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.
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Department of Infectious Diseases, Xiehe Hospital, Tongji Medical University, Wuhan.
Ferencik
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Grosse-Wilde
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D000483:Alleles; D000818:Animals; D001483:Base Sequence; D017931:DNA Primers; D004285:Dogs; D005838:Genotype; D006649:Histocompatibility Antigens / Q000235:genetics*; D015395:Histocompatibility Antigens Class I; D008969:Molecular Sequence Data; D016133:Polymerase Chain Reaction; D012150:Polymorphism, Restriction Fragment Length
D017931:DNA Primers; D006649:Histocompatibility Antigens; D015395:Histocompatibility Antigens Class I; C039692:histocompatibility antigen DLA