Analysis of multiple single nucleotide polymorphisms closely positioned in the ovine PRNP gene using linear fluorescent probes and melting curve analysis.
BACKGROUND
Resistance and susceptibility to scrapie has been associated with single nucleotide polymorphisms located within codons 136, 154 and 171 of the ovine prion protein gene (PRNP). Dual-labelled HyBeacon probes were developed to analyse single and clustered polymorphisms within these and neighbouring codons.


METHODS
Extracted DNAs and unpurified blood samples were genotyped with respect to polymorphisms in PRNP codons 136, 141, 154 and 171. PCR amplicons were investigated using a LightTyper instrument, measuring the stability of probe/target hybridisation through peak melting temperatures and determining the sequence of nucleotides at polymorphic sites.


RESULTS
The performance of HyBeacon assays was evaluated in a validation study comparing genotypes with those obtained using a primer extension assay (Sequenom MassEXTEND) analysed on a MALDI-ToF mass spectrometer. Over 12,000 sheep samples were successfully genotyped, reliably detecting A136, V136, T136, T137, L141, F141 R154, H154, L168, R171, Q171, H171 and K171 sequence variants using only 4 HyBeacon probes.


CONCLUSIONS
HyBeacon assays provide an extremely robust and accurate method for the analysis of single and clustered PRNP polymorphisms in a high-throughput format. The flexibility of the diagnostic tests ensures that samples are correctly genotyped even in the presence of additional sequence variations that flank the polymorphisms of interest. Such sequence variations may also be neutralised using universal bases such as 5-nitroindole if required.
French
David J
DJ
LGC, Queens Road, Teddington, Middlesex, TW11 0LY, UK. david.french@lgc.co.uk
Jones
Dominic
D
McDowell
David G
DG
Thomson
Jim A
JA
Debenham
Paul G
PG
D000818:Animals; D003062:Codon; D004252:DNA Mutational Analysis / Q000379:methods* / Q000662:veterinary; D017931:DNA Primers; D015342:DNA Probes; D005456:Fluorescent Dyes; D005838:Genotype; D013058:Mass Spectrometry; D017354:Point Mutation; D016133:Polymerase Chain Reaction; D020641:Polymorphism, Single Nucleotide; D018620:PrPSc Proteins / Q000235:genetics*; D012608:Scrapie / Q000235:genetics*; D012756:Sheep; D044366:Transition Temperature
D003062:Codon; D017931:DNA Primers; D015342:DNA Probes; D005456:Fluorescent Dyes; D018620:PrPSc Proteins