Real-time closed tube single nucleotide polymorphism (SNP) quantification in pooled samples by quencher extension...

Quencher extension (QEXT) is a novel single step closed tube real-time method to quantify SNPs using reporters and quenchers in combination with primer extension. A probe with a 5'-reporter dye is single base extended with a dideoxy nucleotide containing a quencher dye if the target SNP allele is present. The extension is recorded from the quenching (reduced fluorescence) of the reporter dye. This avoids the influence of the unincorporated dye-labeled nucleotides, resulting in high accuracy and a high signal-to-noise ratio. The relative amount of a specific SNP allele is determined from the nucleotide incorporation rate in a thermo-cycling reaction. We tested the QEXT assay using five SNPs in the Listeria monocytogenes inlA gene as a model system. The presence of the target SNP alleles was determined with high statistical confidence (P < 0.0005). The quantitative detection limits were between 0 and 5% for the targeted SNP alleles on a background of other SNP alleles (P < 0.05). The QEXT method is directly adaptable to current real-time PCR equipment and is thus suited for high throughput and a wide application range.