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Susanne Hummel· Ancient DNA Typing Methods, Strategies and Applications

Springer-Verlag Berlin Heidelberg GmbH

Susanne Hummel

Ancient DNA Typing Methods, Strategies and Applications

With 152 Figures and 34 Tables

Springer

Dr.

SUSANNE HUMMEL

Historische Anthropologie und Humanokologie Institut fUr Zoologie und Anthropologie Georg August Universitat Gottingen Biirgerstra6e 50 37073 Gottingen Germany

Library of Congress Cataloging. in-Publication Data Hummel, Susanne Ancient DNA typing: methods, strategies, and applications 1 Susanne Humel. p.cm. Includes bibliographical references and index. \. DNA, Fossil--Analysis. 2. Mitochondrial DNA--Analysis. 3. Nucleotide sequence. 4. Polymerase chain reaction.!. Title. This work is subject to copyright. All rights reserved, whether the whole or part of the material is concerned, specifically the rights of translation, reprinting, reuse of illustrations, recitation, broadcasting, reproduction on microfilm or in any other way, and storage in data banks. Duplication of this publication or parts thereof is permitted only under the provisions of the German Copyright Law of September 9,1965, in its current version, and permission for use must always be obtained from Springer-Veriag. Violations are liable for prosecution under the German Copyright Law. http://www.springer.de ISBN 978-3-642-07705-0 ISBN 978-3-662-05050-7 (eBook) DOl 10.1007/978-3-662-05050-7 © Springer-Verlag Berlin Heidelberg 2003 Originally published by Springer-Verlag Berlin Heidelberg New York in 2003. Solleover reprint of the hardcover 1st edition 2003

The use of general descriptive names, registered names, trademarks~ etc. in this publication does not imply, even in

the absence of a specific statement, that such names are exempt from the relevant protective laws and regulations and therefore free for general use. Cover design: Design & Production, Heidelberg Typesetting: Cameraready by the author SPIN 10645234 31/3150 - 5432 10- Printed on acid-free paper

De~icate~

to m~ parents

Preface

Who does not remember the genetic identification of the skeletons of the Romanov family or the investigation of the putative clothing of the famous Kaspar Hauser? Those are highlights in the analysis of ancient DNA, a field that has come a long way in the last decade. The topic of ancient DNA analysis and the possible future implications inspire both the public and the scientific community. This book addresses scientific practitioners and graduate students from different disciplines interested in ancient and degraded DNA research. The book is divided into chapters about methods and strategies, protocols and applications, and serves both as a laboratory manual and as a readable textbook in a meanwhile highly specialized field. It was possible to write this book because of the experience accumulated in ancient DNA typing and the committed efforts of many people who worked in the G6ttingen laboratory for ancient DNA research over the last decade. It was mainly these colleagues who carried out experiments for their Diploma and Ph.D. theses; many others worked on their post-doctoral research or were temporary guests in our lab. Therefore, I want to sincerely thank my colleagues, Nancy Banko, Dr. Heike Baron, Andrea Bartels, Ruth Bollongino, Dr. Barbara Bramanti, Dr. Joachim Burger, Dr. Julia Gerstenberger, Birgit GroSkopf, Karin Haack, Uta Immel, Oliver Krebs, Dr. Cadja Lassen, Dr. Kristin Launhardt, Dr. Odile Loreille, Annette Muller, Boris Muller, Dr. Gabriele Nordsiek, Dirk Peters, Dr. Jens Rameckers, Dr. Wera Schmerer, Diane Schmidt, Dr. Tobias Schmidt, Dr. Tobias Schultes, Dr. Peter Spencer, Dr. Lingxia Zhao, and Holger Zierdt. For most reliable technical assistance in the laboratory my thanks go to Melanie Kahle, Nicole Weber, and Birgit Zeike as well as to the trainees Annegret Becker, Simone Haffner, and Katrin Lasch. I would also like to thank Sabine Becker for preparing microdissections, Eberhardt George for taxidermic work, Sibylle Hourticolon for object photography, and Gerlinde Tavakolian for the administration of our research grant money. For cooperation and discussion, I thank Prof. Bernd Brinkmann, Director of the Institute of Legal Medicine, Munster, Dr. Lothar Kaup of the Landeskriminalamt, Hannover, Dr. Martin Oppermann of the Department of Immunolgy, G6ttingen, and Dr. Herman Schmitter of the Bundeskriminalamt, Wiesbaden.

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Nothing could have been done without sample materials. For supporting our research work through continuous access to the invaluable skeletal materials of the Lichtenstein cave, I am grateful to Dr. Stefan Flindt, archaeologist in OsterodelHarz. Likewise, the support of Prof. Gisela Grupe, Dr. Thomas Kapeghiy, Dr. Lothar Klappauf, Dr. Andje Knaack, Regine Krull, Helmut Mayer, Dr. Helmut Rohlfing, Dr. Reinhold Schoon, Dr. Holger Schutkowski, Dr. Angela Simons, Dr. Susi Ullrich, Christian Velde, Dr. Joachim Wahl, and the Customs Office of the Frankfurt Airport is gratefully appreciated. The following institutions are acknowledged for funding our research work: the Bundesministerium fur Bildung und Forschung ("Neue Technologien in den Geisteswissenschaften," KFA Jiilich), the Ministerium fUr Wissenschaft und Kunst, Niedersachsen, the Deutsche Forschungsgemeinschaft, the Stiftung Volkswagenwerk, and the Niedersachsische Umweltstiftung. Further, my thanks go to Dr. Peter Spencer, Perth, who spent quite some time "translating" major parts of the manuscript from "German English" to "English English". Also, I am grateful for the advice and help of Dr. Alexander Fabig and Holger Zierdt in handling the "template" for preparing camera-ready manuscripts for Springer-Verlag and finding out more about many special features of additional software that was necessary for this task. Dr. Dieter Czeschlik of Springer-Verlag, Heidelberg, is acknowledged for incorporating this book into the Springer publishing program, his support, and his patience. For stimulating scientific discussion throughout the last 2 years and much help in the preparation of the manuscript, I sincerely thank Diane Schmidt. Last, but not least, I want to express my special thanks to Prof. Bernd Herrmann who has offered me - and still does - constant encouragement and support throughout my scientific career. In the 1980s, it was Prof. Herrmann who initiated the first ancient DNA experiments in G6ttingen and who gave me the chance to enter this particular scientific field. His ideas are inspiring and truly belong to the scientific avant-garde.

Contents

1 Introduction ............................................................................................. 1 References ............................................................................................. 10 2 DNA markers ........................................................................................ 19 2.1 Mitochondrial DNA ........................................................................ 20 2.1.1 The hypervariable regions and control region V ..................... 23 2.1.2 Cytochrome b .......................................................................... 25 2.2 Chromosomal DNA ........................................................................ 26 2.2.1 Amelogenin ............................................................................. 29 2.2.2 Autosomal STRs ...................................................................... 30 2.2.3 Y-chromosomal STRs ............................................................. 39 2.2.4 X-chromosomal STRs ............................................................ .42 2.2.5 ABO blood group genes ......................................................... .43 2.2.6 CCR5 ....................................................................................... 46 2.2.7 ~F508 ...................................................................................... 47 References ............................................................................................. 48 3 aDNA extraction ................................................................................... 57

3.1 Comparison of extraction methods ................................................. 58 3.2 aDNA yield ..................................................................................... 63 3.3 aDNA preservation ......................................................................... 66 3.3.1 aDNA degradation patterns ..................................................... 66 3.3.2 The age ofaDNA ..................................................................... 68 3.3.3 Fragment lengths of aDNA ...................................................... 73 3.3.4 Storage of aDNA extracts ........................................................ 76 References ............................................................................................. 78 4 PCR ........................................................................................................ 81 4.1 Basic PCR mechanism .................................................................... 82 4.2 Number of amplification cycles ...................................................... 85 4.3 Cycling parameters ......................................................................... 86 4.3.1 Annealing temperature ............................................................ 86 4.3.2 Denaturation temperature ........................................................ 87

x 4.3.3 Elongation temperature ............................................................ 88 4.3.4 Durations of the cycling steps .................................................. 89 4.4 Primer design .................................................................................. 90 4.4.1 Matching primers ..................................................................... 91 4.4.2 Primer energy profile ................................................................ 92 4.4.3 Annealing temperature, primer dimer and hairpins ................. 93 4.4.4 Mismatch primers .................................................................... 95 4.4.5 Software packages ................................................................... 96 4.4.6 Primer design - step by step .................................................... 96 4.5 Multiplex PCR .............................................................................. 101 4.6 PCR failures .................................................................................. 102 4.6.1 DNA degradation ................................................................... 102 4.6.2 Inhibition ............................................................................... 104 4.6.3 DNA overload and overcycling ............................................. 105 References ........................................................................................... 107

5 Analysis of peR products .................................................................. 111 5.1 Fragment length analysis .............................................................. 112 5.1.1 Agarose gel electrophoresis ................................................... 113 5.1.2 Automated fragment length determination ............................ 115 5.2 Sequence analysis ......................................................................... 120 5.2.1 Direct sequencing vs. sequencing clones ............................... 122 5.2.2 Overlapping sequences vs. a multiplex approach .................. 124 5.3 RFLP analysis ............................................................................... 126 References ........................................................................................... 128

6 Authenticity of results ........................................................................ 131 6.1 Contamination sources .................................................................. 132 6.1.1 Contamination through handling ........................................... 13 3 6.1.2 Contamination in reaction tubes ............................................ 136 6.1.3 Cleaning the PCR reaction tubes ........................................... 144 6.1.4 PCR in glass tubes ................................................................. 147 6.2 Validating aDNA results ............................................................... 150 6.3 Ring exercises ............................................................................... 155 References ........................................................................................... 155

7 Applications ......................................................................................... 159 7.1 Sex determination ......................................................................... 160 7.1.1 The children of Aegerten ....................................................... 160 7.1.2 Sex determination by multiplex amplification ....................... 164 7.2 Identification ................................................................................. 165 7.2.1 A disturbed burial site ............................................................ 165

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7.2.2 Duke Christian II ................................................................... 168 7.2.3 Pieces of parchment.. ............................................................. 170 7.2A Sheep, goats and cattle ........................................................... 172 7.2.5 Species, sex and individual identification ............................. 173 7.2.6 Dog or fox .............................................................................. 174 7.2.7 Elephants and rhinoceroses .................................................... 176 7.2.8 Beef in the sausage ................................................................ 179 7.2.9 Rock-wallabies on Depuch Island ......................................... 181 7.3 Kinship .......................................................................................... 183 7.3.1 A Merovingian family ........................................................... 183 7.3.2 The earls of Konigsfeld ......................................................... 185 7.3.3 The Lichtenstein cave - a place of sacrifice or a burial site? 188 7.3.4 Widukind ............................................................................... 193 7.3.5 Orangutans ............................................................................. 197 7 A Population genetics ....................................................................... 200 7 A.l Marriage patterns in Weingarten ........................................... 200 7 A.2 Expert knowledge in Goslar .................................................. 204 7A.3 ABO Blood groups ................................................................. 207 7AA Ll32ccr5 already in the Bronze Age ....................................... 211 7.4.5 LlF 508 in Alia ........................................................................ 214 7 A.6 Y -chromosomal haplotypes and the region of origin ............ 216 References ........................................................................................... 21 7 8 Protocols .............................................................................................. 225 8.1 Collection and storage of samples ................................................ 225 8.2 Sample preparation ....................................................................... 226 8.3 Phenol-based DNA extraction ...................................................... 227 8A DNA extraction from saliva .......................................................... 230 8.5 PCR primers and protocols ........................................................... 231 8.6 Agarose gel electrophoresis .......................................................... 250 8.7 Taq-cyc1e sequencing ................................................................... 253 8.8 PAA gel electrophoresis ............................................................... 254 8.9 RFLP digestion ............................................................................. 258 8.1 0 Basic chemistry and reagents ...................................................... 259 References ........................................................................................... 268 Appendix ................................................................................................. 271 I Laboratory equipment ...................................................................... 271 11 Pre-PCR laboratory equipment ................................................. 272 12 PCR laboratory .......................................................................... 272 1.3 Post-PCR laboratory .................................................................. 273 IA Photographic documentation ..................................................... 273

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II peR trouble shooting ...................................................................... 274 III Internet sites of interest... ............................................................... 278 Subject index........................................................................................... 287