Genomics

Dr Mike Starkey, Animal Health Trust, Newmarket, Suffolk, UK. Dr Ramnath Elaswarapu, Oxford Gene Technology Ltd, Yarnton, Oxford, UK.

• List of Contributors xiPreface xv1 High-Resolution Analysis of Genomic Copy Number Changes 1Mario Hermsen, Jordy Coffa, Bauke Ylstra, Gerrit Meijer, Hans Morreau, Ronald van Eijk, Jan Oosting and Tom van Wezel1.1 Introduction 11.2 Methods and approaches 21.2.1 Oligonucleotide aCGH 21.2.2 SNP aCGH 151.2.3 Multiple ligation-dependent probe amplification (MLPA) 191.3 Troubleshooting 28References 292 Identification of Polymorphic Markers for Genetic Mapping 33Daniel C. Koboldt and Raymond D. Miller2.1 Introduction 332.2 Methods and approaches 342.2.1 Repositories of known genetic variants 342.2.2 Targeted resequencing for variant discovery 352.3 Troubleshooting 452.3.1 Primer design 452.3.2 PCR amplification 452.3.3 Working with binary trace files 462.3.4 Phred/Phrap 46References 463 Genotyping and LOH Analysis on Archival Tissue Using SNP Arrays 49Ronald van Eijk, Anneke Middeldorp, Esther H. Lips, Marjo van Puijenbroek, Hans Morreau, Jan Oosting and Tom van Wezel3.1 Introduction 493.2 Methods and approaches 503.2.1 Arrays 503.2.2 Genotyping 503.2.3 Linkage and association analysis 513.2.4 Formalin-fixed, paraffin-embedded tissue 513.2.5 Loss of heterozygosity 583.3 Troubleshooting 63References 644 Genetic Mapping of Complex Traits 67Nancy L. Saccone4.1 Introduction 674.2 Methods and approaches 684.2.1 Association methods: unrelated case–control samples 684.2.2 Association methods: family-based samples 814.2.3 Linkage methods: parametric LOD score analysis 824.2.4 Linkage methods: non-parametric methods 834.2.5 Summary and conclusions 844.3 Troubleshooting 844.3.1 Combining datasets 84References 855 RNA Amplification Strategies: Toward Single-Cell Sensitivity 91Natalie Stickle, Norman N. Iscove, Carl Virtanen, Mary Barbara, Carolyn Modi, Toni Di Berardino, Ellen Greenblatt, Ted Brown and Neil Winegarden5.1 Introduction 915.1.1 The need for amplification 915.1.2 Amplification approaches 935.2 Methods and approaches 1005.2.1 T7 RNA polymerase-based in vitro transcription 1005.2.2 Global-RT-PCR 1075.3 Troubleshooting 115References 1166 Real-Time Quantitative RT-PCR for mRNA Profiling 121Stephen A. Bustin and Tania Nolan6.1 Introduction 1216.2 Methods and approaches 1226.2.1 Sample selection 1226.2.2 RNA extraction 1236.2.3 Clinical and environmental samples 1276.2.4 Reverse transcription 1306.2.5 qPCR using SYBR green I dye detection 1346.2.6 qPCR using labeled oligonucleotide probe detection 1376.2.7 Quantification methods 1406.2.8 RT-qPCR standardization 1436.3 Troubleshooting 1446.3.1 No/Poor/Late amplification 1446.3.2 No-template, negative control yields an amplification product 1476.3.3 No reverse transcriptase control yields an amplification product 1486.3.4 Primer dimers formed 1486.3.5 Multiple peaks in SYBR green I melt curve 1486.3.6 Standard curve is unreliable (correlation coefficient <0.98 over at least 5 log dilution and with samples repeated in triplicate) 1496.3.7 Erratic amplification plots/high well-to-well variation 149References 1497 Gene Expression in Mammalian Cells 155Félix Recillas-Targa, Georgina Guerrero, Martín Escamilla-del-Arenal and Héctor Rincón-Arano7.1 Introduction 1557.1.1 Artificial chromosomes and transgenesis 1577.1.2 Gene transfer and expression problems 1577.1.3 Position effects and chromatin 1577.1.4 Tissue-specific regulatory elements 1587.1.5 Sustained expression and chromatin insulators 1587.2 Methods and approaches 1597.2.1 Site-specific chromosomal integration in mammalian cells 1597.2.2 Plasmid requirement 1617.2.3 Chromosome transfer 1637.3 Troubleshooting 169Acknowledgments 169References 1708 Using Yeast Two-Hybrid Methods to Investigate Large Numbers of Binary Protein Interactions 173Panagoula Charalabous, Jonathan Woodsmith and Christopher M. Sanderson8.1 Introduction 1738.2 Methods and approaches 1748.2.1 Producing large numbers of bait or prey clones 1748.2.2 Generating recombination-compatible inserts for gap repair cloning 1778.2.3 Performing gap repair reactions 1798.2.4 Identifying positive transformants 1818.2.5 Yeast colony PCR 1818.2.6 Bait and prey auto-activation tests 1838.2.7 Targeted ‘matrix’-style Y2H screens 1848.3 Troubleshooting 188References 1899 Prediction of Protein Function 191HonNianChua9.1 Introduction 1919.2 Methods and approaches 1919.2.1 Annotation schemes 1929.2.2 Working with multiple protein identifier systems 1959.2.3 Sequence homology 1969.2.4 Phylogenetic relationships 1999.2.5 Sequence-derived functional and chemical properties 2029.2.6 Protein–protein interaction maps 2039.3 Troubleshooting 205References 20510 Elucidating Gene Function through Use of Genetically Engineered Mice 211Mary P. Heyer, Cátia Feliciano, João Peca and Guoping Feng10.1 Introduction 21110.2 Methods and approaches 21210.2.1 Principles of targeted gene deletion in mice 21210.2.2 Strategies for gene targeting in mice 21510.2.3 Retrieval of DNA from BAC by recombineering 21710.2.4 ES and MEF cell culture 22210.2.5 Mating of chimeras and downstream applications 24410.3 Troubleshooting 245References 24611 Delivery Systems for Gene Transfer 249Charlotte Lawson and Louise Collins11.1 Introduction 24911.2 Methods and approaches 25011.2.1 The ideal gene therapy vector 25011.2.2 Plasmid design 25111.2.3 Viral vectors 25211.2.4 Non-viral DNA vectors 26311.2.5 Assessing the physical properties of a non-viral vector 26711.2.6 Optimizing in vitro gene delivery 26811.2.7 Optimization strategies 27111.2.8 Reporter genes and assays 27111.2.9 Cytotoxicity assays 27211.2.10 Future steps for non-viral vector development 27211.3 Troubleshooting 27311.3.1 General points 273References 27412 Gene Therapy Strategies: Constructing an AAV Trojan Horse 283M. Ian Phillips, Edilamar M. de Oliveira, Leping Shen, Yao Liang Tang and Keping Qian12.1 Introduction 28312.1.1 General strategies for gene therapy: Basic methods 28412.1.2 Gene therapy strategies: Delivering genes to cells 28712.1.3 Viral delivery 28812.1.4 Production, purification and titration of recombinant adeno-associated virus (rAAV) 29112.2 Methods and approaches 29212.3 Troubleshooting 303References 30413 An Introduction to Proteomics Technologies for the Genomics Scientist 307David B. Friedman13.1 Introduction 30713.2 Methods and approaches 30913.2.1 Gel-based strategies 30913.2.2 LC/MS strategies 31213.2.3 MALDI imaging and profiling 31413.3 Troubleshooting 31613.3.1 Number of resolved features and modifications 31613.3.2 Sample consumption, protein identification and depth of coverage 31713.3.3 Statistical power 31713.3.4 Conclusions 318References 318Index 325