Why are my realtime PCR amplification plots hook-shaped?

During the late phase of PCR, as a lot of PCR product has been generated, there is strong competition between hybridization of the probe to the target strand and re-association of the two complementary product strands. For some primer–probe combinations, re-association occurs more quickly than probe hybridization towards the end of the PCR. Therefore, the yield of PCR product seems to decrease after reaching a peak. For accurate quantification, fluorescence data is measured during the log-linear phase of the reaction, i.e., during the initial increase of fluorescence, before this phenomenon occurs.

Can’t find what you are looking for?

Browse the FAQ base with our FAQ search.