In real-time PCR (qPCR) using TaqMan probes, fluorescence signal increases are due to which molecular mechanism?

• A Intercalation of SYBR Green dye into double-stranded PCR product
• B 5'-to-3' exonuclease activity of Taq polymerase degrading the probe and separating fluorophore from quencher ✓
• C Fluorescent primer labelling whose fluorescence increases on polymerisation
• D FRET between two TaqMan probes binding adjacent sequences

Explanation

TaqMan probes are dual-labelled oligonucleotides with a 5' fluorophore (e.g., FAM) and a 3' quencher (e.g., BHQ). When intact and in solution, the quencher suppresses fluorescence via FRET. During PCR extension, if the probe is annealed to the template between primers, the advancing Taq polymerase's 5'-to-3' exonuclease activity degrades the probe, physically separating fluorophore from quencher — fluorescence is released. Signal is proportional to amplicon quantity. This mechanism makes TaqMan highly specific (requires probe hybridisation to target, not just dsDNA) versus SYBR Green (any dsDNA including primer-dimers).

Reference: Harper's Illustrated Biochemistry, 32nd ed.

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