簡介
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
目錄
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
