HyBeacon probes for rapid DNA sequence detection and allele discrimination.

HyBeacon probes are single-stranded oligonucleotides with one or more internal base(s) labeled with a fluorescent dye. When a probe forms a duplex with its target sequence, the level of fluorescence emission increases considerably. HyBeacons have been developed as new tools for rapid sequence detection and discrimination and have been employed in a wide variety of applications including infectious diagnostics and analysis of human polymorphisms. Single-labeled (FVG1) and dual-labeled (FVG11) probes were designed to analyze the factor V Leiden (R506Q) polymorphism which causes an increased risk of deep vein thrombosis and pulmonary embolism. Detection and identification of factor V alleles is performed by melting curve analysis and determination of probe melting temperature (T(m)). HyBeacon hybridization to the glutamine allele (Q) causes the formation of mismatched DNA duplexes that are detected through decreases in T(m). HyBeacon probes are included in homogeneous PCR assays to genotype samples with respect to the factor V polymorphism within 20 min, using purified DNAs and unpurified saliva/blood samples. This paper describes the preparation of homogeneous PCR assays, LightCycler target amplification, and subsequent melting curve analysis. This chapter also describes the use of homologous oligonucleotides and melting curve analysis as a method for probe evaluation.

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 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. CONCLUSION: 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.

Molecular basis of action of HyBeacon fluorogenic probes: a spectroscopic and molecular dynamics study.

HyBeacons, novel DNA probes for ultra-rapid detection of single nucleotide polymorphisms, contain a fluorophore covalently attached via a linker group to an internal nucleotide. As the probe does not require a quencher or self-complementarity to function, this study investigates the molecular-level mechanism underlying the increase of fluorescence intensity on hybridization of HyBeacons with target DNA. Spectroscopic ultraviolet-visible and fluorimetric studies, combined with molecular dynamics simulations, indicate projection of the fluorophore moiety away from the target-probe duplex into aqueous solution, although specific linker-DNA interactions are populated. Based on evidence from this study, we propose that for HyBeacons, the mechanism of increased fluorescence on hybridization is due to disruption of quenching interactions in the single-stranded probe DNA between the fluorophore and nucleobases. Hybridization leads to an extended linker conformation, removing the fluorophore from the immediate vicinity of the DNA bases.

Synthesis of HyBeacons and dual-labelled probes containing 2'-fluorescent groups for use in genetic analysis.

An FMOC-protected 2'-hydroxyethyl uridine phosphoramidite has been used to synthesise fluorescein-labelled HyBeacon probes and "FAM-ROX" dual-labelled fluorogenic oligonucleotides.

Real-time detection of genetically modified soya using Lightcycler and ABI 7700 platforms with TaqMan, Scorpion, and SYBR Green I chemistries.

A comparative cross platform evaluation of real-time polymerase chain reaction detection of DNA sequences present in Roundup Ready soya was undertaken using the ABI 7700 and Roche Lightcycler detection systems in combination with 3 different detection chemistries: TaqMan, Scorpion primers, and SYBR Green I fluorescent dye. Various copy numbers of a plasmid containing the soya lectin sequence were used to determine the sensitivity and reproducibility of the different technology combinations and to examine both inter and intra machine variability. To examine the relative accuracy of each technology, the genetically modified soya content of baked products containing known amounts of Roundup Ready soya was determined by detection of lectin and the EPSPS transgene. It was determined that the combination of TaqMan detection chemistry and the ABI 7700 platform represented the best method for quantitative detection of genetically modified organisms in terms of both precision and accuracy.