David J. French

Genotyping for CYP2C9 and VKORC1 alleles by a novel point of care assay with HyBeacon® probes

BACKGROUND Coumarin anticoagulants such as warfarin are used to treat and prevent thromboembolic events in patients. The required dosage is difficult to predict and the risk of over or under anticoagulation are dependent on several environmental and clinical factors, such as concurrent medication, diet, age and genotype for polymorphisms in two genes CYP2C9 and VKORC1. METHODS A novel fluorescent PCR genotyping assay using HyBeacon® probes, was developed to enable clinical staff to genotype the CYP2C9*2 and CYP2C9*3 alleles and the VKORC1 G-1639A polymorphism directly from unextracted blood samples. A prototype PCR instrument, Genie 1, suitable for point of care use was developed to carry out the assays. The panel of tests was validated by analysing blood samples from 156 individuals and comparing genotypes with data obtained using DNA samples from the same individuals. The accuracy of genotypes obtained with the Genie 1 was compared against results from well validated real time PCR and PCR-restriction fragment length polymorphism analysis. RESULTS Identical results were obtained for the newly developed HyBeacon® method and the validation method in all cases except for one where no result was obtained for the VKORC1 polymorphism on the Genie instrument. The samples used for validation represented all six possible *2 and *3 allele-related CYP2C9 genotypes and all three VKORC1 G-1639A genotypes. CONCLUSIONS We observed excellent accuracy for the newly developed method which can determine genotype in less than 2 h.

Six-colour HyBeacon probes for multiplex genetic analysis.

The analysis of single-point mutations in DNA (nucleotide deletions, insertions and substitutions) has transformed human diagnostics and pharmacogenetics, enabling the development of safer drug treatment regimes for diseases such as cancer, hypercholesterolemia and thrombosis. In these fields, rapid and precise feedback is essential, and to this end we are developing HyBeacon probes for use in the clinic. HyBeacons are linear oligonucleotides possessing one or more fluorescent dyes tethered to internal nucleotides. The inherent fluorescence-quenching properties of the DNA bases and attached fluorophores cause fluorescence emission to be low in the unstructured single-stranded probe. Upon hybridization to their DNA target, the rigid double strand removes the fluorophores from the vicinity of the nucleobases and increases the separation between the dyes. The resultant increase in fluorescence can be used to discriminate between DNA sequences that differ by only a single base (wild-type/mutant, single nucleotide polymorphisms (SNPs)) by melting-curve analysis. This powerful technology has recently become increasingly important due to the emergence of high-resolution DNA melting platforms. Unlike many other fluorogenic probes, HyBeacons do not possess internal secondary structure, so clean and reproducible melting transitions are obtained. Until now, HyBeacon assays have been limited in fluorophore choice to fluorescein and its hexachloro derivative Hex, thus precluding their use in the more complex multiplex assays that are vitally important in a diagnostic context. We now report a six-colour PCR-based HyBeacon system and demonstrate its use in the analysis of mutations in the cystic fibrosis transmembrane conductance regulator (CFTR) gene of human DNA, ABCC7. Alternative systems based on real-time PCR also exist. Taqman and displacement probes are capable of detecting three biallelic SNPs simultaneously, but require two probes for each genotype. High-resolution melting (HRM) with DNA-binding dyes can provide melting-temperature analysis but is limited to a single colour. Hybridization probes and molecular beacons have been used in threeand four-colour systems, respectively, but melting analysis with the latter is complicated by secondary structure. In previous studies, HyBeacons were labelled with various fluorescein dT phosphoramidite monomers during oligonucleotide synthesis. However, the phosphoramidites of other fluorescent nucleotides are not generally available, so in our latest study, oligonucleotide probes containing 5-aminoalkyland 2’-aminoethoxythymidine were labelled with the commercially available N-hydroxysuccinimide (NHS) esters of a series of fluorophores. Labelling in the 5-position places the fluorophore in the major groove of the DNA duplex, whereas 2’-labelling directs the dye towards the minor groove. By using this strategy a series of six HyBeacon probes was designed to target the R516G mutation in the CFTR gene. Each probe contained two additions of the amino-modified T monomer (Scheme 1), with a spacing of three, five or seven nucleotides (Table 1).

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

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 A136, V136, T136, T137, L141, F141 R154, H154, L168, R171, Q171, H171 and K171 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.

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.

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

Abstract 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 fluorimetrie 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.

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.

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. We describe an economic two-stage method for probe synthesis, and a new method for nucleic acid sequence analysis which together considerably reduce costs. The analysis method utilises three-strand and four-strand hybridisation complexes for the detection and identification of nucleic acid target sequences by real-time PCR and fluorescence melting.

Development of HyBeacon® probes for specific mRNA detection using body fluids as a model system.

HyBeacons are linear oligonucleotides which incorporate fluorescent dyes covalently linked to internal nucleotides. They have previously been used with PCR and isothermal amplification to interrogate SNPs and STRs in fields as diverse as clinical diagnostics, food authentication, and forensic DNA profiling. This work explores their use for the identification of expressed gene sequences through mRNA profiling. The use of mRNA is becoming increasingly common in forensic casework to identify body fluids on evidence items, as it offers higher specificity and fewer false positives than current chemical presumptive testing methods. The work presented here details the development of a single-step one-tube RT-PCR assay to detect the presence of body fluids of forensic interest (saliva, blood, seminal fluid, vaginal fluid and menstrual blood) using HyBeacon® probes and melt curve analysis. Each assay shows a high degree of specificity to the target body fluid mRNA suggesting there is no requirement to remove genomic DNA prior to analysis. Of the five assays developed, four were able to detect between 10 and 100 copies of target cDNA, the fifth 1000 copies of target. The results presented here demonstrate that such an approach can be optimised for non-expert users and further areas of work are discussed.