High-resolution HLA-DQB1 typing by combination of group-specific amplification and restriction fragment length...

A reliable method for high-resolution HLA-DQB1 typing using the combination of group-specific amplification and RFLP analysis is described. Group-specific amplification was carried out for the alleles of two groups using the two primer pairs under the same PCR conditions. One group contains DQ5 and DQ6 specificities and the other DQ2, DQ3, and DQ4 specificities. Computer analysis on cleavage patterns for 19 alleles of the DQB1 gene showed that the 11 alleles of the former group could be distinguished with five restriction enzymes and the eight alleles of the latter group could be distinguished with four enzymes. We could reduce the number of restriction endonucleases required compared with the number used in previous studies because we selected appropriate restriction enzymes which had at least one recognition site in almost all DQB1 alleles as a form of internal control. Moreover, DQB1*0602 and 0603, which were indistinguishable using the previously reported PCR-RFLP methods, could be distinguished by the present method. The results of typing of 100 samples from Japanese individuals by this method showed no discrepancy with the results obtained by serologic methods. The calculated allele frequencies showed good agreement with those reported at the 11th International Histocompatibility Workshop.