Analysis of multiple single nucleotide polymorphisms closely positioned in the ovine PRNP gene using linear fluorescent probes and melting curve analysis

<p>Abstract</p> <p>Background</p> <p>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 (<it>PRNP</it>). Dual-labelled HyBeacon probes were developed to analyse single and clustered polymorphisms within these and neighbouring codons.</p> <p>Methods</p> <p>Extracted DNAs and unpurified blood samples were genotyped with respect to polymorphisms in <it>PRNP </it>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.</p> <p>Results</p> <p>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, F¹⁴¹R¹⁵⁴, H¹⁵⁴, L¹⁶⁸, R¹⁷¹, Q¹⁷¹, H¹⁷¹and K¹⁷¹sequence variants using only 4 HyBeacon probes.</p> <p>Conclusion</p> <p>HyBeacon assays provide an extremely robust and accurate method for the analysis of single and clustered <it>PRNP </it>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.</p

Rapid typing of STRs in the human genome by HyBeacon® melting

A new method based on DNA melting has been developed for the rapid analysis of STRs in the human genome. The system is based on homogeneous PCR followed by fluorescence melting analysis and utilises a HyBeacon® probe combined with a PCR primer-blocker oligonucleotide. The use of blockers of different length permits identification of the full range of common D16S539 repeats enabling detection of 99.8% of known alleles. The interrogation of STRs can be carried out on standard genetic analysis platforms and could be applied to other loci to form the basis of a bespoke high-throughput system for use in forensic analysis, particularly as fluorescent genetic analysis platforms are now available for high-resolution melting. This methodology may be suitable for rapid forensic DNA analysis at the point-of-arrest or in a custody suite where it is important to identify an individual from a small group of suspects/detainees

Analysis of multiple single nucleotide polymorphisms closely positioned in the ovine gene using linear fluorescent probes and melting curve analysis-1

<p><b>Copyright information:</b></p><p>Taken from "Analysis of multiple single nucleotide polymorphisms closely positioned in the ovine gene using linear fluorescent probes and melting curve analysis"</p><p>http://www.biomedcentral.com/1471-2334/7/90</p><p>BMC Infectious Diseases 2007;7():90-90.</p><p>Published online 3 Aug 2007</p><p>PMCID:PMC1994165.</p><p></p>ectively. (B) Tis detected as a melting peak at approximately 56.9°C, enabling clear differentiation of genotypes such as ARR/VRQ and ARR/TRQ

Analysis of multiple single nucleotide polymorphisms closely positioned in the ovine gene using linear fluorescent probes and melting curve analysis-0

<p><b>Copyright information:</b></p><p>Taken from "Analysis of multiple single nucleotide polymorphisms closely positioned in the ovine gene using linear fluorescent probes and melting curve analysis"</p><p>http://www.biomedcentral.com/1471-2334/7/90</p><p>BMC Infectious Diseases 2007;7():90-90.</p><p>Published online 3 Aug 2007</p><p>PMCID:PMC1994165.</p><p></p>ectively. (B) Tis detected as a melting peak at approximately 56.9°C, enabling clear differentiation of genotypes such as ARR/VRQ and ARR/TRQ

Rapid detection of diagnostic targets using isothermal amplification and HyBeacon probes – a homogenous system for sequence-specific detection

Isothermal amplification is a rapid, simple alternative to PCR, with amplification commonly detected using fluorescently labelled oligonucleotide probes, intercalating dyes or increased turbidity as a result of magnesium pyrophosphate generation. SNP identification is possible but requires either allele-specific primers or multiple dye-labelled probes, but further downstream processing is often required for allelic identification. Here we demonstrate that modification of common isothermal amplification methods by the addition of HyBeacon probes permits homogeneous sequence detection and discrimination by melting or annealing curve analysis. Furthermore, we demonstrate that isothermal amplification and sequence discrimination is possible directly from a crude sample such as an expressed buccal swab