COMPREHENSIVE TYPING OF DQB1 ALLELES BY PCR-RFLP

The protocols represented in this report can resolve all 22 DQB1 allel es. The second exon of DQB1 was subjected to PCR using two group-speci fic primers to obtain DQB1 group 1 (DQ5 and DQ6) and group 2 (DQ2, DQ3 , DQ4) specific amplified products, respectively. Three endonucleases, ApaI, BssHII and NciI, can provide typing of DQ5 and DQ6 based on eas y-to-read uncleaved, cleaved and a combination of uncleaved/cleaved pa tterns. Similarly, two endonucleases, FokI and BgII can define the spe cificities DQ2, DQ3 and DQ4. Moreover, all 13 group 1 DQB1 alleles and all but one of their 78 possible heterozygotes can be unambiguously r esolved using an extended panel of 10 endonucleases. The remaining pai r of heterozygotes, DQB105031/0603 and 05032/0608, can however be res olved by double digestion with BsmFI and SfaNI. RsaI splits the previo usly unresolved alleles DQB10602 and 0603 in the amplified products o f the modified primer SDQ-01. Fnu4HI can resolve DQB10606 from 0605. DQB10603, 0607 and 0608 can be resolved by SfaNI and the new endonucl ease BsmFI. The comprehensive typing of group 2 DQB1 alleles can be ac hieved using five endonucleases. All 9 group 2 DQB1 alleles and all bu t one pair (DQB10301/0302 from DQB1*03032/0304) of 36 possible hetero zygotes can be resolved. Thus, PCR-RFLP remains a simple, inexpensive and reliable method for DQB1 typing. The PCR-RFLP can be used for comp rehensive DQB1 typing either independently or to complement the PCR-SS P and PCR-SSOP methods.