Determination of sheep prion gene polymorphisms from paraffin-embedded tissue.
Amino acid polymorphisms of the prion protein (PrP) 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 that is important for scrapie control programs. Determination of sheep PrP alleles typically has required extraction of DNA from host tissues that are freshly derived or stored frozen. We describe 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 studied. The DNA sequence identity was confirmed in 87 of 94 matched samples of PET and frozen tissue specimens. DNA from brainstem PET of 2 sheep, from which fresh tissue was not available, was amplified and sequenced after formalin fixation for 7-70 days. This method will allow retrospective analysis of PrP genetics of sheep subsequent to postmortem diagnosis of scrapie when nonfixed tissue is unavailable for DNA extraction; however, it is not recommended that submission of fixed tissue supplant collection of fresh tissues for the purpose of determining PrP gene polymorphisms.
Kunkle
Robert A
RA
National Animal Disease Center, USDA, Agricultural Research Service, 2300 Dayton Avenue, PO Box 70, Ames, IA 50010, USA. rkunkle@nadc.ars.usda.gov
Miller
Janice M
JM
Alt
David P
DP
Cutlip
Randall C
RC
Cockett
Noelle E
NE
Wang
Shiquan
S
Richt
Juergen A
JA
Thomsen
Bruce V
BV
Hall
S Mark
SM
D000818:Animals; D001933:Brain Stem / Q000737:chemistry; D004247:DNA / Q000235:genetics / Q000302:isolation & purification; D020022:Genetic Predisposition to Disease; D005838:Genotype; D016612:Paraffin Embedding / Q000662:veterinary; D016133:Polymerase Chain Reaction / Q000379:methods / Q000662:veterinary; D020641:Polymorphism, Single Nucleotide; D011328:Prions / Q000235:genetics*; D012189:Retrospective Studies; D012608:Scrapie / Q000235:genetics*; D012756:Sheep / Q000235:genetics*
D011328:Prions; D004247:DNA