DLA-DRB1 histocompatibility genotyping using RT-nested PCR and cycle sequencing

Class-II histocompatibility genes are associated with predisposition to aut oimmune diseases in many mammal species. We have developed a technique usin g reverse transcriptase and nested-PCR for amplification from blood samples of expressed sequences encoded by canine DLA-DRB1 loci. In the first polym erase chain reaction (PCR), we utilize primers DR-SP and DR-STOP as develop ed by Sarmiento et al. (1990), In the nested PCR, we utilize two additional primers, namely primer 57 [5'-TCTTGGAGGCTCCTGGATGACAGC-3'] and primer 367 [5'-CACAACTACGGGGTGATTGAGAGC-3'] to produce a 334 bp amplified product. Aft er digestion with restriction endonucleases, some of the alleles can be ide ntified by restriction fragment length polymorphism (RFLP). The increasing information on new DLA-DRB1 alleles over the last two years renders the DLA -DRB1 too diverse for convenient use of RFLP. However, the expressed sequen ces amplified by our protocol can be conveniently identified by cycle seque ncing. This RT n-PCR protocol will suffice for the genotyping of individual dogs at the DLA-DRB1 locus. (C) 1999 Elsevier Science B.V. All rights rese rved.