Common probes and fluorescent dyes for real-time PCR

Common probes and dyes for real-time PCR

The green lightning bolt is the excitation light. The green circles are fluorophores, the dark-red circles are quenchers, and the black circles are dark quenchers. The large gray circle is a polymerase with 5′-to- 3′ exonuclease activity. The thin black squares are blockers, and the orange cylinders are minor groove binders

A( Double-stranded DNA dyes show a significant increase in fluorescence when bound to DNA.

B( Hydrolysis probes between a fluorescent reporter and a quencher are cleaved by polymerase activity, resulting in increased fluorescence.

C( Dual-hybridization probes change color by resonance energy transfer when hybridized.

D( The molecular beacon hairpin quenches fluorescence until target binding that separates the quencher from the fluorophore.

E( Scorpion probes are quenched in the native conformation but increase in fluorescence when the original hairpin loop is hybridized to its extension product.

F( Dark quencher probes are initially quenched by a minor groove binder and the dark quencher. Hybridization to the target releases the fluorescence.

G( The short strand of partially double-stranded probes is displaced in the presence of target, releasing fluorescence from quenching.

Reference: Henry’s Clinical Diagnosis and Management by Laboratory Methods 24th Edition