NR AXNK
AU Granato,A.; Caldon,M.; Colamonico,R.; Vaccari,G.; Aufiero,G.M.; Vascellari,M.; Mutinelli,F.
TI Genotyping of Ovine Prion Protein Gene Variants by PCR with Melting Curve Analysis
QU International Conference - Prion 2007 (26.-28.9.2007) Edinburgh International Conference Centre, Edinburgh, Scotland, UK - Book of Abstracts: Pathology and Pathogenesis P03.11
IA http://www.prion2007.com/pdf/Prion Book of Abstracts.pdf
PT Konferenz-Poster
AB
Background: Scrapie, a transmissible spongiform encephalopathy (TSE) which affects sheep and goats, is characterised by the conversion of the cellular prion protein (PrPc) into the pathological form PrPsc. It is known that the susceptibility of sheep to scrapie is conditioned by polymorphisms in the PrP gene. In particular, specific allelic variants of the PrP gene determined by polymorphisms at codons 136, 154 and 171, are associated with susceptibility or resistance to both natural and experimental scrapie.
Aim: To evaluate the use of fluorescence resonance energy transfer (FRET) based real time PCR for studying the polymorphisms at codons 136, 154 and 171 of ovine PrP gene.
Methods: The analysis of PrP polymorphisms was performed on whole blood DNA samples of 2,000 sheep by real time PCR (LightCycler 2.0, Roche), using FRET probes and successive allelic discrimination by determination of melting temperature. The
allelic discrimination technique based on FRET probes allows distinguishing between alleles of a single polymorphism.
Results: We observed the known polymorphisms at codon 136 (GCC or GTC) and at codon 154 (CGT or CAT), which encode for Ala or Val, or for Arg or His, respectively. The analysis of codon 171 which encodes for Arg, Gln, His or Lys (CGG, CAG, CAT or AAG), showed 3 out of 2,000 examined sheep with melting temperature of 51.8°, just between 53°, which corresponds to H, and 50.5° to K (Lys), respectively. The sequencing of these samples revealed the presence of a double heterozygosis A/C and G/T (MAK) on the first and the third nucleotide, respectively. Moreover in 23 samples we observed that also the mutation at the second nucleotide of codon 168 (CCA or CTA) can affect the melting temperature at codon 171. In fact, in these cases a melting temperature of approximately 52° (between His and Lys) was recorded and the sequencing of samples revealed Gln at codon 171 and Leu at codon 168.
Conclusions: The FRET-based PrP genotyping method is rapid, cheap and can differentiate all genotypes at each locus in one capillary. Based on our experience, however, a disadvantage, when using the FRET analysis, is the possible
misinterpretation of results attributable to additional sequence variation lying within the target sequence of the probes. In these cases, even if rare, the melting curve analysis is unable to define unambiguously the genotype at some polymorphic codon, requiring a further confirmatory analysis.
AD A. Granato, M. Caldon, R. Colamonico, G.M. Aufiero, M. Vascellari, F. Mutinelli, Istituto Zooprofilattico Sperimentale delle Venezie, Histopathology Department, Italy; G. Vaccari, Istituto Superiore di Sanità, Department of Veterinary Public Health, Italy
SP englisch
PO Schottland