The investigator will develop new technology to saturate specifically targeted regions of the genome with a novel type of PCR detectable marker. The significance of the technology lies in its promise to accelerate the assembly of physical maps which in turn would expedite genome sequencing and gene identification. The ease of making a physical map depends on the local density of genetically mapped markers. Even for the mouse genome, with almost 6,000 markers mapped, laborious chromosome """"""""walks"""""""" must often be undertaken. The goal of saturation mapping is to expedite physical mapping by isolating markers at intervals substantially smaller than the size of a Yeast Artificial Chromosome clone. Mapping tightly linked markers requires an efficient system for high throughput genotyping because a large number of strains must be screened to uncover infrequent recombination events. The investigator proposes to saturate regions with Restriction Site Polymorphism (RSP) markers, which have attributes that make them well suited for this type of analysis. RSPs are codominant PCR detectable makers, are detectable by hybridization, are amenable to detection by agarose gel electrophoresis or non-electrophoretic methods, are well suited for automated high throughput genotyping, and can be disseminated by distributing primer sequences. The technology the investigator will develop is based on Targeted RFLP Subtraction, a method that the investigator recently used to efficiently isolate region specific markers in a model organism. Specifically, the investigator will (1) modify RFLP Subtraction for isolating the versatile RSP markers and (2) optimize and demonstrate the use of Targeted RFLP Subtraction for saturating a region of the mouse genome with the PCR detectable RSPs. The Targeted RFLP Subtraction method will be useful in any organism in which controlled matings are possible. Saturating regions of the human genome with the aid of rodent-human hybrid cell lines or in the context of linkage disequilibrium studies may enhance identification of human disease genes.
