A research on DLA-DRB1 genotyping by PCR-RFLP. I. To select a appropriate oligonucleotide primer pair.

In order to study the DLA (Dog Leucocyte Antigen) class II region we utilized the polymerase chain reaction based restriction fragment length polymorphism (PCR-RFLP) method, which has been reported previously as an efficient and simple technique for accurate definition of the HLA class II alleles. To search for a appropriate primer pair a series of amplifications with 4 different primer pairs DLA-DR-SP/Stop, DLA-DR-SP/P3, HLA-DRB-GH46/50 and HLA-DRB-AMP-A/B were provided. Only one satisfactory amplification was obtained with the primer pair HLA-DRB-AMP-A/B. The analogous sequences of the primer pair are found in the sequence of HLA-DRB-cDNA. The amplification region of the primer pair includes also the three hypervariable regions (HVR) in the sequence of DLA-DRB cDNA. Southern blot hybridization analysis confirmed the specificity of the primer pair HLA-DRB-AMP-A/B. The results of Hae III and Hinfl digestion show high polymorphism in DLA-D region and allele specific polymorphic patterns. Therefore, it is suggested that the primer pair HLA-DRB-AMP-A/B is at present the only available and usefull primer pair in PCR-RFLP study of DLA-DRB1 gene.