GENETIC MAPPING AND CLONING OF THE HD GENE USING YACS

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The Huntington's disease gene (HD) lies in the telomeric chromosome band 4p16.3, within a 2.5 megabase region flanked by genetic markers D4S125 and D4S168 (Whaley et al., 1991; Bates et al., 1991). The enormity of this region suggests the need for further refinement of the candidate region through linkage disequilibrium studies, which for example have been invaluable in the cloning of the cystic fibrosis gene. It is expected that nonrandom association of certain marker alleles with the HD mutation may provide the genetic information necessary for more precise placement of the HD gene. The first part of this proposal will seek to identify highly polymorphic (dC-dA).(dG-dT) dinucleotide repeats in the candidate region. At least four such repeats have already been identified and are being subcloned from phages and cosmids that have been mapped in this region. A PCR-based assay is further proposed to facilitate the identification of these (CA)n markers directly from yeast artificial chromosomes (YACs) isolated for the candidate region; (CA)n containing inter-Alu PCR amplification products will be identified by Southern hybridization to a (CA)n probe and the sequences flanking the (CA)n repeat block will be directly determined by sequencing the gel- purified PCR product using Alu primers or (CA)n and (GT)n oligomers. The second part of the proposal will seek to identify and characterize candidate genes for HD. This will entail several approaches: a) utilizing gel-purified YACs as probes to directly screen cDNA libraries, b) immobilizing gel-purified YACs onto membrane filters and applying PCR- amplified cDNA inserts as probes.