DNA Fingerprinting

An Introduction

Breakthroughs In Molecular Biology DNA Fingerprinting is the second volume to appear in this exciting new series of high quality, affordable books on the fields of molecular biology and immunology. This series is dedicated to the rapid publication of the latest breakthroughs and cutting edge technologies as well as syntheses of major advances within molecular biology.

Other volumes in the series include:

PCR Technology: Principles and Applications for DNA Amplification edited by H. Erlich

Lymphokines: The Molecular Biology of Regulators of Immune and Inflammatory Responses by K.-I. Arai and J. de Vries

Homologous Recombination and Gene Targeting by J. Sedivy and A. Joyner

Signal Transduction in Biological Systems by Y. Kaziro and K.-I. Arai

YAC Libraries: Construction and Use edited by B. Brownstein

DNA Fingerprinting

An Introduction

Lorne T. Kirby

M Pal grave Macmillan

©Stockton Press, 1990

All rights reserved. No part of this publication may be reproduced, or transmitted, in any form or by any means, without permission.

Published in the United States and Canada by Stockton Press 15 East 26th Street, New York, N.Y. 10010

Library of Congress Cataloglng·ln·Publicatlon Data

DNA fingerprinting: an introduction/Lorne T. Kirby. p. cm.-(Breakthroughs in molecular biology)

Includes bibliographical references.

ISBN 978·0·935859·94·2 (soft)

1. DNA fingerprints. I. Kirby, Lorne T., 1938- . II. Series. [DNLM: 1. DNA-genetics. 2. Forensic Medicine. au 58 062825]

RA1057.55.D635 1990 614'.1-dc20 DNLMlDLC for Library of Congress

ISBN 978-0-935859-94-2

Published in the United Kingdom by MACMILLAN PUBLISHERS LTD (Journals Division), 1990

British Library Cataloguing In Publication Data DNA Fingerprinting 1. Forensic medicine. Role of organisms I. Kirby, Lorne T. 1938-614.1

ISBN 978-0-333-54024-4 ISBN 978-1-349-12040-6 (eBook) 00110.1007/978-1-349-12040-6

987654321

90-9527 CIP

ISBN 978-0-333-54024-4 ISBN 978-1-349-12040-6 (eBook)DOI 10.1007/978-1-349-12040-6

To William and Mary Kirby, Sarah-Dean, Christopher, and Marcia Louise

CONTENTS

1. INTRODUCTION ................................................................................ 1

DEFINITION ........................................................................................ 1 mSTORY ............................................................................................. 2 A CASE FOR DNA .............................................................................. 3 APPLICATIONS .................................................................................. 3 REFERENCES ...................................................................................... 5

2. GENETIC PRINCIPLES ..................................................................... 7

DNA STRUCTURE .............................................................................. 8 Nuclear DNA .................................................................................... 8 Organelle DNA ............................................................................... 10

DNA FUNCTION ............................................................................... 11 REPRODUCTION .............................................................................. 13 LINKAGE ........................................................................................... 19 PEDIGREE ANALYSIS ..................................................................... 20 MUTATION ....................................................................................... 20 RESTRICTION ENDONUCLEASES ................................................ 23 CONCEPT OF POLyMORPHISM .................................................... 24 RECOMBINANT DNA ...................................................................... 26 BASE SEQUENCE DETERMINATION ........................................... 29 OLIGONUCLEOTIDE SYNTHESIS ................................................ 30 REPETITIVE SEQUENCE ORGANIZATION ................................. 31 NOMENCLATURE OF ARBITRARY DNA SEGMENTS .............. 33 REFERENCES .................................................................................... 33

vii

viii Contents

3. LABORATORY ORGANIZATION ................................................. 35

LABORATORY SECTIONS ............................................................. 36 Office .............................................................................................. 36 Specimen Documentation .............................................................. 36 Specimen Processing ...................................................................... 36 General Preparation ........................................................................ 39 Vector Handling ............................................................................. 39 Amplification by PCR .................................................................... 39 Hybridization and Radioisotope Handling ..................................... 39 Darkroom ........................................................................................ 45 Result Interpretation ....................................................................... 45 Tissue Culture ................................................................................. 45 Wash-up Area ................................................................................. 45

EQUIPMENT SUMMARY ................................................................ 46 OPERATION ...................................................................................... 47

Personnel ........................................................................................ 47 WorkFlow ...................................................................................... 48 Safety .............................................................................................. 49

REFERENCES .................................................................................... 50

4. SPECIMENS ........................................................................................ 51

COLLECTION .................................................................................... 52 STORAGE AND TRANSPORT ........................................................ 55 DNA EXTRACTION .......................................................................... 55

Principles ........................................................................................ 55 Automated System ......................................................................... 56 Manual System Organic Solvent Extraction .................................. 56

Whole blood (Method 1) • Whole blood (Method 2) • Tissue culture, amniotic fluid, and buccal cells· Hair roots, biopsy, and autopsy tissues· Fixed tissues· Stains· Sperm and female-cell mixtures· Plant tissues

Manual System Nonorganic Solvent Extraction ............................ 65 Whole blood· Stains • Sperm and female-cell mixtures

DIALySIS ........................................................................................... 66 RNAASE TREATMENT ...................................................................... 67 QUANTITATION ............................................................................... 67 DNA CONCENTRATION ................................................................. 68 QUALITY DETERMINATION ......................................................... 69

ix

EFFECTS OF ENVIRONMENTAL FACTORS INCLUDING CONTAMINATING DNA ON DNA PROFILES ...... 69 ANALYSIS REAGENTS ................................................................... 70 REFERENCES .................................................................................... 73

5. DNA AMPLIFICATION .................................................................... 75

POLYMERASE CHAIN REACTION ............................................... 76 Amplified Fragment Length Polymorphisms (AMP-FLPs) ........... 77 Advantages of PCR ........................................................................ 78 Pitfalls in PCR ................................................................................ 78 A PCR Protocol .............................................................................. 78

PROBE AMPLIFICATION ................................................................ 79 TISSUE CULTURE ............................................................................ 82 REFERENCES .................................................................................... 89

6. ANALYSIS TECHNIQUES ............................................................... 91

RESTRICTION ENZYME CLEAVAGE ........................................... 94 GEL-BLOT ORGANIZATION .......................................................... 95

Forensic .......................................................................................... 95 Parentage ........................................................................................ 95 Size Markers ................................................................................... 96

ELECTROPHORESIS ........................................................................ 96 Equipment ...................................................................................... 97 Reagents ......................................................................................... 97 Procedure ........................................................................................ 98

PHOTOGRAPHY ............................................................................... 99 DNA RECOVERY ............................................................................ 100 MEMBRANE TRANSFER .............................................................. 100

Southern Blots .............................................................................. 100 Dot-(Slot-) Blots ........................................................................... 104

RADIOISOTOPE PROBE LABELING ........................................... 104 NONRADIOISOTOPE PROBE LABELING .................................. 109 HYBRIDIZATION ........................................................................... 110

Principles ...................................................................................... 111 Procedures .................................................................................... 112 PCR Products ............................................................................... 115

PRINT DETECTION ........................................................................ 115

x Contents

BLOT STRIPPING AND REPROBING .......................................... 116 DATA PROCESSING ...................................................................... 116

Allele Fragment Size Determination ............................................ 116 Digital Analysis ............................................................................ 118 The FBI System ............................................................................ 118 Interpretation of Profile Matches ................................................. 119

PROFILE REP<)RT .......................................................................... 125 DNA PROFILE DATA BASES ....................................................... 126 P<)TENTIAL PITFALLS .................................................................. 127 TROUBLESHOOTING GUIDE ...................................................... 130 ANALYSIS COST ............................................................................ 130 ANALYSIS REAGENTS ................................................................. 130 REFERENCES .................................................................................. 131

7. PROBES, ALLELE MUTATIONS, AND RESTRICTION ENZYMES ............................................................ 135

PROBES ............................................................................................ 136 Polymorphism .............................................................................. 136 Tandem Repeat Single-Locus vs. Multilocus Probes ................... 13 7 Oligonucleotide Probes ................................................................ 139 Amplification and Stability .......................................................... 139 Availability ................................................................................... 141 Probes Used in Forensic Analysis ................................................ 141

MUTATIONS ................................................................................... 142 Allele Formation .......................................................................... 142 Genotype Analysis ....................................................................... 142

RESTRICTION ENZYMES ............................................................. 143 Recognition Site Features ............................................................. 143 Fragment Resolution .................................................................... 143 Sensitivity to Inhibitors and Stability ........................................... 143 Price and Availability ................................................................... 143 The Enzymes Selected for Forensic Analysis .............................. 143

REFERENCES .................................................................................. 145

8. PROBABILITY AND STATISTICAL ANALYSIS ...................... 149

STATISTICAL METHODOLOGY ................................................. 150 Random Sampling ........................................................................ 150 Data Presentation .......................................................................... 150

xi

Summary Statistics ....................................................................... 150 Measures of central tendency· Measures of dispersion

Statistical Inference ...................................................................... 153 Point estimation • Interval estimation • Calculation of allele frequency distributions· Sample size determina­tion • Simultaneous confidence intervals· Simultaneous allele frequency determination • Binning

Hypothesis testing ........................................................................ 160 Testing for proportions· Example· Chi squared goodness of fit test· Examples

PROBABILITY ................................................................................ 164 Probability of Combined Events: Addition and Multiplication Rilles ..................................................................... 164 Bayes' Theorem and Revision of Probabilities ............................ 165 Random Variables and Distributions ........................................... 167 Genetic Applications of Probability ............................................. 168

Hardy-Weinberg law • Wahlund'sprinciple· Forensicprob-ability determination· Likelihood of paternity • Estimation of probabilities formulti1ocus multiallele fmgerprint systems

REFERENCES .................................................................................. 176

9. QUALITY CONTROL ..................................................................... 179

DNA TEST SYSTEM STANDARDS .............................................. 180 CORRECT SPECIMEN .................................................................... 181 DNA QUALITY ............................................................................... 181 DNA CONTAMINATION ............................................................... 182 ANALYSIS CONTROL ................................................................... 182 SHEWHART SYSTEM .................................................................... 185 RESULT INTERPRETATION ......................................................... 187 REFERENCES .................................................................................. 187

10. LEGAL AND ETHICAL CONSIDERATIONS ............................. 189 Kenneth E. Melson

THE IMPACT OF DNA TYPING ................................................... 189 THE LEGAL STANDARDS FOR ADMISSIBILITY OF DNA TYPING .................................................................................. 192

The Frye Test ............................................................................... 192 Ascertaining General Acceptance ................................................ 195

xii Contents

Proper Application of the Technique ........................................... 196 The Relevancy Test of Admissibility ........................................... 197 Another Approach ........................................................................ 198 Legislated Admissibility of DNA Typing .................................... 199 DNA Data Banking ...................................................................... 200

LEGAL FOUNDATION FOR GENERAL ADMISSIBILITY ....... 201 Obtaining Known and Unknown Biological Samples ................. 202 Chain of Custody .......................................................................... 203 Proper Laboratory Procedures ...................................................... 204

PROCEDURAL AND ETIDCAL CONCERNS .............................. 207 Procedural Safeguards .................................................................. 207 Ethical Concerns .......................................................................... 209

CONCLUSION ................................................................................. 210 NOTES .............................................................................................. 210

11. CASE APPLICATIONS ................................................................. 217

FORENSIC ....................................................................................... 218 Cases ............................................................................................. 221

Switched specimens· A polio victim· No mistaken identity· The widowed pensioner· A burglar's fall· The bus driver· Crematorium bloodstain· Amplified evidence • Finally exonerated· 67,500,000,000: 1 • A small town murder

PARENTAGE ................................................................................... 226 Cases ............................................................................................. 226

Arranged killing· A victim's estate • An abandoned baby· The real parents· An immigration test

MEDICAL ........................................................................................ 229 Applications .................................................................................. 229

Posttransplant cell population identification • Twin zygosity determination • Tumor analysis • Identification of microorganisms • Tissue culture cell line identification· Paternity determination· CVS analysis

ANIMAL SCIENCES ....................................................................... 233 Cases ............................................................................................. 235

Canine and feline ID • The stud· Domesticated fowl· Falcons and cranes • Old Orange Band • Laundered macaws· Sexually liberated· A whale of a tale

xiii

WILDLIFE POACHING .................................................................. 243 The Smoking Gun ........................................................................ 243

PLANT SCIENCES .......................................................................... 252 FUTURE ........................................................................................... 252 REFERENCES .................................................................................. 252

APPENDIX I GUIDELINES FOR A QUALITY ASSURANCE PROGRAM FOR DNA RESTRICTION FRAGMENT LENGTH POLYMORPHISM ANALYSIS ..................... 261

II THE COMBINED DNA INDEX SYSTEM (CODIS): A THEORETICAL MODEL ............................................. 279

ill SUPPLIERS OF DNA ANALYSIS EQUIPMENT, PRODUCTS, OR SERVICES ••.•.....•••••... 319

GLOSSARY ••••••••••••••.••••••.•..••..•••.•..••••••••••..•..••••...•...•••••.•.........•••..•... 325

ABBREVIATIONS AND SYMBOLS .•••••••.•.••••.•••..•..•.•••.•••••••...•.••• 345

NAME INDEX ................................................................................... 351

SUBJECT IND EX ............................................................................. 359

PREFACE

Each individual, except identical twins, possesses a unique genetic signature that can be visualized using recombinant DNA techniques. The signatures resemble the scanner bar codes on supermarket products.

These DNA "fingerprints," or profiles, have broad applications in forensic analysis, paternity testing, diagnostic medicine, plant and animal sciences, and wildlife poaching. Where applicable-for example in homicide, rape, and assault cases-the use of the new procedures should considerably reduce the weight of eyewitness testimony, which many believe is responsible for more miscarriages of justice than any other single type of evidence. As suggested by Judge Joseph Harris (1988): County of Albany in the State of New York, this new technology could be the greatest single advance in the search for truth, conviction of the guilty, and acquittal of the innocent since the advent of cross-examination.

I had two objectives in writing this introductory text. First, there appeared to be a need at the field, laboratory, and courtroom levels for a summary of the principles and basic methodology underlying DNA analysis to identify tissues and individuals. As with any new technology, considerable care must be exercised during transfer from the experimental stage to service applications. Analysts involved in establishing and operating DNA laboratories and those individuals engaged in the legal aspects of forensic, paternity, and other identity cases should find this book a valuable source of information. A number of organizations, including government,

*People v. Wesley, 140 Misc. 2d 306,533 N.Y.S. 2d 643 (Co. Ct. 1988).

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xvi Preface

universities, law enforcement agencies, and private laboratories are attempting to standardize DNA fingerprinting protocols for human forensic application. In North America this process is being sponsored by the Federal Bureau of Investigation through the Technical Working Group on DNA Analysis Methods (TWGDAM). The standardization includes quality assurance, analysis materials and methods, and the assimilation of DNA profile databases. The recommendations available at the time of this printing are included in the methods sections and in Appendices I and IT.

My second objective was to provide a text for students in biological and medical sciences. Students not intimately familiar with recombinant DNA technology can read this book, learn how to use the techniques, and make informed judgments on issues concerning application of this new approach to identity testing.

The chapters have been organized to first provide background principles, then practical methodology, and then case applications. An extensive glossary has been included to assist those readers not familiar with DNA fingerprinting terminology. Detailed reference lists have also been compiled; these include the articles cited in each chapter and other references relevant to the subject area. Chapter 10 was con­tributed by Kenneth E. Melson, Esquire, Federal prosecutor in Alexandria, V A and Lecturer at the National Law Center, George Washington University, Washington, DC. Responsibility and credit for this contribution is given to Mr. Melson. The chapter provides an up-to-date presentation of legal issues related to DNA typing in the United States.

I am most indebted to Professor Martin L. Puterman for his considerable con­tribution to Chapter 8, Probability and Statistical Analysis. I am grateful to the Literary Executor of the late Sir Ronald A. Fisher, F.R.S. to Dr. Frank Yates, F.R.S. and the Longman Group Ltd, London for permission to reprint Table 8-1 from their book Statistical Tables for Biological, Agricultural and Medical Research (6th Edition 1974).

I thank the Department of Pathology, University of British Columbia, for support, including secretarial assistance with typing of the manuscript; the extra effort by the secretaries was most appreciated. I also thank Vicky Earle of Biochemical Communications for preparing a number of excellent illustrations.

lowe a special debt of thanks to Dr. Bruce Budowle and Dr. Ron Fourney for reviewing the manuscript and for making extremely helpful, detailed suggestions. I am also grateful to Molly Creery, Dr. Adrian Drobines, Dr. Peter Gill, Mr. Mike Luchemko, and Mr. Rick Miller for a number of suggestions. Thanks are due to Chris Munro and Pat Good for preparing the camera-ready materials and to Paula Pinter for those short periods of emergency assistance.

Lastly, what would authors do without the wisdom of editors like Dr. Jim Miller, who carried out ongoing edits during the manuscript preparation, and Dr. Ingrid Krohn, my perspicacious publisher whose support and enthusiasm, like DNA fingerprints, can only be described as unique?