In YAC Protocols experienced researchers offer a comprehensive collection of their easily reproducible and proven methods for analyzing complex human, animal, and plant genomes. The step-by-step protocols cover all aspects of yeast artificial chromosomes, ranging from the construction of YAC libraries to their storage, screening, and database management, and to their use in such specialist applications as the transfer of YACs to mammalian cells and the isolation of coding sequences from YACs. YAC Protocols allows workers to add promising approaches adapted from yeast genetics to their personal repertoires and to introduce novel techniques emerging from the accumulating experience with YACs as a tool for genome analysis. Its extensive notes and background information not only significantly enhance the effective use of the methods, but also provide a valuable hands-on resource for those wishing to adapt protocols to suit their own applications.
Generation of Large Insert YAC Libraries. YAC Library Storage and Transport. YAC Library Screening I: Preparation of Hybridization Filters and PCR Pools. YAC Library Screening II: Hybridization and PCR-Based Screening Protocols. Cloning of Human Telomeres in Saccharomyces cerevisiae. Purification of YAC-Containing Total Yeast DNA. Restriction Analysis of YACs. RARE-Cleavage Analysis of YACs. YAC Localization by Fluorescence In Situ Hybridization. Alu-PCR Fingerprinting of YACs. Detection of Chimerism in YAC Clones. Amplification with Arbitrary Primers. End Rescue from YACs Using the Vectorette. Isolation of YAC Ends by Plasmid Rescue. End-Rescue of YAC Clone Inserts by Inverse PCR. Covering YAC-Cloned DNA with Phages and Cosmids. Fragmentation and Integrative Modification of YACs. Targeting Mutations to YACs by Homologous Recombination. Reconstruction of Large Genomic Segments of DNA by Meiotic Recombination Between YACs. Genomic Reconstruction by Mitotic Recombination of YACs. Amplification of the Copy Number of YACs. Transfer of YAC Clones to New Yeast Hosts. Use of ACEDB as a Database for YAC Library Data Management. YAC Transfer into Mammalian Cells by Cell Fusion. YAC Transfer by Microinjection. Transfection of Mammalian Cells via Lipofection. The Isolation of cDNAs by Hybridization of YACs to cDNA Libraries. cDNA Selection with YACs. Markers Selection and Media in YAC Cloning. Index.