A robust, low- to medium-throughput prnp genotyping system in sheep

In this study we combined the amplification refractory mutation system (ARMS) with standard fluorescent based fragment length analyses method to develop a prnp genotyping method (PRNP ARMS).By optimised primer design it was possible to type the 4 relevant single nucleotide polymorphisms (SNPs) in the prnp simultaneously in one multiplex reaction. Automated fragment length analysis enabled automated allele designation. Suitability of the PRNP ARMS for routine application was proven by typing samples with known genotypes and larger sample numbers from half-sib families.The ARMS PRNP typing method established in this study is universally suited for a broad range of typing projects with different requirements. It provides an efficient and inexpensive diagnostic mutation analysis that will improve the quality of prnp genotyping compared with other low-cost methods. It can be implemented by most molecular genetic laboratories using standard equipment.Scrapie is a contagious prion disease [1] of sheep and goat. In contrast to BSE, there is no evidence for a transmission to humans. Nevertheless, BSE is experimentally transmittable to sheep and the resulting disease can not be distinguished from scrapie [2,3]. Even though BSE has not been found in farmed sheep yet, the possibility that BSE occurs in "scrapie" diseased sheep can not be excluded. Therefore, scrapie control programs were implemented in many countries. Since no vaccine or therapeutic means is currently available, these programs rely on selective breeding for scrapie resistance. Susceptibility to scrapie is largely controlled by three polymorphic amino acid positions (136, 154, 171) of the ovine prion protein gene (prnp) [4] and reliable genotyping of the corresponding DNA polymorphism is required as a basis for selection decisions.For determination of prnp alleles or haplotypes, there are several typing techniques applied today. Direct sequencing covering the region of exon 2 encoding amino acid positions 136, 1