Gene Cloning and DNA Analysis

Terry Brown, University of Manchester, Faculty of Life Sciences, UK

Preface to the Sixth Edition. Part 1: The Basic Principles of Gene Cloning and DNAAnalysis. 1 Why Gene Cloning and DNA Analysis are Important. 1.1 The early development of genetics. 1.2 The advent of gene cloning and the polymerase chainreaction. 1.3 What is gene cloning? 1.4 What is PCR? 1.5 Why gene cloning and PCR are so important. 1.6 How to find your way through this book. 2 Vectors for Gene Cloning: Plasmids andBacteriophages. 2.1 Plasmids. 2.2 Bacteriophages. 3 Purification of DNA from Living Cells. 3.1 Preparation of total cell DNA. 3.2 Preparation of plasmid DNA. 3.3 Preparation of bacteriophage DNA. 4 Manipulation of Purified DNA. 4.1 The range of DNA manipulative enzymes. 4.2 Enzymes for cutting DNA restrictionendonucleases. 4.3 Ligation joining DNA molecules together. 5 Introduction of DNA into Living Cells. 5.1 Transformation the uptake of DNA by bacterialcells. 5.2 Identification of recombinants. 5.3 Introduction of phage DNA into bacterial cells. 5.4 Identification of recombinant phages. 5.5 Introduction of DNA into non-bacterial cells. 6 Cloning Vectors for E. coli. 6.1 Cloning vectors based on E. coli plasmids. 6.2 Cloning vectors based on M13 bacteriophage. 6.3 Cloning vectors based on l bacteriophage. 6.4 and other high capacity vectors enable genomiclibraries to be constructed. 6.5 Vectors for other bacteria. 7 Cloning Vectors for Eukaryotes. 7.1 Vectors for yeast and other fungi. 7.2 Cloning vectors for higher plants. 7.3 Cloning vectors for animals. 8 How to Obtain a Clone of a Specific Gene. 8.1 The problem of selection. 8.2 Direct selection. 8.3 Identification of a clone from a gene library. 8.4 Methods for clone identification. 9 The Polymerase Chain Reaction. 9.1 The polymerase chain reaction in outline. 9.2 PCR in more detail. 9.3 After the PCR: studying PCR products. 9.4 Real time PCR enables the amount of starting material to bequantified. Part 2: The Applications of Gene Cloning and DNA Analysis inResearch. 10 Sequencing Genes and Genomes. 10.1 The methodology for DNA sequencing. 10.2 How to sequence a genome. 11 Studying Gene Expression and Function. 11.1 Studying the RNA transcript of a gene. 11.2 Studying the regulation of gene expression. 11.3 Identifying and studying the translation product of acloned gene. 12 Studying Genomes. 12.1 Genome annotation. 12.2 Studies of the transcriptome and proteome. Part 3: The Applications of Gene Cloning and DNAAnalysis in Biotechnology. 13 Production of Protein from Cloned Genes. 13.1 Special vectors for expression of foreign genes in E.coli. 13.2 General problems with the production of recombinant proteinin E. coli. 13.3 Production of recombinant protein by eukaryotic cells. 14 Gene Cloning and DNA Analysis in Medicine. 14.1 Production of recombinant pharmaceuticals. 14.2 Identification of genes responsible for human diseases. 14.3 Gene therapy. 15 Gene Cloning and DNA Analysis in Agriculture. 15.1 The gene addition approach to plant geneticengineering. 15.2 Gene subtraction. 15.3 Problems with genetically modified plants. 16 Gene Cloning and DNA Analysis in Forensic Science andArchaeology. 16.1 DNA analysis in the identification of crime suspects. 16.2 Studying kinship by DNA profiling. 16.3 Sex identification by DNA analysis. 16.4 Archaeogenetics using DNA to study humanprehistory. Glossary. Index.