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 casecontrol 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 Proteinprotein 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
