Quencher Extension for Single Nucleotide Polymorphism Quantification in Bacterial Typing and Microbial Community Analyses

Abstract

Quencher extension is a novel single-step closed tube real-time method to quantify single nucleotide polymorphisms (SNPs) in combination with primer extension. A probe with a 5′-reporter is single-base extended with a dideoxy nucleotide containing a quencher if the target SNP allele is present. The reaction is measured from the quenching (reduced fluorescence) of the reporter. The relative amount of a specific SNP allele is determined from the nucleotide incorporation rate in a thermocycling reaction. The quencher extension protocol presented was developed for SNP allele quantification in Listeria monocytogenes and for microbial community analyses.