The detection chemistry we’re using is a dual labeled probe with a 5’ nuclease a
ID: 97108 • Letter: T
Question
The detection chemistry we’re using is a dual labeled probe with a 5’ nuclease activity, which corresponds to Figure3c in Wong and Medrano (2005). In PAL5, the reporting fluorophore (R) is 5’Fam (fluoresces in the blue range) and the quencher (Q) is 3’ Iowa black. For the -tubulin, the reporting fluorophore (R) is 5’Hex (fluoresces in the green range) and the quencher (Q) is 3’ Iowa black. In this chemistry, the quenchers reduce the reporters’ fluorescence by FRET (fluorescence resonance energy transfer) until the reporter fluorophore is released. When is this reporter fluorophore being released in this reaction?
Explanation / Answer
Dual-labeled oligonucleotide probes is widely used in assays for genetic analysis like nucleic acid detection/ amplification methods and is based on Fluorescence resonance energy transfer (FRET) mechanism. This fluorogenic probes are labelled with both a reporter and a quencher dye. Reporter fluorophore is only released when the two dyes are physically separated through hybridization or nuclease activity.