Measurement of PCR product during amplification
Here we discuss the method used in the PE Applied Biosystems PRISM 7700 instrument.
The product itself is not actually measured. Rather, DNA polymerase, in the process of making product, collides with a quenched fluorescent oligonucleotide hybridized to the template. The polymerase has 5' exonuclease activity, which degrades the oligonucleotide holding the fluorochrome in proximity to the quencher. The liberated fluorochome provides the signal. Specificity is assured because the polymerase can only degrade an oligonucleotide if that oligonucleotide is hybridized to the template the polymerase is copying.