Project 4 involves the analysis, mapping and application of human short tandem repeat polymorphisms (STRPs). Primers will be synthesized and tested for short tandem repeat sequences, mostly (dC-dA)n(dG-dT)n sequences, that have been previously determined. The framework linkage map for chromosome 19 will be completed by filling remaining gaps. Cosmid clones from the Livermore National Laboratory which map to the gap regions will be used to develop the necessary polymorphisms. At least one cosmid clone from the Livermore collection will be identified which contains each polymorphic marker. The technology for typing STRPs will be improved through minimizing reagent costs and maximizing typing rates. Reagent costs will be reduced primarily by dropping PCR volumes. Genotyping rates will be improved by enhancing image analysis and by applying fluorescence- based technology. STRPs developed in Projects 1 and 2 will be typed through CEPH reference families. Approximately 100,000 reference family genotypes will be determined each grant year. Genotyping data will be sent to Project 5 for linkage map construction and detection of probable typing errors. Typing errors will be corrected through reexamination of autoradiographs and if necessary through repeat amplification and electrophoresis. Sample misidentification will be avoided between Projects 3 and 4 by duplicate typing of about 5% of markers. If resources and time permit, genetic and physical maps will be connected by identifying large insert genomic clones containing the STRPs. Mutation rates and mechanisms for STRPs will be studied by identifying rare mutation events occurring within the reference families. Attempts will be made to distinguish in vivo from in vitro events. Evolution of short tandem repeats will be investigated primarily by sequencing amplified DNA for specific polymorphisms from various primate species. Locations of short tandem repeat sequences relative to Alu and other interspersed repetitive elements will be determined. The large amounts of data generated within the Center will provide an unprecedented opportunity to reexamine several basic human meiotic parameters. Interference, sexual dimorphism in recombination rates, age dependence of recombination, distance relationships between genetic and physical maps and distributions of STRPs will all be studied. The hypothesis that interference is nearly complete for relatively small genetic intervals will receive particular attention because of the large impact of this question on clinical DNA testing. Genes responsible for human disease will be mapped through collaborative linkage mapping projects. Markers and maps developed within the Center will be made available to visitors who will bring to Marshfield DNA from their kindreds for brief, intense genotyping efforts. Emphasis will be placed on heritable disorders which affect primarily women or minorities.
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