HLA-DRB1 GENOTYPING BY MODIFIED PCR-RFLP METHOD COMBINED WITH GROUP-SPECIFIC PRIMERS

We previously introduced HLA-DQA1,-DPB1 and DQB1 genotyping with the modified PCR-RFLP method using some informative restriction enzymes which have either a single cleavage site or alternatively no cleavage site in the amplified DNA region, depending on the HLA alleles, making reading of RFLP band patterns much easier. In this study, 43 HLA-DRB1 alleles, excluding DRB1*1103 and *1104 for which no restriction enzymes are available to distinguish each from the other, could be defined by this modified PCR-RFLP method combined with 7 pairs of group-specific primers. It is impossible to distinguish DRB1*0701 and DRB1*0702 as they are identical for the second exon of DRB1. For DR1-DRB1, DR2-DRB1, DR4-DRB1, DR7-DR1, DR9-DRB1, DRw10-DRB1 or DRw52 associated antigens (DR3, w11, w12, w13, w14, and DRw8)-DRB1 gene amplification, the second exon of the DRB1 gene was selectively amplified using each group-specific primer from genomic DNAs of 70 HLA-homozygous B-cell lines and healthy Japanese by PCR. Amplified DNAs were digested with restriction endonucleases and then subjected to electrophoresis assaying simply for cutting, or no cutting, of the DNA, although some alleles can be distinguished only after examination of RFLP band patterns generated and in some cases using double digestion technique with two restriction enzymes. This modified PCR-RFLP method can be successfully applied to all possible DRB1 heterozygotes, despite the fact that 15 pairs of heterozygotes among them cannot be distinguished theoretically by the PCR-SSO method, because the PCR-RFLP method can tell whether two polymorphic sites are linked to each other (cis position) or located on a different chromosome (trans position) by checking the length of RFLP bands generated with double digestion. Thus, the PCR-RFLP method is technically simple, practical and inexpensive for determination of the HLA-DRB1 alleles for routine HLA typing work.