How does a qPCR exactly work with binding dyes and fluorescent probes?
In: Biology
Do you know how regular PCR works? Assuming you do for this.
Intercalating dyes bind between the strands of DNA, and only give off light when they’re bound in this fashion. As the amount of DNA in the sample increases with replication, the amount of bound dye increases, and the fluorescence increases. It’s exact intensity is measured every replication cycle, so that the cycle number that it passes a certain threshold can be recorded, and that correlates to the amount of DNA present in the initial sample.
Fluorescent probes are pieces of single-stranded DNA complementary to a region in the middle of the region you’re amplifying. They have a fluorophore on one end, and a quencher on the other end. The fluorophore can’t glow when it’s in proximity to the quencher. As the reaction progresses, and the enzyme adds bases in the region between the two primers, the probe is in the way, and the enzyme chews it up, releasing the pieces into the solution. This creates a greater distance between the fluorophore and the quencher, so that it can glow and it’s intensity can be recorded as above.
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