DNA sequences that characterize the class I and class II HLA alleles can be identified by hybridization with oligonucleotide probes. In the present studies we will use selective amplification of genomic DNA by the polymerase chain reaction (PCR) followed by hybridization with sequence-specific probes. Locus-specific and group-specific PCR will be used. The latter will be preferred when otherwise correct assignment would be difficult, due to the sharing of polymorphic sequences between alleles. Because of the large number of polymorphisms and the complex sharing of sequences, typing methods will be developed by groups of related alleles. Radioactive and non-radioactive batch-mode procedures will be used, and a method oriented to typing of single samples, using an enzyme-linked immunoassay with magnetic beads, will be explored. The latter approach will make use of 96-well trays pre-loaded with oligonucleotide probes and will be suitable for automation of the typing procedure. Population studies will include North American Caucasoids, American Blacks, Mexican-Americans, and North American Indians. Variant alleles prevalent in each of these populations will be analyzed. it is anticipated that some new alleles will be described on the basis of hybridization patterns. They will be confirmed by sequencing. The functional role of the polymorphic sequences determined by oligonucleotide hybridization, will be investigated using primary mixed lymphocyte cultures (MLC) and T cell clones. In these experiments we will attempt to determine the correlations between polymorphic residues and T cell recognition. Detailed information to be developed will be useful to understand the immune response, HLA associated diseases and the impact of HLA polymorphisms in clinical transplantation.
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