AAT Bioquest

Why are quenchers used in real time PCR?

Posted August 14, 2023


In real-time PCR, quenchers are used to prevent background fluorescence from interfering with the accurate measurement of DNA or RNA in the sample. 

In PCR reactions, a fluorescent dye is typically used to tag DNA or RNA molecules. The dye works by emitting light when it is excited by a specific wavelength. However, this emitted light can be detected even in the absence of the target DNA or RNA, which can compromise the accuracy of the PCR results. Adding a quencher to the reaction can help resolve this problem. 

Quenchers are molecules that reduce the amount of emitted light when they are close to the probe. When the quencher and fluorescent dye are in close proximity to each other, the quencher absorbs the emitted light, preventing its detection. Then, when the DNA or RNA molecule of interest is replicated during the PCR reaction, the dye gets separated from the quencher and starts to emit light again. 

Researchers measure the amount of DNA or RNA in a sample by monitoring the differences in fluorescence intensity over the course of the reaction. The increase in fluorescence indicates that the target molecule is present in the sample, while a decrease in fluorescence indicates that the target molecule is not present in the sample.

Additional resources

Multiplex quantitative PCR using self-quenched primers labeled with a single fluorophore

Real-Time PCR (qPCR)

DABCYL succinimidyl ester [4-((4-(Dimethylamino)phenyl)azo)benzoic acid, succinimidyl ester] *CAS 146998-31-4*

Tide Quencher™ 2WS succinimidyl ester [TQ2WS, SE]