Genomics: Essential Methods

Genomics research has made significant advances in recent years. In this book, a team of internationally-renowned researchers share the most up-to-date information in a field that has in recent years switched emphasis from gene identification to functional genomics and the characterization of genes and gene products. This volume approaches this complex subject with a broad perspective to supply the reader with a vital overview of genomics and its derivative fields, with a focus on pivotal issues such as data analysis. Expansive and current, this book is a comprehensive research guide that describes both the key new techniques and more established methods. Every chapter discusses the merits and limitations of the various approaches and then provides selected tried-and-tested protocols, as well as a plethora of good practical advice for immediate use at the bench. Key features: Provides a broad introduction to current practices and techniques for lab-based research in genomics Explains clearly and precisely how to carry out selected techniques in addition to background information on the various approaches Chapters are written by a leading international authorities in the field and cover both well-known and new, tried and tested, methods for working in genomics Includes troubleshooting guide and reviews of alternative techniques An essential laboratory manual for students and researchers at all levels

Table of 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 1.1 Introduction 1 1.2 Methods and approaches 2 1.2.1 Oligonucleotide aCGH 2 1.2.2 SNP aCGH 15 1.2.3 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. Miller 2.1 Introduction 33 2.2 Methods and approaches 34 2.2.1 Repositories of known genetic variants 34 2.2.2 Targeted resequencing for variant discovery 35 2.3 Troubleshooting 45 2.3.1 Primer design 45 2.3.2 PCR amplification 45 2.3.3 Working with binary trace files 46 2.3.4 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 3.1 Introduction 49 3.2 Methods and approaches 50 3.2.1 Arrays 50 3.2.2 Genotyping 50 3.2.3 Linkage and association analysis 51 3.2.4 Formalin-fixed, paraffin-embedded tissue 51 3.2.5 Loss of heterozygosity 58 3.3 Troubleshooting 63 References 64 4 Genetic Mapping of Complex Traits 67 Nancy L. Saccone 4.1 Introduction 67 4.2 Methods and approaches 68 4.2.1 Association methods: unrelated case–control samples 68 4.2.2 Association methods: family-based samples 81 4.2.3 Linkage methods: parametric LOD score analysis 82 4.2.4 Linkage methods: non-parametric methods 83 4.2.5 Summary and conclusions 84 4.3 Troubleshooting 84 4.3.1 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 Introduction 91 5.1.1 The need for amplification 91 5.1.2 Amplification approaches 93 5.2 Methods and approaches 100 5.2.1 T7 RNA polymerase-based in vitro transcription 100 5.2.2 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 6.1 Introduction 121 6.2 Methods and approaches 122 6.2.1 Sample selection 122 6.2.2 RNA extraction 123 6.2.3 Clinical and environmental samples 127 6.2.4 Reverse transcription 130 6.2.5 qPCR using SYBR green I dye detection 134 6.2.6 qPCR using labeled oligonucleotide probe detection 137 6.2.7 Quantification methods 140 6.2.8 RT-qPCR standardization 143 6.3 Troubleshooting 144 6.3.1 No/Poor/Late amplification 144 6.3.2 No-template, negative control yields an amplification product 147 6.3.3 No reverse transcriptase control yields an amplification product 148 6.3.4 Primer dimers formed 148 6.3.5 Multiple peaks in SYBR green I melt curve 148 6.3.6 Standard curve is unreliable (correlation coefficient <0.98 over at least 5 log dilution and with samples repeated in triplicate) 149 6.3.7 Erratic amplification plots/high well-to-well variation 149 References 149 7 Gene Expression in Mammalian Cells 155 Félix Recillas-Targa, Georgina Guerrero, Martín Escamilla-del-Arenal and Héctor Rincón-Arano 7.1 Introduction 155 7.1.1 Artificial chromosomes and transgenesis 157 7.1.2 Gene transfer and expression problems 157 7.1.3 Position effects and chromatin 157 7.1.4 Tissue-specific regulatory elements 158 7.1.5 Sustained expression and chromatin insulators 158 7.2 Methods and approaches 159 7.2.1 Site-specific chromosomal integration in mammalian cells 159 7.2.2 Plasmid requirement 161 7.2.3 Chromosome transfer 163 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. Sanderson 8.1 Introduction 173 8.2 Methods and approaches 174 8.2.1 Producing large numbers of bait or prey clones 174 8.2.2 Generating recombination-compatible inserts for gap repair cloning 177 8.2.3 Performing gap repair reactions 179 8.2.4 Identifying positive transformants 181 8.2.5 Yeast colony PCR 181 8.2.6 Bait and prey auto-activation tests 183 8.2.7 Targeted ‘matrix’-style Y2H screens 184 8.3 Troubleshooting 188 References 189 9 Prediction of Protein Function 191 HonNianChua 9.1 Introduction 191 9.2 Methods and approaches 191 9.2.1 Annotation schemes 192 9.2.2 Working with multiple protein identifier systems 195 9.2.3 Sequence homology 196 9.2.4 Phylogenetic relationships 199 9.2.5 Sequence-derived functional and chemical properties 202 9.2.6 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 10.1 Introduction 211 10.2 Methods and approaches 212 10.2.1 Principles of targeted gene deletion in mice 212 10.2.2 Strategies for gene targeting in mice 215 10.2.3 Retrieval of DNA from BAC by recombineering 217 10.2.4 ES and MEF cell culture 222 10.2.5 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 11.1 Introduction 249 11.2 Methods and approaches 250 11.2.1 The ideal gene therapy vector 250 11.2.2 Plasmid design 251 11.2.3 Viral vectors 252 11.2.4 Non-viral DNA vectors 263 11.2.5 Assessing the physical properties of a non-viral vector 267 11.2.6 Optimizing in vitro gene delivery 268 11.2.7 Optimization strategies 271 11.2.8 Reporter genes and assays 271 11.2.9 Cytotoxicity assays 272 11.2.10 Future steps for non-viral vector development 272 11.3 Troubleshooting 273 11.3.1 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 Introduction 283 12.1.1 General strategies for gene therapy: Basic methods 284 12.1.2 Gene therapy strategies: Delivering genes to cells 287 12.1.3 Viral delivery 288 12.1.4 Production, purification and titration of recombinant adeno-associated virus (rAAV) 291 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. Friedman 13.1 Introduction 307 13.2 Methods and approaches 309 13.2.1 Gel-based strategies 309 13.2.2 LC/MS strategies 312 13.2.3 MALDI imaging and profiling 314 13.3 Troubleshooting 316 13.3.1 Number of resolved features and modifications 316 13.3.2 Sample consumption, protein identification and depth of coverage 317 13.3.3 Statistical power 317 13.3.4 Conclusions 318 References 318 Index 325