The overall objective for this proposal is to develop a system for HLA phenotyping for the HLA-A, -B, -C antigens based on nucleotide sequence variations in the coding region of the individual HLA alleles. Conventional serological typing for HLA does not detect all known HLA alleles. Serological techniques supplemented by cellular assays for histocompatibility testing or biochemical detection of the HLA gene products by isoelectric focusing gel electrophoresis or by two-dimensional gel electrophoresis are impractical for routine applications in clinical histocompatibility testing, and many HLA alleles are still undetected. Recent progress in the definition of HLA alleles by their unique combination of nucleotide sequences provide an extensive database from which a DNA-based HLA typing system can be developed. The application of gene specific DNA amplification utilizing the polymerase chain reaction (PCR) combined with sequence specific oligonucleotide probes (SSOP) and allele specific oligonucleotide probes (ASOP) have made it possible to develop HLA typing procedures for HLA class II alleles encoded by the DRB, DQA, DQB, DPA and DPB genes. A similar systematic approach for DNA-based allele specific typing for HLA-A, -B and -C genes has not yet been developed. The present proposal focuses on the development of a PCR-based DNA typing procedure with SSOP and ASOP. Presently available nucleotide sequence data for more than sixty HLA-A, -B and -C alleles will be used within the first specific aim to design a system for HLA class I typing. Universal and locus specific primers will be designed for the HLA-A, -B and -C alleles and optimal conditions for their application will be determined. A series of SSOPs and ASOPs will be developed and conditions for a uniform hybridization pattern will be determined. The hybridization pattern will be evaluated on an HLA well-defined highly selected cell panel and a computer algorism to assist in allele assignment will be designed. The second specific aim concerns the determination of nucleotide sequences for HLA-A, -B and -C variants which can be detected by 1D-IEF. Full-length cDNA clones encoding the class I genes will be obtained using a PCR-based technology. An expres- sion vector which can be used for sequencing as well as transfection will be used for cloning. The cDNA clones will be expressed in a human B cell line deficient for its HLA class I antigen expression and the HLA specificity of the isolated gene will be determined by 1D-IEF. Nucleotide sequence data obtained within specific aim #2 together with other novel sequences reported will be included in the database and applied for refining the SSOP and ASOP developed in the first specific aim. This strategy should contribute significantly to developing a reliable DNA-based HLA typing system for large-scale HLA class I phenotyping of unrelated individuals.
Showing the most recent 10 out of 13 publications
