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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