We propose to develop and apply a set of techniques, based on the target dependent legation of oligonucleotides, for the automated analysis of DNA sequence polymorphisms. These techniques will speed up and increase the resolution of genetic linkage analysis, extend the range of clinical conditions that may be mapped to a chromosomal location and aid in the identification of the disease gene loci. The applied aspects of this proposal will permit a comparison between the genetic and physical measures of distance in the loci encoding the alpha and beta chains of the T-cell antigen receptor and will provide information regarding the prevalence, distribution and nucleotide sequence of human DNA polymorphisms in these genomic regions. The analysis of the T-cell receptor alpha chain locus on chromosome 14 will be extended to include a set of linkage markers spanning the length of the chromosome for realistic test of the efficiency of automated linkage analysis. We will develop an instrument capable of rapidly analyzing large numbers of polymorphic sequences from minute tissue samples in an automated fashion. These sequence information required for the design of allele-specific reagents will be obtained by sequence analysis of established linkage markers in addition to scanning genomic regions for polymorphic sequences using denaturing gradient gel electrophoresis. Linkage maps will be established for the T-cell receptor loci and for chromosome 14 by the study of segregation of alleles in amplified marker loci from sperm cells, containing haploid chromosomal complement.
