Ultra-rapid DNA analysis using HyBeacon probes and direct PCR amplification from saliva

We describe a novel probe technology, termed HyBeacons, which provides a new homogeneous method for fluorescence-based sequence detection and allele discrimination. Employing a single nucleotide polymorphism located in the N-acetyltransferase 2 gene as a model system, we demonstrate the utility of HyBeacon probes for rapid and reliable sequence analysis. We also demonstrate that homozygous and heterozygous samples may be accurately identified using a single HyBeacon oligonucleotide. Polymorphic DNA sequences were detected and differentiated by real-time PCR and melt peak methodologies, without performing extraction of genomic DNA prior to target amplification. Employing a combination of homogeneous HyBeacon analysis, the rapid thermal cycling conditions of the LightCycler and direct amplification from saliva, allowed samples to be genotyped within 30 min. Such rapid non-invasive diagnostic technologies may permit 'point-of-care' genetic testing to be performed in hospitals and doctor's surgeries.