Hello,
when is the light measurement taking place in qPCR? during the annealing or during the extension?
Thank you
Unless you have a special purpose, you typically measure fluorescence during extension. During annealing it would not be very practical, as a lot of your DNA structures would be a long-ish single strand attached to a short primer, and therefore would provide low level of fluorescence
In quantitative polymerase chain reaction (qPCR), the measurement of fluorescence is typically performed during the extension phase of each amplification cycle. Specifically, it occurs after the annealing step and before the denaturation step.
During the qPCR process, after the DNA template and primers have annealed to their complementary sequences, the DNA polymerase extends the primers by incorporating fluorescently labeled nucleotides into the newly synthesized DNA strand. These fluorescent labels emit fluorescence signals that can be measured to determine the amount of amplified DNA.
The fluorescence measurement is usually carried out using a specialized qPCR instrument that includes a light source (such as a laser or LED) and a detector. The instrument monitors the fluorescence emitted by the labeled DNA during the extension phase of each cycle. The level of fluorescence is proportional to the amount of amplified DNA present in the reaction.
It's worth noting that different qPCR chemistries and instruments may have slight variations in the exact timing and duration of the fluorescence measurement, but generally, it takes place during the extension phase of the qPCR amplification cycle.
Luigi Marongiu
the light measurement in qPCR is typically performed during the extension phase, after the annealing of primers and before the subsequent denaturation step in the next PCR cycle.
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