Complete HLA-A DNA typing using the PCR-RFLP method combined with allele group- and sequence-specific amplification.
We have established a practical method of complete high-resolution typing for all HLA-A alleles using the polymerase chain reaction (PCR)-restriction fragment-length polymorphism (RFLP) technique combined with allele group- and sequence-specific amplification. The second and third exons of the HLA-A gene, in which most allelic variations are observed, were separately amplified by PCRs with 3 and 4 group-specific primer pairs, respectively. Each PCR-amplified product was digested by allele-specific restriction endonucleases and then subjected to electrophoresis on a 10% polyacrylamide gel. In this way, 62 out of 79 HLA-A alleles could be discriminated by the RFLP patterns derived from the genetic polymorphism in the exon 2 and 3 domains. The remaining 17 alleles could be defined unequivocally by either PCR-RFLP analysis after exon 4 amplification or PCR analysis with sequence-specific primers (SSP). By this method, complete HELA-A genotyping for all homozygous and heterozygous combinations can be accomplished, establishing technically simple, economical and practical routine typing of the HLA-A gene, especially for small samples.
Moribe
T
T
Shionogi Biomedical Laboratory, Diagnostic Science Division, Shionogi & Co., Ltd., Settsu, Osaka, Japan.
Kaneshige
T
T
Inoko
H
H
D000483:Alleles; D015234:HLA-A Antigens / Q000235:genetics*; D006650:Histocompatibility Testing; D006801:Humans; D016133:Polymerase Chain Reaction / Q000379:methods*
D015234:HLA-A Antigens