Preface
1 Introduction
2 Basic molecular biology
2.1 Nucleic acid structure
2.2 What is a gene?
2.3 Information flow: gene expression
2.4 Gene structure and organization
3 How to clone a gene
3.1 What is cloning?
3.2 Overview of the procedures
3.3 Gene libraries
3.4 Hybridization
3.5 Polymerase chain reaction
3.6 Extraction and purification of nucleic acids
3.7 Detection and quantitation of nucleic acids
3.8 Gel electrophoresis
4 Cutting and joining DNA
4.1 Restriction endonucleases
4.2 Ligation
4.3 Modification of restriction fragment ends
4.4 Other ways of joining DNA molecules
5 Vectors
5.1 Plasmid vectors
5.2 Vectors based on the lambda bacteriophage
5.3 Cosmids
5.4 M13 vectors
CONTENTS
5.5 Expression vectors
5.6 Vectors for cloning and expression in eukaryotic cells
5.7 Supervectors: YACs and BACs
5.8 Summary
6 Genomic and cDNA libraries
6.1 Genomic libraries
6.2 Growing and storing libraries
6.3 cDNA libraries
6.4 Random, arrayed and ordered libraries
7 Finding the right clone
7.1 Screening libraries with gene probes
7.2 Screening expression libraries with antibodies
7.3 Subcloning
7.4 Characterization of plasmid clones
8 Polymerase chain reaction
8.1 The PCR reaction
8.2 PCR in practice
8.3 Cloning PCR products
8.4 Long-range PCR
8.5 Reverse-transcription PCR
8.6 Rapid amplification of cDNA ends
8.7 Quantitative PCR
8.8 Applications of PCR
9 Characterization of a cloned gene
9.1 DNA sequencing
9.2 Databank entries and annotation
9.3 Sequence analysis
9.4 Sequence comparisons
9.5 Protein structure
9.6 Confirming gene function
10 Analysis of gene expression
10.1 Analysing transcription
10.2 Methods for studying the promoter
10.3 Regulatory elements and DNA-binding proteins
10.4 Translational analysis
CONTENTS
11 Products from native and manipulated cloned genes
11.1 Factors affecting expression of cloned genes
11.2 Expression of cloned genes in bacteria
11.3 Expression in eukaryotic host cells
11.4 Adding tags and signals
11.5 In vitro mutagenesis
11.6 Vaccines
12 Genomic analysis
12.1 Genome sequencing
12.2 Analysis and annotation
12.3 Comparing genomes
12.4 Genome browsers
12.5 Relating genes and functions: genetic and physical maps
12.6 Transposon mutagenesis and other screening techniques
12.7 Conclusion
13 Analysis of genetic variation
13.1 Single nucleotide polymorphisms
13.2 Larger-scale variations
13.3 Other methods for studying variation
13.4 Human genetic diseases
13.5 Molecular phylogeny
14 Post-genomic analysis
14.1 Analysing transcription; transcriptomes
14.2 Array-based methods
14.3 Translational analysis; proteomics 3
14.4 Post-translational analysis: protein interactions
14.5 Integrative studies; systems biology
15 Modifying organisms; transgenics
15.1 Modification of bacteria and viruses: live vaccines
15.2 Transgenesis and cloning
15.3 Animal transgenesis
15.4 Applications of transgenic animals
15.5 Transgenic plants and their applications
Glossary
Bibliography
Index
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