Comprehensive typing of HLA-DPB1 alleles by polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP)

Abstract

Comprehensive typing of 53 HLA-DPB1 alleles was performed by polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP) method using 78 polymerase chain reaction-sequence-specific oligonucleotide probe (PCR-SSOP) defined DNA specimens (14 retrospective, 64 prospective). A single primer pair was used to amplify the second exon to obtain DPB1-specific amplified product of 294 bp. A combination of RFLPs and cleaved/uncleaved patterns of various endonucleases was employed to resolve DPB1 alleles. A panel of 13 endonucleases (RsaI, Sau96I, BsrBI, DdeI, BsaJI, BssHII, ScaI, ScaI, BbvI, BsgI, FokI, Bsp1286I and BstUI) yielded unique RFLP patterns for all but 2 pairs of DPB1 alleles. However, these remaining 2 pairs of rare alleles could be resolved by an additional digestion with AciI (DPB1*3901 from 4001 and DPB1*4901 from 5301). The unique RFLP patterns of 21 DPB1 alleles using PCR-SSOP typed DNA specimens had been verified. Of the 1,378 possible heterozygotic patterns, 69 pairs and a triplet had been identified that would yield identical RFLP patterns. However, all but one pair, DPB1*3901/5301 from 4001/4901, of these heterozygotes could be resolved by double digestions with appropriately selected endonucleases from the panel used here. Thus, PCR-RFLP remains a simple and effective method for high resolution DPB1 typing.