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A comprehensive, up-to-date guide on all aspects of synthesis and screening of genetic libraries
Detailed step-by-step protocols and extensive hands-on advice gleaned from the authors' experience
Perfectly suited for experienced researchers and graduate students using these technologies in the lab
In recent years, it has seemed as though classical cloning strate gieswere taking something of a back seat to new advances in the development of applied polymerase chain reaction strategies. The tried and established methods of library construction and recombinant DNAcloning pioneered during the 1970sand early 1980s provided many of the bedrock approaches used to find cDNA and genomic clones of significance and made possible accessible DNA sequencing technologies that did not require exotic or even hazardous chemistry. Today, these technologies are enjoying arenaissance asthey find newutilityin novelguises combinedwith the newer approaches and technologies based on polymerase chain reaction. Although it isunlikely that anyscien tist todaywould employ all of the technologies presented in this volume, every scientist working in the disciplines of molecular biology and genetics shouldbe familiar with all ofthem andwill likelyneed aworkingknowledge or havean application for some of them. This volume is designed to provide an easily accessible introduction to what are rapidlybecoming core strategies in the application of modern recombinant DNA technologies. These approaches should be of value to both experienced scientists seeking to apply these technologies to their systems aswell as to scientists and graduate students,in the more formative stages of development as molecular biologists, wishing to apply these powerful technologies. The first section of this volume, encompassing the first five chapters,addresses directlythe applications ofpolymerasechain reaction to avarietyofproblems in DNAcloning that are,or have been, extremely challenging using more traditional approaches and technologies.
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.