DEVELOP PCRS TO DETECT POLYMORPHISMS AT MAPPED RFLP LOCI

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Human RFLP probes which have been genetically mapped will be converted into PCR-based markers. Mapped RFLP probes are an important tool for research in many areas. A subset of about 150 RFLP probes has been selected and is currently being used for mapping genetic disease genes and studying loss of heterozygosity in tumor cells. Converting the subset probes into PCR-based markers will greatly facilitate these studies because PCR-based genotyping methods are a significant improvement over currently used genomic Southern hybridization methods. The oligonucleotides developed in this research will also be an important research product for constructing physical maps for human chromosomes. In some cases they can also be used for the diagnosis of genetic diseases and tumors, and for identity testing. In phase I feasibility studies, two PCR-markers involving CA dinucleotide repeats, which are in the vicinity of the originally mapped RFLP loci, will be developed. In addition, the region flanking a dimorphic RFLP will be subcloned and sequenced using a random approach. In phase II, PCRs will be developed for the entire subset of RFLP probes. Other methods such as DGGE (denaturing gradient gel electrophoresis), SSCP (single-stranded conformation polymorphism), inverse PCR and vectorette PCR will also be used to achieve the overall goal.