PHYSICAL MAPPING OF HUMAN CHROMOSOME 12 BY FISH

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The overall objective of this project is to assist in the construction of a detailed physical and genetic map of human chromosome 12 using fluorescence in situ hybridization (FISH) in consort with high resolution optical imaging microscopy. Our initial goal will be to physically map a set of 200-300 cosmid clones to chromosome 12 using FISH on metaphase chromosome spreads. These clones will provide the initial framework map of chromosome 12 and nucleation sites for further clone isolation. Alu- PCR products will be generated from each of these clones and used to screen a gridded array of Alu-PCR products, made with the same Alu primer, from individual YAC clones in the CEPH large insert YAC libraries. Individual YACs and YAC contigs identified via this screening process will be FISH mapped on metaphase chromosomes to both confirm their chromosomal location and to determine the possible presence of chimeric clones. Contig clones will be first mapped as a pool; when necessary individual clones from contigs exhibiting chimerisms will be mapped separately in order to identify problem clones. Multicolor FISH analysis will then be used to define the relative order of contigs along the chromosome and to orient the member clones relative to the telomere and centromere. The size of gaps between contigs will be measured by cohybridization of end clones to extended chromatin fibers produced by the treatment of cells with the topoisomerase inhibitor N-[4- (acridenylamino)-3-methoxyphenyl]-methanesulfonamide or by histone depletion of isolated nuclei. The latter studies are designed to assist in closure of the physical map and to establish whether a cosmid, P-1 or YAC size clone will be required to cover the gap interval. Finally, Alu- PCR products from the terminal clones of adjacent YAC contigs will be prepared and used to screen an appropriate library in order to identify clones than span such gap intervals. Multicolor FISH to extended chromatin fibers will be used to confirm closure.