HLA-DR8 Subtyping by Polymerase Chain Reaction (PCR) - DNA Conformation Polymorphism (DCP) Analysis : a simple and practical genotyping method
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PCR (polymerase chain reaction)
Subcloning, HLA (human leukocyte antigen)
SSO (sequence-specific oligonucleotide) typing
Sanger's dideoxy sequencing method
Two hundred Japanese panels were serologically typed for human leukocyte antigen (HLA) - DR to assign 65 HLA-DRS haplotypes, which were then subdivided into two genotypes, i.e., DRB1 *0802 and DRB1 *0803, by a polymerase chain reaction (PCR) - based, simple, and practical method. The panels possessing DR8 specificity were firstly subjected to PCR with a couple of primers specifically to amplify their DR52 associated group - DRB1 genes. PCR products were then denatured in the presence of formamide, electrophoresed in a non-denaturing polyacrylamide gel, and visualized by silver staining. The same DRB1 products of these samples were also mixed with the DRB1 *1302, and simultaneously analyzed by the same procedure. Electrophoretic mobilities of the samples were compared with those of the typing standards to genotype their DRS-DRB1 alleles by using the characteristic polymorphism in the single-stranded DNAs and the heteroduplexes. This method, designated PCR - DNA conformation polymorphism (DCP) analysis, allowed for genotyping of the DR8-DRB1 alleles without using sequence - specific oligonucleotide probes (SSOP) or restriction endonucleases. The entire process after PCR was completed within a few hours. The tested panels were also genotyped for DRB1 gene by the PCR-SSOP method for comparison with results obtained by the PCR-DCP method. Satisfactory coincidence was achieved and it represented how accurately the new system genotyped DRB1 *0802 and DRB1 *0803. PCR-DCP analysis was thus shown to be practical and useful for subtyping of serologically defined DR8 specificities.
This work was supported in part by grants-in-aid from the Ministry of Education, Culture and Science, Japan, and research grants from the Ministry of Health and Welfare, Japan.
Hiroshima Journal of Medical Sciences
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Hiroshima University Medical Press
Departmental Bulletin Paper
Departmental Bulletin Papers
Graduate School of Biomedical Science