loci Summarize DNA types Determine Allele Frequencies for Each Locus Perform Statistical Tests on Data
Ethnic/ Racial Group 1

Usually >100 per group (see Table 20.1)

Get IRB approval Often anonymous samples from a blood bank

See Chapter 5 (STR kits available) and Chapter 15 (STR typing/interpretation)

See Table 20.2 and Appendix II

Hardy-Weinberg equilibrium for allele independence Linkage equilibrium for locus independence Examination of genetic distance between populations

Ethnic/ Racial Group 2

Use Database(s) to Estimate an Observed DNA Profile Frequency

See Chapter 21 62

Figure 20.1, J.M. Butler (2005) Forensic DNA Typing, 2nd Edition 2005 Elsevier Academic Press

U.S. Population Samples

(Appendix II)
0.450 0.400 0.350

Frequency

0.300 0.250 0.200 0.150 0.100 0.050 0.000

**
8 9 10 11 12 13 14 15 D13S317 Allele
Caucasians (N=302) Hispanics (N=140)
63
Figure 20.3, J.M. Butler (2005) Forensic DNA Typing, 2nd Edition 2005 Elsevier Academic Press

African Americans (N=258)

How Statistical Calculations are Made

Generate data with set(s) of samples from desired population 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 the rarity 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
64

Assumptions with Hardy-Weinberg Equilibrium

None of these assumptions are really true

65
Table 20.6, J.M. Butler (2005) Forensic DNA Typing, 2nd Edition 2005 Elsevier Academic Press

Individual Genotypes Are Summarized and Converted into Allele Frequencies

The 11,12 genotype was seen 54 times in 302 samples (604 examined chromosomes)
66
Table 20.2, J.M. Butler (2005) Forensic DNA Typing, 2nd Edition 2005 Elsevier Academic Press

Allele Frequency Tables

Butler et al. (2003) JFS 48(4):908-911 Einum et al. (2004) JFS 49(6)

Allele frequencies denoted with an asterisk (*) are below the 5/2N minimum allele threshold recommended by the National Research Council report (NRCII) The Evaluation of Forensic DNA Evidence published in 1996.

D3S1358
Allele 11 12 13 14 Most common 15 allele 15.2 16 17 18 19 20

Caucasian

Caucasian

African American N=258 Allele 11 12 13 14 15 15.2 16 17 18 19 20 --0.0019* 0.0892 0.3023 0.0019* 0.3353 0.2054 0.0601 0.0039*

African American

N= 302
0.0017* 0.0017* -0.1027 0.2616 -0.2533 0.2152 0.15232 0.01160 0.0017*

N= 7,636
0.0009 0.0007 0.0031 0.1240 0.2690 -0.2430 0.2000 0.1460 0.0125 0.0001*

N= 7,602
0.0003* 0.0045 0.0077 0.0905 0.2920 0.0010 0.3300 0.2070 0.0630 0.0048
67

Illustrative calculations

68

DNA Profile Frequency with all 13 CODIS STR loci

AmpFlSTR Identifiler (Applied Biosystems) TH01 D19 D3 AMEL D8 D5 VWA D21 TPOX D13 FGA D7 D16 D18 CSF D2

What would be entered into a DNA database for searching: 16,1717,1821,2212,1428,3014,1612,1311,149,99,116,68,810,10

Locus D3S1358 VWA FGA D8S1179 D21S11 D18S51 D5S818 D13S317 D7S820 D16S539 THO1 TPOX CSF1PO

allele 16 17 21 12 28 14 12 11 9 9 6 8 10

value 0.2533 0.2815 0.1854 0.1854 0.1589 0.1374 0.3841 0.3394 0.1772 0.1126 0.2318 0.5348 0.2169

allele 17 18 22 14 30 16 13 14 11

value 0.2152 0.2003 0.2185 0.1656 0.2782 0.1391 0.1407 0.0480 0.3212

1 in 9.17 8.87 12.35 16.29 11.31 26.18 9.25 30.69 31.85 13.8 18.62 3.50 21.28

Combined 9.17 81 1005 16,364 185,073 4,845,217 44,818,259 1.38 x 109 4.38 x 1010 6.05 x 1011 1.13 x 1013 3.94 x 1013 8.37 x 1014

P R O D U C T R U L E

The Random Match Probability for this profile in the U.S. Caucasian population 69 12 is 1 in 837 trillion (10 )

The Same 13 Locus STR Profile in Different Populations

1 in 837 trillion 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)

These values are for unrelated individuals

assuming 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) 70

*http://www.csfs.ca/pplus/profiler.htm

STR Cumulative Profile Frequency with Multiple Population Databases

21 1014 to 1071
D.N.A. Box 21.1, J.M. Butler (2005) Forensic DNA Typing, 2nd Edition 2005 Elsevier Academic Press

Example Calculations with Population Substructure Adjustments

72

Example Calculations with Corrections for Relatives

73