HLA-DQ GENOTYPING USING A MODIFIED PCR-RF LP METHOD - APPLICATION TO THE HLA-DQA1 GENE

Since 1989, several HLA-DQA1 PCR-RFLP genotyping protocols have been p ublished. These methods require complete digestion of the PCR products and determination of the restriction fragments lenght. The HLA-DQA1 P CR-RFLP genotyping protocol describe here uses one amplification step through PCR, digestion of the PCR-products with 8 restriction endonucl eases, and determination of the fragments size after polyacrylamide ge l electrophoresis. Five of the enzymes, having no more than one restri ction site in each allele (ApaL1, HphI; BsaJI FokI and MboII), allow d istribution of all the genotypes in 13 allelic-combination groups on t he base of the digestion pattern: cut/not rut. Three additional enzyme s (MnlI DdeI and RsaI), having at least 2 restriction sites in each al lele, ave used to assign the genotype in each allelic-combination grou p on the base of the restriction fragments length observed. Eight of t he 13 alleles, 36 of the 31 HLA-DQA1 genotypes could be characterized. Four to 8 samples could be characterized each day, including DNA extr action. The number of endonucleases used could act as internal control of enzymatic activities and the genotyping protocol can include new H LA-DQA1 alleles without modification of the experimental steps. This p rotocol can be applied easily in a laboratory, without specific techni cal training or specific equipment.