Designed as an introductory text the authors cover all core strategies in the application of modern recombinant DNA technology. The first chapters directly address the applications of polymerase chain reaction to a variety of problems in DNA cloning that are, or have been, extremely challenging using more traditional approaches and technologies. These include cDNA cloning and transcript mapping, mutagenesis as well as the cloning of very long transcripts and protocols using limiting amounts of total RNA. Further chapters describe approaches to subtractive cloning technologies as well as novel specialized expression cloning and library screening strategies. The handbook contains detailed step-by-step protocols and extensive hands-on advice.

Inhalt

PCR and cDNA Cloning Techniques.- 1 Strategies for cDNA Cling and Mapping RNA Transcripts.- 2 Cloning PCR Products.- 3 Site Directed Deletion, Insertion, and Substitution Using PCR.- 4 Long RT-PCR Cloning - Amplification of Full-Length Enterovirus Genomes.- 5 Construction of cDNA Libraries from Small Quantities of Total RNA Using Template Switching Catalyzed by M-MLV Reverse Transcriptase.- Subtractive and Differential Display Techniques.- 6 Subtractive Hybridization and cDNA Cloning.- 7 Differential Display to Identify Steroid-Induced Genes in Endocrine Cells.- Specialized Cloning and Library Screening Strategies.- 8 Two Hyrid cDNA Cloning.- 9 Yeast One and Two Hyrid cDNA Cloning.- 10 High-Throughput Library Screening by Fluorescent Hybridisations on Gridded Membranes.- 11 Identification of Cell Targeting Ligands Using Random Peptide-Presenting Phage Libraries.