HLA-DR8 subtyping by polymerase chain reaction (PCR)-DNA conformation polymorphism (DCP) analysis: a simple and practical genotyping method.
Two hundred Japanese panels were serologically typed for human leukocyte antigen (HLA) - DR to assign 65 HLA-DR8 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 DR8--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.
2nd Department of Surgery, Hiroshima University School of Medicine, Japan.
Y
Y
Fukuda
A
A
Kimura
S
S
Hoshino
S
S
Shintaku
T
T
Sakaguchi
T
T
Asahara
M
M
Sakaki
R
R
Sumimoto
H
H
Tashiro
M
M
Furukawa
H
H
Ohdan
T
T
Yoshida
K
K
Dohi
D001483:Base Sequence; D002460:Cell Line; D004247:DNA / Q000235:genetics; D005069:Evaluation Studies as Topic; D005838:Genotype; D006684:HLA-DR Antigens / Q000145:classification* / Q000235:genetics*; D059810:HLA-DR Serological Subtypes; D006650:Histocompatibility Testing / Q000379:methods; D006801:Humans; D016133:Polymerase Chain Reaction / Q000379:methods; D018807:Polymorphism, Single-Stranded Conformational
D006684:HLA-DR Antigens; D059810:HLA-DR Serological Subtypes; C041559:HLA-DR8 antigen; D004247:DNA