This project seeks to develop a completely automated method of rapid genotyping based on the multiplex DNA sequence technology developed at the Utah Center for Human Genome Research over the last three years. The proposed method combines direct and continuous blotting of size-separated PCR amplified products onto nylon membranes and the sequential detection of individual marker systems by enzyme-linked fluorescence. The entire multiplex hybridization protocol is automated. Highly polymorphic genetic markers recently developed at Utah for high-resolution genetic maps will be chosen for complete coverage of the human genome and for their equidistant spacing. These PCR-based markers contain polymorphic tetranucleotide repeats. The exclusive use of this class of markers will eliminate the problem of laddering commonly observed with dinucleotide repeat polymorphisms and will enable the genotypes to be scored unambiguously and efficiently by an automated computer program. As new polymorphic STS markers are developed to optimize the start points for the chromosome 17 DNA sequencing effort, they will be mapped with this technology. Likewise, this technology will map new STRs revealed during production-scale DNA sequencing. With multiplexing of probes, researchers will be able to carry out a complete genome scan with a single electrophoretic gel. it is proposed that independent investigators have access to this multiplex technology for large-scale genotyping studies and simultaneously continue to have access to the existing marker-linkage resources at the Utah Center for Human Genome Research. The Technology Access laboratory will also support Project, which will develop capillary gel electrophoresis as an alternative method for rapid genotyping, by supplying polymorphic STR markers and samples of human DNA.
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