COMPREHENSIVE TYPING OF DQB1 ALLELES BY PCR-RFLP

The protocols represented in this report can resolve all 22 DQB1 alleles. The second exon of DQB1 was subjected to PCR using two group-specific 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 easy-to-read uncleaved, cleaved and a combination of uncleaved/cleaved patterns. Similarly, two endonucleases, FokI and BgII can define the specificities DQ2, DQ3 and DQ4. Moreover, all 13 group 1 DQB1 alleles and all but one of their 78 possible heterozygotes can be unambiguously resolved using an extended panel of 10 endonucleases. The remaining pair of heterozygotes, DQB1*05031/0603 and 05032/0608, can however be resolved by double digestion with BsmFI and SfaNI. RsaI splits the previously unresolved alleles DQB1*0602 and 0603 in the amplified products of the modified primer SDQ-01. Fnu4HI can resolve DQB1*0606 from 0605. DQB1*0603, 0607 and 0608 can be resolved by SfaNI and the new endonuclease BsmFI. The comprehensive typing of group 2 DQB1 alleles can be achieved using five endonucleases. All 9 group 2 DQB1 alleles and all but one pair (DQB1*0301/0302 from DQB1*03032/0304) of 36 possible heterozygotes can be resolved. Thus, PCR-RFLP remains a simple, inexpensive and reliable method for DQB1 typing. The PCR-RFLP can be used for comprehensive DQB1 typing either independently or to complement the PCR-SSP and PCR-SSOP methods.