Selective Isolation of Mammalian Genes by TAR Cloning

This unit describes procedures for selective isolation of specific chromosomal regions and entire mammalian genes as circular yeast artificial chromosomes (YACs). The isolation is based on transformation‐associated recombination (TAR) in yeast between genomic DNA and a linearized vector containing targeting sequences homologous to a region of interest. The details preparation of highly competent yeast spheroplasts and transformation of the spheroplasts by genomic DNA along with a TAR vector. Support protocols describe preparation of chromosome‐sized genomic DNA in solid agarose plugs, identification of positive clone pools among primary yeast transformants using PCR, and a quick method for preparing PCR templates from lysed spheroplasts. PCR analysis of each clone in the positive pool is performed to identify individual clones containing the gene of interest. A final support protocol describes retrofitting TAR‐isolated YACs into bacterial artificial chromosomes (BACs).