PowerPoint Slides for Use with Forensic DNA Typing, 2nd Edition

PowerPoint Slides for Use with Forensic DNA Typing, 2nd Edition
Prepared by John M. Butler Available at 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.

Introduction

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

Forensic DNA Typing, 2nd Edition: Biology, Technology, and Genetics of STR Markers
John Butler (not NIST) New Material: 10 additional chapters Statistics (basics with examples) Real-time PCR Serology tests Y-STRs and mtDNA ABI 3100 Expert systems Mass disasters including WTC Example cases for training purposes >500 new reference citations 50 new figures and 45 new tables 688 pages; $79.95 Double the size of the first edition Chapter 1 Overview & History of DNA Typing Chapter 2 DNA Biology Review Chapter 3 Sample Collection, Extraction, Quantitation Chapter 4 PCR Amplification Chapter 5 Common STRs and Commercial Kits Chapter 6 Biology of STRs Chapter 7 Forensic Issues Chapter 8 Single Nucleotide Polymorphisms Chapter 9 Y-Chromosome DNA Tests Chapter 10 Mitochondrial DNA Analysis Chapter 11 Non-Human DNA and Microbial Forensics Chapter 12 DNA Separation Methods Chapter 13 DNA Detection Methods Chapter 14 Instrumentation for STR Typing: ABI 310, ABI 3100, FMBIO Chapter 15 STR Genotyping Issues Chapter 16 Lab Validation Chapter 17 New Technologies, Automation, and Expert Systems Chapter 18 CODIS and DNA Databases Chapter 19 Basic Genetic Principles and Statistics Chapter 20 STR Database Analyses Chapter 21 Profile Frequency Estimates Chapter 22 Statistical Analysis of Mixtures and Degraded DNA Chapter 23 Kinship and Paternity Testing Chapter 24 Mass Disaster DNA Victim Identification Appendix I Reported STR Alleles Appendix II U.S. Population Data-STR Allele Frequencies Appendix III Suppliers of DNA Analysis Equipment Appendix IV DAB QA Standards Appendix V DAB Recommendations on Statistics Appendix VI Application of NRC II to STR Typing Appendix VII Example DNA Cases Jan 2001 Feb 2005 1st Edition 2nd Edition

Available January 2001 $69.95, 350 pp. Available February 2005 $79.95, 688 pp.

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

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

Chapter 1

Multi-Locus Probes ABO blood groups Multiplex STRs
Speed of Analysis (Technology) Power of Discrimination (Genetics) Low High Slow Fast Markers Used (Biology) RFLP Single Locus Probes Multi-Locus Probes ABO blood groups Multiplex STRs DQ single STR D1S80 mtDNA PolyMarker Figure 1.1 Comparison of DNA typing technologies. Forensic DNA markers are arbitrarily plotted in relationship to four quadrants defined by the power of discrimination for the genetic system used and the speed at which the analysis for that marker may be performed. Note that this diagram does not reflect the usefulness of these markers in terms of forensic cases. Figure 1.1, J.M. Butler (2005) Forensic DNA Typing, 2nd Edition © 2005 Elsevier Academic Press

Biology Technology Genetics
Sample Obtained from Crime Scene or Paternity Investigation DNA Extraction Quantitation PCR Amplification of 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 If match occurs, comparison of DNA profile to population databases Generation of Case Report with Probability of Random Match Figure 1.2 Overview of biology, technology, and genetic components of DNA typing using short tandem repeat (STR) markers. Figure 1.2, J.M. Butler (2005) Forensic DNA Typing, 2nd Edition © 2005 Elsevier Academic Press

gender ID A B C D E F G H I J Figure 1.3 Comparison of the DNA profiles for two individuals obtained with multiple short tandem repeat (STR) markers. STR length variation at unique sites on 10 different chromosomes are probed with this DNA test to provide a random match probability of approximately 1 in 3 trillion. A gender identification test also indicates that the top sample is from a male while the bottom sample is from a female individual. These results were obtained from a spot of blood the size of a pin head in less than 5 hours. The DNA size range in base pairs is shown across the top of the plot. Results from each DNA marker are indicated by the letters A-J. Figure 1.3, J.M. Butler (2005) Forensic DNA Typing, 2nd Edition © 2005 Elsevier Academic Press

Chapter 2

A) B) 5’ 3’ 5’end Sugar—Base… 3’end | Phosphate
Figure 2.1 Basic components of nucleic acids: (A) phosphate sugar backbone with bases coming off the sugar molecules, (B) chemical structure of phosphates and sugar molecules illustrating numbering scheme on the sugar carbon atoms. DNA sequences are conventionally written from 5’-to-3’. Figure 2.1, J.M. Butler (2005) Forensic DNA Typing, 2nd Edition © 2005 Elsevier Academic Press

Phosphate-sugar backbone
G  C T = A C  G T C A G 5’ 3’ denatured strands hybridized Hydrogen bonds Phosphate-sugar backbone Figure 2.2 Base pairing of DNA strands to form double-helix structure. Figure 2.2, J.M. Butler (2005) Forensic DNA Typing, 2nd Edition © 2005 Elsevier Academic Press

Human Genome 23 Pairs of Chromosomes + mtDNA
Located in cell nucleus X Y Autosomes 2 copies per cell Located in mitochondria (multiple copies in cell cytoplasm) mtDNA 16,569 bp Mitochondrial DNA Figure 2.3 The human genome contained in every cell consists of 23 pairs of chromosomes and a small circular genome known as mitochondrial DNA. Chromosomes 1-22 are numbered according to their relative size and occur in single copy pairs within a cell’s nucleus with one copy being inherited from one’s mother and the other copy coming from one’s father. Sex-chromosomes are either X,Y for males or X,X for females. Mitochondrial DNA is inherited only from one’s mother and is located in the mitochondria with hundreds of copies per cell. Together the nuclear DNA material amounts to over 3 billion base pairs (bp) while mitochondrial DNA is only about 16,569 bp in length. Sex-chromosomes Nuclear DNA 3.2 billion bp 100s of copies per cell Figure 2.3, J.M. Butler (2005) Forensic DNA Typing, 2nd Edition © 2005 Elsevier Academic Press

Chromosome 12 p Band 3 12p3 (short arm) centromere q (long arm) 12q5
telomere q (long arm) Band 5 Band 3 Chromosome 12 12p3 12q5 Figure 2.4 Basic chromosome structure and nomenclature. The centromere is a distinctive feature of chromosomes and plays an important role during mitosis. On either side of the centromere are “arms” that extend to terminal regions, known as telomeres. The short arm of a chromosome is designated as “p” while the long arm is referred to as “q”. The band nomenclature refers to physical staining with a Giemsa dye (G-banded). Band localization is determined by G-banding the image of a metaphase spread during cell division. Bands are numbered outward from the centromere with the largest values near the telomeres. Figure 2.4, J.M. Butler (2005) Forensic DNA Typing, 2nd Edition © 2005 Elsevier Academic Press

(A) Sequence polymorphism
AGACTAGACATT AGATTAGGCATT (B) Length polymorphism (AATG)(AATG)(AATG) 3 repeats (AATG)(AATG) Figure 2.5 Two forms of variation in DNA: (A) sequence polymorphisms and (B) length polymorphisms. The short tandem repeat DNA markers discussed in this book are length polymorphisms. 2 repeats Figure 2.5, J.M. Butler (2005) Forensic DNA Typing, 2nd Edition © 2005 Elsevier Academic Press

Homologous pair of chromosomes
6 3 4 5 Homologous pair of chromosomes Locus A Locus B Allele 1 Allele 2 Figure 2.6 Schematic representation of two different STR loci on different pairs of homologous chromosomes. The chromosomes with the open circle chromosomes are paternally inherited while the solid centromere chromosomes are maternally inherited. Thus, this individual received the four repeat allele at locus A and the three repeat allele at locus B from their father, and the five repeat allele at locus A and the six repeat allele at locus B from their mother. Figure 2.6, J.M. Butler (2005) Forensic DNA Typing, 2nd Edition © 2005 Elsevier Academic Press

Chapter 3

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

TRANSFER aqueous (upper) phase to new tube
ORGANIC FTA Paper CHELEX Blood stain PUNCH WASH Multiple Times with extraction buffer PERFORM PCR PCR Reagents SDS, DTT, EDTA and proteinase K INCUBATE (56 oC) Phenol, chloroform, isoamyl alcohol QUANTITATE DNA Apply blood to paper and allow stain to dry VORTEX (NO DNA QUANTITATION TYPICALLY PERFORMED WITH UNIFORM SAMPLES) Water INCUBATE (ambient) 5% Chelex INCUBATE (100 oC) REMOVE supernatant Centrifuge TRANSFER aqueous (upper) phase to new tube CONCENTRATE sample (Centricon/Microcon-100 or ethanol precipitation) TE buffer Figure 3.1 Schematic of commonly used DNA extraction processes. Figure 3.1, J.M. Butler (2005) Forensic DNA Typing, 2nd Edition © 2005 Elsevier Academic Press

Perpetrator’s sperm mixed with victim’s epithelial cells
Remove a portion of the mixed stain SDS, EDTA and proteinase K (cell lysis buffer) Incubate at 37 oC Centrifuge Perpetrator’s sperm mixed with victim’s epithelial cells sperm pellet REMOVE supernatant SDS, EDTA and proteinase K + DTT Figure 3.2 Schematic of differential extraction process used to separate male sperm cells from female epithelial cells. DTT lyses sperm heads “Male Fraction” sperm pellet “Female Fraction” Figure 3.2, J.M. Butler (2005) Forensic DNA Typing, 2nd Edition © 2005 Elsevier Academic Press

Differential extraction used to separate sperm (male fraction) from vaginal epithelial cells (female fraction) female Evidence (female fraction) Evidence (male fraction) male Suspect male Victim female The four samples typically associated with a forensic DNA case…

Calibration standards Unknown Samples
20 ng 10 ng 5 ng 2.5 ng 1.25 ng 0.63 ng Calibration standards Unknown Samples ~2.5 ng Figure 3.3 Illustration of a human DNA quantitation result with the slot blot procedure. A serial dilution of a human DNA standard is run on either side of the slot blot membrane for comparison purposes. The quantity of each of the unknown samples is estimated by visual comparison to the calibration standards. For example, the sample indicated by the arrow is closest in appearance to the 2.5 ng standard. Figure 3.3, J.M. Butler (2005) Forensic DNA Typing, 2nd Edition © 2005 Elsevier Academic Press

(A) 1 ng sample (B) Figure 3.4 (A) Range of DNA concentrations reported for a 1 ng DNA sample supplied to 74 laboratories in an interlaboratory study (Kline et al. 2003). Overall the median value was very close to the expected 1 ng level with 50% falling in the boxed region. However, laboratories returned values ranging from 0.1 ng to 3 ng. (B) A target plot examining concordance and apparent precision for the various laboratory methods used. Legend: A=ACES kit; q = Quantiblot with unreported visualization method; E = Quantiblot with chemiluminescent detection; T = Quantiblot with colorimetric detection; 1, 2, 3, 4, and 5 represent methods used by only one lab. Figure 3.4, 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) High levels of DNA create interpretation challenges (more artifacts to review) 100 ng -A Too much DNA Off-scale peaks Split peaks (+/-A) Locus-to-locus imbalance +A 10 ng 2.0 ng Well-balanced STR multiplex 1 ng STR Kits Work Best in This Range 0.5 ng 0.1 ng Too little DNA Heterozygote peak imbalance Allele drop-out Locus-to-locus imbalance 0.01 ng Stochastic effect when amplifying low levels of DNA produces allele dropout

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 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 g = 3.08 picograms (pg) A diploid human cell contains ~6 pg genomic DNA 4. One ng of DNA contains the DNA from diploid cells 1 ng genomic DNA (1000 pg)/6pg/cell = ~333 copies of each locus (2 per 167 diploid genomes)

Chapter 4

Typically 25-35 cycles performed during PCR
94 oC 60 oC 72 oC Time Temperature Single Cycle Typically cycles performed during PCR The denaturation time in the first cycle is lengthened to ~10 minutes when using AmpliTaq Gold to perform a “hot-start” PCR Figure 4.1 Thermal Cycling Temperature Profile for PCR. Thermal cycling typically involves 3 different temperatures that are repeated over and over again times. At 94 oC, the DNA strands separate, or “denature”. At 60 oC, primers bind or “anneal” to the DNA template and target the region to be amplified. At 72 oC, the DNA polymerase extends the primers by copying the target region using the deoxynucleotide triphosphate building blocks. The entire PCR process is about 3 hours in duration with each cycle taking ~5 minutes on conventional thermal cyclers: 1 minute each at 94 oC, 60 oC, and 72 oC and about 2 minutes ramping between the 3 temperatures. Figure 4.1, J.M. Butler (2005) Forensic DNA Typing, 2nd Edition © 2005 Elsevier Academic Press

Separate strands (denature)
Add primers (anneal) Make copies (extend primers) Repeat Cycle, Copying DNA Exponentially Starting DNA Template 5’ 3’ Forward primer Reverse primer Figure 4.2 DNA Amplification Process with the Polymerase Chain Reaction. In each cycle, the two DNA template strands are first separated (denatured) by heat. The sample is then cooled to an appropriate temperature to bind (anneal) the oligonucleotide primers. Finally the temperature of the sample is raised to the optimal temperature for the DNA polymerase and it extends the primers to produce a copy of each DNA template strand. For each cycle, the number of DNA molecules (with the sequence between the two PCR primers) doubles. Figure 4.2, J.M. Butler (2005) Forensic DNA Typing, 2nd Edition © 2005 Elsevier Academic Press

(A) Simultaneous amplification of three locations on a DNA template
Locus A Locus C Locus B (B) Resolution of PCR products with size-based separation method Figure 4.3 Schematic of Multiplex PCR. A multiplex PCR makes use of two or more primer sets within the same reaction mix. Three sets of primers, represented by arrows, are shown here to amplify three different loci on a DNA template (A). The primers were designed so that the PCR products for locus A, locus B, and locus C would be different sizes and therefore resolvable with a size-based separation system (B). A C B small large Figure 4.3, J.M. Butler (2005) Forensic DNA Typing, 2nd Edition © 2005 Elsevier Academic Press

Polymerization and Strand Displacement
Q Forward primer Reverse primer 3’ 5’ TaqMan probe Polymerization and Strand Displacement Forward primer Reverse primer 3’ 5’ Q R Probe Cleavage (release of reporter dye) Fluorescence occurs when reporter dye and quencher dye are no longer in close proximity Figure 4.4 Schematic of TaqMan (5’ nuclease) assay. Forward primer Reverse primer 3’ 5’ Q R Completion of Polymerization Figure 4.4, J.M. Butler (2005) Forensic DNA Typing, 2nd Edition © 2005 Elsevier Academic Press

Exponential product growth
Cycle Number Normalized Fluorescence 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 Figure 4.5 Real-time PCR output and example standard curve used to determine quantity of input DNA. 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). Figure 4.5, J.M. Butler (2005) Forensic DNA Typing, 2nd Edition © 2005 Elsevier Academic Press

Chapter 5

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 Schematic of minisatellite and microsatellite (STR) DNA markers. PCR primers are designed to target invariant flanking sequence regions. The number of tandem repeat units in the repeat regions varies among individuals making them useful markers for human identification. Figure 5.1, J.M. Butler (2005) Forensic DNA Typing, 2nd Edition © 2005 Elsevier Academic Press

5’-TTTCCC TCAT TCAT TCAT TCAT TCAT TCAT TCACCATGGA-3’
5’-TTTCCC TCAT TCAT TCAT TCAT TCAT TCAT TCACCATGGA-3’ 3’-AAAGGG AGTA AGTA AGTA AGTA AGTA AGTA AGTGGTACCT-5’ Figure 5.2 Example of the DNA sequence in a STR repeat region. Note that using the top strand versus the bottom strand results in different repeat motifs and starting positions. In this example, the top strand has 6 TCTA repeat units while the bottom strand has 6 TGAA repeat units. Under ISFH recommendations (Bar et al. 1997), the top strand from GenBank should be used. Thus, this example would be described as having [TCAT] as the repeat motif. Repeat numbering, indicated above and below the sequence, proceeds in the 5’-to-3’ direction as illustrated by the arrows. Figure 5.2, J.M. Butler (2005) Forensic DNA Typing, 2nd Edition © 2005 Elsevier Academic Press

Find representative alleles spanning population variation
Separate PCR products from various samples amplified with primers targeted to a particular STR locus Combine Re-amplify Find representative alleles spanning population variation Polyacrylamide Gel Figure 5.3 Principle of allelic ladder formation. STR alleles from a number of samples are separated on a polyacrylamide gel and compared to one another. Samples representing the common alleles for the locus are combined and re-amplified to generate an allelic ladder. Each allele in the allelic ladder is sequenced since it serves as the reference material for STR genotyping. Allelic ladders are included in commercially available STR kits. Figure 5.3, J.M. Butler (2005) Forensic DNA Typing, 2nd Edition © 2005 Elsevier Academic Press

PowerPlex 16 kit (Promega Corporation)
PCR product size (bp) 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 standard CXR (Red) FL (Blue) JOE (Green) TMR (Yellow) AmpFlSTR Identifiler™ kit (Applied Biosystems) 6-FAM (Blue) VIC (Green) NED (Yellow) PET (Red) D8S1179 D21S11 D7S820 CSF1PO D3S1358 TH01 D13S317 D16S539 D2S1338 D19S433 D18S51 TPOX VWA AMEL D5S818 FGA GS500 LIZ size standard LIZ (Orange) Figure 5.4 Commercially available STR kit solutions for a single amplification of the 13 CODIS core loci. General size ranges and dye-labeling strategies are indicated. The PowerPlex 16 kit uses 4-dyes while the Identifiler kit uses 5-dyes. Loci with dotted boxes are additional loci specific to each kit. Figure 5.4, J.M. Butler (2005) Forensic DNA Typing, 2nd Edition © 2005 Elsevier Academic Press

ILS600 DNA sizing standard
D3S1358 TH01 D21S11 D18S51 Penta E D5S818 D13S317 D7S820 D16S539 Penta D CSF1PO Amelogenin (sex-typing) VWA D8S1179 TPOX FGA blue panel Overlay of all 4 colors (including internal size standard) yellow panel green panel red panel ILS600 DNA sizing standard 200 bp 300 bp 100 bp 400 bp 500 bp 325 350 375 425 450 475 225 250 275 120 140 160 180 Figure 5.5 PowerPlex® 16 result from 1 ng genomic DNA. Figure 5.5, J.M. Butler (2005) Forensic DNA Typing, 2nd Edition © 2005 Elsevier Academic Press

LIZ-labeled GS500 DNA sizing standard
D3S1358 (8 alleles) VWA (14 alleles) D16S539 (9 alleles) D2S1338 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 250 bp* 139bp 200 bp 160 bp 300 bp 340 bp 350 bp 150 bp LIZ-labeled GS500 DNA sizing standard 100 bp Red panel D19S433 (15 alleles) D5S818 TPOX D13S317 D7S820 CSF1PO AMEL (2 alleles) Figure 5.6 AmpFlSTR® Identifiler allelic ladders (Applied Biosystems). A total of 205 alleles are included in this set of allelic ladders used for genotyping a multiplex PCR reaction involving 15 STR loci and the amelogenin sex-typing test. Figure 5.6, J.M. Butler (2005) Forensic DNA Typing, 2nd Edition © 2005 Elsevier Academic Press

Fluorescent dye at 5’end
For each linker unit added, there is an apparent migration shift of ~2.5 bp Primer sequence 3’-end 5’-end Non-nucleotide linkers (mobility modifiers) PCR amplification generates a labeled PCR product containing the mobility modifiers Figure 5.7 Illustration of mobility modifiers used in Applied Biosystems’ Identifiler STR kit. Non-nucleotide linkers are synthesized into the primer between the fluorescent dye and 5’end of the primer sequence. During PCR amplification, the dye and linker are incorporated into the amplicon. With the added non-nucleotide linker, the mobility of the generated STR allele will be shifted to a larger apparent size during electrophoresis. This shift of STR alleles for a particular locus then enables optimal inter-locus spacing for STR loci labeled with the same fluorescent dye without having to alter the PCR primer binding positions (see Figure 5.8). Figure 5.7, J.M. Butler (2005) Forensic DNA Typing, 2nd Edition © 2005 Elsevier Academic Press

(A) COfiler kit (B) Identifiler kit Size overlap 6 15 CSF1PO 6 D7S820
allele relative size ranges 6 15 CSF1PO JOE-labeled (green) bp bp 6 D7S820 15 NED-labeled (yellow) bp bp (B) Identifiler kit allele relative size ranges 10 non-nucleotide linkers = ~ +25 bp shift Figure 5.8 Illustration of how non-nucleotide linkers attached to CSF1PO PCR products in the Identifiler STR kit help with inter-locus spacing between D7S820 and CSF1PO. In the COfiler kit (A), CSF1PO and D7S820 are labeled with different colored fluorescent labels and thus do not interfere with one another. However, in the Identifiler kit (B), both D7S820 and CSF1PO are labeled with the same dye and would therefore have overlapping STR alleles unless primer positions were changed or mobility modifiers were used. A ~25 bp shift of the CSF1PO PCR products is accomplished by the addition of 10 non-nucleotide linkers. PCR product sizes for allelic ladder ranges displayed here are from the COfiler and Identifiler kit user’s manuals. Note that sizes for D7S820 alleles do not match exactly because different dye labels are used with both the PCR products and the internal size standard thus impacting their relative mobilities. 6 D7S820 15 6 15 CSF1PO 6FAM-labeled (blue) 6FAM-labeled (blue) bp bp bp bp Figure 5.8, J.M. Butler (2005) Forensic DNA Typing, 2nd Edition © 2005 Elsevier Academic Press

(A) PowerPlex® 1.1 Kit (B) PowerPlex® 16 Kit +30 bp shift in size
CSF1PO forward primer CSF1PO reverse primer (AGAT)6-15 (A) PowerPlex® 1.1 Kit 91 bp 128 bp TMR-labeled PCR product sizes = bp (B) PowerPlex® 16 Kit 13 bp 238 bp JOE-labeled PCR product sizes = bp Figure 5.9 Variation in CSF1PO primer positions between (A) PowerPlex 1.1 and (B) PowerPlex 16 STR kits. The base pair (bp) numbers in bold indicate the distance between the repeat region and 3’end of the pertinent primer. The overall PCR product size for CSF1PO is shifted +30 bp with the primer changes from PowerPlex 1.1 to PowerPlex 16. +30 bp shift in size Figure 5.9, J.M. Butler (2005) Forensic DNA Typing, 2nd Edition © 2005 Elsevier Academic Press

Figure 5.10 SE33 (ACTBP2) allelic ladder from PowerPlex ES kit produced by Promega Corporation.
Figure 5.10, J.M. Butler (2005) Forensic DNA Typing, 2nd Edition © 2005 Elsevier Academic Press

X Y X = 212 bp Y = 218 bp X = 106 bp Y = 112 bp AmpFlSTR kits
6 bp deletion X = 212 bp Y = 218 bp X = 106 bp Y = 112 bp AmpFlSTR kits and PowerPlex 16 PowerPlex 1.1 Figure 5.11 Schematic of the amelogenin sex-typing assay. The X and Y chromosomes contain a high degree of sequence homology at the amelogenin locus. The primer sets depicted here target a 6 bp deletion that is present only on the X chromosome. The presence of a single peak indicates that the sample comes from a female while two peaks identifies the sample’s source as male. The primers to amplify the 106/112 bp fragments are used in the AmpFlSTR kits while the PowerPlex 1.1 kit uses the larger primer set. Female: X, X 1:1 Mixture: 3X + 1Y Male: X, Y 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 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 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 Figure 5.12 Homepage for STRBase, an internet-accessible database of information on STR markers used in forensic DNA typing. STRBase may be accessed via the URL: and contains among other things a comprehensive listing of all papers relating to STR typing for human identity testing purposes now numbering over 2,000 references. Figure 5.12, J.M. Butler (2005) Forensic DNA Typing, 2nd Edition © 2005 Elsevier Academic Press

Chapter 6

Relative Fluorescence Units
D21S11 D18S51 D8S1179 DNA Size (bp) Relative Fluorescence Units Stutter Product 6.3% 6.2% 5.4% Allele Figure 6.1 STR alleles shown with stutter products (indicated by arrows). Only the stutter percentage for the first allele from each locus is noted. Figure 6.1, J.M. Butler (2005) Forensic DNA Typing, 2nd Edition © 2005 Elsevier Academic Press

Primary mechanism for stutter product formation
GATA CTAT 3’ 5’ 1 2 3 4 5 6 (A) Normal replication GATA CTAT 3’ 5’ 1 2 3 4 5 6 2’ (B) Insertion caused by backward slippage GATA CTAT 3’ 5’ 1 2 3 5 6 (C) Deletion caused by forward slippage 4 C T A Figure 6.2 Illustration of slipped-strand mispairing process that is thought to give rise to stutter products. (A) During replication the two DNA strands can easily come apart in the repeat region and since each repeat unit is the same, the two strands can reanneal out of register such that the two strands are off-set by a single repeat unit. (B) If a repeat unit bulges out on the new synthesized strand during extension then an insertion results in the next round of amplification. (C) If on the other hand, the repeat unit bulge occurs in the template strand, then the resulting synthesized strand is one repeat unit shorter than the full length STR allele. The frequency at which this process occurs is related to the flanking sequence, the repeat unit, and the length of the allele being amplified. Generally for tetranucleotide STR loci, stutter occurs less than 15% of the time and is observed as a small peak one repeat shorter than the STR allele. Primary mechanism for stutter product formation Figure 6.2, J.M. Butler (2005) Forensic DNA Typing, 2nd Edition © 2005 Elsevier Academic Press

TH01 TPOX CSF1PO D7S820 D5S818 VWA D3S1358 D13S317 D8S1179 FGA D16S539
Alleles LOCI Figure 6.3 Stutter Percentages for 13 CODIS STR Loci (data adapted from AmpFlSTR manuals). Alleles for each STR locus are shown from smallest to largest. Each locus has a different average stutter percentage but all loci show the trend of increasing stutter with larger alleles (longer number of repeats). Figure 6.3, J.M. Butler (2005) Forensic DNA Typing, 2nd Edition © 2005 Elsevier Academic Press

D8S1179 A OR (-A form) (+A form) (A) (B) 5’ 3’ Reverse Primer Forward
Polymerase extension Reverse Primer Forward A OR (-A form) (+A form) Shoulder peak -A +A Split peak (A) (B) Full-length allele (n) allele + 1 base (n+1) Measurement Result with dye labeled DNA strand Incomplete adenylation D8S1179 Figure 6.4 Schematic of non-template nucleotide addition shown (A) with illustrated measurement result (B). DNA polymerases add an extra nucleotide beyond the 3’-end of the target sequence extension product. The amount of non-template addition is dependent on the sequence of the 5’-end of the opposing primer. In the case of dye labeled PCR products where the fluorescent dye is on the forward primer, the reverse primer sequence is the critical one. Figure 6.4, J.M. Butler (2005) Forensic DNA Typing, 2nd Edition © 2005 Elsevier Academic Press

D3S1358 VWA FGA DNA Size (bp) -A 10 ng template (overloaded) +A
2 ng template (suggested level) DNA Size (bp) Relative Fluorescence (RFUs) off-scale Figure 6.5 Incomplete non-template addition with high levels of DNA template. In the top panel, partial adenylation (both –A and +A forms of each allele) is seen because the polymerase is overwhelmed due to an abundance of DNA template. Note also that the peaks in the top panel are off-scale and flat-topped in the case of the smaller FGA allele. When the suggested level of DNA template is used, all alleles are fully adenylated (bottom panel). Figure 6.5, J.M. Butler (2005) Forensic DNA Typing, 2nd Edition © 2005 Elsevier Academic Press

Figure 6. 6 Detection of a microvariant allele at the STR locus FGA
Figure 6.6 Detection of a microvariant allele at the STR locus FGA. The sample in the bottom panel is compared to the allelic ladder shown in the top panel using Genotyper 2.5 software. Peaks are labeled with the allele category and the calculated fragment sizes using the internal sizing standard GS500-ROX. 1 = S25-L25 = = bp 2 = SOL - L28 = = bp c = |1 -2| = | | = 0.99 bp 28.1 Figure 6.6, J.M. Butler (2005) Forensic DNA Typing, 2nd Edition © 2005 Elsevier Academic Press

TPOX (A) AMEL D8S1179 D21S11 D18S51 (B)
Figure 6.7 Tri-allelic patterns observed at (A) TPOX and (B) D18S51 from different samples. Allele calls are listed underneath each peak. (A) The TPOX result was obtained with the Identifiler STR kit and run on the ABI (B) This DNA sample was amplified with the Profiler Plus STR kit, separated on the ABI Prism 310 Genetic Analyzer and viewed with Genotyper software. Only the green dye-labeled PCR products are shown here for simplicity’s sake. Figure 6.7, J.M. Butler (2005) Forensic DNA Typing, 2nd Edition © 2005 Elsevier Academic Press

* A) B) C) STR Repeat region Example: TH01 9.3 allele
(-A in 7th repeat) D18S allele (+AG in 3’-flanking region) Rare VWA allele amplified with AmpFlSTR primers (A-to-T in 2nd base from 3’end of forward primer) Repeat region Reverse primer binding region 3’flanking region 5’flanking region Forward primer binding region STR Figure 6.8 Possible sequence variation in or around STR repeat regions and the impact on PCR amplification. The asterisk symbolizes a DNA difference (base change, insertion or deletion of a nucleotide) from a typical allele for a STR locus. In situation (A), the variation occurs within the repeat region and should have no impact on the primer binding and the subsequent PCR amplification (although the overall amplicon size may vary slightly). In situation (B), the sequence variation occurs just outside the repeat in the flanking region but interior to the primer annealing sites. Again, PCR should not be affected although the size of the PCR product may vary slightly. However, in situation (C) the PCR can fail due to a disruption in the annealing of a primer because the primer no longer perfectly matches the DNA template sequence. Figure 6.8, J.M. Butler (2005) Forensic DNA Typing, 2nd Edition © 2005 Elsevier Academic Press

Heterozygous alleles are well balanced
6 8 Imbalance in allele peak heights 6 8 * 8 Figure 6.9 Impact of a sequence polymorphism in the primer binding site illustrated with a hypothetical heterozygous individual. Heterozygous allele peaks may be well-balanced (A), imbalanced (B), or exhibit allele dropout (C). * Allele 6 amplicon has “dropped out” Figure 6.9, J.M. Butler (2005) Forensic DNA Typing, 2nd Edition © 2005 Elsevier Academic Press

(a) (b) 14,18 15,17 14,18 15,17 15,18 13,17 Figure 6.10 Mutational event observed in family trios. Normal transmission of alleles from a STR locus (a) is compared here to mutation of paternal allele 14 into the child’s allele 13 (b). Figure 6.10, J.M. Butler (2005) Forensic DNA Typing, 2nd Edition © 2005 Elsevier Academic Press

Mutation Rates for Common STR Loci
J.M. Butler (2005) J. Forensic Sci., in press Appendix 2 STR System Maternal Meioses (%) Paternal Meioses (%) Number from either Total Number of Mutations Mutation Rate CSF1PO 95/304,307 (0.03) 982/643,118 (0.15) 410 1,487/947,425 0.16% FGA 205/408,230 (0.05) 2,210/692,776 (0.32) 710 3,125/1,101,006 0.28% TH01 31/327,172 (0.009) 41/452,382 (0.009) 28 100/779,554 0.01% TPOX 18/400,061 (0.004) 54/457,420 (0.012) 100/857,481 VWA 184/564,398 (0.03) 1,482/873,547 (0.17) 814 2,480/1,437,945 0.17% D3S1358 60/405,452 (0.015) 713/558,836 (0.13) 379 1,152/964,288 0.12% D5S818 111/451,736 (0.025) 763/655,603 (0.12) 385 1,259/1,107,339 0.11% D7S820 59/440,562 (0.013) 745/644,743 (0.12) 285 1,089/1,085,305 0.10% D8S1179 96/409,869 (0.02) 779/489,968 (0.16) 364 1,239/899,837 0.14% D13S317 192/482,136 (0.04) 881/621,146 (0.14) 485 1,558/1,103,282 D16S539 129/467,774 (0.03) 540/494,465 (0.11) 372 1,041/962,239 D18S51 186/296,244 (0.06) 1,094/494,098 (0.22) 466 1,746/790,342 0.22% D21S11 464/435,388 (0.11) 772/526,708 (0.15) 580 1,816/962,096 0.19% Penta D 12/18,701 (0.06) 21/22,501 (0.09) 24 57/41,202 Penta E 29/44,311 (0.065) 75/55,719 (0.135) 59 163/100,030 D2S1338 15/72,830 (0.021) 157/152,310 (0.10) 90 262/225,140 D19S433 38/70,001 (0.05) 78/103,489 (0.075) 71 187/173,490 SE33 (ACTBP2) 0/330 (<0.30) 330/51,610 (0.64) None reported 330/51,940 0.64%

Chapter 7

Agarose yield gel results
Degraded DNA sample D5S818 D13S317 D7S820 D16S539 CSF1PO Penta D Agarose yield gel results Smear of degraded DNA fragments High molecular weight DNA in a tight band (A) (B) Good quality DNA Degraded DNA Figure 7.1 Impact of degraded DNA on (A) agarose yield gel results and (B) STR typing. (A) Degraded DNA is broken up into small pieces that appear as a smear on a scanned yield gel compared to good quality DNA possessing intact high molecular weight DNA. (B) Signal strength is generally lost with larger size PCR products when STR typing is performed on degraded DNA, such as is shown from the green dye-labeled loci in the PowerPlex 16 kit. Thus, 180 bp D13S317 PCR products have a higher signal than 400 bp Penta D amplicons because more DNA molecules are intact in the 200 bp versus the 400 bp size range. Figure 7.1, J.M. Butler (2005) Forensic DNA Typing, 2nd Edition © 2005 Elsevier Academic Press

miniSTRs: new tool for degraded DNA
STR repeat region miniSTR primer Conventional PCR primer (A) (B) Conventional STR test (COfiler™ kit) MiniSTR assay (using Butler et al primers) Smaller PCR products work better with low copy number or fragmented DNA templates Figure 7.2 (A) MiniSTRs, or reduced sized amplicons for STR typing, are created by designing PCR primers that anneal closer to the repeat region than conventional STR kit primers. (B) PCR product sizes, such as demonstrated here with D16S539, can be reduced by over 150 bp relative to conventional tests. MiniSTR assays can produce the same typing result as those from larger STR amplicons produced by kits often with greater success on degraded DNA samples. 150 bp smaller Figure 7.2, J.M. Butler (2005) Forensic DNA Typing, 2nd Edition © 2005 Elsevier Academic Press

J. Forensic Sci. Sept 2003 issue
Describes new primer sequences for all CODIS loci and initial assays developed Big Mini TH01 Allelic Ladder TH01 TPOX CSF1PO D21S11 D7S820 FGA PCR product size (bp) -71 bp -33 bp -117 bp -105 bp -191 bp -148 bp Size relative to ABI kits

(a) (b) Heterozygous peak region >70% <15% Stutter region
100% Heterozygous peak region 85% MIXTURE REGION 9% Higher than typical stutter product (>15%) 60% 10% 25% Wrong side of allele to be typical stutter product Smaller peak area than normally seen with heterozygote partner alleles(<70%) (a) (b) Figure 7.3 Illustration of typical single source (a) versus mixed sample (b) heterozygote peak patterns. The relative peak areas due to the measured fluorescent signal are useful indicators to decipher the presence of a sample mixture. If the highest peak at a locus is set at 100%, then heterozygous alleles should have peak areas and peak heights that are greater than 70% of the highest alleles. Stutter products are typically less than 15% of their corresponding allele peak and shorter by four base pairs for tetranucleotide repeats. Figure 7.3, J.M. Butler (2005) Forensic DNA Typing, 2nd Edition © 2005 Elsevier Academic Press

Step #1 Step #2 Step #3 Step #4 Step #5 Step #6
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 Figure 7.4 Steps in the interpretation of mixtures (Clayton et al. 1998). Figure 7.4, J.M. Butler (2005) Forensic DNA Typing, 2nd Edition © 2005 Elsevier Academic Press

D5S818 D13S317 D7S820 D8S1179 D21S11 D18S51 Amel VWA FGA D3S1358 blue panel green panel yellow panel DNA Size (bp) Relative Fluorescence Units Figure 7.5 Mixture of male and female DNA typical of what might be seen in a forensic case involving mixed samples. Profiler Plus multiplex STR data is displayed here with GeneScan 3.1. Sample mixture indicators include an imbalance in the amelogenin X and Y alleles as well as greater than two peaks at multiple STR markers. Figure 7.5, J.M. Butler (2005) Forensic DNA Typing, 2nd Edition © 2005 Elsevier Academic Press

amelogenin X-Y peak imbalance
D 3 peaks at D8S1179 4 peaks at D21S11 4 peaks at D18S51 X Y DNA Size (bp) RFUs Figure 7.6 Peak areas for green panel data from example mixture in Figure Mixture ratio calculations for these STR markers are shown in Table 7.3. RFUs = relative fluorescence units. Figure 7.6, J.M. Butler (2005) Forensic DNA Typing, 2nd Edition © 2005 Elsevier Academic Press

2 = major component 1 = major component 1 2 AA BC AB BC BC AC AB AC
CC AB BB AC 2 = major component 1 = major component 1 2 A B C Figure 7.7 Expected peak profiles for 2:1 mixture combinations involving three peaks. These peak profiles assume no overlap with stutter products and homozygote alleles that possess twice the signal strength of each heterozygote allele. Figure 7.7, J.M. Butler (2005) Forensic DNA Typing, 2nd Edition © 2005 Elsevier Academic Press

Common Casework Challenges
DEGRADED DNA D5S818 D13S317 D7S820 D16S539 CSF1PO Penta D Loss of signal at larger size loci D3S1358 TH01 D13S317 D16S539 D2S1338 MIXTURES More than two alleles at multiple loci From Butler, J.M. (2004) Short tandem repeat analysis for human identity testing. Current Protocols in Human Genetics, John Wiley & Sons, Hoboken, NJ, Unit 14.8, (Supplement 41), pp

Forensic Casework DNA Profiles
Profiler Plus STR kit results Crime scene evidence (MIXTURE of victim and perpetrator) suspect All of the suspect’s alleles are included in the evidentiary DNA profile

Chapter 8

Protocol Steps for Allele-Specific Primer Extension SNP Assay
Genomic DNA sample (Multiplex) PCR ExoSAP Digestion Amplification Add SNP primer(s) and SNaPshot mix SNP Extension (cycle sequencing) SAP treatment Primer Extension Sample prep for 310/3100 Run on ABI 310/3100 Data Analysis (GeneScan) Analysis Figure 8.1 Steps in allele-specific primer extension SNP detection (e.g., minisequencing or SNaPshot) assay. The boxed portions illustrate additional steps performed in this assay relative to STR typing. Add GS120 LIZ size standard Use E5 filter (5-dye) and POP4 standard conditions Type Sample (Genotyper or GeneMapperID) Figure 8.1, J.M. Butler (2005) Forensic DNA Typing, 2nd Edition © 2005 Elsevier Academic Press

(A) TT CT CC (B) Sample 1 Sample 2
Figure 8.2 Allele-specific primer extension results using four autosomal SNP markers on two different samples (A). SNP loci are from separate chromosomes (1, 6, 14, and 20) and therefore unlinked. Electrophoretic resolution of the SNP primer extension products occurs due to poly-T tails that are 5 nucleotides different from one another (B). (TTTTT)–(TTTTT)-primer2(chromosome 6)-ddC/ddT (TTTTT)–primer1(chromosome 20)-ddT/ddT (TTTTT)–(TTTTT)–(TTTTT)-primer3(chromosome 14)-ddC/ddT (TTTTT)–(TTTTT)–(TTTTT)–(TTTTT)-primer4(chromosome 1)-ddC/ddC (B) Figure 8.2, J.M. Butler (2005) Forensic DNA Typing, 2nd Edition © 2005 Elsevier Academic Press

Two possible alleles “long” (+) allele “short” (-) allele
Human Alu Repeat (~300 bp) AluI 400 bp 100 bp “long” (+) allele “short” (-) allele Figure 8.3 Schematic of the Alu element insertion PCR assay. The Alu sequence, which is approximately 300 bp in size, may either be present or absent at a particular location in the human genome. When the flanking region of the Alu repeat is targeted with primers, PCR amplification will result in products that are 400 bp if the Alu element is inserted or 100 bp if it is absent. A simple ethidium bromide-stained agarose gel may be used to genotype individuals. Individuals that are homozygous for the insertion will amplify a 400 bp DNA fragment. Those who are heterozygous for the insertion will amplify both 400 bp and 100 bp fragments, and individuals that are homozygous for the lack of the Alu insertion element will exhibit only the 100 bp DNA fragment. Figure 8.3, J.M. Butler (2005) Forensic DNA Typing, 2nd Edition © 2005 Elsevier Academic Press

Chapter 9

(passed on complete, but only by sons)
Lineage Markers Autosomal (passed on in part, from all ancestors) Figure 9.1 Illustration of inheritance patterns from recombining autosomal genetic markers and the lineage markers from the Y-chromosome and mitochondrial DNA. Y-Chromosome (passed on complete, but only by sons) Mitochondrial (passed on complete, but only by daughters) Figure 9.1, J.M. Butler (2005) Forensic DNA Typing, 2nd Edition © 2005 Elsevier Academic Press

Y-Chromosome STR Profile
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 Figure 9.2 Schematic illustrating the types of autosomal or Y-STR profiles that might be observed with sexual assault evidence where mixtures of high amounts of female DNA may mask the STR profile of the perpetrator. Y-STR testing permits isolation of the male component without having to perform a differential lysis. Figure 9.2, J.M. Butler (2005) Forensic DNA Typing, 2nd Edition © 2005 Elsevier Academic Press

? uncle 3rd cousin Figure 9.3 An example pedigree showing patrilineal inheritance where all shaded males have the same Y-chromosome barring any mutations. To help identify the person in question, any of the other males with the same patrilineage could provide a reference sample to assist in a missing persons investigation, mass disaster victim identification, or deficient paternity test (boxed region) where the father is deceased or not available for testing. Figure 9.3, J.M. Butler (2005) Forensic DNA Typing, 2nd Edition © 2005 Elsevier Academic Press

X Y (A) (B) 154 Mb 50 Mb Yp Yq Heterochromatic region (not sequenced)
Pseudoautosomal Region 1 (PAR1) Pseudoautosomal Region 2 (PAR2) Non-Recombining Portion of Y Chromosome (NRY) Male-specific region of the Y (MSY) 50 Mb X Y 154 Mb Yp Yq Heterochromatic region (not sequenced) ~30 Mb Euchromatic region (23 Mb) centromere recombination Figure 9.4 (A) Schematic of X and Y sex chromosomes. The two tips of the Y-chromosome known as the pseudoautosomal region 1 (PAR1) and 2 (PAR2) recombine with the tips of the X-chromosome. The remaining 95% of the Y-chromosome is referred to as the non-recombining portion of the Y-chromosome (NRY) or male-specific region of the Y (MSY). (B) The Y-chromosome is composed of both euchromatic and heterochromatic regions of which only the 23 megabases of euchromatin has been sequenced. Figure 9.4, J.M. Butler (2005) Forensic DNA Typing, 2nd Edition © 2005 Elsevier Academic Press

Duplicated regions are 40,775 bp apart and facing away from each other
DYS385 a/b Multi-Copy (Duplicated) Marker a = b a  b a b Duplicated regions are 40,775 bp apart and facing away from each other F primer R primer (B) DYS389 I/II Single Region but Two PCR Products (because forward primers bind twice) I II F primer R primer Figure 9.5 Schematic illustration of how multiple PCR primer binding sites give rise to multi-copy PCR products for (A) DYS385 a/b and (B) DYS389I/II. Arrows represent either forward “F” or reverse “R” primers. In the case of DYS385 a/b, the entire region around the STR repeat is duplicated and spaced about 40,775 bp apart on the long arm of the Y-chromosome. Thus, amplification with a single set of primers gives rise to one peak if the “a” repeat region is equal in size to the “b” repeat region or separate peaks if “a” and “b” differ in length. DYS389 possess two primary repeat regions that are flanked on one side by a similar sequence. Widely used forward primers bind adjacent to both repeats generating amplicons that differ in size by ~120 bp. Note that DYS389II is inclusive of the DYS389I repeat region and therefore some analyses subtract DYS389II-DYS389I repeats. DYS389I DYS389II Figure 9.5, J.M. Butler (2005) Forensic DNA Typing, 2nd Edition © 2005 Elsevier Academic Press

DYS391 DYS19 DYS385 DYS389II a b DYS393 DYS390 Y-PLEX™ 6 Kit
PCR product size (in nucleotides relative to GS500 ROX) Relative fluorescence units Figure 9.6 Result from the commercial Y-STR kit Y-PLEX™ 6. Figure 9.6, J.M. Butler (2005) Forensic DNA Typing, 2nd Edition © 2005 Elsevier Academic Press

(A) (B) PCR product size (bp)
Figure 9.7 Alleles present in (A) PowerPlex® Y and (B) Y-PLEX™ 12 allelic ladders for the DYS385 a/b locus. Figure 9.7, J.M. Butler (2005) Forensic DNA Typing, 2nd Edition © 2005 Elsevier Academic Press

(A) 9-14 16-21 1 2 3 18-23 (B) (C) (D) N14 N4 N3 N7 N20 N10
ATCTATCTTGAATTAATAGATTCAAGGTGATAGATATACAGATAGATAGATACATAGGTGGAGACAGATAGATGATAAATAGAAGATAGATAGATAGATAGATAGATAGATAGATAGATAGATAGATAGATAGATAGATA 9-14 [GATA] 16-21 1 2 3 N4 N14 N3 N7 [ATCT] 18-23 [AGAT] N20 N10 (B) (C) Figure 9.8 Various published allele nomenclatures for DYS439. (A) Sequence from DYS439 spanning the repeat regions used in the various nomenclatures; (B) Schematic of allele designation by Ayub et al. 2000—repeat range is 9-14; (C) Schematic of allele designation by Grignani et al. 2000—repeat range is 16-21; (D) Schematic of allele designation by Gonzalez-Neira et al. 2001—repeat range is The most widely accepted designation and what is used in commercial Y-STR kits is (B)--that of Ayub et al (D) Figure 9.8, J.M. Butler (2005) Forensic DNA Typing, 2nd Edition © 2005 Elsevier Academic Press

M42 M60 RPS4Y (M130) M168 M89 M45 M201 M52 M170 M172 A B C D E E3a F* G H I J2 K* L M N M175 M5 M11 P36 O Q R R1b M9 P* M91 YAP M174 M2 M96 12f2 J LLY22g M207 M173 P25 M269 R1b3 R1 Figure 9.9 Y Chromosome Consortium Tree with 18 major haplogroups (A-R). Representative Y-SNP markers that define each haplogroup are listed next to the branch point. The most common African American haplogroup is E3a. The most common Caucasian (European) haplogroup is R1b/R1b3. Figure 9.9, J.M. Butler (2005) Forensic DNA Typing, 2nd Edition © 2005 Elsevier Academic Press

Historical Investigation DNA Study
Thomas Jefferson II Field Jefferson Peter Jefferson President Thomas Jefferson Eston Hemings Thomas Woodson Different Y Haplotype Same Y Haplotype Jefferson Y Haplotype ? Figure 9.10 Ancestry of Thomas Jefferson and Eston Hemings male lines. The shaded boxes represent the samples tested by Foster et al. (1998) in their Jefferson Y chromosome study. A male descendant of Eston Hemings, son of Thomas Jefferson’s slave Sally Hemings, was found to have a Y chromosome haplotype that matched male descendants of Field Jefferson, President Thomas Jefferson’s uncle. A male descendant of Thomas Woodson, claimed by some to be descended from Jefferson, had a different Y haplotype and therefore could not have been a Jefferson. Figure 9.10, J.M. Butler (2005) Forensic DNA Typing, 2nd Edition © 2005 Elsevier Academic Press

Modern Use of Y-STR Testing
Captured December 13, 2003 Uday and Qusay Hussein Killed July 22, 2003 Matching Y-STR Haplotype Used to Confirm Identity (along with allele sharing from autosomal STRs) Is this man really Sadaam Hussein? Butler, J.M. (2005) Forensic DNA Typing, 2nd Edition, Box 23.1, p. 534

Some Reported Casework Examples
J.M. Butler (2005) Forensic DNA Typing, 2nd Edition; Table 9.7 Kit/Loci Used Reference Comments In-house assay with DYS19, DYS390, DYS389I/II Prinz et al. (2001) Forensic Sci. Int. 120: In one year at the New York City Office of the Chief Medical Examiner, Y-STR testing was performed in more than 500 cases with over 1000 evidence and reference samples examined. A full or partial profile was obtained on 81% of all tested evidence samples (740 worked/915 samples tested). Mixtures of at least two males were observed in 97 instances. In male/female mixtures of up to 1:4000, the male component could be cleanly detected. In-house assay with 9 Y-STR loci amplified in 3 PCR reactions Dekairelle and Hoste (2001) Forensic Sci. Int. 118: Y-STR typing was attempted on 166 semen traces from 89 cases that failed to yield a detectable male autosomal profile following differential extraction. About half of the cases had sufficient DNA to produce a Y-STR profile. In-house assay with DYS393, DYS389I/II Sibille et al. (2002) Forensic Sci. Int. 125: Y-STR results could still be obtained more than 48 hours after the sexual assault in 30% of the cases examined. In 104 swabs collected with no evidence of sperm, Y-STRs could be detected in ~29% of the samples tested. Prinz (2003) Forensic Sci. Rev. 15: Six case studies are reviewed along with advantages and disadvantages of Y-STR testing in each case: (1) different semen donors on vaginal swab and underwear; (2) possible oligospermic perpetrator gave a nice Y-STR profile but failed to have a “male” fraction with differential extraction; (3) oral intercourse with no autosomal results—not possible to enrich male cell fraction with differential extraction in cases involving saliva; (4) presence of multiple semen donors created a complex autosomal mixture that could be sorted out with Y-STR results; (5) sperm cell fraction lacked amelogenin Y-specific peak due to known deletion—Y-STR results confirmed that the sperm cell fraction DNA was of male origin; and (6) Y-STR testing was used to rapidly screen 18 semen stains for comparison to 5 suspects and thus save the time of performing the differential extraction Y-PLEX 6 and Y-PLEX 5 kits Sinha (2003) Forensic Sci. Rev. 15: Five cases are reviewed: (1) criminal paternity case with a male fetus where the alleged father could not be excluded as the biological father; (2) autosomal STR test resulted in an uninterpretable mixture—suspect was excluded at 3 of the 7 Y-STR loci tested; (3) Y-PLEX 6 STR profile matched suspect with sweat stains on cloth found at crime scene; (4) fingernail cuttings from a victim matched a suspect at 11 Y-STR loci while another suspect was excluded at 2 loci; (5) semen positive stain with no sperm cells produced a Y-PLEX 6 profile consistent with the male suspect Sinha et al. (2004) J. Forensic Sci. 49: Seven cases are reviewed (some are the same as Sinha 2003) and a list of cases where Y-STR results have been accepted in U.S. courts is provided.

Chapter 10

“16,569” bp 22 tRNAs 2 rRNAs Heavy (H) strand 13 genes
Control region (D-loop) 1/16,569 cyt b ND5 ND6 ND4 ND4L ND3 COIII ATP6 ATP8 COII 12S rRNA 16S ND1 ND2 COI OH 9-bp deletion OL F V L1 I Q M W A N C Y S1 D K G R H S2 L2 E P T HV1 HV2 16024 16365 73 340 576 “16,569” bp 1 22 tRNAs 2 rRNAs 13 genes Heavy (H) strand Light (L) strand Figure 10.1 Schematic showing the circular mitochondrial DNA genome (mtGenome). The heavy (H) strand is represented by the outside line and contains a higher number of C-G residues than the light (L) strand. The 37 RNA and protein coding gene regions are abbreviated around the mtGenome next to the strand from which they are synthesized (see Table 10.2). Most forensic mtDNA analyses presently examine only HV1 and HV2 in the non-coding control region or displacement loop shown at the top of the figure. Due to insertions and deletions that exist around the mtGenome in different individuals, it is not 16,569 bp. Figure 10.1, J.M. Butler (2005) Forensic DNA Typing, 2nd Edition © 2005 Elsevier Academic Press

MtDNA Haplotype Groups:
1 2 3 5 4 12 11 10 9 6 7 8 18 17 15 16 13 14 MtDNA Haplotype Groups: 2,3,6,8,11,13,15,16 4,9,10 14,17,18 A B C D E F G Figure 10.2 Illustration of maternal mitochondrial DNA inheritance for 18 individuals in a hypothetical pedigree. Squares represent males and circles females. Each unique mtDNA type is represented by a different letter. Figure 10.2, J.M. Butler (2005) Forensic DNA Typing, 2nd Edition © 2005 Elsevier Academic Press

AFDIL “mini-primer” set AFDIL primer set
HV1 HV2 HV3 16024 16365 73 340 438 574 16519 16569/1 342 bp 268 bp 137 bp F15989 R16251 F16190 R16410 VR1 VR2 C-stretch AFDIL “mini-primer” set PSI (263 bp) PSII (221 bp) F15 R285 F155 R381 PSIII (271 bp) PSIV (227 bp) MPS1A (170 bp) MPS1B (126 bp) MPS2A (133 bp) MPS2B (143 bp) MPS3A (126 bp) MPS3B (132 bp) MPS4A (142 bp) MPS4B (158 bp) AFDIL primer set Figure 10.3 The three hypervariable (HV) regions of the mtDNA control region. HV1 spans nucleotide positions (342 bp), HV2 spans positions (268 bp), and HV3, which is rarely examined in forensic testing, spans positions (137 bp). The general positions for variable regions VR1 and VR2 are noted although these are rarely used. PCR primer sets (PS) commonly used by the Armed Forces DNA Identification Laboratory (AFDIL) are illustrated. Primer nomenclature designates the 5’-nucleotide for each primer. PCR product sizes for each set of primers is noted in parentheses. The bottom section shows “mini-primer” PCR product sizes that are used with highly degraded DNA samples to enable greater recovery of sequence information (see Gabriel et al. (2001a)). Figure 10.3, J.M. Butler (2005) Forensic DNA Typing, 2nd Edition © 2005 Elsevier Academic Press

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 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 Figure 10.4 Process for evaluation of mtDNA samples. The evidence or question (Q) sample may come from a crime scene or a mass disaster. The reference or known (K) sample may be a maternal relative or the suspect in a criminal investigation. In a criminal investigation, the victim may also be tested and compared to the Q and K results. Compare Q and K sequences Compare with database to determine haplotype frequency Figure 10.4, J.M. Butler (2005) Forensic DNA Typing, 2nd Edition © 2005 Elsevier Academic Press

3’-TAAATGATTCC-5’ A AT ATT ATTT ATTTA ATTTAC ATTTACT ATTTACTA
ATTTACTAA ATTTACT ATTTAC ATTT ATTTA AT ATTTACTA ATTTACTAAG ATTTACTAAGG A DNA template 5’ 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) Figure 10.5 DNA sequencing process with fluorescent ddNTPs. A primer that has been designed to recognize a specific region of a DNA template anneals and is extended with a polymerase. Because a mixture of dNTPs and ddNTPs exist for each of the four possible nucleotides, some of the extension products are halted by incorporation of a ddNTP while other molecules continue to be extended. Each ddNTP is labeled with a different dye that enables each extension product to be distinguished by color. A size-based separation of the extension products permits the DNA sequence to be read provided that sufficient resolution is present to clearly see each base. Figure 10.5, J.M. Butler (2005) Forensic DNA Typing, 2nd Edition © 2005 Elsevier Academic Press

Hypervariable Region I
GAAAAAGTCT TTAACTCCAC CATTAGCACC CAAAGCTAAG ATTCTAATTT AAACTATTCT CTTTTTCAGA AATTGAGGTG GTAATCGTGG GTTTCGATTC TAAGATTAAA TTTGATAAGA CTGTTCTTTC ATGGGGAAGC AGATTTGGGT ACCACCCAAG TATTGACTCA CCCATCAACA GACAAGAAAG TACCCCTTCG TCTAAACCCA TGGTGGGTTC ATAACTGAGT GGGTAGTTGT C C A ACCGCTATGT ATTTCGTACA TTACTGCCAG CCACCATGAA TATTGTACGG TACCATAAAT TGGCGATACA TAAAGCATGT AATGACGGTC GGTGGTACTT ATAACATGCC ATGGTATTTA ACTTGACCAC CTGTAGTACA TAAAAACCCA ATCCACATCA AAACCCCCTC CCCATGCTTA TGAACTGGTG GACATCATGT ATTTTTGGGT TAGGTGTAGT TTTGGGGGAG GGGTACGAAT CAAGCAAGTA CAGCAATCAA CCCTCAACTA TCACACATCA ACTGCAACTC CAAAGCCACC GTTCGTTCAT GTCGTTAGTT GGGAGTTGAT AGTGTGTAGT TGACGTTGAG GTTTCGGTGG T T C G C CCTCACCCAC TAGGATACCA ACAAACCTAC CCACCCTTAA CAGTACATAG TACATAAAGC GGAGTGGGTG ATCCTATGGT TGTTTGGATG GGTGGGAATT GTCATGTATC ATGTATTTCG C CATTTACCGT ACATAGCACA TTACAGTCAA ATCCCTTCTC GTCCCCATGG ATGACCCCCC GTAAATGGCA TGTATCGTGT AATGTCAGTT TAGGGAAGAG CAGGGGTACC TACTGGGGGG TCAGATAGGG GTCCCTTGAC CACCATCCTC CGTGAAATCA ATATCCCGCA CAAGAGTGCT AGTCTATCCC CAGGGAACTG GTGGTAGGAG GCACTTTAGT TATAGGGCGT GTTCTCACGA FBI A1 (L15997) Roche (F15975) HV1 16093 16126 16129 HVI C-stretch Roche IA Roche ID Roche IC Roche IE FBI B1 (H16391) Roche (R16418) Hypervariable Region I 342 bp examined SSO Probes 16093 16126 16129 16270 16278 16304 16309 16311 16362 Only 9 sites examined Figure 10.6

Hypervariable Region II
GATCACAGGT CTATCACCCT ATTAACCACT CACGGGAGCT CTCCATGCAT TTGGTATTTT CTAGTGTCCA GATAGTGGGA TAATTGGTGA GTGCCCTCGA GAGGTACGTA AACCATAAAA G CGTCTGGGGG GTATGCACGC GATAGCATTG CGAGACGCTG GAGCCGGAGC ACCCTATGTC GCAGACCCCC CATACGTGCG CTATCGTAAC GCTCTGCGAC CTCGGCCTCG TGGGATACAG C T C GCAGTATCTG TCTTTGATTC CTGCCTCATC CTATTATTTA TCGCACCTAC GTTCAATATT CGTCATAGAC AGAAACTAAG GACGGAGTAG GATAATAAAT AGCGTGGATG CAAGTTATAA G C T G ACAGGCGAAC ATACTTACTA AAGTGTGTTA ATTAATTAAT GCTTGTAGGA CATAATAATA TGTCCGCTTG TATGAATGAT TTCACACAAT TAATTAATTA CGAACATCCT GTATTATTAT A ACAATTGAAT GTCTGCACAG CCACTTTCCA CACAGACATC ATAACAAAAA ATTTCCACCA TGTTAACTTA CAGACGTGTC GGTGAAAGGT GTGTCTGTAG TATTGTTTTT TAAAGGTGGT AACCCCCCCT CCCCCGCTTC TGGCCACAGC ACTTAAACAC ATCTCTGCCA AACCCCAAAA TTGGGGGGGA GGGGGCGAAG ACCGGTGTCG TGAATTTGTG TAGAGACGGT TTGGGGTTTT ACAAAGAACC CTAACACCAG CCTAACCAGA TTTCAAATTT TATCTTTTGG CGGTATGCAC TGTTTCTTGG GATTGTGGTC GGATTGGTCT AAAGTTTAAA ATAGAAAACC GCCATACGTG TTTTAACAGT CACCCCCCAA CTAACACATT ATTTTCCCCT CCCACTCCCA TACTACTAAT AAAATTGTCA GTGGGGGGTT GATTGTGTAA TAAAAGGGGA GGGTGAGGGT ATGATGATTA HV2 73 146 150 HV2 C-stretch Roche IIA Roche IID Roche IIB Roche IIC FBI D1 (H408) Roche (RR429) FBI C1 (L048) Roche (F15) 195 189 152 247 200 198 Hypervariable Region II 73-340 268 bp examined SSO Probes 73 146 150 152 189 195 198 200 247 Only 9 sites examined Figure 10.6

Primer strategies typically used with C-stretch containing samples (A)
HV1 C-stretch 16189T Poor quality sequence (two length variants out of phase) Good quality sequence Primer strategies typically used with C-stretch containing samples (A) (B) (C) C-stretch Use of internal primers Double reactions from the same strand Fig Comparison of a sample with (A) 16189T (no HV1 C-stretch) to (B) one with the C-stretch. Notice how the sequence quality quickly drops after the string of cytosine residues due to the presence of two or more length variants that creates a situation where the extension products are out of phase or register with one another. Different primer combinations are typically used on samples containing a C-stretch as illustrated in (C) to recover sequence information from both strands or to provide a double read of the same strand. Figure 10.7, J.M. Butler (2005) Forensic DNA Typing, 2nd Edition © 2005 Elsevier Academic Press

(A) mtDNA Sequences Aligned with rCRS (positions 16071-16140)
ACCGCTATGT ATTTCGTACA TTACTGCCAG CCACCATGAA TATTGTACGG TACCATAAAT rCRS ACCGCTATGT ATCTCGTACA TTACTGCCAG CCACCATGAA TATTGTACAG TACCATAAAT Q K (B) Reporting Format with Differences from rCRS Sample Q 16093C 16129A Sample K 16093C 16129A Figure 10.8 (A) Comparison of sequence alignments for hypothetical Q and K samples with (B) conversion to the revised Cambridge Reference Sequence (rCRS) differences for reporting purposes. Figure 10.8, J.M. Butler (2005) Forensic DNA Typing, 2nd Edition © 2005 Elsevier Academic Press

16093 (C/T) 16086 16101 Figure 10.9 (A) Sequence heteroplasmy at position possessing both C and T nucleotides compared to (B) the same region (positions ) on a different sample containing only a T at position Figure 10.9, J.M. Butler (2005) Forensic DNA Typing, 2nd Edition © 2005 Elsevier Academic Press

K: 1-1-1-1-1-1-1-1-1-1 Q: 1-2-3-2-0-1-4-2-2-w1 IA IC ID IE IIA IIB IIC
IID 189 16093 HVI HVII 3 4 5 6 Ref 7 K Q (A) “blank” Figure (A) Results schematically displayed of a known (K) reference and a question (Q) sample that do not match one another using the Roche LINEAR ARRAY mtDNA HVI/HVII Region-Sequence Typing Strips. (B) Types are reported as a string of numbers representing the LINEAR ARRAY probe results. Failure of the PCR product to bind to a probe region (e.g., HVIE in sample Q) is referred to as a “blank”, is reported as a zero in the string of numbers, and is due to polymorphisms in the sample near the probe site that disrupt hybridization. Weak signals such as indicated by the arrow for 189 in sample Q are also due to a closely spaced polymorphism that disrupts full hybridization of the PCR product to the sequence-specific probe present on the LINEAR ARRAY. (B) Reported Types K: Q: w1 Figure 10.10, J.M. Butler (2005) Forensic DNA Typing, 2nd Edition © 2005 Elsevier Academic Press

Chapter 11

SRY (male) 4 7 Male cat 1 5 2 9 10 3 6 8 11 4 7 Female cat 2 5 10 1 6 9 8 3 11 Figure 11.1 DNA profiles produced from male (top panel) and female (bottom panel) cat DNA using a multiplex STR typing assay dubbed the “MeowPlex” (Butler et al. 2002). This test examines 11 autosomal STRs and a region of the SRY gene contained on the Y-chromosome that can be used for sex determination. Figure 11.1, J.M. Butler (2005) Forensic DNA Typing, 2nd Edition © 2005 Elsevier Academic Press

Chapter 12

- + Top view Side view Gel Buffer Voltage Loading well anode cathode
Gel lanes DNA bands Buffer Figure 12.1 Schematic of a gel electrophoresis system. The horizontal gel is submerged in a tank full of electrophoresis buffer. DNA samples are loaded into wells across the top of the gel. These wells are created by a “comb” placed in the gel while it is forming. When the voltage is applied across the two electrodes, the DNA molecules move towards the anode and separate by size. The number of lanes available on a gel is dependent on the number of teeth in the comb used to define the loading wells. At least one lane on each gel is taken up by a molecular weight size standard that is used to estimate the sizes of the sample bands in the other lanes. Figure 12.1, J.M. Butler (2005) Forensic DNA Typing, 2nd Edition © 2005 Elsevier Academic Press

Bisacrylamide cross-linker
Acrylamide monomer Bisacrylamide cross-linker Figure 12.2 Illustration of polyacrylamide gel polymerization. The pore size of a gel is controlled by its degree of cross-linking, which depends on the proportions of acrylamide and bisacrylamide in the gel. Polymerization is typically induced by free radicals resulting from the chemical decomposition of ammonium persulfate. TEMED, a free radical stabilizer, is also added to the gel mixture to stabilize the polymerization process. A polyacrylamide gel is poured between two glass plates that define its dimensions. A spacer is placed between the plates to define the thickness of the gel. A typical gel size is 17 cm x 43 cm x 0.4 cm. DNA molecules flow through the polyacrylamide gel matrix along the z-axis going into the page, much like flowing through a chain link fence with various sized holes. Figure 12.2, J.M. Butler (2005) Forensic DNA Typing, 2nd Edition © 2005 Elsevier Academic Press

Capillary filled with polymer solution
Laser Inlet Buffer Capillary filled with polymer solution 5-20 kV - + Outlet 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 Figure 12.3 Schematic of capillary electrophoresis instruments used for DNA analysis. The capillary is a narrow glass tube approximately 50 cm long and 50 microns in diameter. It is filled with a viscous polymer solution that acts much like a gel in creating a sieving environment for DNA molecules. Samples are placed into a tray and injected onto the capillary by applying a voltage to each sample sequentially. A high voltage (e.g., 15,000 volts) is applied across the capillary after the injection in order to separate the DNA fragments in a matter of minutes. Fluorescent dye-labeled products are analyzed as they pass by the detection window and are excited by a laser beam. Computerized data acquisition enables rapid analysis and digital storage of separation results. Figure 12.3, J.M. Butler (2005) Forensic DNA Typing, 2nd Edition © 2005 Elsevier Academic Press

(a) (b) Ogston Sieving Reptation Gel Gel
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 Figure 12.4 Illustration of DNA separation modes in gel electrophoresis. Separation according to size occurs as DNA molecules pass through the gel, which acts as a molecular sieve (a). Ogston sieving and reptation are the two primary mechanisms used to describe the movement of DNA fragments through a gel (b). Figure 12.4, J.M. Butler (2005) Forensic DNA Typing, 2nd Edition © 2005 Elsevier Academic Press

Chapter 13

S’1 S1 So      ex max em max (a) hex hem energy (b) Emission
Excitation Emission Wavelength (nm)   ex max em max Fluorescence (b) Stokes shift Figure 13.1 Illustration of the fluorescence process (a) and excitation/emission spectra (b). In the first step of the fluorescence process, a photon (hex) from a laser source excites the fluorophore (dye molecule) from its ground energy state (So) to an excited transition state (S’1). The fluorophore then undergoes conformational changes and interacts with its environment resulting in the relaxed singlet excitation state (S1). During the final step of the process, a photon (hem) is emitted at a lower energy. Because energy and wavelength are inversely related to one another, the emission photon has a higher wavelength than the excitation photon. Figure 13.1, J.M. Butler (2005) Forensic DNA Typing, 2nd Edition © 2005 Elsevier Academic Press

(a) Ethidium bromide DNA labeled with intercalating dye Unlabeled DNA SYBR Green Intercalator inserts between base pairs on double-stranded DNA (b) Fluorescent dNTPs are incorporated into both strands of PCR product Figure 13.2 Methods for fluorescently labeling DNA fragments. Double-stranded DNA molecules may be labeled with fluorescent intercalating dyes (a). The fluorescence of these dyes is enhanced upon insertion between the DNA bases. Alternatively a fluorescent dye may be attached to a nucleotide triphosphate and incorporated into the extended strands of a PCR product (b). The most common method of detecting STR alleles is the use of fluorescent dye labeled primers (c). These primers are incorporated into the PCR product to fluorescently label one of the strands. (c) One strand of PCR product is labeled with fluorescent dye Fluorescent dye labeled primer Figure 13.2, J.M. Butler (2005) Forensic DNA Typing, 2nd Edition © 2005 Elsevier Academic Press

FAM JOE TAMRA ROX (blue) (green) (yellow) (red)
Figure 13.3 ABI dyes used in four-color STR detection. The circle portion of the dye highlights the succinimidyl ester, which is used for dye attachment to the fluorescent oligo. In the AmpFlSTR kits, TAMRA has been replaced by NED (also a yellow dye). PowerPlex 1.1 and 2.1 STR kits use fluorescein and TMR, which are very similar to FAM and TAMRA, respectively. The red dye ROX is used to label an internal sizing standard and is the same as CXR used in PowerPlex kits. Figure 13.3, J.M. Butler (2005) Forensic DNA Typing, 2nd Edition © 2005 Elsevier Academic Press

with color contributions Normalized Fluorescent Intensity
520 540 560 580 600 620 640 WAVELENGTH (nm) 100 80 60 40 20 310 Filter Set F with color contributions 5-FAM JOE NED ROX Laser excitation (488, nm) Normalized Fluorescent Intensity Figure 13.4 Fluorescent emission spectra of ABI dyes used with AmpFlSTR kits. ABI 310 Filter Set F is represented by the 4 boxes centered on each of the 4 dye spectra. Each dye filter contains color contributions from adjacent overlapping dyes that must be removed by a matrix deconvolution. The dyes are excited by an argon ion laser, which emits light at 488 and nm. Figure 13.4, J.M. Butler (2005) Forensic DNA Typing, 2nd Edition © 2005 Elsevier Academic Press

Relative Fluorescent Intensity
Wavelength (nm) 505 585 650 fluorescein TMR CXR Laser excitation (532 nm) Relative Fluorescent Intensity Figure 13.5 Schematic of three-color detection using the Hitachi FMBIO Scanner System. These dyes and spectral files (shown with boxes) are used for detection of STR alleles amplified with the Promega PowerPlex systems. Figure 13.5, J.M. Butler (2005) Forensic DNA Typing, 2nd Edition © 2005 Elsevier Academic Press

Figure 13.6 Example of a matrix file table from an ABI Prism 310 instrument. These values are used by the GeneScan Analysis Software to separate the various dye colors from one another. The letters B, G, Y, and R represent the dye colors Blue, Green, Yellow, and Red, respectively. All matrix files should have values of 1.0 on the diagonal from top left to bottom right. The other values in the table should all be less than These values represent the amount of spectral overlap observed for each dye in each virtual detection filter. Matrix file values differ between instruments and even run conditions on a single instrument. Thus, a unique matrix file must be made for an instrument and a particular set of run conditions. Figure 13.6, J.M. Butler (2005) Forensic DNA Typing, 2nd Edition © 2005 Elsevier Academic Press

(a) (b) Scan number Region shown below Relative Fluorescence Units
DNA size in base pairs Relative Fluorescence Units Figure 13.7 STR Data from ABI Prism 310 Genetic Analyzer. This sample was amplified with the AmpFlSTR SGM Plus kit. Raw data prior to color separation (a) compared with GeneScan 3.1 color separated allele peaks (b). The red-labeled peaks are from the internal sizing standard GS500-ROX. (b) Figure 13.7, J.M. Butler (2005) Forensic DNA Typing, 2nd Edition © 2005 Elsevier Academic Press

Sample Interpretation
Mixture of dye-labeled PCR products from multiplex PCR reaction Sample Separation Sample Detection CCD Panel (with virtual filters) Argon ion LASER (488 nm) Color Separation Fluorescence ABI Prism spectrograph Capillary Sample Injection Size Processing with GeneScan/Genotyper software Sample Interpretation Figure 13.8 Schematic illustration of the separation and detection of STR alleles with an ABI Prism 310 Genetic Analyzer. Figure 13.8, J.M. Butler (2005) Forensic DNA Typing, 2nd Edition © 2005 Elsevier Academic Press

Profiler Plus multiplex STR result
DNA size (bp) COfiler multiplex STR result FGA D21S11 D18S51 D8S1179 VWA D13S317 D5S818 Amel D3S1358 D7S820 TH01 D16S539 CSF1PO TPOX Figure 13.9 AmpFlSTR Profiler Plus and COfiler STR Data Collected on an ABI 310 Capillary Electrophoresis System. The STR loci that are surrounded by a box are common to both multiplex mixes and are therefore useful as a quality assurance measure to demonstrate sample concordance. Figure 13.9, J.M. Butler (2005) Forensic DNA Typing, 2nd Edition © 2005 Elsevier Academic Press

Sample Detection (Post-Electrophoresis)
DNA samples are loaded onto a polyacrylamide gel STR alleles separate during electrophoresis through the gel Sample Separation Sample Detection (Post-Electrophoresis) 505 nm scan to detect fluorescein-labels 585 nm scan to detect TMR-labels Figure Schematic of gel separation and FMBIO II detection of STR alleles. Figure 13.10, J.M. Butler (2005) Forensic DNA Typing, 2nd Edition © 2005 Elsevier Academic Press

Figure PowerPlex® 16 BIO data collected on a Hitachi FMBIO III plus Fluorescence Imaging System. Color-separated data for this same gel is contained in Figure Figure courtesy of Margaret Kline, NIST. Figure 13.11, J.M. Butler (2005) Forensic DNA Typing, 2nd Edition © 2005 Elsevier Academic Press

Chapter 14

Mechanical stepper motor
Autosampler Tray Pump Block capillary Detection window Syringe filled with POP-4 polymer Injection electrode Heated plate for temperature control Buffer (inlet) (outlet) Mechanical stepper motor Deionized water sample tubes Figure 14.1 Schematic of ABI Prism 310 Genetic Analyzer. The capillary stretches between the pump block and the injection electrode. The mechanical stepper motor pushes polymer solution in the syringe into the pump block where it enters and then fills the capillary. Samples placed in the autosampler tray are sequentially injected onto the capillary. Electrophoretic separation occurs after each end of the capillary is placed in the inlet and outlet buffer and a voltage is applied across the capillary. A laser (not shown) is used to detect fluorescently labeled DNA fragments as they pass by the capillary detection window. Figure 14.1, J.M. Butler (2005) Forensic DNA Typing, 2nd Edition © 2005 Elsevier Academic Press

ELECTROPHORESIS and DETECTION steps are simultaneous
Replace capillary Refill syringe with polymer solution Fill buffer vials Prepare samples (denature, cool, and mix with size standard) Prepare sample sheet and injection list Automated Sample Injection, Electrophoresis and Data Collection Genotype STR alleles Size DNA Fragments Perform Data Analysis GeneScan Software Genotyper Software Manually inspect the data Performed only once per batch of ~96 samples Allelic ladder every tenth injection ELECTROPHORESIS and DETECTION steps are simultaneous Figure 14.2 Sample processing steps using ABI 310 Genetic Analyzer. Figure 14.2, J.M. Butler (2005) Forensic DNA Typing, 2nd Edition © 2005 Elsevier Academic Press

Data Collection Scan Number Relative Fluorescence Units Figure 14.3 Results from ABI 310 using the same sample that has been diluted in different formamide solutions. Formamide solutions with higher conductivities (larger number of S) result in less DNA being injected into the capillary. The sample is the GS350 ROX-labeled sizing standard. Figure courtesy of Bruce McCord, Ohio University. Figure 14.3, J.M. Butler (2005) Forensic DNA Typing, 2nd Edition © 2005 Elsevier Academic Press

Capillaries Electrodes for Injection
Figure 14.4 Photo of a 3100 capillary array illustrating the 16 glass capillaries (top) and the electrodes surrounding the capillaries (bottom) that enable electrokinetic injection of DNA samples from 2 columns of an 8x12 96well microtiter plate. Electrodes for Injection Figure 14.4, J.M. Butler (2005) Forensic DNA Typing, 2nd Edition © 2005 Elsevier Academic Press

ABI 3100 Matrix (Spectral Calibration) Sample
Blue (5FAM) Green (JOE) Yellow (NED) Red (ROX) Separate samples run for each dye color Each sample contains multiple peaks All peaks labeled with the same dye color ABI 310 Matrix Samples ABI 3100 Matrix (Spectral Calibration) Sample Single sample run containing all dye colors Only one peak per dye color Injected into each capillary of the array (A) (B) A separate spectral calibration file is created for each capillary Figure 14.5 Comparison of matrix samples used for spectral calibration in (A) ABI 310 and (B) ABI 3100 instruments. Figure 14.5, J.M. Butler (2005) Forensic DNA Typing, 2nd Edition © 2005 Elsevier Academic Press

Gel placed here Figure 14.6 Schematic of FMBIO II Fluorescence Imagining System. Following electrophoretic separation of STR alleles, a gel is placed in the FMBIO and scanned to reveal to presence of fluorescently labeled PCR products. The FMBIO utilizes a solid state, green laser (1) to excite fluorophores at 532 nm using a polygon scanning mirror (2). The resulting fluorescent light signals emitted from the excited fluorophores attached to DNA fragments are then collected by two optical fiber arrays (3). Specific fluorescent dye signals are isolated using separate interference filters (4) and are converted to electrical signals with two photomultiplier tubes (5). Figure used with permission from Hitachi Genetic Systems web page. Figure 14.6, J.M. Butler (2005) Forensic DNA Typing, 2nd Edition © 2005 Elsevier Academic Press

FMBIO III Gel Imager System
Penta D Penta E FGA CSF1PO D18S51 D16S539 TPOX D7S820 D8S1179 D21S11 D13S317 TH01 VWA D5S818 D3S1358 Amelogenin PowerPlex 16 BIO

ELECTROPHORESIS and DETECTION steps are separate
SCAN GEL Size DNA Fragments Genotype STR alleles Pour Gel Load Samples (allelic ladder every third lane) Electrophoresis STaR Call™ Genotyping Software FMBIO II Fluorescence Imaging System Perform Data Analysis FMBIO® Analysis Software ReadImage Software Manually inspect the data Post-Electrophoresis ELECTROPHORESIS and DETECTION steps are separate Prepare samples: denature, cool, and mix with loading dye Figure 14.7 Sample processing steps using the Hitachi FMBIO II or III. Multiple gels can be prepared simultaneously and then quickly scanned after electrophoresis to improve sample throughput. Figure 14.7, J.M. Butler (2005) Forensic DNA Typing, 2nd Edition © 2005 Elsevier Academic Press

PowerPlex® 1.1 PowerPlex® 2.1 505 nm scan 585 nm scan Penta E
Fluorescein-labeled TMR-labeled Penta E 505 nm scan 585 nm scan ladders ladders FGA samples CSF1PO D18S51 D16S539 TPOX D7S820 TPOX D21S11 Amelogenin D8S1179 D13S317 TH01 9.3/10 TH01 Figure 14.8 (left) PowerPlex 1.1 STR data collected from the 505 nm (fluorescein-labeled alleles) and 585 nm (TMR-labeled alleles) scans of a gel containing allelic ladders and PCR-amplified samples. (right) PowerPlex 2.1 STR data collected on the same two samples. The three marked loci—TH01, TPOX, and VWA—are common to both multiplex kits and are therefore useful as a quality assurance measure to demonstrate concordance from two separate PCR amplifications of the same DNA sample. Note that the allelic ladders for VWA and TH01 have different alleles for the PowerPlex 1.1 and 2.1 systems yet the samples shown here provide the same genotype and thus demonstrate full concordance of PCR amplifications from both kits. The 9.3 and 10 alleles for the PowerPlex 2.1 TH01 allelic ladder are resolved (see boxed area) indicating that single base resolution can be achieved in that portion of the gel electrophoretic separation. Original data courtesy of Hitachi Genetic Systems. VWA D5S818 VWA D3S1358 Figure 14.8, J.M. Butler (2005) Forensic DNA Typing, 2nd Edition © 2005 Elsevier Academic Press

Fluorescein Scan JOE Scan Rhodamine Red-X Scan Texas Red-X Scan
200 100 225 250 275 180 160 140 120 300 325 350 375 400 425 450 TPOX D8S1179 VWA FGA Amelogenin Penta D CSF1PO D7S820 D13S317 D5S818 D16S539 Penta E D18S51 D21S11 TH01 D3S1358 Figure 14.9 FMBIO III+ color-separated STR data collected from four scans of a gel containing PowerPlex® 16 BIO PCR-amplified samples. Each scan detects the PCR products labeled with one of three dyes: fluorescein, JOE, and Rhodamine Red-X. The ILS600 size standard is labeled with the fourth dye Texas Red-X and contains DNA fragments ranging from 100 bp to 600 bp in 20 or 25 bp increments (the upper bands are not captured on this gel). The STR loci amplified are indicated above the alleles observed. Allelic ladders are on the far left and right of each scan. The combined color image for the same gel may be seen in Figure Figure courtesy of Margaret Kline, NIST. Figure 14.9, J.M. Butler (2005) Forensic DNA Typing, 2nd Edition © 2005 Elsevier Academic Press

Chapter 15

Expert Systems under Development (e.g., True Allele)
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 or FMBIO Analysis software Genotyper or StaR Call Internal sizing standard (e.g., GS500-ROX) Matrix file Allelic ladder sample GeneMapperID Expert Systems under Development (e.g., True Allele) Figure 15.1 Genotyping process for STR allele determination. Software packages for DNA fragment analysis and STR genotyping perform much of the actual analysis, but extensive review of the data by trained analysts/examiners is often required. Figure 15.1, J.M. Butler (2005) Forensic DNA Typing, 2nd Edition © 2005 Elsevier Academic Press

Peak height in relative fluorescence units (RFUs)
COfiler STR data GeneScan view Genotyper view Allele call (repeat number) determined by comparison of peak size (bp) to allelic ladder allele peak sizes run under the same electrophoretic conditions Peak height in relative fluorescence units (RFUs)

(a) (b) DNA Size Data Point
35 50 75 100 139 160 200 250 300 340 350 400 450 490 500 150 DNA fragment peaks in sample DNA Size Data Point bp bp DNA fragment peaks are sized based on the sizing curve produced from the points on the internal size standard (a) (b) Figure 15.2 Peak sizing with DNA fragment analysis. An internal size standard, such as GS500-ROX (a), is analyzed along with the DNA sample and used to calibrate the peak data points to their DNA size (b). This standard is labeled with a different color fluorescent dye, in this case ROX (detected as red), so that it can be spectrally distinguished from the STR alleles which are labeled in other colors. Figure 15.2, J.M. Butler (2005) Forensic DNA Typing, 2nd Edition © 2005 Elsevier Academic Press

Green Yellow Blue Figure 15.3 Genotype results on the two samples displayed in Figure 1.3 obtained with AmpFlSTR SGM Plus STR kit amplification and Genotyper 2.5 analysis. Figure 15.3, J.M. Butler (2005) Forensic DNA Typing, 2nd Edition © 2005 Elsevier Academic Press

Incomplete adenylation Biological (PCR) artifacts
Deciphering Artifacts from the True Alleles D3S1358 Stutter products 6.0% 7.8% Incomplete adenylation D8S1179 -A +A Biological (PCR) artifacts Dye blob STR alleles stutter Pull-up (bleed-through) spike Blue channel Green channel Yellow channel Red channel Figure 15.4 Hypothetical electropherogram displaying several artifacts often observed with STR typing. Figure 15.4, J.M. Butler (2005) Forensic DNA Typing, 2nd Edition © 2005 Elsevier Academic Press

Three Possible Outcomes
Butler, J.M. (2005) Forensic DNA Typing, 2nd Edition, p. 385 Match – Peaks between the compared STR profiles have the same genotypes and no unexplainable differences exist between the samples. Statistical evaluation of the significance of the match is usually reported with the match report (see Chapter 21). Exclusion (Non-match) – The genotype comparison shows profile differences that can only be explained by the two samples originating from different sources. Inconclusive – The data does not support a conclusion as to whether the profiles match. This finding might be reported if two analysts remain in disagreement after review and discussion of the data and it is felt that insufficient information exists to support any conclusion.

Chapter 16

ASTM and American Academy of Forensic Sciences Springer-Verlag
Figure 16.1 Common journals for publications on forensic DNA methods, population genetics, and validation studies. Elsevier Science Elsevier Science Figure 16.1, J.M. Butler (2005) Forensic DNA Typing, 2nd Edition © 2005 Elsevier Academic Press

Chapter 17

Separation Time (minutes)
2.2 2.0 1.8 1.6 1.4 Separation Time (minutes) D5S818 D13S317 D7S820 D16S539 vWA TH01 TP0X CSF1P0 11 12 13 15 19 9.3/10 (a) (b) Fluorescein scan TMR scan Fluorescence (arb.) Figure 17.1 Rapid microchip CE separation of the 8 STR loci from PowerPlex 1.1 (Schmalzing 1999). The electropherograms from scanning each color of a simultaneous two-color analysis are divided. The PCR-amplified sample is mixed with the allelic ladders prior to injection to provide a frame of reference for genotyping the sample. The allele calls for each locus are listed next to the corresponding peak. Figure courtesy of Dr. Daniel Ehrlich, Whitehead Institute. Figure 17.1, J.M. Butler (2005) Forensic DNA Typing, 2nd Edition © 2005 Elsevier Academic Press

(fluorescent dye attached)
Capture Probe (attached to chip) Reporter Probe (fluorescent dye attached) Hybridization between probes and STR allele STR allele containing 6 repeat units (b) 9 7 6 5 4 3 8 Locus D Locus C Locus B Locus A Alleles (Repeat #) Figure Schematic of Nanogen STR hybridization chip assay. The assay illustrated in (a) involves a capture probe oligonucleotide that is attached at a unique location on the chip (b). The capture probe hybridizes to the appropriate STR allele by binding to the repeat region and bases of the flanking region. A reporter probe containing 1 to 3 repeat units, some flanking sequence and a fluorescent dye hybridizes to the STR allele and generates a fluorescent signal at the probe site that can be interpreted to yield the sample’s genotype (c). Multiple STR loci may be probed on the same chip with one probe site existing for each allele. (c) Locus A = 6,8 Locus B = 3,4 Locus C = 6,8 Locus D = 6,6 Figure 17.2, J.M. Butler (2005) Forensic DNA Typing, 2nd Edition © 2005 Elsevier Academic Press

Under Vacuum (<10-6 torr)
Sample Plate (Target) y x + Pulsed UV Laser Flight Tube target Detector Ion optics Under Vacuum (<10-6 torr) m/z Matrix Sample DNA +20 kV Field-free drift region (ions separate by size) Figure 17.3 Schematic of MALDI time-of-flight mass spectrometry. DNA samples are mixed with a matrix compound and dried as individual spots on a sample plate. The sample plate is then introduced to the vacuum environment of the mass spectrometer. A small portion of the sample is ionized by a UV laser pulse and the generated ions are accelerated through the ion optics. The ions separate by size as they pass down the flight tube. The mass-to-charge ratio (m/z) of each ion is detected as it impacts the detector. Figure 17.3, J.M. Butler (2005) Forensic DNA Typing, 2nd Edition © 2005 Elsevier Academic Press

Figure 17.4 Mass spectrum of a TH01 allelic ladder obtained with a time-of-flight mass spectrometer (Butler 1998). The ladder contains alleles 5, 6, 7, 8, 9, 9.3, and 10. It was generated by re-amplifying an AmpFlSTR® Green I allelic ladder mix using primers that bind close to the repeat region. The PCR product size of allele 10 is 83 bp with a measured mass of 20,280 Da. The separation time in the mass spectrometer for allele 10 is only 204 microseconds! The allele 9.3 and allele 10 peaks, which are a single nucleotide apart, differ by 1.5 microseconds on a separation time scale and can be fully resolved with this method. Figure 17.4, J.M. Butler (2005) Forensic DNA Typing, 2nd Edition © 2005 Elsevier Academic Press

STRess2 Pendulum Sequencer data
TrueAllele or Genescan/Genotyper or GeneMapperID STRess2 Pendulum Batch validity assessment Ladder sizing / designation A u d i t n g Mixture analysis Convicted offender interpretation Crimestain interpretation Complex interpretation Complex search algorithms Cross-contamination check Figure 17.5 Modules in STRess2 showing data flow. Figure courtesy of Martin Bill, Forensic Science Service. Locomation Evidential algorithms Results Database Court report Figure 17.5, J.M. Butler (2005) Forensic DNA Typing, 2nd Edition © 2005 Elsevier Academic Press

Chapter 18

(Tallahassee, Florida)
NDIS (FBI Laboratory) SDIS (Richmond, Virginia) (Tallahassee, Florida) LDIS (Tampa) (Orlando) (Broward County) (Roanoke) (Norfolk) (Fairfax) National Level State Level Figure 18.1 Schematic of the three tiers in the Combined DNA Index System (CODIS). DNA profile information begins at the local level, or Local DNA Index System (LDIS), and then can be uploaded to the state level, or State DNA Index System (SDIS), and finally to the national level, or National DNA Index System (NDIS). Each local or state laboratory maintains its portion of CODIS while the FBI Laboratory maintains the national portion (NDIS). Local Level Figure 18.1, J.M. Butler (2005) Forensic DNA Typing, 2nd Edition © 2005 Elsevier Academic Press

Chapter 19

Collect data and calculate the test statistic (S)
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) Figure 19.1 Flow chart illustrating the steps in hypothesis testing. The null hypothesis (H0) is mutually exclusive of the alternative hypothesis (H1). Adapted from Graham (2003). Figure 19.1, J.M. Butler (2005) Forensic DNA Typing, 2nd Edition © 2005 Elsevier Academic Press

Truth about the population Decision based on sample examined
(A) Hypothesis Testing Decisions Truth about the population Decision based on sample examined H0 True H1 True Correct decision Type II error Type I error Accept H0 Reject H0 (Accept H1) (B) Example Figure 19.2 (A) Comparison of decisions based on hypothesis testing and the relationship of type I and type II errors. (B) Example demonstrating how type I and type II errors correlate to false positive and false negative results. Adapted from Graham (2003). Defendant Not Guilty Guilty Courtroom Verdict Correct decision Wrongfully acquitted Wrongfully accused Not Guilty Guilty Figure 19.2, J.M. Butler (2005) Forensic DNA Typing, 2nd Edition © 2005 Elsevier Academic Press

Father gametes (sperm)
Resulting genotype combinations and frequencies Mother gametes (egg) AA Aa p2 2pq aa q2 A a p q AA aA A p2 qp p Father gametes (sperm) a Aa aa q pq q2 Figure 19.3 A cross-multiplication (Punnett) square showing Hardy-Weinberg frequencies resulting from combining two alleles A and a with frequencies p and q, respectively. Note that p + q = 1 and that the Hardy-Weinberg genotype proportions are simply a binomial expansion of (p+q)2, or p2 + 2pq + q2. Punnett square Freq (A) = p Freq (a) = q p + q = 1 (p + q)2 = p2 + 2pq + q2 Figure 19.3, J.M. Butler (2005) Forensic DNA Typing, 2nd Edition © 2005 Elsevier Academic Press

AA aa p2 q2 Aa 2pq Frequency of A allele (p) 1.0 0.8 0.6 0.4 0.2 0.0
Frequency of genotype in population 0.4 Figure 19.4 Graph depicting genotype frequencies for AA, Aa, and aa when Hardy-Weinberg equilibrium conditions are met. The highest amount of heterozygotes Aa are observed when alleles frequencies for both A and a are 0.5. Adapted from Hartl and Clark (1997). 0.2 0.0 0.2 0.4 0.6 0.8 1.0 Frequency of a allele (q) Figure 19.4, J.M. Butler (2005) Forensic DNA Typing, 2nd Edition © 2005 Elsevier Academic Press

Chapter 20

Decide on Number of Samples and Ethnic/Racial Grouping
Gather Samples Get IRB approval 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 independence Linkage equilibrium for locus independence Usually >100 per group (see Table 20.1) Use Database(s) to Estimate an Observed 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) and Chapter 15 (STR typing/interpretation) Examination of genetic distance between populations Figure 20.1 Steps in generating and validating a population database that can then be used to estimate the frequency of an observed DNA profile in the population. Figure 20.1, J.M. Butler (2005) Forensic DNA Typing, 2nd Edition © 2005 Elsevier Academic Press

Figure 20.2 Screenshot of the DNATYPE computer program developed by Ranajit Chakraborty, David Stivers, and Yixi Zhong in the 1990’s and widely used for original analysis of much of the RFLP and STR data generated by the North American forensic community. It is a DOS based program that has an Microsoft Windows user interface added by Snehit Cherian and Robert Gaensslen through funding by the National Institute of Justice. Tests performed with this program include checking for duplicate sample types in databases, single locus tests for Hardy-Weinberg equilibrium, multiple locus tests for linkage equilibrium, and genetic distances between pairs of population databases. Figure 20.2, J.M. Butler (2005) Forensic DNA Typing, 2nd Edition © 2005 Elsevier Academic Press

Figure 20.3 Histogram comparison of allele frequencies for three different U.S. population groups for alleles observed at the STR locus D13S317 (see Appendix II). Alleles are indicated by repeat number. Note that a single observation was made of an allele 15 in both the Caucasian and Hispanic sample sets (asterisks). Since these are below the minimum allele threshold of 5 observations, then the value used in performing frequency estimates would be [5/2(302)] for the Caucasians and [5/2(140)] for the Hispanics. * * Figure 20.3, J.M. Butler (2005) Forensic DNA Typing, 2nd Edition © 2005 Elsevier Academic Press

How Statistical Calculations are Made
Generate data with set(s) of samples from desired population group(s) – Generally only 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

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

Individual Genotypes Are Summarized and Converted into Allele Frequencies
The 11,12 genotype was seen 54 times in 302 samples (604 examined chromosomes) Table 20.2, J.M. Butler (2005) Forensic DNA Typing, 2nd Edition © 2005 Elsevier Academic Press

Allele Frequency Tables
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. Allele Frequency Tables Butler et al. (2003) JFS 48(4): Einum et al. (2004) JFS 49(6) D3S1358 Caucasian Caucasian African American African American Allele 11 13 14 15 15.2 16 17 18 19 12 N= 302 N= 7,636 Allele 11 13 14 15 15.2 16 17 18 19 12 N=258 N= 7,602 0.0017* 0.0009 -- 0.0003* 0.0007 0.0017* -- 0.0045 0.0031 -- 0.0019* 0.0077 Most common allele 0.1027 0.1240 0.0892 0.0905 0.2616 -- 0.2690 -- 0.3023 0.2920 0.0019* 0.0010 0.2533 0.2430 0.3353 0.3300 0.2152 0.2000 0.2054 0.2070 0.1460 0.0601 0.0630 0.0125 0.0039* 0.0048 0.0001* 20 0.0017* 20

Chapter 21

DNA Profile Frequency with all 13 CODIS STR loci
AMEL D3 TH01 TPOX D2 D19 FGA D21 D18 CSF D16 D7 D13 D5 VWA D8 AmpFlSTR® Identifiler™ (Applied Biosystems) What would be entered into a DNA database for searching: 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 Locus allele value 1 in Combined D3S1358 16 0.2533 17 0.2152 9.17 VWA 0.2815 18 0.2003 8.87 81 FGA 21 0.1854 22 0.2185 12.35 1005 D8S1179 12 14 0.1656 16.29 16,364 D21S11 28 0.1589 30 0.2782 11.31 185,073 D18S51 0.1374 0.1391 26.18 4,845,217 D5S818 0.3841 13 0.1407 9.25 44,818,259 D13S317 11 0.3394 0.0480 30.69 1.38 x 109 D7S820 9 0.1772 31.85 4.38 x 1010 D16S539 0.1126 0.3212 13.8 6.05 x 1011 THO1 6 0.2318 18.62 1.13 x 1013 TPOX 8 0.5348 3.50 3.94 x 1013 CSF1PO 10 0.2169 21.28 8.37 x 1014 P R O D U C T L E The Random Match Probability for this profile in the U.S. Caucasian population is 1 in 837 trillion (1012)

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): ( *

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

Example Calculations with Population Substructure Adjustments

Example Calculations with Corrections for Relatives

Chapter 22

D13S317 A1 A2 A3 Possible Alleles Suspect Included Suspect Excluded 15
Evidence 11,14 11,12 11,11 11,13 A1 14 13 A2 12 A3 11 Figure 22.1 Hypothetical example of a mixture with STR data at the D13S317 locus. The crime scene evidence sample possesses alleles 11, 12, and 14. 10 Allele 13 is not possible based on evidence showing only alleles 11, 12, and 14 9 8 Figure 22.1, J.M. Butler (2005) Forensic DNA Typing, 2nd Edition © 2005 Elsevier Academic Press

D13S317 PE = 2pq + q2 A1 A2 A3 Let p = A1 + A2 + A3 (with q = 1-p)
Possible Alleles A Freq (p) PE = 2pq + q2 15 * Evidence A1 14 p = p(A11) + p(A12) + p(A14) = = q = 1-p = = 13 A2 12 A3 11 Figure 22.2 Calculation of probability of exclusion with a mixture example. PE = 2( )( ) + ( )2 = = 10 9 8 From Table 20.2 Figure 22.2, J.M. Butler (2005) Forensic DNA Typing, 2nd Edition © 2005 Elsevier Academic Press

FGA Figure 22.3 Mixture profile at the STR locus FGA from a 3-person mixture. Genotypes and peak heights in relative fluorescence units are shown underneath each peak. The peak height variation observed can be used to tentatively associate alleles 20 and 22 as coming from the same individual, alleles 18.2 and 23 as coming from the second contributor, and alleles 24 and 27 as coming from the third contributor. Figure 22.3, J.M. Butler (2005) Forensic DNA Typing, 2nd Edition © 2005 Elsevier Academic Press

Chapter 23

? ? (A) Parentage (Paternity) Testing Random man Alleged father Mother
(known parent) ? Rules of Inheritance Child has two alleles for each autosomal marker (one from mother and one from biological father) Child will have mother’s mitochondrial DNA haplotype (barring mutation) Child, if a son, will have father’s Y-chromosome haplotype (barring mutation) child (B) Reverse Parentage Testing (Missing Persons Investigation) Alleged father Alleged mother Figure 23.1 Illustration of question being asked with (A) parentage testing and (B) reverse parentage testing. The most common form of parentage testing, namely paternity testing, uses results from a mother and a child to answer the question if the alleged father could have fathered the child versus a random man. With a reverse parentage test, DNA types from one or both parents are used to determine if an observed type could have resulted from a child of the alleged father and mother. ? Missing child Figure 23.1, J.M. Butler (2005) Forensic DNA Typing, 2nd Edition © 2005 Elsevier Academic Press

(A) Mendelian Inheritance mother father
C,D A,B B,C mother father child (B) Example 11,14 8,12 12,14 11,12 8,14 8,11 Obligate paternal allele 14 11 Figure 23.2 (A) Mendelian inheritance patterns with a mother possessing alleles A and B contributing one of them to the child while the father who possesses alleles C and D also contributes one of his alleles to the child. (B) An example pedigree for a family where the parents possess different alleles enabling identification of the obligate paternal allele in each of the children. This scenario can become more complicated to interpret if mutations occur or maternal and paternal alleles are shared. Figure 23.2, J.M. Butler (2005) Forensic DNA Typing, 2nd Edition © 2005 Elsevier Academic Press

Chapter 24

(toothbrush believed to have belonged to the victim)
DNA profile from mass disaster victim direct reference (toothbrush believed to have belonged to the victim) (A) Direct comparison (B) Kinship analysis D5S818 D13S317 D7S820 D16S539 CSF1PO Penta D ? son wife victim 10,10 9,10 9,13 8,9 8,14 11,13 10,12 8,10 8,12 9,12 9,9 11,14 12,13 ?,10 9,? ?,13 ?,14 11,? or ?,13 victim (father) actual profile Predicted victim profile mass disaster victim profile Figure 24.1 Example demonstrating the use of reference samples in mass disaster victim identification using DNA typing. (A) Direct comparison involves analysis of a direct reference sample from some kind of personal effect of the victim. (B) Kinship analysis utilizes close biological relatives, such as those illustrated in Figure 24.2, to reconstruct a victim’s DNA profile. Figure 24.1, J.M. Butler (2005) Forensic DNA Typing, 2nd Edition © 2005 Elsevier Academic Press

Missing Individual Spouse Son Daughter Brother Sister Father Mother
Niece Nephew Grandmother Uncle Aunt Cousin Figure 24.2 Direct biological relatives that can provide valuable reference samples for aiding identification of the missing individual. Ideally samples are available from multiple relatives to help establish robust kinship. A sample from a spouse is only valuable in connection with a child’s sample in order to determine the expected alleles coming from the missing individual. Of course, more extended family members, with direct maternal or paternal linkage, can provide helpful samples when mitochondrial DNA or Y-chromosome testing is performed. Samples that would be valuable for the maternally transmitted mitochondrial DNA are indicated in dashed boxes. Figure 24.2, J.M. Butler (2005) Forensic DNA Typing, 2nd Edition © 2005 Elsevier Academic Press

Identification of 229 victims
1277 crash scene samples 229 VICTIMS 310 Relative Reference Samples 89 Personal Effects Collections using FTA paper (from 22 countries) RELATIONAL DATABASE 9 STR loci typed (Profiler Plus) Sorted groups 9 STR loci tested (Profiler Plus) 4 additional STR loci tested (COfiler) 4 additional STR loci tested (COfiler) Identification of 229 victims Database query Figure 24.3 Strategy for disaster victim identification in the Swissair Flight 111 crash scene investigation. Most samples were tested with 9 STRs and amelogenin using the AmpFlSTR Profiler Plus kit and then 4 additional STRs were added with the COfiler kit as needed to obtain a higher power of discrimination between closely related individuals. The relational database compared genotypes of 1277 crash scene samples to genotypes of all 229 victims and family relatives, a total of 71,490 genotype comparisons (Leclair et al. 1999). Figure 24.3, J.M. Butler (2005) Forensic DNA Typing, 2nd Edition © 2005 Elsevier Academic Press

(A) Material Flow NYSP (Albany) OCME Myriad Bode Orchid Cellmark
Figure 24.4 Illustration of (A) material and (B) data flow between laboratories involved in processing World Trade Center samples. Laboratories included Office of the Chief Medical Examiner (OCME, New York City), New York State Police (NYSP, Albany, NY), Myriad Genetics (Salt Lake City, UT), Orchid Cellmark (Dallas, TX), Celera Genomics (Rockville, MD), and Bode Technology Group (Springfield, VA). Physical materials shipped between laboratories included DNA extracts (solid red line), buccal swabs from biological relative reference samples (dashed red line), personal effects (dotted red line), recovered bones (solid blue line), and recovered tissue (dashed blue line). Note that most of the DNA samples were extracted at the NYSP and OCME laboratories although some tissue and bone were shipped directly to Myriad and Bode. All bones were extracted at Bode. Figure courtesy of National Institute of Justice World Trade Center Kinship and Data Analysis Panel and New York City Office of the Chief Medical Examiner. Celera Extract Swab Personal Effect Bone Tissue Figure 24.4, J.M. Butler (2005) Forensic DNA Typing, 2nd Edition © 2005 Elsevier Academic Press

(B) Data Flow NYSP (Albany) OCME Myriad Bode Orchid Cellmark Celera QC
Figure 24.4 (cont.) SEE PREVIOUS SLIDE FOR FIGURE CAPTION QC Check QC Check WTC CODIS M-FISys Figure 24.4, J.M. Butler (2005) Forensic DNA Typing, 2nd Edition © 2005 Elsevier Academic Press

Appendices I-VII

PCR product size (bp) Figure A7.1 Profiler Plus green loci for the victim, suspect, and differential extraction female and male fractions from Case A. A mixture with a minor component consistent with the suspect is observed in the epithelial-rich (female) fraction (see third panel). Arrows indicate where stutter filters have removed allele calls. Figure A7.1, J.M. Butler (2005) Forensic DNA Typing, 2nd Edition © 2005 Elsevier Academic Press

(A) Y-PLEX 6 (FAM-labeled loci)
PCR product size (bp) Figure A7.2 (A) Y-PLEX 6 results from FAM-labeled (blue) loci on evidence, victim, and suspect samples in Case B. No PCR products were observed in the female victim sample since it does not contain a Y-chromosome. Figure A7.2, J.M. Butler (2005) Forensic DNA Typing, 2nd Edition © 2005 Elsevier Academic Press

(B) Y-PLEX 6 (TAMRA-labeled loci)
PCR product size (bp) Figure A7.2 (B) Y-PLEX 6 results from TAMRA-labeled (yellow) loci on evidence, victim, and suspect samples in Case B. Figure A7.2, J.M. Butler (2005) Forensic DNA Typing, 2nd Edition © 2005 Elsevier Academic Press

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