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Open Access Methodology article

Ovine reference materials and assays for prion genetic testing

Michael P Heaton1*, Kreg A Leymaster1, Theodore S Kalbfleisch2, Brad A Freking1, Timothy PL Smith1, Michael L Clawson1 and William W Laegreid13

Author Affiliations

1 USDA, ARS, United States Meat Animal Research Center (USMARC) State Spur 18D, P.O. Box 166, Clay Center, NE 68933, USA

2 University of Louisville, Center for Genetics and Molecular Medicine, 580 South Preston Street, Louisville, KY 40202, USA

3 University of Illinois, Department of Veterinary Pathobiology, 2001 S. Lincoln Avenue, Urbana, IL 61802, USA

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BMC Veterinary Research 2010, 6:23  doi:10.1186/1746-6148-6-23

Published: 30 April 2010

Abstract

Background

Genetic predisposition to scrapie in sheep is associated with several variations in the peptide sequence of the prion protein gene (PRNP). DNA-based tests for scoring PRNP codons are essential tools for eradicating scrapie and for evaluating rare alleles for increased resistance to disease. In addition to those associated with scrapie, there are dozens more PRNP polymorphisms that may occur in various flocks. If not accounted for, these sites may cause base-pair mismatching with oligonucleotides used in DNA testing. Thus, the fidelity of scrapie genetic testing is enhanced by knowing the position and frequency of PRNP polymorphisms in targeted flocks.

Results

An adaptive DNA sequencing strategy was developed to determine the 771 bp PRNP coding sequence for any sheep and thereby produce a consensus sequence for targeted flocks. The strategy initially accounted for 43 known polymorphisms and facilitates the detection of unknown polymorphisms through an overlapping amplicon design. The strategy was applied to 953 sheep DNAs from multiple breeds in U.S. populations. The samples included two sets of reference sheep: one set for standardizing PRNP genetic testing and another set for discovering polymorphisms, estimating allele frequencies, and determining haplotype phase. DNA sequencing revealed 16 previously unreported polymorphisms, including a L237P variant on the F141 haplotype. Two mass spectrometry multiplex assays were developed to score five codons of interest in U.S. sheep: 112, 136, 141, 154, and 171. Reference tissues, DNA, trace files, and genotypes from this project are publicly available for use without restriction.

Conclusion

Identifying ovine PRNP polymorphisms in targeted flocks is critical for designing efficient scrapie genetic testing systems. Together with reference DNA panels, this information facilitates training, certification, and development of new tests and knowledge that may expedite the eradication of sheep scrapie.