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 ( Cafa-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 Rsa I, Mbo I, Taq I and Asp HI. 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 amino acid residues included in the side-chain pockets of the antigen-binding site of the DR molecule are also described.