As an adjunct to the gene mapping projects described in this application and as a technique development project by the Genetic Resources CORE, we propose to refine and apply new methods for the detection of DNA polymorphisms. We believe that these methods will permit the detection of nearly all base substitutions in selected regions of the genome and will provide a new database of polymorphisms for linkage studies. These techniques should simplify and reduce the cost of DNA analysis not only for mapping studies but for such applications as paternity testing, carrier detection of inherited disorders, prenatal diagnosis, and identification of somatic mutations in various neoplastic disorders. In addition, these methods should allow a more thorough determination of the extent of variation in human populations. Briefly, small regions of DNA will be amplified by the polymerase chain reaction (PCR) technique and assayed for specific base substitutions. First, PCR amplified DNA would be tested for polymorphisms with frequent cutting restriction endonucleases not generally acrylamide gels either directly following amplification or after mixing and reannealing with amplified DNA from individuals with known alleles. Allelic differences or mismatches between dissimilar strands would be identified by their relative melting positions. Lastly, selected samples would be compared by direct sequencing of the PCR amplified products. These various approaches would be compared (particularly the first and second) to determine the optimum method for detecting variation. On the basis of this information we would develop a new database of polymorphisms at particular chromosomal locations. This would be augmented by designing amplifying primers based on known polymorphic sequences and by determining the sequence around polymorphic sites in probes currently used for RFLP analysis. Two classes of PCR identifiable polymorphisms (PIPs) are expected to emerge, those detected by restriction enzyme digestion and others that will be detected most easily using allele specific oligos (ASOs). The combined library of PIPs would then be applied to the mapping projects described in the accompanying projects.
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