Analysis of multiple single nucleotide polymorphisms closely positioned in the ovine PRNPgene using linear fluorescent probes and melting curve analysisReportar como inadecuado




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BMC Infectious Diseases

, 7:90

First Online: 03 August 2007Received: 15 November 2006Accepted: 03 August 2007

Abstract

BackgroundResistance 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.

MethodsExtracted 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.

ResultsThe 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 A, V, T, T, L, F R, H, L, R, Q, H and K sequence variants using only 4 HyBeacon probes.

ConclusionHyBeacon 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.

Electronic supplementary materialThe online version of this article doi:10.1186-1471-2334-7-90 contains supplementary material, which is available to authorized users.

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Autor: David J French - Dominic Jones - David G McDowell - Jim A Thomson - Paul G Debenham

Fuente: https://link.springer.com/







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