Genomics -- Contents -- List of Contributors -- Preface -- 1 High-Resolution Analysis of Genomic Copy Number Changes -- 1.1 Introduction -- 1.2 Methods and approaches -- 1.2.1 Oligonucleotide aCGH -- 1.2.2 SNP aCGH -- 1.2.3 Multiple ligation-dependent probe amplification (MLPA) -- 1.3 Troubleshooting -- References -- 2 Identification of Polymorphic Markers for Genetic Mapping -- 2.1 Introduction -- 2.2 Methods and approaches -- 2.2.1 Repositories of known genetic variants -- 2.2.2 Targeted resequencing for variant discovery -- 2.3 Troubleshooting -- 2.3.1 Primer design -- 2.3.2 PCR amplification -- 2.3.3 Working with binary trace files -- 2.3.4 Phred/Phrap -- References -- 3 Genotyping and LOH Analysis on Archival Tissue Using SNP Arrays -- 3.1 Introduction -- 3.2 Methods and approaches -- 3.2.1 Arrays -- 3.2.2 Genotyping -- 3.2.3 Linkage and association analysis -- 3.2.4 Formalin-fixed, paraffin-embedded tissue -- 3.2.5 Loss of heterozygosity -- 3.3 Troubleshooting -- References -- 4 Genetic Mapping of Complex Traits -- 4.1 Introduction -- 4.2 Methods and approaches -- 4.2.1 Association methods: unrelated case-control samples -- 4.2.2 Association methods: family-based samples -- 4.2.3 Linkage methods: parametric LOD score analysis -- 4.2.4 Linkage methods: non-parametric methods -- 4.2.5 Summary and conclusions -- 4.3 Troubleshooting -- 4.3.1 Combining datasets -- References -- 5 RNA Amplification Strategies: Toward Single-Cell Sensitivity -- 5.1 Introduction -- 5.1.1 The need for amplification -- 5.1.2 Amplification approaches -- 5.2 Methods and approaches -- 5.2.1 T7 RNA polymerase-based in vitro transcription -- 5.2.2 Global-RT-PCR -- 5.3 Troubleshooting -- References -- 6 Real-Time Quantitative RT-PCR for mRNA Profiling -- 6.1 Introduction -- 6.2 Methods and approaches -- 6.2.1 Sample selection -- 6.2.2 RNA extraction.

6.2.3 Clinical and environmental samples -- 6.2.4 Reverse transcription -- 6.2.5 qPCR using SYBR green I dye detection -- 6.2.6 qPCR using labeled oligonucleotide probe detection -- 6.2.7 Quantification methods -- 6.2.8 RT-qPCR standardization -- 6.3 Troubleshooting -- 6.3.1 No/Poor/Late amplification -- 6.3.2 No-template, negative control yields an amplification product -- 6.3.3 No reverse transcriptase control yields an amplification product -- 6.3.4 Primer dimers formed -- 6.3.5 Multiple peaks in SYBR green I melt curve -- 6.3.6 Standard curve is unreliable (correlation coef.cient <0.98 over at least 5 log dilution and with samples repeated in triplicate) -- 6.3.7 Erratic amplification plots/high well-to-well variation -- References -- 7 Gene Expression in Mammalian Cells -- 7.1 Introduction -- 7.1.1 Artificial chromosomes and transgenesis -- 7.1.2 Gene transfer and expression problems -- 7.1.3 Position effects and chromatin -- 7.1.4 Tissue-specific regulatory elements -- 7.1.5 Sustained expression and chromatin insulators -- 7.2 Methods and approaches -- 7.2.1 Site-specific chromosomal integration in mammalian cells -- 7.2.2 Plasmid requirement -- 7.2.3 Chromosome transfer -- 7.3 Troubleshooting -- Acknowledgments -- References -- 8 Using Yeast Two-Hybrid Methods to Investigate Large Numbers of Binary Protein Interactions -- 8.1 Introduction -- 8.2 Methods and approaches -- 8.2.1 Producing large numbers of bait or prey clones -- 8.2.2 Generating recombination-compatible inserts for gap repair cloning -- 8.2.3 Performing gap repair reactions -- 8.2.4 Identifying positive transformants -- 8.2.5 Yeast colony PCR -- 8.2.6 Bait and prey auto-activation tests -- 8.2.7 Targeted 'matrix'-style Y2H screens -- 8.3 Troubleshooting -- References -- 9 Prediction of Protein Function -- 9.1 Introduction -- 9.2 Methods and approaches -- 9.2.1 Annotation schemes.

9.2.2 Working with multiple protein identifier systems -- 9.2.3 Sequence homology -- 9.2.4 Phylogenetic relationships -- 9.2.5 Sequence-derived functional and chemical properties -- 9.2.6 Protein-protein interaction maps -- 9.3 Troubleshooting -- References -- 10 Elucidating Gene Function through Use of Genetically Engineered Mice -- 10.1 Introduction -- 10.2 Methods and approaches -- 10.2.1 Principles of targeted gene deletion in mice -- 10.2.2 Strategies for gene targeting in mice -- 10.2.3 Retrieval of DNA from BAC by recombineering -- 10.2.4 ES and MEF cell culture -- 10.2.5 Mating of chimeras and downstream applications -- 10.3 Troubleshooting -- References -- 11 Delivery Systems for Gene Transfer -- 11.1 Introduction -- 11.2 Methods and approaches -- 11.2.1 The ideal gene therapy vector -- 11.2.2 Plasmid design -- 11.2.3 Viral vectors -- 11.2.4 Non-viral DNA vectors -- 11.2.5 Assessing the physical properties of a non-viral vector -- 11.2.6 Optimizing in vitro gene delivery -- 11.2.7 Optimization strategies -- 11.2.8 Reporter genes and assays -- 11.2.9 Cytotoxicity assays -- 11.2.10 Future steps for non-viral vector development -- 11.3 Troubleshooting -- 11.3.1 General points -- References -- 12 Gene Therapy Strategies: Constructing an AAV Trojan Horse -- 12.1 Introduction -- 12.1.1 General strategies for gene therapy: Basic methods -- 12.1.2 Gene therapy strategies: Delivering genes to cells -- 12.1.3 Viral delivery -- 12.1.4 Production, purification and titration of recombinant adeno-associated virus (rAAV) -- 12.2 Methods and approaches -- 12.3 Troubleshooting -- References -- 13 An Introduction to Proteomics Technologies for the Genomics Scientist -- 13.1 Introduction -- 13.2 Methods and approaches -- 13.2.1 Gel-based strategies -- 13.2.2 LC/MS strategies -- 13.2.3 MALDI imaging and profiling -- 13.3 Troubleshooting.

13.3.1 Number of resolved features and modifications -- 13.3.2 Sample consumption, protein identification and depth of coverage -- 13.3.3 Statistical power -- 13.3.4 Conclusions -- References -- Index.