A rapid HLA-DRB1*04 subtyping method using PCR and DNA heteroduplex generators.

We describe here a rapid polymerase chain reaction (PCR)-based method for the identification of HLA-DRB10401-0412 alleles. The method is based on the generation of specific DNA heteroduplex patterns between PCR products derived from selective group-specific amplification of the various DRB104 alleles and PCR products derived from two synthetic DNA heteroduplex generator (DHG) molecules following non-denaturing polyacrylamide minigel electrophoresis. One DHG was designed to detect DRB10401, *0405, *0407, *0408, and 0409 alleles, whilst the other was designed to detect DRB10402, *0403, *0404, *0406, 0410, 0411, and 0412 alleles. Characteristic heteroduplex patterns were obtained for all DRB104 alleles tested both in homozygous and heterozygous situations. Both DHG and PCR-SSP (sequence-specific primer) typing were performed on 41 DRB104 positive DNAs from Singaporean Chinese blood donors and complete concordance in results was obtained. HLA-DRB10403, 0405, and 0406 were found to account for 95% of the DRB104 alleles in the population studied. The DHG technique described is technically simple and rapid since it essentially involves only two PCR amplifications per individual subtyping. The technique is particularly useful for resolving DRB104 combinations which are indistinguishable by PCR-SSO (sequence-specific oligonucleotide) or PCR-SSP subtyping.