A RAPID HLA-DRB1-ASTERISK-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-DR810401-*0412 alleles. The method is b ased on the generation of specific DNA heteroduplex patterns between P CR products derived from selective group-specific amplification of the various DRB104 alleles and PCR products derived from two synthetic D NA heteroduplex generator (DHG) molecules following non-denaturing pol yacrylamide minigel electrophoresis. One DHG was designed to detect DR B10401, *0405, *0407, *0408, and *0409 alleles, whilst the other was designed to detect DRB10402, *0403, *O404, *0406, *0410, *0411, and * 0412 alleles. Characteristic heteroduplex patterns were obtained for a ll DRB 104 alleles tested both in homozygous and heterozygous situati ons. Both DHG and PCR-SSP (sequence-specific primer) typing were perfo rmed on 41 DRB104 positive DNAs from Singaporean Chinese blood donors and complete concordance in results was obtained. HLA-DRB10403, *040 5, and 0406 were found to account for 95% of the DRB1*04 alleles in t he population studied. The DHG technique described is technically simp le and rapid since it essentially involves only two PCR amplifications per individual subtyping. The technique is particularly useful for re solving DRB104 combinations which are indistinguishable by PCR-SSO (s equence-specific oligonucleotide) or PCR-SSP subtyping.