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

VERONICA IT-CAROLLO, EGLE VILLEGAS CASTAGNASSO,SILVINA DIAZ, GUILLERMO GIOVAMBATTISTA

Tokio

Congreso; 29TH INTERNATIONAL CONFERENCE ON ANIMAL GENETICS; 2004

International Society of Genetics

The polymorphism of the canine major histocompatibility complex class II DRB1 gene was analyzed. Genomic DNA of whole blood samples was extracted from 64 Shar-pei dogs (many closely related) and 13 Argentinean mastiff. The exon 2 of DLA-DRB1 gene was analysed by PCR-RFLP and PCR-SSCP methods. Fifty -four DLA-DRB1 exon 2 previously reported sequences were analysed for HaeIII, RsaI and MspI restriction sites. This analysis showed: 1) nine possible restriction patterns for HaeIII; (2) ten restriction patterns for RsaI; (3) six restriction patterns for MspI. Consequently, thirty three PCR-RFLP defined alleles could be detected by this method. In the screened dog sample, the PCR-RFLP analysis revealed eight HaeIII patterns, six RsaI patterns and four MspI patterns. On the other hand, PCR-SSCP analysis exhibited thirteen different band patterns, and the number of bands per animal ranged from one to four. Both methods showed few alleles within each breed and different alleles among breeds. Our results suggested that SSCP method is more accurate to detect DLA polymorphism than RFLP technique. The improvement of both should provide a simple and rapid technique for an accurate definition of DLA-DRB1 typing in dogs. In the future this will be useful in the development of strategies to study the association between the MHC and many dog diseases.