Preface 1 Introduction 2 Basic Molecular Biology 2.1 Nucleic Acid Structure 2.2 Gene Structure and Organization 2.3 Information Flow:Gene Expression 3 How to Clone a Gene 3.1 What is Cloning? 3.2 Wverview of the Procedures 3.3 Gene Libraries 3.4 Hybridization 3.5 Polymerase Chain Reaction 4 Purification and Separation of Nucleic Acids 4.1 Extraction and Purification of Nucleic Acids 4.2 Detection and Quantitation of Nucleic Acids 4.3 Gel Electrophoresis 5 Cutting and Joining DNA 5.1 Restriction Endonucleases 5.2 Ligation 5.3 Alkaline Digests 5.4 Double Digests 5.5 Modification of Restriction Fragment Ends 5.6 Other Ways of Joining DNA Molecules 5.7 Summary 6 Vectors 6.1 Plasmid Vectors 6.2 Vectors Based on the Lambda Bacteriophage 6.3 Cosmids 6.4 M13 Vectors 6.5 Expression Vectors 6.6 Vectors for Cloning and Expression in Eukaryotic Cells 6.7 Supervectors:YACs and BACs 6.8 Summary 7 Genomic and cDNA Libraries 7.1 Genomic Libraries 7.2 Growing and Storing Libraries 7.3 cDNA Libraries 7.4 Random,Arrayed and Ordered Libraries 8 Finding the Right Clone 8.1 Screening Libraries with Gene Probes 8.2 Screening Expression Libraries with Antibodies 8.3 Rescreening 8.4 Subcloning 8.5 Characterization of Plasmid Clones 9 Polymerase Chain Reaction(PCR) 9.1 The PCR Reaction 9.2 PCR in Practice 9.3 Cloning PCR Products 9.4 Long-range PCR 9.5 Reverse-transcription PCR 9.6 Rapid Amplification of cDNA Ends(RACE) 9.7 Applications of PCR 10 DNA Sequencing 10.1 Principles of DNA Sequencing 10.2 Automated Sequencing 10.3 Extending the Sequence 10.4 Shotgun Sequencing:Contig Assembly 10.5 Genome Sequencing 11 Analysis of Sequence Data 11.1 Analysis and Annotation 11.2 Databanks 11.3 Sequence Comparisons 12 Analysis of Genetic Variation 12.1 Nature of Genetic Variation 12.2 Methods for Studying Variation 13 Analysis of Gene Expression 13.1 Analysing Transcription 13.2 Comparing Transcriptomes 13.3 Methods for Studying the Promoter 13.4 Translational Analysis 14 Analysis of Gene Function 14.1 Relating Genes and Functions 14.2 Genetic Maps 14.3 Relating Genetic and Physical Maps 14.4 Linkage Analysis 14.5 Transposon Mutagenesis 14.6 Allelic Replacement and Gene Knock-outs 14.7 Complementation 14.8 Studying Gene Function through Protein Interactions 15 Manipulating Gene Expression 15.1 Factors Affecting Expression of Clonde Genes 15.2 Expression of Cloned Genes in Bacteria 15.3 Expression in Eukaryotic Host Cells 15.4 Adding Tags and Signals 15.5 In vitro Mutagenesis 16 Medical Applications,Present and Future 16.1 Vaccines 16.2 Detection and Identification of Pathogens 16.3 Human Genetic Diseases 17 Transgenics 17.1 Transgenesis and Cloning 17.2 Animal Transgenesis and its Applications 17.3 Transgenic Plants and their Applications 17.4 Summary Bibliography Glossary Index
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