A simple and rapid method for HLA-DRB and -DQB typing by digestion of PCR-amplified DNA with allele specific restriction endonucleases

Abstract

The polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP) method, which we previously reported as an efficient and convenient typing technique for accurate definition of the HLA-DQA1 and -DPB1 alleles, is now extended and applied to HLA-DRB and -DQB typing. The second exon of the HLA-DRB (B1 and B3 or B4) and DQB (B1 and B2) genes was selectively amplified from genomic DNAs of 70 HLA-homozygous B cell lines by PCR. Amplified DNAs were digested with the restriction endonucleases, which can recognize allelic variations specific for HLA-DR, -DQ, and -Dw allospecificities and then subjected to electrophoresis in polyacrylamide gel. Of DRB genes, FokI, HinfI, HhaI, HphI, KpnI and SacII were selected and the 20 different polymorphic patterns of the restriction fragments thus obtained were found to correlate with each HLA-DR and -Dw type defined by serological and cellular typing. Of the DQB genes, FokI, HaeIII, HhaI, RsaI and Sau3AI produced nine different polymorphic patterns of the restriction fragments, correlating with the HLA-DQ and -Dw types. This PCR-RFLP method provides a simple and rapid technique for accurate definition of the HLA-DR, -DQ and -Dw types at the nucleotide level, eliminating the need for radioisotope as well as allele specific oligonucleotide probes.