The overall objective of this project is the development of a simple, rapid, and precise method of typing the polymorphism at the HLA class II (in particular, DRB1, DQB1, DPB1, and DQA1) and the class I (HLA-A, B, and C) loci using the polymerase chain reaction (PCR) method of DNA amplification. Our experimental strategy is to use, when possible, locus specific PCR primers and a panel of sequence specific oligonucleotide probes. Our goal is to develop a simple and convenient """"""""reverse dot blot"""""""" assay for each locus which involves the hybridization of a labeled PCR product to a strip containing a defined array of immobilized probes in a single reaction, as well as an automated approach to """"""""reading"""""""" the genotype from the pattern of probe reactivity. This project will involve the sequence analysis of class I and class II genes from non-Caucasian populations and families to identify new alleles and haplotypes as well as the determination of allele, phenotype, and genotype frequencies in various human populations using oligonucleotide typing. Unlike immunological approaches or RFLP analysis, methods for detecting coding sequence polymorphisin, such as oligonucleotide typing, are valuable because they indicate where and how alleles differ. Individual residues are known to elicit allogeneic T cell responses; consequently, this typing approach should prove valuable for transplantation. In particular, it should help us determine the nature and location of mismatches which are important in determining the clinical outcome of bone marrow transplants. In addition, these assays will continue to help us identify specific class II polymorphic residues which may be implicated in disease susceptibility. They should also prove valuable for studies of peptide, MHC, and T cell receptor interactions.
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