|Miller, Janice - ARS RETIRED|
|Cutlip, Randall - ARS RETIRED|
|Cockett, Noelle - UTAH STATE UNIVERSITY|
|Thomsen, Bruce - USDA-APHIS-NVSL, AMES, IA|
|Hall, S Mark - USDA-APHIS-NVSL, AMES, IA|
Submitted to: Journal of Veterinary Diagnostic Investigation
Publication Type: Peer Reviewed Journal
Publication Acceptance Date: March 19, 2006
Publication Date: September 1, 2006
Citation: Kunkle, R.A., Miller, J.M., Alt, D.P., Cutlip, R.C., Cockett, N.E., Wang, S., Richt, J.A., Thomsen, B.V., Hall, S.M. 2006. Determination of sheep prion gene polymorphisms from paraffin-embedded tissue. Journal of Veterinary Diagnostic Investigation. 18(5):443-447. Interpretive Summary: The genetics of the normal prion protein greatly influences the susceptibility of sheep to scrapie. Flock breeding programs to increase the proportion of scrapie resistant sheep are essential to lower the incidence of the disease. Current methods use blood or other fresh tissues to determine resistance. In instances where only formalin-fixed tissues are available for examination, this information is lost. This study describes development of a method to extract DNA from formalin-fixed sheep tissues to determine prion gene sequences. The method enables accurate sequencing of the prion gene from sheep. Application of this method will allow genetic testing in instances where only fixed tissues are available. Also, because this method permits scrapie diagnosis and genetic testing from a single sample, there is no possibility of tissue identification error. Working with formalin-fixed tissues could be used as a less expensive method of testing than those using tissues requiring refrigeration or freezing.
Technical Abstract: Amino acid polymorphisms of the PrP protein greatly influence the susceptibility of sheep to scrapie. Selective breeding to increase the prevalence of PrP gene alleles associated with scrapie resistance is a flock management practice important to scrapie control programs. Determination of sheep PrP alleles typically has required extraction of DNA from host tissues that are freshly derived or stored frozen. This report describes application of a DNA extraction procedure for formalin-fixed, paraffin-embedded tissues (PET) for the purpose of PCR amplification and nucleotide sequencing of relevant codons (136-171) of the sheep PrP gene. Tissues derived from 96 sheep were utilized in this study. There was successful PCR amplification of DNA from 94/96 sheep brainstem PET samples. DNA sequence identity was confirmed in 90/94 matched samples of PET and fresh tissue samples. DNA from brainstem PET from 2 sheep, from which fresh tissue was not available, was amplified and sequenced following formalin-fixation for 7-70 days.