Three Sections of the Columbia Genome Center, Physical Mapping, DNA Sequencing, and Informatics will serve as a genomics Core to assist in gene localization, gene discovery, mutation analysis and functional characterizations. The physical mapping laboratory will identify genomic clones YACs, BACs and/or PACs from redundant (>8X) genomic libraries that map to 6q. The components of the physical maps to be generated will depend on the need to extend the 6q physical map beyond its current boundaries. Maps will be assembled by identifying superimposed YAC and PAC/BAC contigs, the latter selected from a >8X human PAC or BAC library.. The contig maps will be annotated by identification of transcribed genomic sequences, by exon-trapping, cDNA selection, or exon predictions. The ends of the clones will be sequenced to enrich the map with polymorphic markers, and STS/PCR primmer sequences. The Integrated Mapping Package software will be used to integrate all published map data, including D markers, assigned sequences, and known genes that have been previously mapped to the site. Three strategies will be used to identify candidate genes: single or double pass sequencing, exon trapping and cDNA selection. The PAC/BAC contigs will be refined into tiling paths by restriction analysis or fingerprinting. Clones aligned into minimal paths will be subjects to single or double mass sequencing and analysis by the latest versions of GRAIL &/or GENEFINDER, to identify putative exons. The tiling path clones will also serve as targets for exon trapping, and the trapped products will be sequenced to an accuracy of 99.95%. The appropriate PACs/BACs will also be used to select cDNA from normalized libraries. Candidate genes will be analyzed for mutations by direct sequencing of PCR products of DNA or RNA obtained from normal and tumor tissue (.e.g., lymphoma, breast and prostatic carcinomas). DNA sequencing services will also be provided to project 2, to define the PTEN mutations that mediate progression in a variety of tumors. Human mutation analysis will be based on the defined intron-exon boundaries. In support of functional analysis of PTEN, the mouse homologue will be mapped and sequenced.
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