A Guide to Forensic DNA Profiling 1st Edition

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The increasingly arcane world of DNA profiling demands that those needing to understand at least some of it must find a source of reliable and understandable information. Combining material from the successful Wiley Encyclopedia of Forensic Science with newly commissioned and updated material, the Editors have used their own extensive experience in criminal casework across the world to compile an informative guide that will provide knowledge and thought-provoking articles of interest to anyone involved or interested in the use of DNA in the forensic context.

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AGUIDETO FORENSIC DNAPROFILING

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AGUIDETO FORENSIC DNAPROFILING Editors Allan Jamieson Scott Bader TheForensicInstituteGlasgowUK

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This edition frst published 2016 © 2016 John Wiley Sons Ltd Registeredoffce John Wiley Sons Ltd The Atrium Southern Gate Chichester West Sussex PO19 8SQ United Kingdom For details of our global editorial offices for customer services and for information about how to apply for permission to reuse the copyright material in this book please see our website at www.wiley.com. The right of the authors to be identifie as the authors of this work has been asserted in accordance with the Copyright Designs and Patents Act 1988. All rights reserved. No part of this publication may be reproduced stored in a retrieval system or transmitted in any form or by any means electronic mechanical photocopying recording or otherwise except as permitted by the UK Copyright Designs and Patents Act 1988 without the prior permission of the publisher. Wiley also publishes its books in a variety of electronic formats. Some content that appears in print may not be available in electronic books. Designations used by companies to distinguish their products are often claimed as trademarks. All brand names and product names used in this book are trade names service marks trademarks or registered trademarks of their respective owners. The publisher is not associated with any product or vendor mentioned in this book. This publication is designed to provide accurate and authoritative information in regard to the subject matter covered. It is sold on the understanding that the publisher is not engaged in rendering professional services. If professional advice or other expert assistance is required the services of a competent professional should be sought. The Publisher and the Authors make no representations or warranties with respect to the accuracy or completeness of the contents of this work and specificall disclaim all warranties including without limitation any implied warranties offtness for a particular purpose. The advice and strategies contained herein may not be suitable for every situation. In view of ongoing research equipment modifications changes in governmental regulations and the constant fl w of information relating to the use of experimental reagents equipment and devices the reader is urged to review and evaluate the information provided in the package insert or instructions for each chemical piece of equipment reagent or device for among other things any changes in the instructions or indication of usage and for added warnings and precautions. The fact that an organization or Website is referred to in this work as a citation and/or a potential source of further information does not mean that the author or the publisher endorses the information the organization or Website may provide or recommendations it may make. Further readers should be aware that Internet Websites listed in this work may have changed or disappeared between when this work was written and when it is read. No warranty may be created or extended by any promotional statements for this work. Neither the Publisher nor the Author shall be liable for any damages arising herefrom. Chapters whose authors are US Government employees are © US Government in North America and © John Wiley Sons in the rest of the world. The views expressed by those authors who are US Government employees do not necessarily reflec the views of the US Government Agencies they work for. LibraryofCongressCataloging-in-PublicationData Names: Jamieson Allan editor. | Bader Scott editor. Title: A guide to forensic DNA profilin / edited by Allan Jamieson Scott Bader. Description: Hoboken : Wiley 2016. | Includes bibliographical references and index. Identifiers LCCN 2015040516 print | LCCN 2015040674 ebook | ISBN 9781118751527 hardback | ISBN 9781118751503 pdf | ISBN 9781118751510 epub Subjects: LCSH: DNA fngerprinting. Classification LCC RA1057.55.G85 2016 print | LCC RA1057.55 ebook | DDC 614/.1–dc23 LC record available at http://lccn.loc.gov/2015040516 A catalogue record for this book is available from the British Library. Cover Image: Bart Sadowski/Getty Typeset in 9.5∕11.5 pt Times by SPi Global Chennai India Printed and bound in Singapore by Markono Print Media Pte Ltd. This book is printed on acid-free paper responsibly manufactured from sustainable forestry in which as least two trees are planted for each one used for paper production.

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Contents Contrib utors ix F or ew ord xiii Pr eface xv Glossary xvii Ab br e viations and Acr onyms xxi P art A: Backgr ound 1 1 Introduction to Forensic Genetics ScottBader 3 2 DNA: An Overview EleanorAlisonMayGraham 9 3DNA SimonJ.Walsh 29 4 Introduction to Forensic DNA Profilin – The Electropherogram epg AllanJamieson 37 5 Biological Stains PeterR.Gunn 51 6 Sources of DNA Sally-AnnHarbison 59 7 Identificatio and Individualization ChristopheChampod 69 8 Transfer GeorginaE.Meakin 73 9 Laboratory Accreditation AllanJamieson 79

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vi Contents 10 Validation CampbellA.Ruddock 85 P art B: Analysis Inter pr etation 97 11 Extraction CampbellA.Ruddock 99 12 Quantitation RobertI.O’Brien 107 13 Amplificatio CampbellA.Ruddock 115 14 Interpretation of Mixtures Graphical AllanJamieson 119 15 DNA Mixture Interpretation DanE.Krane 133 16 Degraded Samples JasonR.Gilder 141 17 Ceiling Principle: DNA SimonJ.Walsh 147 18 Y-Chromosome Short Tandem Repeats JackBallantyneandErinK.Hanson 149 19 Expert Systems in DNA Interpretation HindaHanedandPeterGill 155 20 Paternity Testing BurkhardRolfandPeterWiegand 163 21 Observer Effects WilliamC.Thompson 171 P art C: A pplications 175 22 Databases SimonJ.Walsh 177 23 Missing Persons and Paternity: DNA BruceS.Weir 185 24 Familial Searching KlaasSlootenandRonaldMeester 195 25 Single Nucleotide Polymorphism ClausBørstingVaniaPereiraJeppeD.AndersenandNielsMorling 205 26 Mini-STRs MichaelD.CobleandRebeccaS.Just 223

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Contents vii 27 Phenotype TonyFrudakis 229 28 Mitochondrial DNA: Profilin TerryMelton 245 29 Geographical Identificatio by Viral Genotyping HiroshiIkegayaPekkaJ.SaukkoYoshinaoKatsumataandTakehikoTakatori 251 30 Microbial Forensics BruceBudowleandPhillipC.Williamson 259 31 Wildlife Crime LucyM.I.Webster 271 P art D: Court 277 32 DNA Databases – The Significanc of Unique Hits and the Database Controversy RonaldMeester 279 33 DNA Databases and Evidentiary Issues SimonJ.WalshandJohnS.Buckleton 287 34 Communicating Probabilistic Forensic Evidence in Court JonathanJ.Koehler 297 35 Report Writing for Courts RhondaM.Wheate 309 36 Discovery of Expert Findings RhondaM.Wheate 315 37 Ethical Rules of Expert Behavior AndreA.Moenssens 323 38 Verbal Scales: A Legal Perspective TonyWard 329 39 Direct Examination of Experts AndreA.Moenssens 335 40 Cross-Examination of Experts AndreA.Moenssens 339 41 DNA in the UK Courts RhondaM.Wheate 343 42 Legal Issues with Forensic DNA in the USA ChristopherA.Flood 355 43 Issues in Forensic DNA AllanJamieson 369 44 Future Technologies and Challenges AllanJamieson 381 Index 393

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Contributors Jeppe D . Andersen UniversityofCopenhagenCopenhagenDenmark Chapter 25: Single Nucleotide Polymorphism Scott Bader TheForensicInstituteGlasgowUK Chapter 1: Introduction to Forensic Genetics Jack Ballantyne UniversityofCentralFloridaandNationalCenterforForensicScienceOrlando FLUSA Chapter 18: Y-Chromosome Short Tandem Repeats Claus Børsting UniversityofCopenhagenCopenhagenDenmark Chapter 25: Single Nucleotide Polymorphism John S. Buckleton InstituteofEnvironmentalScienceandResearchLtd.AucklandNewZealand Chapter 33: DNA Databases and Evidentiary Issues Bruce Budo wle UniversityofNorthTexasHealthScienceCenterFortWorthTXUSA Chapter 30: Microbial Forensics Christophe Champod UniversityofLausanneInstitutdePoliceScientifqueLausanneSwitzerland Chapter 7: Identificatio and Individualization Michael D . Coble TheArmedForcesDNAIdentifcationLaboratoryRockvilleMDUSA Chapter 26: Mini-STRs Christopher A. Flood FederalDefendersofNewYorkInc.NewYorkNYUSA Chapter 42: Legal Issues with Forensic DNA in the USA Tony Frudakis DNAPrintGenomicsInc.SarasotaFLUSA Chapter 27: Phenotype Jason R. Gilder ForensicBioinformaticsFairbornOHUSA Chapter 16: Degraded Samples Peter Gill NorwegianInstituteofPublicHealthOsloNorway UniversityofOsloOsloNorway Chapter 19: Expert Systems in DNA Interpretation

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x Contrib utors Eleanor Alison May Graham NorthumbriaUniversityNewcastleuponTyneUK Chapter 2: DNA: An Overview Peter R. Gunn UniversityofTechnologySydneyBroadwayNewSouthWalesAustralia Chapter 5: Biological Stains Hinda Haned NetherlandsForensicInstituteTheHagueTheNetherlands Chapter 19: Expert Systems in DNA Interpretation Erin K. Hanson University of Central Florida and National Center for Forensic Science OrlandoFLUSA Chapter 18: Y-Chromosome Short Tandem Repeats Sally-Ann Harbison InstituteofEnvironmentalScienceandResearchLtd.AucklandNewZealand Chapter 6: Sources of DNA Hir oshi Ik egaya KyotoPrefecturalUniversityofMedicineKyotoJapan Chapter 29: Geographical Identificatio by Viral Genotyping Allan Jamieson TheForensicInstituteGlasgowUK Chapter 4: Introduction to Forensic DNA Profilin – The Electropherogram epg Chapter 9: Laboratory Accreditation Chapter 14: Interpretation of Mixtures Graphical Chapter 43: Issues in Forensic DNA Chapter 44: Future Technologies and Challenges Rebecca S. Just TheArmedForcesDNAIdentifcationLaboratoryRockvilleMDUSA Chapter 26: Mini-STRs Y oshinao Katsumata NationalInstituteofPoliceScienceTokyoJapan NagoyaIsenNagoyaJapan Chapter 29: Geographical Identificatio by Viral Genotyping Jonathan J . Koehler NorthwesternUniversitySchoolofLawChicagoILUSA Chapter 34: Communicating Probabilistic Forensic Evidence in Court Dan E. Krane WrightStateUniversityDaytonOHUSA Chapter 15: DNA Mixture Interpretation Geor gina E. Meakin UniversityCollegeLondonLondonUK Chapter 8: Transfer Ronald Meester VUUniversityAmsterdamAmsterdamTheNetherlands Chapter 24: Familial Searching Chapter 32: DNA Databases – The Significanc of Unique Hits and the Database Controversy

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Contrib utors xi Terry Melton MitotypingTechnologiesStateCollegePAUSA Chapter 28: Mitochondrial DNA: Profilin Andr e A. Moenssens UniversityofMissouriatKansasCityKansasCityMOUSA UniversityofRichmondRichmondVAUSA Chapter 37: Ethical Rules of Expert Behavior Chapter 39: Direct Examination of Experts Chapter 40: Cross-Examination of Experts Niels Morling UniversityofCopenhagenCopenhagenDenmark Chapter 25: Single Nucleotide Polymorphism Robert I. O’Brien NationalForensicScienceTechnologyCenterNFSTCLargoFL USA Chapter 12: Quantitation Vania Per eira UniversityofCopenhagenCopenhagenDenmark Chapter 25: Single Nucleotide Polymorphism Burkhard Rolf EurofnsMedigenomixForensikGmbHEbersbergGermany Chapter 20: Paternity Testing Campbell A. Ruddock OklahomaCityPoliceDepartmentForensicDNAunitOklahomaCity OKUSA Chapter 10: Validation Chapter 11: Extraction Chapter 13: Amplificatio Pekka J . Saukk o UniversityofTurkuTurkuFinland Chapter 29: Geographical Identificatio by Viral Genotyping Klaas Slooten VUUniversityAmsterdamAmsterdamTheNetherlands NetherlandsForensicInstituteTheHagueTheNetherlands Chapter 24: Familial Searching Tak ehik o Takatori NationalInstituteofPoliceScienceTokyoJapan Chapter 29: Geographical Identificatio by Viral Genotyping William C. Thompson UniversityofCaliforniaIrvineCAUSA Chapter 21: Observer Effects Simon J . Walsh AustralianFederalPoliceCanberraACTAustralia Chapter 3: DNA Chapter 17: Ceiling Principle: DNA Chapter 22: Databases Chapter 33: DNA Databases and Evidentiary Issues

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xii Contrib utors Tony Ward UniversityofHullHullUK Chapter 38: Verbal Scales: A Legal Perspective Lucy M.I. Webster ScienceandAdviceforScottishAgricultureEdinburghUK Chapter 31: Wildlife Crime Bruce S. Weir UniversityofWashingtonSeattleWAUSA Chapter 23: Missing Persons and Paternity: DNA Rhonda M. Wheate TheForensicInstituteGlasgowUK Chapter 35: Report Writing for Courts Chapter 36: Discovery of Expert Findings Chapter 41: DNA in the UK Courts Peter Wiegand UniversityHospitalofUlmUlmGermany Chapter 20: Paternity Testing Phillip C. Williamson UniversityofNorthTexasHealthScienceCenterFortWorthTXUSA Chapter 30: Microbial Forensics

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Foreword My contact with Professor Jamieson and Dr Bader or Allan and Scott as I now know them and shall refer to them began in the seminal trial of Sean Hoey in relation to the Omagh Bombing in Northern Ireland in 2007. This was the firs serious challenge in the United Kingdom to the use of low copy number LCN DNA profiling the frst form of what has become more generally known as low template DNA profiling Although Mr Hoey was primarily acquitted as a result of reservations surrounding the way in which key exhibits were seized stored and examined the learned trial judge the Honorable Mr Justice Weir raised concerns in relation to the reliability of interpreting LCN DNA. Such concerns were no doubt borne out of the points we advanced on behalf of Mr Hoey which were in turn borne out of the concerns of those from The Forensic Institute. From the outset The Forensic Institute expressed the strongest of reservations as to the reliability of interpreting such minute amounts of DNA found on the relevant exhibits none of which were from the Omagh incident as it happens. These concerns caused a seismic response in scientifi and legal circles which continues to this day. Toward the end of 2014 I had the pleasure of working with Allan and Scott in a murder trial at the Old Bailey. They were instructed on behalf of the defense to comment upon the reliability and inter- pretability of low template DNA recovered from a murder scene. In this recent case part of the argu- ment focused upon the reliability of the methods employed by the prosecution to quantify the probative value of such low amounts of DNA. One of the methods employed involved the use of software which was said to overcome the complex nature of the results another was the “counting method.” Following the cross-examination of one of the prosecution’s lead forensic scientists the Crown withdrew the DNA evidence in the case. There is no doubt that for lawyers DNA profilin can present a daunting challenge. This is not only in understanding the science involved but also in knowing how best to present the results in a way that can be easily understood. I am indebted to Allan and Scott for guiding our legal teams through the morass of graphs statistics and terminology enabling us to be able to properly represent our clients on the most serious of allegations. I am optimistic that the clarity of their approach and the appreciation of the needs of the non-specialist will be reflecte in the content of this book. I am also delighted to learn that this will be one of the few books that brings together the scientifi and legal aspects of DNA profilin in such a comprehensive approach. That is not an easy task but I know that the Editors had assistance from Professor Andre Moenssens of the Wiley Encyclopedia of Forensic Science where many of the articles in this book originate. Needless to say I write this having not read all of the articles in this work but I am confiden that if the skills I have taken advantage of in our casework are taken into the production of this work then it will provide a valuable resource for both lawyers and experts alike in the continuing quest to tackle the increasingly complex issues involved in forensic DNA profiling A lawyer writing a preface for a book written by scientists Progress indeed. Kieran Vaughan QC Garden Court Chambers

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Preface Forensic DNA profilin has revolutionized forensic science. However from relatively simple beginnings using what would now be regarded as huge amounts of sample e.g. bloodstain not only has the underlying technology changed i.e. RFLP to STRs and SNPs but the complexity of the interpretation of the analytical results has increased in the quest to get more information from smaller and more complex samples. Most of these developments are published and debated in the scientifi literature although some are guarded for ostensibly commercial reasons or sometimes it seems simply to avoid showing one’s hand to the other side in an adversarial legal system. Much of the scientifi and statistical debate remains active and there is no settled position. Indeed it could be contended that in many of these arguments each side has a rational and reasoned position simply different to their opponents. This book does not seek to provide or claim to have the fnal answer on any of these because for many issues there is none. In recognition of the state of fux within parts of the discipline we have not sought to provide only our view or indeed the view of any author as the fina word and therefore no article can be taken to represent the view of anyone other than the authors of the article at the time of writing. Views in some articles may contradict views in others that is a reflectio of the state of the art and is common in science. Although some articles in this work were created specificall with this book in mind the vast majority of articles are from the Wiley Encyclopedia of Forensic Science. 1 The consequence of this is that there is inevitably some duplication of information. However because we intend that each article can stand alone we consider that such duplication as exists simply adds to the utility of the book. Forensic science operates by definition within a legal context. This creates several problems in creating a volume like this one. Different jurisdictions may have different legal requirements of the expert and even the experts may have local practices that differ from other localities nationally or internationally. Even within the United States and United Kingdom depending on the level of court there are widely differing expectations and standards for the admissibility of scientifi evidence e.g. Frye Daubert or none. We cannot expect to cover all of the variances and so the articles other than where specificall addressing jurisdictional issues should be taken as informing on the generality of practices. The dichotomy between legal and scientifi standards is perhaps best illustrated in the NAS report of 2009 “The bottom line is simple: In a number of forensic science disciplines forensic science professionals have yet to establish either the validity of their approach or the accuracy of their conclusions and the courts have been utterly ineffective in addressing this problem. For a variety of reasons – including the rules governing the admissibility of forensic evidence the applicable standards governing appellate review of trial court decisions the limitations of the adversary process and the common lack of scientifi expertise among judges and lawyers who must try to comprehend and evaluate forensic evidence – the legal system is ill-equipped to correct the problems of the forensic science community.” For those reasons and others e.g. availability of other evidence we would caution as have others against using any court decision as validation or invalidation of any scientifi test. It is not unknown for different courts 1 www.wileyonlinelibrary.com/ref/efs

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xvi Pr eface within the same jurisdiction to rule both ways on the same science for example the use of low template DNA in New York City. Thus this volume sets out to provide a comprehensive introduction to the scientific statistical and legal issues within the context of forensic DNA profiling The rate of development of the feld is so great that almost any publication will be out of date within a very short time. However the information provided here will provide a solid foundation from which future developments can be understood and evaluated. Allan Jamieson Scott Bader July 2015

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Glossary accr editation recognition of procedural management at an institution allele one of alternative forms of a genetic marker component/DNA type amplificatio increase in amount of sample DNA created by PCR process amylase enzyme of saliva and to lesser extent some other body fluid AP Acid Phosphatase detected by presumptive test for seminalfuid base pair building block unit of DNA baseline the experimental zero value on the x-axis of analytical results bin part of the epg showing known allelic sizes body flui usually refers to any biological material from which DNA can be obtained b uccal derived from mouth cavity cell smallest living structure of biological organism chr omosome structure containing DNA including many genes inherited as a single unit from cell to cell and generation to generation coancestry coefficien a measure of the relatedness of two people Daubert legal standard for admissibility of expert evidence in some US states degraded DN A partially destroyed DNA usually indicated by lower or absent amounts of longer DNA components diploid possessing two alleles at each locus dr op-in appearance of DNA component in a profil due to background contamination dr op-out disappearance of DNA component from a profil due to random sampling of low level quantity electr ophor esis movement of chemical through a matrix under the force of electrical fiel

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xviii Glossary extraction in DNA casework the removal of DNA from cells Fry e legal standard for admissibility of expert evidence in some US states genotype genetic composition of an individual comprising both alleles at each/all loci haploid possessing only one allele at each locus haplotype genetic composition of an individual comprising one allele at each/all loci linked together as a inherited group hemizygous only one allele component present at a locus heter ozygous two alleles at one locus are different types homozygous two alleles at one locus are the same type HWE Hardy Weinberg Equilibrium stable frequency of alleles ISO17025 accreditation for the general requirements for the competence to carry out tests and/or calibrations including sampling ladder allelic quality control sample containing alleles of known size and run separately to other samples locus/loci pl. specifi location/entity of DNA marker or gene on a chromosome area of DNA tested in profil lo w copy number LCN very low amount of DNA in sample specificall also the increased amplificatio cycle number used for PCR method lo w template very low amount of DNA in sample micr o one millionth 10 −6 milli one thousandth 10 −3 mitochondrion intracellular structure containing mitochondrial DNA mixtur e more than one contributor DNA profiling multiplex chemistry analysing many loci mutation alteration in genetic component nano one thousand millionth 10 −9 nucleus intracellular structure containing nuclear DNA used in standard DNA profiling odds number of favourable outcomes/number of unfavourable outcomes

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Glossary xix partial pr ofil one in which all of the components do not appear Phadebas presumptive test for saliva detects amylase activity phenotype expressed/observed biological characteristic controlled by combination of alleles in genotype pico one million millionth 10 −12 polygenic controlled by several genes polymerase chemical that creates the amplificatio of DNA by PCR polymor phic many forms population in statistics any set of items under study pr esumpti v e suggestive not definit ve primer chemical that binds to specifi site locus of sample DNA to enable amplificatio in PCR pr obability number of favourable outcomes/number of possible unfavourable outcomes pull-up artifact seen in another part of DNA profil due to presence of a DNA component in one part of the profil quantitation measurement of the amount of a sample rfu relative fluorescenc unit measurement of peak height in an electropherogram sali v a body flui produced by salivary glands in mouth containing salivary amylase semen body flui produced by male ejaculation including seminal fuid and sperm cells seminal flui nutrient body flui secreted by prostate gland of males for transmission of sperm cells in ejaculate sensiti vity a a measure of how small an amount of material a technique can detect b the effect on the signal or measurement of a change in an input ability to detect and measure a sample specificit ability to discriminate an individual component of a sample sperm male sexual cell present in semen produced by testes carrying haplotype of individual stochastic effect due to random variation caused by sampling of low level sample stochastic thr eshold approximate level at which random sampling effects can be expected

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xx Glossary stutter artifact seen in DNA profil as smaller peak adjacent to main peak of real DNA component v alidation evidence of compliance/effica y for a process being fi for purpose with demonstration of capabilities and limits x-axis the horizontal axis of a graph y-axis the vertical axis of a graph

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Abbreviations and Acronyms A adenine AAFS American Academy of Forensic Sciences ABC American Board of Criminalistics ABI Applied Biosystems ACPO Association of Chief Police Offcers ADO allele dropout AIMs ancestry informative markers AP acid phosphatase APA American Psychological Association ASCLD/LAB American Society of Crime Laboratory Directors/Laboratory Accreditation Board BKV BK virus bps base pairs C cytosine CCD charged coupled device CE capillary electrophoresis CF cystic fibrosi CODIS Combined Offender DNA Index System CPI Combined Paternity Index CPI combined probability of inclusion CZE capillary zone electrophoresis DAB DNA Advisory Board ds double-stranded DTT dithiothreitol EBV Epstein–Barr virus EDNAP European DNA Profilin Group emPCR emulsion PCR ENFSI European Network of Forensic Science Institutes EPG electropherogram ESS European Standard Set EVC externally visible characteristics FBI Federal Bureau of Investigation FSS forensic science service G guanine Hb heterozygote balance ratio HBV hepatitis B virus HHV-1 human herpes virus type 1 HIV-1 human immunodeficien y virus type 1 HLA human leukocyte antigen HPHR heterozygous peak height ratio

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xxii Ab br e viations and Acr onyms HPLC high-performance liquid chromatography HPV human papillomavirus HV hypervariable HWE Hardy Weinberg Equilibrium IAI International Association for Identificatio IBD identical-by-descent IISNP individual identificatio SNP indel insertion/deletion ISFG International Society for Forensic Genetics ISO International Standards Organization JCV polyomavirus JC LCN low copy number LDO locus dropout LMD laser microdissection LoCIM locus classificatio and inference of the major LR likelihood ratio LT low-template LTDNA low template deoxyribonucleic acid MALDI/TOF matrix-assisted laser desorption/ionizationtime-of-f ight MCMC Monte Carlo Markov Chain MDA multiple displacement amplificatio MGF maternal grandfather MGM maternal grandmother MHC major histocompatibility complex MLE most likely estimate MLP multilocus probing MP match probability mRNA messenger RNA mtDNA mitochondrial deoxyribonucleic acid MW molecular weight NAS National Academy of Science USA NCIDD National Criminal identificatio DNA Database NDIS National DNA Index System NDNAD National DNA Database NFI Netherlands Forensic Institute NGS next-generation sequencing NOAA National Oceanic and Atmospheric Administration NRC National Research Council nuDNA nuclear deoxyribonucleic acid OCME Offic of the Chief Medical Examiner PCR polymerase chain reaction PE probability of exclusion PGF paternal grandfather PGM paternal grandmother PHR peak height ratio PHT peak-height threshold PML progressive multifocal leukoencephalopathy PoD probability of detection POI person of interest PSA prostate-specifi antigen

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Ab br e viations and Acr onyms xxiii QA/QC quality assurance and quality control QAS quality assurance standard rCRS revised Cambridge Reference Sequence RFID radio frequency identificatio RFLP restriction fragment length polymorphism RFU relative fuorescence unit RHC red hair color RMNE random man not excluded RMP random match probability SBE single-base extension SDIS State DNA Identificatio System SDS sodium dodecyl sulfate SFGR spotted fever group Rickettsia SGM second generation multiplex SLP single locus probes SNP single nucleotide polymorphism SOP standard operating protocol SSM slipped strand mispairing STR short tandem repeat SWG scientifi working group SWGDAM scientifi working group for DNA analysis methods T thymine UV ultraviolet VNTR variable number of tandem repeat WGA whole genome amplificatio WTC World Trade Center YHRD Y chromosome haplotype reference database

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PAR T A Backgr ound

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Chapter 1 Intr oduction to F or ensic Genetics Scott Bader TheForensicInstituteGlasgowUK The I deal F or ensic Material – Indi vidualization Forensic genetics has been touted as the gold stan- dard of forensic analysis. This is because DNA fulfil many of the criteria that make the perfect forensic tech- nology to establish a person’s presence at a scene of crime. Most forensic disciplines concerned with offences against the person and some other crimes try to estab- lish a link between items found at the scene and items found on or associated with a suspect. In other words to establish whether the recovered items could have originated from the same source. This process can be summarized as 1. Establishing a match 2. Calculating the significanc of the match The perfect conclusion of this exercise is to unequiv- ocally establish that the material from the crime scene could only have come from exactly the same source as that found on or associated with the suspect and no other source. The goal of most forensic matching is to reduce the potential population from which an item could have come to one individual within the popu- lation. This extreme is the definitio of identification The process that we are more interested in because of its more common application is that ofindividualiza- tion. This is the process of individualization. Individ- ualization is a population problem as it is necessary to be able to demonstrate how many people in a popu- lation may have the match characteristics discovered by the investigator. Therefore modern scientifi indi- vidualization techniques recognize that most if not AGuidetoForensicDNAProfling Edited by Allan Jamieson and Scott Bader © 2016 John Wiley Sons Ltd. ISBN: 9781118751527 all evidence is probabilistic which is to say that we attempt to establish a probability or likelihood that two items had a common origin. The ideal forensic material must enable matching and probability calcu- lations. There are other qualities that a forensically useful material should have.Ideally the material should be 1. Unique 2. Not change over time i.e. during normal use 3. Likely to be left at a scene in sufficien quantity to establish a match 4. Not change after being left at the scene and during subsequent examination In this book we shall see that DNA meets many but not all of these criteria and how the limitations are handled. So what makes DNA a good material forensically DN A – The Molecule DNA is sometimes called theblueprintoflife and has characteristics that are appropriate to its role. Many if not all of these characteristics are important in Forensic Genetics which is simply genetics in a legal context. These characteristics include its simplicity and yet complexity both of which are incorporated within the polymeric chemical structure of the back- bone molecule and the varied sequence of sidechain bases the so-called letters of its information content arranged in a double helix see DNA: An Overview. The molecule is made from a relatively small number of building blocks yet contains a vast amount and range of information that can defin the nature of the biolog- ical cell and ultimately the multicellular organism

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4 Backgr ound within which the DNA is located. The double helix structure is relatively stable in time yet is adaptable enough to “open up” to allow a living cell to use the contained information to go about its life func- tions transcription or to make copies of itself repli- cation. DNA is stable so as to enable transfer of the genetic information from generation to generation after replication with cell division and mating where relevant yet it can also change to varying extents. Some of the changes are important to only an indi- vidual organism and may be deleterious e.g. muta- tion giving rise to a cancer or are the basis for indi- vidual variation e.g. mutation giving rise to a new variant and the haploid segregation of chromosomes in gametes with the return of diploid pairing at fertil- ization to produce a new individual. Some changes affect a subpopulation e.g. lineages and even even- tually an entire population e.g. natural selection of mutations and new diploid combinations leading to evolutionary change. The chapter on DNA describes some fundamental concepts about DNA and genetics. In summary the genetic material of humans comprises about 3 billion nucleotides or building blocks and is present in two copies per cell so about 6 billion in total. This DNA is found within the nucleus of all cells other than red blood cells in total it is called thegenome and contains the genes that encode the proteins created by the cell to defin the cell’s type and characteristics and ulti- mately the entire organism of the human individual. It also contains other DNA sequences that are regula- tory i.e. affect the temporal or quantitative expression of the genes structural i.e. affect intracellular pack- aging and stability of DNA or are as yet of unknown function or may even be foreign to a normal human cell e.g. a viral infection. All of these elements are contained within 23 separate lengths of DNA the chro- mosomes. The concept that DNA contains the information for biological life using a genetic code encoded within the sequence of bases along the double helix molecule means that if we as forensic scientists can “read” that code we can question and determine the source of a given sample of DNA. The general DNA structure and constituents are the same so that with the right analyt- ical toolkits we are able to answer that question. So we could test not only whether the DNA is from a human horse cannabis plant or soil microbe but in theory identify the individual human. Scientists are able to take advantage of the “adaptable stability” of DNA and mimic the process of replication so as to make multiple copies of a DNA sample using the poly- merase chain reaction PCR see method. The ampli- fie DNA is then processed and the data interpreted accordingly. DN A in P opulations The frst main concept to elaborate upon is that of Mendelian genetics see Mendel mentioned inDNA. For a simple biological example I will use the ABO blood group system. Here there is a single gene involved that define a person’s blood group. The gene controls the production of a chemical on the surface of blood cells. The gene exists within the human popu- lation in one of three forms or variants: A B and O and when referring to the gene it is written italicized. The existence of variable forms within the population is called a polymorphism and these genetic variants are known scientificall as alleles. They control the production of a protein that exists respectively as either protein variant A variant B or is not produced i.e. absent and thus called O for null. In any individual the genes that encode everything that eventually produces a human being are present in two copies not including the X and Y chromosomes one inherited from mother and one inherited from father. It is the combination of the two copies of all the genes that will determine the fina characteristics of the individual. So while there might be just the one gene for the blood cell protein described above there will be two copies of the gene in each person. All of the possible genetic combinations seen in different individuals are thereforeAABBOOABAOBOand where the variants are the same the person is called homozygous where they are different the person is calledheterozygous. Going back to the description of the proteins that would be produced from the genetic variants they are as follows in the table: Gene variants Protein variants Blood group AA A only A BB B only B OO Nothing O AB AandB AB AO A only A BO B only B

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Intr oduction to F or ensic Genetics 5 The combination of gene variants possible in any human are called the genotypes firs column and the fnal observed biological characteristics in this instance the blood group are called the phenotypes. By way of illustrating the difference – the genotypes AO and AA both have the phenotype A because only A is actually observed in blood group testing the O is “silent.” When both variants in an individual are the same the genotype is called homozygous and when differentheterozygous. In this example the gene variantsA andB are what is termed codominant in that they are both observ- able in the phenotype. The O gene variant is termed recessive in that when it is present with something else the something else takes precedence and is the only characteristic observable. Here the “recessive- ness” is simply becauseO produces nothing whereas A andB produce A and B proteins. Note that while we can determine the blood group phenotype of a person from his or her genotype going in the other direction is not so simple. So a person of blood group B for example may be eitherBB orBO genotype. Knowing the frequency of the different variants present in the population allows us to predict the percentage chance that a blood group B person has eitherBB orBO geno- type. As we now analyze at the DNA level we do not need to study actual biological traits although these are under development also see Phenotype like the ABO blood group and can analyze highly poly- morphic nongene sequences see DNA DNA: An Overview. This has the advantage of comprising a much larger proportion of the genome from which to select for analysis and showing much greater variability. This greater variability leads to greater individualization such that we can identify a sample as coming from a very small handful of people if not an individual as opposed to the large sections of the population using previous technology. So modern DNA profilin kits use multiplex PCR methods to analyze several STR areas called loci at the same time. In the United Kingdom in recent years until 2014 there was a standard kit called AmpFlSTR SGM Plus used to analyze 10 loci as well as the sex-determining region but this has now been super- seded by use of one of several kits collectively called DNA17 covering the European standard 17 loci. The United States uses a set of 13 loci for criminal justice purposes the Combined DNA Index System CODIS marketed and tested in two main commercial kits AmpFlSTR Identifler and Power- Plex 16. For paternity or kinship testing purposes seeMissingPersonsandPaternity:DNA data from these loci are supplemented by other kits STR or single nucleotide polymorphisms SNPs covering even more than the core sets used in criminal justice systems. The Scientifi Expert This is a time of increasing stringency in the require- ments of experts to establish the reliability of the tech- niques that they use as well as their authority in the use of those. Many systems have been and are being developed that aim to assist the court in assessing such claims. However most of these have been by a group of experts in the same fiel forming themselves into some sort of group and deciding for themselves whether it is expertise and who they will register or endorse. External validation should be a feature of any such system. To claim a scientifi basis for an expertise the expert should be able to demonstrate for the tech- niques that they use as a minimum seeLaboratory AccreditationValidation: 1. Reliability studies of analytical technique valida- tion. The scientifi approach to this problem is normally to put a sample through the system to see howoften thesampleproducesthesameresult. This is the reproducibility of the technique its ability to provide consistent results when applied to the same samples. 2. Establishment of false positive and false negative rates. Even if the technique is perfect in many systems there is not a clear difference in the measurement when it is used to separate two or more groups. A false positive occurs when something that does not have the characteristic being sought is classifie as actually having them whereas a false negative is when something should be placed in a particular class but isn’t. Generally systems are designed to minimize the number of false positives and false negatives. However in many systems the two are inextricably linked and as one attempts to minimize one error the other increases. 3. Define match criteria. This requires a specificatio for the degree of simi- larity that two items must have before we declare a

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6 Backgr ound match. The specificit or discriminating power of a technique is “The ability of an analytical proce- dure to distinguish between two items of different origin.” 4. Probability of any match being a “true” match. Given that all matches are probable matches this leads to the further requirement to know the prob- ability that the match is to that item or material and no other. F o r ensic DN A DNA is present in many types of biological substance see Biological Stains Sources of DNA that can be analyzed by nuclear DNA profilin methods see DNA: An Overview. These substances include body fluid such as blood or semen that can be seen by the naked eye if in suffcient quantity as well as invisible amounts of the same fluid or of other substances such as skin cells in sweat or fingermark see Sources of DNA. The chemical stability of DNA is useful for forensic genetics because it means that the DNA of a biological sample may be analyzed long after it was deposited at a crimescene. This has been very useful for example in the re-examination of evidence in cold cases long after storage and with the advent of new analytical methods. If preserved under the right conditions and using the appropriate methods DNA has even been studied from ancient samples such as Egyptian pharaohs and woolly mammoths. DNA is not stable forever and again depending on circumstances after deposition at a crimescene and following collection and handling by forensic scientists it can degrade and affect the ability to get useable results seeDegradedSamples. Wherever possible the nature of a biological deposit is identifie or at least suggested so as to assist the evaluation of the significanc of a DNA profil once it has been produced. When a body flui suspected to be blood semen or saliva is present in sufficien amount so as to be visible to the naked eye the location of the substance to be tested is clear and sampling can proceed. After the stain is located it is usually subjected to a chemical test to try to determine what sort of stain it may be. These tests usually only suggest rather than indicate the presence of a biological type requiring at least one other test to be done to confir the result. For example microscopic examination of a suspected semen sample by acid phosphatase analysis that find sperm cells thus confirm a sample to be or to contain semen. Often these confirmator tests are not done to save on time and expense and the evidential weight of a sample is left as “probably an X stain using the relevant presumptive test with a DNA profil matching Mr Y .”. When there is little or nothing visible of a biological stain a search for the possible location of substances must be made. The recovered biological substance is then chem- ically processed so as to release whatever DNA is present seeExtraction. The procedure of extraction must be able to extract the DNA with minimal loss of sample due to the usually small sample collected and can be done manually or automated. The extracted and purifie DNA is then neither in suffcient quan- tity nor in a form so as to be studied directly hence the succeeding stages of quantitation and amplifica tion see Quantitation Amplifcation. The quantifi cation step determines how much DNA is present in the sample so that an appropriate amount can be used in the amplificatio step – suffcient to be analyzed and not so much to overload the system. The amplificatio step involves a method calledPCR and makes multiple copies of the relevant areas of DNA being profile while “marking” them chemi- cally with markers or labels to enable detection by machine. The amplifie and labeled DNA fragments are at this point in a liquid mixture that must be sepa- rated to enable detection and measurement. It is there- fore forced by an electrical voltage through a tube containing a molecular sieve or gel a process called electrophoresis. This process separates the pieces of DNA according to size length such that smaller pieces pass more quickly through the gel than large ones. As the pieces pass a specifi point in the tube they are illuminated by light of a define wavelength. This so-called incident or excitatory light causes the chemical labels added to the sample DNA during amplificatio to release light of a different wavelength fluorescence Different areas of DNA profile have one of a small number of chemical labels so that depending on which fuorescent light is released at what time during electrophoresis it is possible to know what area of DNA is being detected. The amount of fluo rescence detected is used a measure of the amount of DNA passing through and thus representative of the amount of DNA in the original forensic sample. The information is captured as electronic data and analyzed using software to produce a profil seeIntroduction

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Intr oduction to F or ensic Genetics 7 toForensicDNAProfling – TheElectropherogram epg. Setting aside some of the difficultie in assessing whether a DNA component is truly present or not a DNA profil can be matched to another DNA profil with 100 accuracy interpretation and with a preci- sion dependent on the number of loci used in the match. The “match criteria” are defined the numbers must matchexactly. Another parameter that may be useful to know when assessing an identificatio system is the sensitivity. In this context this means how little of the material from the individual that needs to be available to enable an identification The technology enables the profilin of the DNA from a single cell but in forensic genetics the presence of mixtures and degraded samples can render that ability a vice rather than a virtue see InterpretationofMixturesGraphical DNAMixture Interpretation andDegradedSamples. Many matches today are enabled by the creation of DNA databases that store the DNA profile of people selected by a process depending on the jurisdictional rules seeDatabases. However there are a number of scientifi and social issues that arise from the use of these databases see DNA Databases – The Signif- canceofUniqueHitsandtheDatabaseControversy DNA Databases and Evidentiary Issues. Other emerging technologies have also caused debate see PhenotypeFamilialSearching. Having established the degree to which a match exists between the crime material and the suspect the forensic scientist must now evaluate the significanc of the match see Identifcation and Individualization Communicating Probabilistic Forensic Evidence in Court. The starting point for all such calculations includes the frequency of the particular components within the population. Those frequencies for the DNA components used in forensic DNA profilin have been measured. The use to which those frequencies are put varies with the type of profil obtained and the choice of method for calculating the significanc of the evidence. This is the increasingly arcane topic of statistics which has become so complex that some have introduced probabilistic genotyping software because the calculations are claimed to be too complex to be undertaken manually see Issues in Forensic DNA. An important concept underlying the use of frequen- cies in forensic work is called the Hardy–Weinberg equilibrium HWE. The HWE is a state in which the allelic frequencies do not change. Its forensic signifi cance is that in the process of calculating the expected frequencies of genotypes in the population this equi- librium or steady state is assumed as a starting basis. Given that any person will normally have two alleles at each locus although both may be the same the allelic frequencies can be used to calculate genotype frequencies paired combinations of alleles by multi- plication. In a simple example if there are only two alleles A or B possible at a locus then there are only three types of individuals: AA BBand AB.Ifthe frequency of alleleA isp and the frequency of allele B is q it is possible to calculate the frequency of each type of person in the population assuming the Hardy–Weinbergrules. These rules are 1. No selection 2. No mutation 3. No migration 4. Random mating 5. An infinitel large population If these rules are not met the population is not in equilibrium and the allele frequencies will be changing. There is some debate on how to accom- modate this and other possibilities into calculating the statistics of the random match probability RMP for a profil seeDNAMixtureInterpretation. Most commonly a correction factor is incorporated into the calculation for genotype frequencies to allow for population substructures. One of the HWE rules is “random mating.” The alleles exist in pairs within men and women but the pairs separate in the formation of sperm and egg so in effect the next population is a sample of drawing two alleles together one from the male and one from the female. If p and q are the frequencies of alleles A and B respectively and A and B are the only two alleles at that locus then it is quite simple algebra to calculate the result of one round of “random mating” in a population where the population of parents have the frequenciesp andq forA andB: p+q×p+qp+q 2 p 2 + 2pq+q 2 This gives the frequency of AA children as p 2 AB children as 2pqandBB children asq 2 .

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8 Backgr ound A table can show the same thing perhaps more easily: Fathers pq Mothers pp×pp 2 p×qpq qp×qpq p×qq 2 Thus an allelic frequency database can be used to calculate an RMP to an individual: the probability that one could pick a person who would have the same genotype profile at random from a population of unrelated individuals. Most professional codes of practice for forensic scientists demand that the scientist is an impar- tial participant in the legal process. Unfortunately while science may be impartial in a very restricted sense it would appear from a considerable body of research that scientists are not. There is an increasing awareness within the forensic community that biases are not only possible but also probable. We merely introduce the topic here see Observer Effects. Similarly although routine DNA profilin of human DNA dominates the perception of forensic DNA there are other techniques using different sources of DNA see Mitochondrial DNA: Profling Wildlife Crime Microbial Forensics and different technologies see Single Nucleotide Polymorphism in addition to the more extreme application of the routine methods see IssuesinForensicDNA. The legal process may be terminated before a case gets to court but the forensic scientist is frequently required to provide evidence for use in court. The penultimate chapters are devoted to the use of DNA profilin in court. Finally we consider the current debates arising from DNA profilin and consider where the future may lie for this technology that has revolutionized crime investigation and arguably the entire feld of forensic science. Related Articles in EFS Online Short T andem Repeats: Inter pr etation

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Chapter 2 DN A: An Ov er view Eleanor Alison May Graham NorthumbriaUniversityNewcastleuponTyneUK History of DN A Pr ofilin DNA profiling as we know it today was developed thanks to two independent breakthroughs in molecular biology that occurred at the same time on different sides of the Atlantic. In the United States the poly- merase chain reaction PCR was developed by Kary Mullis of Cetus Corporation 1–3. Almost simul- taneously the individual-specifi banding patterns observed after restriction fragment-length polymor- phism RFLP analysis of repeated DNA sequences were discovered by Professor Sir Alec Jeffreys at the University of Leicester 4–6. In its earliest incarna- tion this technique termed as DNA fngerprinting by its creators was performed by restriction of 0.5–10μg of extracted DNA using the restriction enzymeHinFI followed by Southern blotting hybridization with probes termed33.533.6and33.15 designed to bind to multiple “minisatellites” present in the restricted DNA 6. This multilocus probing MLP technique would result in the binding of probes to multiple independent DNA fragments at the same time giving rise to the traditional “bar code” pattern that is often visualized discussing DNA profiling even today. Differences in the number of times the probe sequence is repeated in each DNA fragment form the basis of the individual patterns observed on the autoradiogram image. The Mendelian inheritance of these markers was established by the pedigree analysis of 54 related persons 4 and the individual nature of the banding pattern was further established by the examination of 20 unrelated persons 6. The probability of two unrelated individuals carrying the same fingerprin AGuidetoForensicDNAProfling Edited by Allan Jamieson and Scott Bader © 2016 John Wiley Sons Ltd. ISBN: 9781118751527 Also published in EFS online edition DOI: 10.1002/9780470061589.fsa532.pub2 was calculated from these data and was estimated as 3× 10 −11 for probe 33.15 alone provided 15 bands could be resolved in the 4–20-kb size range on the autoradiogram. The potential application of this tech- nique to maternity/paternity disputes and to forensic investigation was recognized immediately by Jefferys et al. and was demonstrated by DNA fngerprinting of forensic-type samples such as bloodstains and semen in the same year 5. Diffculties in interpreta- tion of MLP images quickly resulted in single locus probes SLP with variable number of tandem repeat VNTR loci becoming the markers of choice for DNA profiling Thefrst report concerning the use of DNA profilin in a criminal investigation was published in 1987 7. This investigation used two unpublished SLPs to link semen stain samples collected from two rape and murder cases that had occurred 3 years apart in 1983 and 1986 in Leicestershire United Kingdom. The probability of this match occurring by chance was calculated as 5.8× 10 −8 . This result not only linked the two crimes but also exonerated an innocent man impli- cated in the murders and led to thefrst mass screening project undertaken for DNA profilin in the world 8. The potential of DNA analysis for forensic science had now been demonstrated the technology now required statistical validation by analysis of popula- tion frequencies and application to casework samples before it could progress. Early evaluation studies on MLP 33.15 provided optimistic support for the use of DNA for the personal identificatio and the iden- tificatio of male rapists from a mixed male/female sample 9. It does however also begin to uncover the limitations of this method. A mean success rate of only 62 for the DNA fngerprinting of donated vaginal swabs was observed and no typing was possible for blood or semen stains that had been stored for 4 years at room temperature and diffculty in directly

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10 Backgr ound comparing related samples run on different gels was also cited as a potential problem 9. Similar studies and European collaborations were undertaken on SLPs such as YNH24 and MS43a 10 11. Difficultie were again observed when interpreting gel images with only 77.9 of 70 samples distributed between nine laboratories producing matching results when a 2.8 “window” for size differences between gel runs and laboratories was used 10. It was recognized that subtle differences between laboratory protocols were responsible for some of the observed discrepancies leading to a requirement for the standardization of laboratory methodology 10 and DNA profil interpretation 12 13. Such standardizations could improve the repro- ducibility of DNA typing results for MLP and SLP marker systems but in order to be applicable to forensic investigation DNA systems must be robust and must be applicable to samples of a less than pris- tine nature or that which consists of only a few cells. PCR was firs applied to forensic DNA profilin for the investigation of the HLA-DQ-1 gene a polymor- phic gene that encodes a human leukocyte antigen cell surface protein located in the major histocompatibility complex MHC class II region on chromosome 6 14. Two big breakthroughs occurred between the late 1980s and early 1990s that would form the basis of DNA profilin techniques that are recognized today. An alternative class of DNA marker the microsatellite or short tandem repeat STR marker was described by Weber et al. 15 and an alternative method for DNA visualization PCR amplification and fuores- cent labeling of VNTR markers was also introduced 16 17. STR Analysis STR markers are similar to the VNTR markers that were originally identifie and utilized in DNA fnger- printing and SLP profiling The difference between the classes of DNA marker lies in the length of the tandemly repeated DNA sequence. VNTRs contain 10–33-bp hypervariable repeat motifs 4 that must be observed over a size range of 4–20 kb 9 and SLP markers that are observed over a size range of 1–14 kb 10. An STR marker repeat is composed of 1–6-bp repeat motifs 18 making the region of DNA that must be scrutinized very short 1 kb. This length reduction is immediately beneficia to one of the problems encountered in SLP profiling diffculty in analyzing degraded DNA 5. The use of the multiplex PCR to amplify target sequences before visualization significantl reduces the amount of DNA required for analysis from microgram to nanogram amounts 18 19. The detection and visual- ization method of polyacrylamide gel electrophoresis and fuorescent detection using an automated DNA sequencer model 370 Applied Biosystems Foster City CA USA in combination with an internal size standard GS2500 Applied Biosystems and GENESCAN 672 software Applied Biosystems also allowed for precise band sizing answering prob- lems of intralaboratory and interlaboratory allele designation discrepancies that had been observed in SLP analysis 12 20 21. One of the earliest multiplexed STR systems developed for forensic DNA analysis was a quadru- plex reaction that amplifie the STR markers HUMVWA31/A HUMTHO1 HUMF131A1 and HUMFES/FPS 22. These particular STR markers were selected from the hundreds of STRs identi- fie throughout the human genome 23 based on a number of important parameters. Each STR must have a high level of allelic variability to maximize the discriminating power of each marker. Markers should have short PCR product length 500 bp to aid the analysis of degraded DNA. The chromosomal location of any potential marker should be checked to avoid closely linked loci and tetranucleotide repeat motifs are preferred due to low artifact production during PCR amplificatio 24. The overall match probability using this system was calculated as 1.3× 10 −4 for white Caucasian populations 18. Validation studies carried out on this quadruplex system determined that 1 ng template DNA was optimal for the amplifica tion and analysis that stutter bands up to 11 were observed at some loci especially HUMVWA21/A and that DNA mixtures in a ratio of 1 : 2 or 2 : 1 could be distinguished using this system 22. Further validation studies were carried out on extremely compromised casework samples collected from the victims of the Waco siege which resulted in the identificatio of several individuals which was not possible by any other means 25 26. The quadruplex system described above was next developed into a septaplex as the application of DNA profilin to forensic casework increased. The new septaplex system coamplifie the tetranucleotide STR loci HUMVWFA31/A vWA HUMTHO1 THO1

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DN A: An Ov er view 11 D8S1179 HUMFIBRA FGA D21S11 and D18S51 27. The nomenclature used for naming these STR markers had been standardized now to allow for easy comparison between different working groups 28. This system also included primers for the amplifi cation of a region of the amelogenin gene which could be used to deduce the sex of the DNA sample being analyzed 29. Optimization and validation studies on this septaplex system reduced the amount of template required for the generation of full DNA profil to just 500 pg with partial profile being gener- ated from as little as 50 pg the equivalent of just 10 diploid cells 27. This septaplex became known as the second-generation multiplex SGM system 30 and was used to populate the firs national criminal intelligence DNA database which became operational in the United Kingdom in April 1995 27. The SGM system was human specifi and highly discriminating with a probability of chance association calculated as 1× 10 −8 31. It could also be applied to degraded DNA samples and was capable of detecting and resolving mixed DNA profile at ratios between 1 : 10 and 10 : 1 30. One fina evolution would take place the inclusion of a further four STR markers D3S1358 D19S433 D16S539 and D2S1338 to produce the STR profilin kit that is currently used in the United Kingdom for forensic DNA profiling population of the national DNA database NDNAD and for paternity testing. TheAmpFlSTR ® SGMPlus™System The AmpFlSTR ® SGM Plus™ system commercially produced by Applied Biosystems ABI a division of Perkin Elmer Foster City California USA was introduced in June 1999 and was validated for use in forensic casework in 2000 32. It was designed to replace the SGM system for forensic casework in the United Kingdom to decrease the probability of a chance match occurring from 1× 10 −8 to one in trillions for unrelated individuals 32. This greatly increased the statistical power of DNA evidence to be taken for scrutiny in the courtroom while being back compatible with DNA profile already stored in the NDNAD. To maintain back-compatibility partial DNA profile must contain data at a minimum of four of the original SGM loci to be considered for uploading to the NDNAD. The statistical power of this new system was deemed so great that instead of calculating the exact match probability for a full DNA profile it was recommended that an arbitrary conservative estimate of one in a billion be reported for the match probability between unrelated individuals one in a million for parent/child relations and 1 in 10 000 for siblings 33. The characteristics of each STR marker are detailed in Table 1. The AmpFlSTR ® SGM Plus™ PCR amplificatio kit can be analyzed by two DNA separation methods: capillary electrophoresis or polyacrylamide gel elec- trophoresis. In this article the analysis was performed by polyacrylamide gel electrophoresis using an ABI Prism™ 377XL DNA Sequencer ABI. The ABI Prism™ 377XL DNA Sequencer was introduced by Applied Biosystems in 1995 and stopped its use in 2001 44. Automated fragment sizing of fluores cently labeled DNA fragments was achieved by the use of a scanning argon ion laser which tracks back and forth across a “read-region” at the lower end of a vertical polyacrylamide gel. As each labeled DNA fragment passes the laser the fluorescen dye is excited resulting in emission of light. This light is then collected and separated by wavelength onto a charged coupled device CCD camera. The camera used on the 377 model is capable of detecting four different wave- lengths simultaneously allowing for the detection of three similarly sized PCR products in a single gel run with inclusion of a separately colored size stan- dard in each lane. The 377XL model was validated for forensic STR analysis in 1996 using the original SGM septaplex system 45. The set of validation exper- iments carried out determined that complete resolu- tion of 1 bp differences between fragments could be achieved up to 350 bp sizing precision was increased twofold and sensitivity was increased by one-third compared to the predeceasing 373A DNA sequencer 45. Gel electrophoresis has now largely if not completely been superseded by the use of capillary electrophoresis in commercial forensic laborato- ries and many research institutes around the world. The market leader for provision of capillary elec- trophoresis equipment Applied Biosystems now part of Life Technologies has produced several instruments that have been adopted for forensic use 46. The current instrument of choice for many laboratories is the 16-capillary 3130XL Genetic Analyser but this may itself soon be replaced by the 3500/3500XL for 8/24-cappillary capacity. The 3500 series of instruments have been designed specificall for the forensic market and include the

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12 Backgr ound Table 1 Characteristics of SGM Plus STR loci a Marker Chromosome location No. of alleles in allelic ladder Size range bp Repeat motif Dye label References D3S1358 3p 8 114–142 TCA TCTG 1–3 TCTA n 5-FAM 34 vWA b 12p12-pter 14 157–209 TCATCTG 3–4 TCTA n 5-FAM 35 D16S539 16q24-qter 9 234–274 AGAT n 5-FAM GenBank G07925 D2S1338 2q35–37.1 14 289–341 TGCC n TTCC n 5-FAM 36 Amelogenin b X p22.1–22.3 NA 107 NA JOE 29 Y p11.2 113 D8S1179 b 8 12 128–172 TCTR n JOE 37 D21S11 b 21q11.2-q21 24 187–172 TCTA n TCTG n TCTA 3 JOE 38 TATCTA 3 TCA TCTA 2 TCCA TA TCTA n D18S51 b 18q21.3 23 262–345 AGAA n JOE 39 D19S433 19q12–13.1 15 106–140 AAGGAAAGAAGG NED 40 TAGGAAGG n THO1 b 11p15.5 10 165–204 AATG n NED 41 FGA b 4q28 28 215–353 TTTC 3 TTTT TTCT NED 42 CTTT n CTCC TTCC 2 a Adapted from AmpFlSTR SGM Plus PCR Amplificatio kit manual and 43 b Loci included in the original SGM kit addition of radio frequency identificatio RFID tags for more effcient consumable monitoring among other features. Although not currently operational in commercial forensic laboratories lab-on-a-chip technologies are being rapidly developed to allow for miniaturization of capillary electrophoresis devices 47 which in the near future may allow for rapid “at scene” DNA profilin to become a reality. Eur opean Standard Set ESS of STRs. Although STRs are used as the DNA marker of choice for forensic DNA analysis and population of NDNADs throughout the world they are not all populated with equivalent data due to the adoption of different STR marker sets in different countries. To increase the discriminatory power of each STR system and decrease the probability of adventitious matches on DNA database searches several new kits have been produced that allow for the analysis of up to 16 STR markers in a single reaction 48 49. Although the new multiplex kits have not been introduced to routine casework in the United Kingdom at the time of writing it is anticipated that they will be adopted in the near future. An additional motivation for the tran- sition to be made comes from the desire to exchange DNA data with member states of the European Union for the resolution of cross-border crime. The Council of the European Union resolution on the exchange of DNA analysis results also known as thePrümTreaty identifie a core set of 12 STR markers that should be used when DNA data exchanges between EU member states see Officia journal. Details of the STR loci included in the new STR multiplex kits are provided in Table 2. AlternativeDNAMarkers Autosomal STR markers have become the most utilized ones in both forensic and paternity DNA profiling However there are numerous alternative markers that can be interrogated when required. Another class of autosomal marker the single nucleotide polymorphism SNP has been inves- tigated for application to forensic casework and identificatio projects. SNPs as the name suggests are alterations of a single base pair and occur on average every few hundred base pairs throughout the human genome 50 51. The major advantage of SNP markers over STRs is the small size of the DNA target making them very useful for degraded DNA analysis and disaster victim identificatio projects 52. Reduced size STR amplicons have however

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DN A: An Ov er view 13 Table 2 STR loci included in newly developed commercial STR profilin kits ESS 12 Loci SGM Plus ® NGM™ NGM Select™ Powerplex ® ESI/X 16 Powerplex ® ESI/X 17 • D3S1358 •• • • • • D8S1179 •• • • • – D16S539 •• • • • • D18S51 •• • • • • D21S11 •• • • • • FGA •• • • • • THO1 •• • • • • vWA •• • • • – D2S1338 •• • • • – D19S433 •• • • • • D1S1656 – •• • • • D2S441 – •• • • • D10S1248 – •• • • • D12S391 – •• • • • D22S1045 – •• • • –SE33 – – • – • – Amelogenin •• • • • been developed to analyze degraded DNA while remaining compatible with current DNA databases to allow easy searching in identificatio projects 53. There are a number of disadvantages associated with the use of SNP markers instead of STRs. The comparatively low discrimination power of each locus requires that approximately 50 SNPs must be inves- tigated to give match probabilities equal to 10 STR loci 54. Mixture analysis is also complicated for SNP analyses are usually bi-allelic but have also been observed to be tri- and even tetra-allelic. The use of a predominantly bi-allelic marker makes the distinction between mixtures and homozygotes diffcult espe- cially for minor contributors which may also display allelic dropout 54. Owing to these problems and the fnancial implications of repopulating NDNADs with SNP profiles it is unlikely that SNP markers play a major role at the forefront of forensic DNA analysis in the foreseeable future. There are still some applications in related felds in which SNP markers are proven to be useful: determination of phenotypic characteristics such as eye color 55 56 and hair color by variation in the melanocortin 1 receptor MC1R gene 57–59. SNP markers can also be used to analyze the uniparentally inherited mito- chondrial DNA mtDNA see Mitochondrial DNA: Profling and Y chromosomes see Y-Chromosome Short Tandem Repeats which are discussed in 60 61. DN A Extraction In order for DNA from any given biological sample to be analyzed by a PCR-based method it must frst be purifie from all organic and inorganic substances with which it is associated see Extraction. This process can become complicated during forensic investiga- tion as DNA is often present on or in materials that are not usually encountered in the molecular biology laboratory. The extraction stage of DNA analysis is also the most susceptible stage to the occurrence of laboratory-induced contamination of sample mate- rial. For this reason DNA extraction protocols no matter which one is employed must always be carried out in a dedicated laboratory physically separated from downstream processes especially PCR. Liter- ature searching for DNA extraction of forensically interesting materials reveals tens of various techniques and variations on each of these. A brief listing of DNA extraction methods for commonly encountered forensic evidentiary samples is given in Table 3. Of the many techniques that have been proposed the most commonly utilized ones are based on three techniques: organic cell lysis with phenol/chloroform purification Chelex ® 100 and silica-based extractions. The organic phenol/chloroform method is the original DNA extraction technique to be applied to forensic and archaeological specimens 5 80 81 107. Although many subtle variations in buffer

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14 Backgr ound Table 3 List of materials commonly collected for DNA analysis during forensic investigations with reference to extraction methods Substrate Type of extraction References Blood Silica 62 63 Chelex ® 64 64 Bone Silica 65–70 Lysis/precipitation 71 Phenol/chloroform 66 67 72 73 Teeth Chelex ® 74 75 Phenol/chloroform 76–79 Hair Phenol/chloroform 80 81 Chelex ® 64 82 Alkaline digestion 83 Silica 82 84 Saliva Chelex ® 64 85–87 Buccal cells Phenol/chloroform 88 89 Silica 90 Maggots Phenol/chloroform 91–93 Feces Silica 94–97 Phenol/chloroform 96 97 Urine Silica 69 98 99 Semen Chelex ® 100 Trace/cells Silica 101 102 Organic 101 103–105 Chelex ® 106 pH chemical concentration incubation time and temperature have been introduced over the years the basis of the technique remains the same. The detergent sodium dodecyl sulfate SDS is used to break down cell walls and lipids present in the sample and proteinase K a strong protease enzyme is used to digest the proteins that protect the DNA molecule in its natural state. An additional component dithiothreitol DTT can also be used to digest more robust materials such as keratinized hair and sperm cells and forms the basis of the differential extraction technique used for vaginal swabs with semen present. The differential extraction of the two cell types is possible as the sperm nuclei are impervious to SDS/proteinase K digestion because the nuclear membrane is reinforced with cross-linked thiol-rich proteins. This allows for the digestion and removal of the female component from the mixture leaving intact sperm nuclei to be digested by the addition of an SDS/proteinase K/DTT mixture 5. Once digested phenol/chloroform is used to separate proteins into organic phenol layer from the nucleic acids in the aqueous chloroform layer. The nucleic acids must then be concentrated and purifie from the hazardous chloroform solvent before further analysis. This is classically achieved by ethanol precipitation but can also be carried out using a commercially available centrifugal filte device such as the Centricon ® 100 system 108. Chelex ® 100 was introduced as a medium for the simple extraction of DNA from forensic materials in 1991 64. This technique was designed specificall for use with forensic specimens and was intended to replace the organic phenol/chloroform technique. The Chelex ® method has three major advantages over the organic phenol/chloroform methods: it is much faster taking only 1 h compared to up to 24 h for the organic it does not require multiple tube transfers reducing the risk of laboratory-induced contamination and it does not require the use of hazardous chem- icals. Chelex ® resin is composed of styrene divinyl- benzene copolymers containing paired iminodiacetate ions which act as chelating groups binding polyva- lent ions such as Mg 2+ 64. In this method Chelex ® resin is added directly to the sample from which DNA is to be extracted. Cell lysis and DNA liberation are achieved by a combination of alkalinity pH 10–11 and heating at 100 ∘ C. Initial testing performed on forensic-type samples such as blood blood stains semen stains and hair demonstrated that the perfor- mance of Chelex ® extraction was equal to that of phenol/chloroform methods and in the case of blood was less likely to allow the carryover of PCR inhibitors such as heme. Similar to organic methods an addi- tional DTT digestion step is required for DNA extrac- tion from sperm 64. Silica particles were frst used for DNA extraction from human serum and urine samples. This method similar to Chelex ® was designed to be more rapid and involves fewer tube transfer stages to reduce the risk of sample contamination or DNA loss than organic methods. The method uses a chaotropic agent guanidinium thiocyanate GuSCN to lyse cells and inactivate nucleases while simultaneously facilitating the binding of the freed nucleic acids to silica particles 69. Once bound the silica–DNA complex can be pelleted allowing cellular debris and other non-DNA components to be removed and discarded. After washing the purifie DNA can then be eluted from the silica in sterile water or TE buffer. This method is extremely sensitive due to the strong binding affinit of silica particles for nucleic acids in the presence of chaotropic agents and is subsequently adapted for use with ancient bone samples 70 109. The method

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DN A: An Ov er view 15 does require some caution as GuSCN produces HCN gas on contact with acids and should therefore be kept in an alkaline solution at all times and disposed of in 10 M NaOH. The silica–DNA binding affinit has been utilized in the development of commercial kits such as the QIAamp ® DNA kits produced by Qiagen Qiagen West Sussex United Kingdom described in QiaAmp and the DNAIQ™ system produced by the Promega Corporation. Both systems have been tested and validated for use with forensic casework samples 62 63 and both systems are compatible with automated robotic workstations 110–112. The versatility and reproducibility of such commercial DNA extraction kits especially QIAamp ® products have made them the choice of the forensic research community over recent years as illustrated by their usage in the majority of research articles published over recent years. To increase sample throughput and reduce operator interaction with forensic samples robotic worksta- tions are now used for the majority of routine samples in commercial DNA laboratories. The frst system to be adopted for mainstream commercial use was the Biorobot EZ1 from Qiagen. This system uses a silica-based DNA extraction methodology that is in principle similar to the QIAamp DNA kits with the silica being bound to magnetic particles instead of forming a solid membrane. Due to the success of this pioneering instrument the market leader for provision of forensic DNA profilin instruments and consumables Applied Biosystems has worked with Tecan Tecan Group Ltd. Mannedorf Switzerland to develop and validate the AutoMate Express™ Forensic DNA Extraction System 113 for use with the PrepFiler Forensic DNA Extraction kit 114 which is another magnetic bead-based DNA extraction system now available for forensic use. DN A Quantificatio Commercially produced DNA profilin kits such as the AmpFlSTR ® SGM Plus™ PCR amplifica tion kit described above are optimized to produce DNA profile from a narrow range of template DNA concentration. AmpFlSTR ® kits produced by Applied Biosystems are optimized to amplify 1–2.5-ng template DNA. The addition of insufficien template DNA can result in stochastic amplification Stochastic amplificatio manifests as unbalanced amplificatio of heterozygote loci and can if severe result in allelic dropout making heterozygotes to appear as homozygotes at the affected loci 115. The addition of template DNA in excess can lead to the production of large stutter peaks that complicate the interpretation of DNA profile The termstutter refers to the produc- tion of natural biological artifacts during the PCR. The currently accepted model for stutter generation is by a “polymerase slippage” model that results in the addition or more often deletion of a single repeat unit from the actual template size. The detection and clas- sificatio of stutter play an important role in profil interpretation especially if a mixture is anticipated or observed. If the stutter characteristics are unknown for a DNA profilin system it is possible that such artifacts may falsely be reported as “actual alleles”. It has been observed that stutter does not usually exceed 10 of the associated allele peak height but with variation between both STR loci and alleles in the locus stutter has been observed to approach 15 116 in some cases. Percentage stutter is calculated using the peak height in relative fluorescen units RFU of the observed peaks. Figure 1 shows a typical stutter pattern at the STR locus FGA. The most commonly observed nonbiological arti- facts caused by the addition of template DNA in excess are termed aspull-up or “bleed through” peaks. Pull-up peaks are produced when the GeneScan/GeneMapper software program is unable to distinguish between the emission spectra of the fuorescent colors in the STR system. This phenomenon is visualized by the appear- ance of false bands in the overamplifie fragment’s size range in differently colored markers. By quantifying all DNA samples before performing DNA profilin PCR the production of such artifacts can be reduced or completely avoided. As with DNA extraction there are numerous methods available to perform this function. The traditional method of DNA quantitation involves measuring the absorbance of the sample at 260 nm on a spectrophotometer. This method is simple to perform and shows little sample-to-sample variation 117 making it a desir- able technique. This technique does however suffer because of its simplicity. The technique is not species specific indicating that any bacterial or fungal DNA that is copurifie with the DNA of interest cannot be distinguished from the human target DNA. Addi- tionally single-stranded DNA and nucleotides cannot be distinguished from double-stranded DNA by this

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16 Backgr ound 240 242 244 246 248 250 252 254 256 23 24 25 26 706 63 57 651 8.8 11 Figur e 1 Example of typical stutter pattern of alleles 24 and 26 at STR locus FGA method which can also lead to falsely high quanti- tation readings 118. A different approach to DNA quantitation is taken by the slot-blot technique. This method is based on the specifi hybridization of a 40-bp probe that binds to the human alpha satellite locus D17Z1 119. Using this technique DNA is quantifie by visual comparison of sample band intensities with the band intensities of standards produced from known amounts of DNA. This compar- ison can be performed either manually – but this allows for the subjectivity of individual interpretation which can lead to differences between operators in quantitation – or electronically by computationally converting the band intensities into numerical values 120. This technique shows a high degree of species specificit human and primate and can detect as little as 150 pg DNA in a standard assay 119. Similar to the development of commercial DNA profilin kits the development of commercial DNA quantitation kits has also progressed. Applied Biosystems produced the Quantiblot ® human DNA quantificatio kit similarly to the original technique of Walsh. Walshetal. use a probe that binds to the D17Z1 locus and has a lower detection limit of 150 pg 119 121 122. An alternative method of DNA quantitation involves the use of afuorescent dye PicoGreen double-stranded ds DNA quantitation reagent. This method was developed to allow high-throughput quantitation of multiple samples concurrently while increasing the sensitivity DNA quantitation methods. PicoGreen is a Hoechst dye that can pass through the lipid membrane of live or fi ed cells to form noncovalent bonds with AT-rich areas in the minor groove of dsDNA 123. The method utilizes the increased fluorescen intensity that is observed when PicoGreen binds to dsDNA. The fluorescen intensity of the PicoGreen dye is measured with a spectrofluoromete capable of producing the excitation wavelength of ∼480 nm and recording at the emission wavelength of ∼520 nm. The DNA is then quantifie by comparison of the sample fuores- cence with the fuorescence of a set of standards that are included in every sample run 124. The biggest disadvantage to this method over the hybridization methods discussed previously is that this method is not specifi for human DNA. Any animal bacterial or fungal DNA copurifie with the human DNA of interest will contribute to the fnal reading and could give a falsely high DNA quantification This method is however much more sensitive than the previously described methods with a reported lower detection limit of 25 pg ml −1 118. The latest development in DNA quantitation is based on the technique of real-time PCR. The method- ology was frst proposed 15 years ago by Higuchi and coworkers 125 who by including ethidium bromide in the PCR reaction were able to continuously monitor the production of dsDNA in “real time”. By capturing the change in fluorescen intensity on video camera the potential of this new development for specifi DNA quantitation was realized 126. There are several different approaches to real-time quantitation of DNA they are all however based on the principle of fluorescen dye binding double-stranded DNA as it accumulates during the PCR process. As the technique is based on the PCR DNA quantitation can be under- taken by targeting any specifi region of template DNA with many systems targeting the multicopyAlu sequence which appears 500 000–1 000 000 times throughout the human genome 127–129. This not only ensures that the technique is species specifi but also has allowed numerous variations many designed to perform additional functions such as independent

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DN A: An Ov er view 17 quantitation of nuclear and mtDNA in a single reaction 130–133 and assessment of DNA quality 134 135. Real-time PCR is the current gold standard for quantificatio of DNA samples prior to downstream processing and is an essential part of the labora- tory process when dealing with low-template DNA LTDNA samples. The importance of DNA quantifi cation was highlighted in the Review of the Science of Low Template DNA Analysis 136 in which it is considered as a “matter of best practice”. As for all other aspects of the DNA laboratory process Applied Biosystems has several kits available for real-time PCR quantificatio of DNA extracts. The most current system available at the time of writing is the Quantifile ® Duo DNA Quantitation kit which allows for simultaneous analysis of human specific via amplificatio of the ribonuclease P RNA compo- nent H1 and male-specifi targeting a region of the sex-determining region on the Y chromosome markers. The reported limit of reproducible detec- tion is in the region of only 11.5 pg μl −1 using this system 137. A competing kit produced by the Promega Corporation the Plexor HY system used the human-specifi RNU2 locus and the male-specifi TSPY gene as alternative DNA targets. The Plexor HY system has a reported reproducible detection limit in the region of 3.8 pg total DNA 138. Both systems also include an internal positive control which can be used to monitor amplificatio effciency of reactions over time and indicate the presence of PCR inhibitors in DNA extracts before DNA profilin is attempted. DN A Pr ofil Inter pr etation The methodologies hardware and software necessary for the generation of STR profile are well established but constantly evolving. Regardless of the exact means of production the interpretation of DNA profile must remain constant. Using the earliest DNA profilin systems based on RFLP analysis and Southern blot hybridization it was proved to be difficul to stan- dardize interpretations between different individuals and different laboratories. Significan differences in band sizing were regularly observed during collabo- rative exercises carried out by DNA testing laborato- ries throughout Europe 10 11. Although measures were taken to standardize as many variables as possible including buffer system gel running time and tempera- ture and DNA visualization method there was still an uncertainty in interpretation uniformity between labo- ratories 10. The breakthrough required for standard- ization of DNA profil interpretation of DNA profile came with the switch to PCR amplificatio 1 2 and the introduction of fuorescent labeling allowing digitization of DNA profilin results 16 17. These advancements paved the way for the development of universal guidelines and validation of commercially produced kits and instruments to allow for the accurate and reliable reproducibility of DNA profilin inter- pretation across independent laboratories worldwide. Validation experiments designed to demonstrate the robustness of the system must be carried out for all commercially produced forensic DNA profilin kits before they can be used for casework. Validation data for the AmpFlSTR ® SGM Plus™ Amplificatio kit used throughout this article were published in 2000 32. The frst step toward standardization was the adoption of a common nomenclature for STR loci including both the name of the marker and the numer- ical designation of each allele observed in the human populations tested. The basis for the currently used naming systems was brought about by the European DNA Profilin Group EDNAP which was formed in 1989 by members of the leading police organizations and universities involved in forensic stain analysis and paternity investigation at that time 139. The next step in achieving complete interlaboratory concordance was the addition of the allelic ladder 140. Allelic ladders are produced by creating a mixture of DNA samples containing all observed allelic variations in the human population and then amplifying this mixture with the primers used for each locus in a given STR kit. Once created the allelic ladder is visu- alized in parallel with every set of samples so that a direct comparison can be made between the amplifie fragment size and the fragment size of previously observed alleles for each allele at each marker. This ensures that slight variations in running conditions such as those observed with Southern hybridization techniques are not misclassifie based on interrun fragment sizing differences 141. The inclusion of allelic ladders also allows for the recognition of rare and novel full and microvariant alleles that are not present in the allelic ladders for each STR kit 142. The fnal leap toward accurate DNA profil interpre- tation was made with the switch from “home brews” to commercially produced STR amplificatio kits and the use of identical or equivalent instruments in

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18 Backgr ound each laboratory throughout the forensic DNA typing community. As a result of the standardization measures described above the interpretation of single-contributor DNA profile when sufficien template is entered into the reaction is a straightforward process. In the forensic setting a large percentage of samples submitted for DNA analysis contain biological material from more than one source and result in mixed STR profiles In order to produce a robust set of interpretation guide- lines for mixed profiles it is vitally important to have a thorough understanding of the amplificatio char- acteristics of biological and stochastic phenomenon in the electropherogram from which the interpretation is made. The most commonly observed artifacts in a DNA profil are termed as stutter bands.Theyare generated during the PCR by slippage of Taq poly- merase resulting in the generation of a fragment one repeat unit shorter or more infrequently longer than the true allele. When the peak height of the amplifie true allele is4000 RFU in the electropherogram stut- tering is unavoidable when amplifying STR loci using Taq polymerase 143. Each STR locus and even each allele in an STR locus has different stuttering char- acteristics but stutter products are rarely observed to exceed a peak height in excess of 15 of the height of the associated allele. Any peak found in a stutter posi- tion with a height 15 of the related allele should therefore be considered as a potential true allele in a mixed DNA profil 143. Another characteristic of the electropherogram that can mystify DNA profil interpretation is the balance of allelic amplificatio at heterozygotic markers. In a perfectly amplifie DNA profile the peak areas of each heterozygote allele should be equal. Differences in the amplificatio effciency of each allele can however result in alleles being unequally amplifie 143. For a single source DNA profil produced from sufficien template 1 ng each shorter allele at a heterozy- gous locus will usually have a peak area 60 of the associated larger allele 143. Heterozygosity balance Hb is calculated as relative peak area differences of heterozygote alleles for each locus. There are two similar methods used to determine Hb for forensic DNA profil interpretation both of which use the peak area in RFU. The frst is calculated as smallest peak area/largest peak area where a result of 0.67 £ 1 indicates that the Hb is in the observed limits 143. This calculation does not however provide any information as to which of the heterozygous alleles has been preferentially amplifie during the PCR. This information is provided when Hb is assessed by the calculation peak area shortest allele/peak area longest allele. In this case a result of 0.671.67 indicates a balanced amplification with values 1 indicating that the shortest allele of the pair has been preferentially amplifie and values 1 indicating the opposite 144. The presence of additional bands in the electro- pherogram can also result from overamplificatio of template DNA causing saturation infuorescent signal that cannot be resolved by the matrix fles of the visu- alization software. These bands are termed aspull-up peaks due to their appearance in the raw data files whereby a smaller peak is observed directly under the overamplifie peak. When the dyes are separated to produce the electropherogram image the smaller pull-up peak appears as an extra band with excess blue signal producing green pull-ups and excess green signal producing yellow pull-ups. Pull-up peaks are easily recognized by the experienced DNA analyst due to their atypical morphology compared to true allele peaks and should not interfere with DNA profil interpretation 143. If a pull-up peak is superim- posed over a suspected true fragment the sample can be diluted and reanalyzed to avoid fuorescent signal saturation. Lo w T emplate DN A Pr ofilin The two biggest drives for developments in forensic DNA profilin are to improve sample throughput and technique sensitivity. Increases in sample throughput are achieved by automation of routine processes such as DNA extraction sample loading and DNA profilin interpretation coupled with effective labo- ratory management and sample tracking. While the sensitivity of DNA profilin reactions can also benefi from technological advances the major advancements are achieved by optimization of reaction chemistries investigation of alternate DNA markers and use of additional stage processes. One area of DNA profilin analysis that has received attention is the inclusion of a pre-PCR stage designed to increase the total amount of template DNA avail- able for STR analysis. A number of techniques similar in their objective but differing in their means can be described under the umbrella term of whole genome amplificatio WGA. As the term indicates

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DN A: An Ov er view 19 these techniques are designed to replicate all DNA present in a given reaction with the intention of generating an unlimited source of template mate- rial for multiple downstream analyses. Techniques of this kind were frst described in 1989 with the publication of a universal DNA amplificatio method which used restriction enzyme digestion and liga- tion into plasmids to achieve its goal 145. The next generation of WGA techniques appeared in the literature during the 1990s and like many other areas of molecular biology it moved away from the cloning techniques to PCR amplification The firs PCR/WGA technique was termed as primer extension preamplifcation polymerase chain reac- tion PEP/PCR 146. This technique utilizes fully degenerate 15-mer oligonucleotide primers and a range of annealing temperatures in a PCR to produce multiple copies of any template DNA present in the reaction. In his introductory article Zhang et al. 146 reported a minimum amplificatio of 30 copies for 78 of the human genome at a 95 confidenc level from a single spermatozoon. The limitations of PEP/PCR were also explored with heterozy- gosity imbalance and locus dropout cited as the main concerns when the technique is applied to single-cell analysis. These observations were confirme in the articles reporting the application of PEP/PCR to preimplantation diagnosis of single cells whereby effects of stochastic sampling and amplificatio were observed for single-cell analysis 147–149. Locus and allele dropout was also observed when PEP/PCR was applied to formalin-fi ed tissues followed by microsatellite analysis 149. Another method designed to perform the function of WGA has been termed as multiple displacement amplifcation MDA. This method wasfrst introduced in 2001 and differs from the previously described techniques by the use of an alternative enzyme 29 DNA polymerase 150. The method of replication is based on a rolling strand-displacement model that proceeds by constantly displacing the newly gener- ated strand of DNA to form a hyper-branched DNA replication structure limited only by the reagents available 151. The promise shown by this method has led to the development of several commercial WGA kits for example GenomiPhi WGA Ampli- ficatio kit GE Healthcare and various publica- tions advocating its use for human genome anal- ysis 152–156. All of these studies however have entered 10 ng DNA into the MDA reaction. Studies conducted using10 ng template DNA have observed unequal amplificatio of genomic DNA when assessed by the use of forensic DNA profilin kits similar or identical to those used in 157–159. Of the numerous WGA method variations that have been published to date none have yet demonstrated the unbiased amplificatio of microsatellite markers when very low template material is available for entry into the WGA reaction 160–162. An alternate approach is the use of a nested PCR strategy which involves the use of two primer sets one designed to prime from positions within the original amplicons. Using this approach the initial PCR performs the function of enriching the template DNA specificall in the regions of ultimate interest. The second internal amplificatio reaction can then be initiated from a greatly increased initial template amount. This approach was successfully applied to the forensic DNA analysis of charred human remains 163. This approach however presents a heightened risk of laboratory-introduced sample contamination by requiring the manipulation of amplifie human DNA breaking a fundamental anticontamination principle of single-direction workfl w during forensic DNA analysis. A far simpler approach to increasing PCR sensitivity has however proven to be extremely successful and become fully integrated into forensic DNA profilin laboratories worldwide. The term low copy number LCN DNA profilin was coined in a publication by Peter Gill of the Forensic Science Service FSS in 2000 and describes the analysis of 100 pg template DNA 116. LCN has come to be associated with the use of increased PCR cycles but there are other techniques that claim to amplify small amounts of DNA. These can be collectively termed as LTDNA analysis. Commer- cially available STR amplificatio kits such as the AmpFlSTR ® SGM Plus™ kit are optimized to produce complete and accurate DNA profile when 1–2.5μl template DNA is amplifie for 28 cycles. Following this standard protocol amplificatio of 100 pg DNA will typically result in severe allelic and locus dropout or even complete failure. The sensitivity of this technique can be radically improved by simply increasing the number of PCR cycles in the case of the AmpFlSTR ® SGM Plus™ kit to 34 cycles 116. The increase in sensitivity opens up the potential of forensic DNA profilin to analyze many more sample types than had ever previously been imagined most notably from fngerprints 164 165

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20 Backgr ound cigarette butts 103 and from the skin surface of manual strangulation victims 166–168 seeSources of DNA for a further description of sources of DNA evidence. The improved sensitivity gained by the use of an extra six PCR cycles does however lead to addi- tional issues in DNA profil interpretation. When undertaking any forensic DNA analysis proce- dure the importance of anticontamination protocols cannot be underestimated. This is especially true when attempting to produce LTDNA profiles for which the template DNA may also be of a degraded nature seeDegradedSamples. The sensitivity of the AmpFlSTR ® SGM Plus™ PCR amplificatio kit when employing 34 cycles is such that laboratory-derived contamination cannot actually be completely avoided 116. The potential to acquire DNA profil informa- tion from minute template amounts can also lead to the production of partial DNA profiles where both allele and locus dropout are observed. To combat these problems along with an increased size of stutter bands and unbalanced amplificatio of heterozygous alleles an entirely new set of DNA profil interpretation rules is required for LTDNA analysis. The majority of the complications observed for LTDNA profile are due to stochastic variation. For example a stutter band generated in an early PCR cycle unassociated contaminating alleles or one of two heterozygous alleles can be preferentially ampli- fie and therefore over-represented in the fina DNA profil 169. The random nature of stochastic varia- tion means that the same stutter products or contami- nating alleles will not be amplifie identically in repli- cate analyses 116 170 leading to the firs rule of LTDNA analysis: an allele can only be reported if it is present in at least two repeated analyses 169. Once this rule has been applied all remaining bands in the DNA profil can now be scrutinized. For stan- dard 28-cycle DNA profilin reactions stutter peaks are not observed to exceed 15 of the associated allele peak height in LCN analysis stutter products are much larger in the range of 20 when associ- ated with peaks 10 000 RFU in area and 40 for associated peaks 10 000 RFU 171. As this cannot be avoided it must always be considered that the stutter peak could be masking an allele of a minor contributor in a mixed profile and as such cannot be discarded simply as a stochastic artifact. Similarly for heterozygosity balance where Hb values are observedtobe 0.67 when using smallest peak area/largest peak area under standard conditions the minimum values observed during LCN analyses for most loci in the AmpFlSTR ® SGM Plus™ PCR kit were observed at a Hb value of 0.2 171. It was also observed that after repeated analysis allelic dropout at known heterozygote loci occurred at a rate of approximately 10 per locus but was not observed when the peak area of alleles was 10 000 RFU 171. By the varying nature of LTDNA work the majority of samples analyzed comprise DNA mixtures of dual or multiple contributors. Mixture analysis of stan- dard or LTDNA profile can be an extremely complex undertaking especially when more than two or three individuals have contributed unequally to a collected sample. In a working forensic casework environment to avoid bias mixture analysis is carried out without any knowledge of reference DNA profile of potential contributors. As the work carried out for inclusion in this thesis was purely research based the interpreta- tion permutations and mathematical rules required for unsighted mixture analysis are not further discussed in this introduction especially as the interpretation of complex mixtures is today more likely to be performed by computational expert systems 172 to increase the speed at which the analysis can be completed and remove any subjectivity that may be introduced by individual DNA analysts. The advantages of using an LTDNA protocol for DNA profil generation are clear when the analysis of minute trace evidence is required. These advantages do not however extend to the analysis of minor contrib- utors to mixed DNA profiles It has been noted that the use of LTDNA profilin does not convey any advan- tage over standard analysis when the minor:major DNA contributor ratio is below 1 : 10 144. This knowledge combined with the additional complica- tions of sporadic contamination increased stutter and heterozygote imbalance ensuring that LTDNA anal- ysis is undertaken only when absolutely necessary. Following criticism of LCN DNA profilin during the Omagh bombing trial R. v Hoey the Association of Chief Police Offcers ACPO suspended the use of the method in England and Wales on 21 December 2007. The method was reinstated on 14 January 2008 following a Review of the Science of Low Template DNA Analysis 136 which found the technique to be fi for purpose. The use of LCN DNA profilin in forensic casework continues to be debated among forensic geneticists around the world 173–177.

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