End-capped HyBeacon probes for the analysis of human genetic polymorphisms related to warfarinmetabolism

Abstract

HyBeacon probes have been used to characterise SNPs in the CYP2C9 and VKORC1 genes associated with variations in the efficiency of warfarin metabolism. PCR amplification of genomic DNA and probe target melting analysis provided a robust and reliable method to differentiate polymorphic sequences at these loci. Probes capped with 5′-trimethoxystilbene were found to exhibit larger fluorescence differences between hybridised and dissociated states than uncapped probes, generating melting peaks of considerably improved height. 3′-Pyrene modifications enhanced probe signal-to-noise and melting peak heights with the additional benefit of acting as PCR stoppers, and 5′,3′-doubly capped probes gave the best analytical results.