HIGH DENSITY GENETIC AND PHYSICAL MAPPING REAGENTS

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This proposal describes methods for producing high density sets of reagents for genetic and physical mapping, using our recently developed methods for preparing chromosome region specific probes and screening entire bacterial artificial chromosome (BAC) libraries in one simple experiment. The probes are prepared by microdissecting the chromosome region of interest from standard cytogenetic preps, and amplification with degenerate primer PCR. The BACs are screened using fluorescence in situ hybridization in a procedure referred to as MICROGENETICS TM: the cells are attached to a microscope slide, either in a random pattern or in a defined array, and replicated; the replica is lysed and fixed in situ, and hybridized with the fluorescent probe. Cells corresponding to positive signals are "picked" from the original slide. We will develop strategies for transferring BAC libraries that currently exist in arrays of multiwell plates onto microscope slides and develop combinatorial hybridization methods so that libraries can be screened with more than one region specific probe simultaneously. After region specific sets of BACs are identified, they are subcloned and screened with repetitive sequence probes; sequences of the repeats are obtained, and primer sets for PCR amplification are developed for polymorphism and genetic analyses. The BACs are assembled into contigs, and the microsatellites are physically positioned along the contig. We propose to develop these reagents for specific regions of species of biomedical and agricultural interest that are unlikely to benefit from a massive genome effort. PROPOSED COMMERCIAL APPLICATION: The commercial potential lies in formation of joint ventures between AGL and the pharmaceutical and agricultural industries, in pursuit of specific genes of either biomedical interest (in the rat) or economic trait loci (in bovine or porcine). Genes of biomedical interest will be for diagnostics and/or therapeutic purposes, and economic trait loci will be for animal breeders primarily for genetic diagnostic purposes (i.e. trait testing). A smaller market for region specific reagents lies in the field of genome research on all these animals.