COMPREHENSIVE TYPING OF HLA-DPB1 ALLELES BY POLYMERASE CHAIN-REACTIONRESTRICTION-FRAGMENT-LENGTH-POLYMORPHISM (PCR-RFLP)

Comprehensive typing of 53 HLA-DPB 1 alleles was performed by polymera se chain reaction-restriction fragment length polymorphism (PCR-RFLP) method using 78 polymerase chain reaction-sequence-specific oligonucle otide probe (PCR-SSOP) defined DNA specimens (14 retrospective, 64 pro spective). A single primer pair was used to amplify the second exon to obtain DPB1-specific amplified product of 294 bp. A combination of RF LPs and cleaved/uncleaved patterns of various endonucleases was employ ed to resolve DPB1 alleles. A panel of 13 endonucleases (RsaI, Sau96I, BsrBI, DdeI, BsaII, BssHII, ScaI, SacI, BbvI, BsgI, FokI, Bsp1286I an d BstUI) yielded unique RFLP patterns for all but 2 pairs of DPB1 alle les. However, these remaining 2 pairs of rare alleles could be resolve d by an additional digestion with AciI (DPB13901 from 4001 and DPB1*4 901 from 5301). The unique RFLP patterns of 21 DPB1 alleles using PCR- SSOP typed DNA specimens had been verified. Of the 1,378 possible hete rozygotic patterns, 69 pairs and a triplet had been identified that wo uld yield identical RFLP patterns. However, all but one pair, DPB1390 1/5301 from 4001/4901, of these heterozygotes could be resolved by dou ble digestions with appropriately selected endonucleases from the pane l used here. Thus, PCR-RFLP remains a simple and effective method for high resolution DPB1 typing.