Synthesis and use of universal sequence probes in fluorogenic multi-strand hybridisation complexes for economical nucleic acid testing.

Analysis of nucleic acid amplification products has become the gold standard for applications such as pathogen detection and characterisation of single nucleotide polymorphisms and short tandem repeat sequences. The development of real-time PCR and melting curve analysis using fluorescent probes has simplified nucleic acid analyses. However, the cost of probe synthesis can be prohibitive when developing large panels of tests.

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.

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.

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.