Mapping genes that influence complex, multigenic or quantitative traits, traits that are relatively common in the population, heterogeneous in etiology,or that result from alleles with low penetrance, represents a principal challenge at the frontier of human genetics. In general, the solution to these genetic problems calls for a method that allows genotypes to be determined at very high resolution, for very large numbers of individuals, at a low cost in labor and materials. This proposal is directed at providing such a method, and making it widely available, convenient, and useful to the human genetics community. Genomic mismatch scanning (GMS) is a new approach to genetic linkage mapping, in which the regions of genetic identity-by-descent between two related individuals are mapped directly using only DNA samples from the two individuals. Each pair of relatives is analyzed in four steps: 1. Genomic DNA is prepared from each individual. 2. Hybrid DNA molecules, containing one strand from each individual, are prepared and isolated. 3. DNA hybrids that are free of mismatches over many thousands of base-pairs are separated from hybrids that contain mismatches. Since allelic sequences not inherited from a common recent ancestor have differences on average every few hundred basepairs, long mismatch-free hybrids are very likely to represent sites of genetic identity by descent. 4. The resulting pool of perfectly-matched DNA hybrids is labelled and used as a probe for in situ hybridization to metaphase chromosomes, or to an ordered array of cloned DNA's each covering a specific map interval. The contiguous intervals to which the probes hybridize correspond to regions of identity-by-descent between the two relatives. By collecting multiple pairs of relatives who share a trait of interest and, for each pair, mapping the regions where they share identity by descent, the genes that influence the trait can be mapped. Compared to methods currently in use, the principal advantages of GMS are: 1. The entire genetic map is surveyed at high resolution in a single, rapid procedure that does not require an electrophoresis step, resulting in a great reduction in cost and labor. 2. Discrete polymorphic markers for specific map intervals are not required but polymorphisms can be detected in virtually any arbitrary interval of interest. 3. Only """"""""affected"""""""" family members need to be analyzed for linkage mapping. GMS can in principle also be used in linkage disequilibrium mapping using """"""""unrelated"""""""" affected individuals to map a trait locus precisely. GMS has been tested successfully in Saccharomyces cerevisiae. The present proposal aims to develop, test and optimize GMS for widespread use in mapping human genes. The key elements of the proposal are: 1. Defining the optimal biochemical methods for GMS in humans. 2. Further developing the enzymatic and hybridization steps in the procedure. 3. Developing PCR- based variations on GMS for affected relative pair and linkage disequilibrium mapping in the human genome.
