Genotyping of the DQA1*4 alleles by restriction enzyme digestion of the PCR product from the AmpliType PM kit.

An earlier study has shown that the three DQA1*4 alleles (0401, 0501 and 0601) can be distinguished by restriction enzyme digestion of the polymerase chain reaction (PCR) product derived from the DQ alpha AmpliType kit (Perikin-Elmer, Norwalk, NJ). We have found that the AmpliType PM kit (Perkin-Elmer, Branchburg, NJ) can also be used to achieve the same goal. In this case, a Bio-Profil image analysis system (Vilber Lourmat, Marne La Vallee, France) is used for evaluating the restricted patterns. After typing the six alleles of DQA1 by the AmpliType HLA DQ alpha Detection Reagent Set (Perkin-Elmer, Branchburg, NJ), the PCR products from the PM kit with allele 4 were digested with Fok I and RsaI, separately. Since the other five fragments from PM kit will conceal the digested fragments of the HLA DQA1 PCR products, we measured the optical density of the pre- and post-digested 242 bp fragments in Fok I digestion, and 214/221 bp fragments in Rsa I digestion to decide the results of enzyme digestion. Out of 136 samples used in this study, 61 contain the DQA1 allele 4 determined by the DQ alpha AmpliType method. All 61 were typed with enzyme digestion, of which there are 2.3%, 19.8% and 8.1% in allele 0401, 0501 and 0601, respectively. Our procedure can thus extend the utilization of AmpliType PM kit and increase the discrimination power of the DQA1 system, especially in populations with high distribution of allele 4.