Canine Mhc DRB1 genotyping by PCR-RFLP analysis

A polymerase chain reaction (PCR) genotyping procedure has been developed for the canine major histocompatibility complex DRB1 gene (Cafo-DRB1), which allows us to distinguish all the DRB1 alleles described to date and reveals the existence of new ones. The polymorphic second exon of the Cafa-DRB1 gene was amplified and the product analysed for restriction fragment length polymorphism (RFLP) with the enzymes RsaI, MboI, TaqI and AspHI. Nine RFLP combinations could be associated with previously known alleles. Two new RFLP combinations corresponded to new alleles and were confirmed by DNA sequencing (Cafa-DRB1*10 and Cafa-DRB1*11). Close associations between RFLPs in the DRB1 second exon and the presence of specific amine acid residues included in the side-chain pockets of the antigen-binding site of the DR molecule are also described.