Genomics Genomics: Essential Methods Edited by Mike Starkey and Ramnath Elaswarapu © 2011 John Wiley & Sons, Ltd. ISBN: 978-0-470-71157-6 Genomics Essential Methods Edited by Mike Starkey Animal Health Trust Ramnath Elaswarapu LGC Ltd. A John Wiley & Sons, Ltd., Publication This edition first published 2011, © 2011 John Wiley & Sons Ltd Wiley-Blackwell is an imprint of John Wiley & Sons, formed by the merger of Wiley’s global Scientific, Technical and Medical business with Blackwell Publishing. Registered office: John Wiley & Sons Ltd, The Atrium, Southern Gate, Chichester, West Sussex, PO19 8SQ, UK Other Editorial Offices: 9600 Garsington Road, Oxford, OX4 2DQ, UK 111 River Street, Hoboken, NJ 07030-5774, USA 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.com/wiley-blackwell The right of the author to be identified as the author 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. 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.
Library of Congress Cataloguing-in-Publication Data Genomics : essential methods/edited by Mike Starkey and Ramnath Elaswarapu.8 6 – dc22 2010028093 A catalogue record for this book is available from the British Library. This book is published in the following electronic formats: ePDF [9780470711620]; Wiley Online Library [9780470711675] Set in 10/12 TimesRoman by Laserwords Private Limited, Chennai, India First Impression 2011 Contents List of Contributors xi Preface xv 1 High-Resolution Analysis of Genomic Copy Number Changes 1 Mario Hermsen, Jordy Coffa, Bauke Ylstra, Gerrit Meijer, Hans Morreau, Ronald van Eijk, Jan Oosting and Tom van Wezel 1.2 Methods and approaches 2 1.3 Multiple ligation-dependent probe amplification (MLPA) 19 1.3 Troubleshooting 28 References 29 2 Identification of Polymorphic Markers for Genetic Mapping 33 Daniel C. Koboldt and Raymond D.2 Methods and approaches 34 2.1 Repositories of known genetic variants 34 2.2 Targeted resequencing for variant discovery 35 2.3 Working with binary trace files 46 2.4 Phred/Phrap 46 References 46 3 Genotyping and LOH Analysis on Archival Tissue Using SNP Arrays 49 Ronald van Eijk, Anneke Middeldorp, Esther H. Lips, Marjo van Puijenbroek, Hans Morreau, Jan Oosting and Tom van Wezel 3.2 Methods and approaches 50 3.2 Genotyping 50 vi CONTENTS 3.3 Linkage and association analysis 51 3.4 Formalin-fixed, paraffin-embedded tissue 51 3.5 Loss of heterozygosity 58 3.3 Troubleshooting 63 References 64 4 Genetic Mapping of Complex Traits 67 Nancy L.2 Methods and approaches 68 4.1 Association methods: unrelated case–control samples 68 4.2 Association methods: family-based samples 81 4.3 Linkage methods: parametric LOD score analysis 82 4.4 Linkage methods: non-parametric methods 83 4.5 Summary and conclusions 84 4.1 Combining datasets 84 References 85 5 RNA Amplification Strategies: Toward Single-Cell Sensitivity 91 Natalie Stickle, Norman N.
Iscove, Carl Virtanen, Mary Barbara, Carolyn Modi, Toni Di Berardino, Ellen Greenblatt, Ted Brown and Neil Winegarden 5.1 The need for amplification 91 5.2 Methods and approaches 100 5.1 T7 RNA polymerase-based in vitro transcription 100 5.2 Global-RT-PCR 107 5.3 Troubleshooting 115 References 116 6 Real-Time Quantitative RT-PCR for mRNA Profiling 121 Stephen A. Bustin and Tania Nolan 6.2 Methods and approaches 122 6.3 Clinical and environmental samples 127 6.5 qPCR using SYBR green I dye detection 134 6.6 qPCR using labeled oligonucleotide probe detection 137 6.8 RT-qPCR standardization 143 6.1 No/Poor/Late amplification 144 6.2 No-template, negative control yields an amplification product 147 CONTENTS vii 6.3 No reverse transcriptase control yields an amplification product 148 6.4 Primer dimers formed 148 6.5 Multiple peaks in SYBR green I melt curve 148 6.6 Standard curve is unreliable (correlation coefficient <0.98 over at least 5 log dilution and with samples repeated in triplicate) 149 6.7 Erratic amplification plots/high well-to-well variation 149 References 149 7 Gene Expression in Mammalian Cells 155 Félix Recillas-Targa, Georgina Guerrero, Martı́n Escamilla-del-Arenal and Héctor Rincón-Arano 7.1 Artificial chromosomes and transgenesis 157 7.2 Gene transfer and expression problems 157 7.3 Position effects and chromatin 157 7.4 Tissue-specific regulatory elements 158 7.5 Sustained expression and chromatin insulators 158 7.2 Methods and approaches 159 7.1 Site-specific chromosomal integration in mammalian cells 159 7.3 Troubleshooting 169 Acknowledgments 169 References 170 8 Using Yeast Two-Hybrid Methods to Investigate Large Numbers of Binary Protein Interactions 173 Panagoula Charalabous, Jonathan Woodsmith and Christopher M.2 Methods and approaches 174 8.1 Producing large numbers of bait or prey clones 174 8.2 Generating recombination-compatible inserts for gap repair cloning 177 8.3 Performing gap repair reactions 179 8.4 Identifying positive transformants 181 8.5 Yeast colony PCR 181 8.6 Bait and prey auto-activation tests 183 8.7 Targeted ‘matrix’-style Y2H screens 184 8.3 Troubleshooting 188 References 189 9 Prediction of Protein Function 191 Hon Nian Chua 9.2 Methods and approaches 191 9.2 Working with multiple protein identifier systems 195 viii CONTENTS 9.5 Sequence-derived functional and chemical properties 202 9.6 Protein–protein interaction maps 203 9.3 Troubleshooting 205 References 205 10 Elucidating Gene Function through Use of Genetically Engineered Mice 211 Mary P. Heyer, Cátia Feliciano, João Peca and Guoping Feng 10.2 Methods and approaches 212 10.1 Principles of targeted gene deletion in mice 212 10.2 Strategies for gene targeting in mice 215 10.3 Retrieval of DNA from BAC by recombineering 217 10.4 ES and MEF cell culture 222 10.5 Mating of chimeras and downstream applications 244 10.3 Troubleshooting 245 References 246 11 Delivery Systems for Gene Transfer 249 Charlotte Lawson and Louise Collins 11.2 Methods and approaches 250 11.1 The ideal gene therapy vector 250 11.4 Non-viral DNA vectors 263 11.5 Assessing the physical properties of a non-viral vector 267 11.6 Optimizing in vitro gene delivery 268 11.8 Reporter genes and assays 271 11.10 Future steps for non-viral vector development 272 11.1 General points 273 References 274 12 Gene Therapy Strategies: Constructing an AAV Trojan Horse 283 M. Ian Phillips, Edilamar M.
de Oliveira, Leping Shen, Yao Liang Tang and Keping Qian 12.1 General strategies for gene therapy: Basic methods 284 12.2 Gene therapy strategies: Delivering genes to cells 287 12.4 Production, purification and titration of recombinant adeno-associated virus (rAAV) 291 CONTENTS ix 12.2 Methods and approaches 292 12.3 Troubleshooting 303 References 304 13 An Introduction to Proteomics Technologies for the Genomics Scientist 307 David B.2 Methods and approaches 309 13.1 Gel-based strategies 309 13.2 LC/MS strategies 312 13.3 MALDI imaging and profiling 314 13.1 Number of resolved features and modifications 316 13.2 Sample consumption, protein identification and depth of coverage 317 13.4 Conclusions 318 References 318 Index 325 List of Contributors Mary Barbara Hon Nian Chua Ontario Cancer Institute, Data Mining Department, Princess Margaret Hospital, Institute for Infocomm Research, University Health Network, 1 Fusionopolis Way, 101 College Street, #21-01 Connexis (South Tower), TMDT, 8-356, Singapore 138632, Toronto, Republic of Singapore ON M5G 1L7, Canada Jordy Coffa Ted Brown Department of Pathology, Samuel Lunenfeld Research Institute, VU University Medical Center, Mount Sinai Hospital, De Boelelaan, 1117, Joseph and Wolf Lebovic Centre, 1081 HV, 60 Murray Street, Amsterdam, 6th Floor, The Netherlands Toronto, ON M5T 3L9, Canada Louise Collins Department of Clinical Sciences, Stephen A. Bustin Kings’s College London School of Medicine, Institute of Cell and Molecular Science, James Black Centre, Barts and The London, 125 Coldharbour Lane, Queen Mary’s School of Medicine London, and Dentistry, SE5 9NU, UK University of London, Whitechapel, London E1 1BB, Edilamar M. de Oliveira UK Laboratory of Biochemistry, School of Physical Education and Sport, Panagoula Charalabous Sao Paulo University, Department of Physiology, Mello Moraes, 65, School of Biomedical Sciences, Cidade Universitária, University of Liverpool, Sao Paulo, Crown Street, 05508-9000, Liverpool L69 3BX, UK Brazil xii LIST OF CONTRIBUTORS Toni Di Berardino Georgina Guerrero Mount Sinai Hospital Centre for Fertility and Instituto de Fisiologı́a Celular, Reproductive Health, Departamento de Genética Molecular, 250 Dundas Street, Universidad Nacional Autónoma de México, West 7th Floor, Apartado Postal 70-242, Toronto, México D. 04510, ON M5T 2Z5, Mexico Canada Mario Hermsen Dept.
Otorrinolaringologı́a, Martı́n Escamilla-del-Arenal Instituto Universitario de Oncologı́a del Instituto de Fisiologı́a Celular, Principado de Asturias, Departamento de Genética Molecular, Edificio H Covadonga 1a Planta Centro, Universidad Nacional Autónoma de México, Lab 2, Apartado Postal 70-242, Hospital Universitario Central de Asturias, México D. 04510, Celestino Villamil s/n, Mexico 33006 Oviedo, Spain Cátia Feliciano Department of Neurobiology, Mary P. Heyer Duke University Medical Center, Department of Neurobiology, 401F Bryan Research Building, Duke University Medical Center, Research Drive, 401F Bryan Research Building, Durham, NC 27710, Research Drive, USA Durham, NC 27710, USA Guoping Feng Norman N. Iscove Department of Neurobiology, Ontario Cancer Institute, Duke University Medical Center, Princess Margaret Hospital, 401F Bryan Research Building, University Health Network, Research Drive, 101 College Street, Durham, NC 27710, TMDT, 8-356, USA Toronto, ON M5G 1L7, David B.
Friedman Canada Vanderbilt University School of Medicine, 215 Light Hall, Daniel C. Koboldt Nashville, Department of Genetics, TN 37232, Washington University School of Medicine, USA 4444 Forest Park Avenue, Box 8501, St. Louis, MO 63108, Ellen Greenblatt USA Mount Sinai Hospital Centre for Fertility and Reproductive Health, Charlotte Lawson 250 Dundas Street, Veterinary Basic Sciences, West 7th Floor, Royal Veterinary College, Toronto, Royal College Street, ON M5T 2Z5, London NW1 0TU, Canada UK LIST OF CONTRIBUTORS xiii Esther H. Lips Tania Nolan Department of Pathology, Sigma-Aldrich House, Leiden University Medical Center, Homefield Business Park, Leiden, Homefield Road, PO Box 9600, Haverhill, 2300RC, Suffolk CB9 8QP, The Netherlands UK Gerrit Meijer Jan Oosting Department of Pathology, Department of Pathology, VU University Medical Center, Leiden University Medical Center, De Boelelaan, 1117, Leiden, 1081 HV, PO Box 9600, Amsterdam, 2300RC, The Netherlands The Netherlands Anneke Middeldorp João Peca Department of Neurobiology, Department of Pathology, Duke University Medical Center, Leiden University Medical Center, 401F Bryan Research Building, Leiden, Research Drive, PO Box 9600, 2300RC, Durham, NC 27710, The Netherlands USA Raymond D.
Ian Phillips Department of Genetics, Keck Graduate Institute, Washington University School of Medicine, Claremont University Colleges, 4566 Scott Ave., 535 Watson Drive, St. Louis, Claremont, MO 63110, CA 91711, USA USA Carolyn Modi Keping Qian University Health Network Microarray Centre, Keck Graduate Institute, 101 College Street, Claremont University Colleges, TMDT, 9-301, 535 Watson Drive, Toronto, Claremont, ON M5G 1L7, CA 91711, Canada USA Hans Morreau Félix Recillas-Targa Department of Pathology, Instituto de Fisiologı́a Celular, Leiden University Medical Center, Departamento de Genética Molecular, Leiden, Universidad Nacional Autónoma de México, PO Box 9600, Apartado Postal 70-242, 2300RC, México D. 04510, The Netherlands Mexico xiv LIST OF CONTRIBUTORS Héctor Rincón-Arano Marjo van Puijenbroek Instituto de Fisiologı́a Celular, Department of Pathology, Departamento de Genética Molecular, Leiden University Medical Center, Universidad Nacional Autónoma de México, Leiden, PO Box 9600, Apartado Postal 70-242, 2300RC, México D. 04510, The Netherlands Mexico Nancy L.
Saccone Tom van Wezel Department of Genetics, Department of Pathology, Division of Human Genetics, Leiden University Medical Center, Washington University School of Medicine, Leiden, St. Louis, PO Box 9600, MO 63110, 2300RC, USA The Netherlands Christopher M.