Direct Isolation of Chromosomal Regions for the Study of Human Genes

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Summary Of Work: Isolation of a single copy gene from a human genome is a laborious process because it relies on the characterization of random clones in libraries comprised of YACs or BACs. Even if a YAC or BAC contains an entire gene, the specific recovery of the gene is an arduous process. Often a gene is available as a contig set of fragments that must be pieced together. We have developed a new novel method for direct cloning of fragment of human chromosomes (TAR cloning). The TAR cloning method is based on Transformation-Associated recombination in yeast. TAR cloning has now been applied for selective isolation of six human genes and loci [breast cancer genes, BRCA1 and BRCA2, the HPRT gene (defective humans have Lesch-Nyhan syndrome), 10q25 neocentromeric locus, the centromeric locus of chromosome Y and the D11S12 locus] and to one mouse v-Ha-ras transgene. We have demonstrated that genes isolated by TAR cloning retain genetic integrity. The TAR cloning can be used in a cycle that provides for specific isolation of human genes and reintroduction of them into human cells. A new procedure for selective cloning of genes by TAR has many utilities for the studying of organization and expression of specific chromosomal regions and genes including the opportunities for construction of human artificial chromosomes and gene therapy. Because only two weeks are required, the TAR cloning provides new opportunities for investigating genes directly from individuals for studying human polymorphism and identification of mutations leading to different diseases. Prior to this work, isolation of specific chromosomal regions would have required the development of a library for each person studied followed by extensive analysis to find the region of interest. These features suggest that TAR cloning can open the way to clinical investigations of entire genes and gene therapy.