Fig. 2

Schematic overview of the Taqman assay. In addition to the genomic DNA template, four additional oligonucleotide components are required to detect the mutation. These include an unlabeled PCR primer pair and two TaqMan probes with a FAM (F) or a VIC (V) dye label on the 5’end, in combination with a minor groove binder (MGB) and a nonfluorescent quencher (Q) on the 3’end (1). The TaqMan probes hybridize to the target DNA after denaturation between the unlabeled PCR primers. The signal from the fluorescent dye on the 5’end of a TaqMan probe is quenched by the quencher on its 3’end through fluorescence resonance energy transfer (FRET) (2). During PCR, the AmpliTaq Gold DNA polymerase extends the unlabeled primers using the genomic DNA template strand. When the DNA polymerase reaches the TaqMan probe, it cleaves the molecule, separating the fluorescent dye from the quencher. The qPCR instrument detects fluorescence from the unquenched FAM or VIC dye in one reaction (3)