Analysis of DLA-DRB1 exon 2 polymorphism in dogs by PCR-RFLP and PCR-SSCP

IT V; VILLEGAS-CASTAGNASSO EE; DIAZ S; GIOVAMBATTISTA G

Japon

Congreso; XXIX International Society for Animal Genetics ISAG; 2004

International Society for Animal Genetics ISAG

The polymorphism of the canine histocompatibility complex (DLA) class II DRB1 gene was analyzed. Genomic DNA of complete blood samples was extracted from 60 Shar-pei dogs (many closely related), 10 Beagles and 10 Argentinean mastiff. The  second exon of DLA-DRB1 gene  was analyzed by PCR-RFLP and PCR-SSCP methods. The fifty-four DLA-DRB1 exon 2 previously reported sequences were analyzed for HaeIII, RsaI and MspI restriction sites. This theorical analysis showed: (1) one to four restriction sites per sequence resulting in nine restriction patterns for HaeIII; (2) zero to three restriction sites per sequence resulting in seven restriction patterns for RsaI; (3) one to five restriction sites per sequence resulting in two to four restriction patterns for MspI. Consequently, twenty eigth PCR-RFLP defined alleles could be detected by this method. In the screened samples, the PCR-RFLP analysis revealed two HaeIII patterns, four RsaI patterns and five MspI patterns. Additional patterns that did not account for known exon 2 DNA sequences were observed, suggesting the existence of novel DLA-DRB1 alleles. PCR-SSCP analysis show up to now nine different band patterns in the sample studied. As expected both methods showed: i) few alleles were detected within each breed; ii) different alleles were observed among breeds. The improvement of this PCR-RFLP  and PCR-SSCP methods should provide a simple and rapid technique for an accurate definition of DLA-DRB1 typing in dogs and studies related to diseases.