HLA-DR8 Subtyping by Polymerase Chain Reaction (PCR) - DNA Conformation Polymorphism (DCP) Analysis : a simple and practical genotyping method

Fukuda Yasuhiko

Kimura Akinori

Hoshino Shuji

Shintaku Sadanori

Sakaguchi Takemasa

Asahara Toshimasa

Sakaki Miyoko

Sumimoto Ryo

Tashiro Hirotaka

Furukawa Masahiro

Ohdan Hideki

Yoshida Tetsuya

Dohi Kiyohiko

Hiroshima Journal of Medical Sciences

[PISSN] 0018-2052

[EISSN] 2433-7668

[NCID] AA00664312

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.

PCR (polymerase chain reaction)

Subcloning, HLA (human leukocyte antigen)

SSO (sequence-specific oligonucleotide) typing

Sanger's dideoxy sequencing method

医学 [ 490 ]

英語

Hiroshima University Medical Press

[ISSN] 0018-2052

[NCID] AA00664312