This project has as its goal the acquisition and analysis of the sequence of genomic DNA that is associated with receptor multigene families and neurological disorders and to understand genomic structure at the sequence level. To accomplish this, sequencing strategies and technologies are being developed that will allow the sequencing of megabase regions of DNA. Our use of four Applied Biosystems automated DNA sequencers allows the generation of up to 48 kilobases of raw sequence per day. A computer network consisting of Macintosh, Sun 4, and Silicon Graphics computers in the laboratory connecting to the NIH Convex and Cray computers is assisting the analysis of large genomic sequences. The use of Taq polymerase and PCR cycle-sequencing reactions has greatly enhanced the quantity and the quality of the sequence data generated. New automation has been implemented in the laboratory by the addition of new robotics which completely automate DNA sequencing reactions. These techniques have been applied to sequencing clones associated with several important neurological disease gene regions of human chromosomes. The laboratory has completed over 2 million bases of DNA sequence this year, including the following projects. Over 15kb from the disease gene for neurofibromatosis I has been sequenced, identifying several new exons. Having the intron-exon boundaries has allowed us to develop PCR probes to sequence exons from NF1 patients in search of point mutations associated with disease symptoms. A three cosmid contig from the Huntington's region of chromosome 4 has been sequenced. This region of approximately 100kb is one of the two largest segments of human chromsomal DNA sequenced to date and is currently being analyzed. The second largest region is a 100kb region of chromosome 19 which has been sequenced. This region is where a number of DNA repair enzymes and the gene for mytonic dystrophy have been mapped. Cosmid clones have been obtained from the human Xq28 region which contains several loci of neurological significance including the alpha3 subunit of the GABA receptor. These clones are currently being processed for sequencing. We have begun sequencing clones obtained from patients with spongiform encephalopathies in collaboration with Lev Goldfarb. This project will involve sequencing about 240 clones from 40 patients. This will require obtaining about 600,000 bases of raw sequence data.
