A new genotyping strategy for efficient scoring of closely positioned SNPs in the ovine prion protein gene.

Amino-acid polymorphisms of the ovine prion protein have been known to influence susceptibility to scrapie for many years. Recently, a role in both classical and atypical scrapie was assigned to new mutations, increasing the overall number of polymorphisms of interest for breeding plans. Besides, the high number and density of polymorphisms in the prion protein gene (PrP) and the presence of unusual mutations in some breeds hampers genotyping methods, making multiplexing difficult and sometimes compromising analytical results. We developed a multiplex genotyping method for the ovine PrP that overcomes the limitations posed by the high number and density of the polymorphisms to interrogate. Nine primers were designed to be compatible in a single primer-extension reaction developed for routine genotyping, with the capacity to identify the following polymorphisms: A136V, M137T, L141F, I142K, R154H, Q171R, Q171H, Q171K and N176K. Site-specific mutations were inserted in primer sequences in order to prevent extension of reciprocally complementary primers. Complete accuracy and repeatability of the assay was assessed with reference to 97 sequenced samples. The presented method constitutes an improved tool for ovine PrP genotyping and a general strategy for the use of primer extension in a genetic context of high density of polymorphisms.