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ID 37845
file
creator
Fukuda, Yasuhiko
Kimura, Akinori
Hoshino, Shuji
Shintaku, Sadanori
Sakaguchi, Takemasa
Asahara, Toshimasa
Sakaki, Miyoko
Sumimoto, Ryo
Furukawa, Masahiro
Ohdan, Hideki
Yoshida, Tetsuya
Dohi, Kiyohiko
subject
PCR (polymerase chain reaction)
Subcloning, HLA (human leukocyte antigen)
SSO (sequence-specific oligonucleotide) typing
Sanger's dideoxy sequencing method
NDC
Medical sciences
abstract
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.
description
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.
journal title
Hiroshima Journal of Medical Sciences
volume
Volume 45
issue
Issue 3
start page
85
end page
92
date of issued
1996-09
publisher
Hiroshima University Medical Press
issn
0018-2052
ncid
language
eng
nii type
Departmental Bulletin Paper
HU type
Departmental Bulletin Papers
DCMI type
text
format
application/pdf
text version
publisher
department
Graduate School of Biomedical Science
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