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United States Department of Agriculture

Agricultural Research Service

Research Project: GENETIC AND BIOLOGICAL DETERMINANTS OF RESPIRATORY DISEASE SUSCEPTIBILITY Title: Ovine Reference Materials and Assays for Prion Genetic Testing

Authors
item Heaton, Michael
item Leymaster, Kreg
item Kalbfleisch, Theodore -
item Freking, Bradley
item Smith, Timothy
item Clawson, Michael
item Laegreid, William -

Submitted to: Meeting Abstract
Publication Type: Abstract Only
Publication Acceptance Date: November 2, 2009
Publication Date: December 9, 2009
Citation: Heaton, M.P., Leymaster, K.A., Kalbfleisch, T.S., Freking, B.A., Smith, T.P., Clawson, M.L., Laegreid, W.W. 2009. Ovine Reference Materials and Assays for Prion Genetic Testing [abstract]. Plant and Animal Genomes XVIII Conference. Abstract No. P577. Available: http://www.intl-pag.org/18/abstracts/

Technical Abstract: Codon variants implicated in scrapie susceptibility or disease progression include those at amino acid positions 112, 136, 141, 154, and 171. Nine single nucleotide polymorphisms (SNPs) determine which residues are encoded by the five implicated codons and accurately scoring these SNPs is essential for eradicating scrapie from diverse populations. In addition, the Prnp coding region contains another 36 known polymorphic sites that may cause base-pair mismatching with oligonucleotides used in DNA testing procedures. Thus, the fidelity of scrapie genetic testing is enhanced by knowing the position and frequency of Prnp SNPs in the target population. A DNA sequencing strategy was developed to determine the full-length Prnp coding sequence for any sheep and, thereby, produce a consensus sequence for any population. The strategy was applied to 953 sheep DNAs, including those from two sets of reference sheep (one set for standardizing Prnp genetic testing and another set for discovering SNPs) estimating allele frequencies and determining haplotype phase. DNA sequencing revealed 16 previously unreported SNPs, including a L237P variant on the F141 haplotype. Two mass spectrometry multi-plex assays were developed to score codons 112, 136, 141, 154, and 171. The predicted amino acids encoded at these sites were 100% concordant with those from Sanger sequencing, and non-Mendelian inheritances were not observed in 96 reference families. These publicly available resources facilitate training, certification, and the development of new tests and knowledge that may expedite the eradication of sheep scrapie.

Last Modified: 12/18/2014
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