Single-nucleotide polymorphism genotyping by melting analysis of dual-labeled probes: examples using factor V Leiden and prothrombin 20210A mutations.

Abstract

The emergence of fluorescence techniques in 96- or 384-well formats has led to high-throughput single-nucleotide polymorphism (SNP) detection methods. Among these, allele discrimination based on real-time PCR technologies has been developed along two main methodologic approaches. In the first approach, a temperature-dependent fluorescent signal is generated by hybridization of a probe at the end of each PCR cycle. The monitoring of fluorescence emission is followed during (1)(2) or at the end of (3)(4) the PCR process. In the latter case, allele detection is achieved by carrying out of a melting curve analysis. In the second approach, exploiting the classic 5′ nuclease assay (TaqMan®) technology, a temperature-independent fluorescence signal is generated during the PCR process by the exonuclease degradation of a hybridized TaqMan probe (5)(6). Except for one method discussed later (3), all of these techniques require the use of two probes for allele differentiation. An allele differentiation study has stressed the risk of artifacts and subsequent erroneous results when a polymorphism is located within the DNA sequence targeted by TaqMan (7). The same study demonstrated the power of methods relying on determination of melting curves. Although other interesting approaches have been reported recently, such as DASH (8), iFret (9), and dipole calculation (10), some of them are technically difficult or time-consuming. In addition, the SYBR Green technique (11) would represent the simplest method to detect point mutations, but it seems to be limited by its lack of ability to differentiate alleles (12). We present here a new methodologic approach that allows high-throughput genotyping of SNPs. Allele differentiation is achieved by the generation of melting curves, with a single dual-labeled probe, at the end of a classic PCR reaction. The reliability of the method is demonstrated for detection of the factor V Leiden …