What are you going to learn?

  • What is qPCR?
  • How does qPCR work?
  • How does qPCR differ from standard PCR?
  • What are the applications of qPCR?
  • terms: probe, quencher, reporter, DNA-binding dye

Quantitative PCR or real-time PCR is a modification of the standard PCR that allows us to monitor the amount of the amplified product after each cycle in real time. When it comes to the components needed for the reaction, it is very similar to the standard PCR: a heat-stable DNA polymerase, primers, dNTPs, the template, and a buffer. However, special kind of thermal cycler containing a laser that illumates PCR tubes is used for the reaction. When illumated, the DNA emits a fluorescent light, which is captured the detector and the information is sent into a computer. The more fluorescence measured, the more DNA in the sample.

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In qPCR, DNA emits a fluorescent light when illuminated by a laser in the thermal cycler. The information is then sent into a computer to monitor the amount of amplified product after each cycle.
standard components of (q)PCR
DNA emits a fluorescent light when illuminated by a laser in the thermal cycler.

There are two commonly-used methods that make DNA emit a fluorescent light. The first method uses a special DNA-binding dye (for example, SYBR® Green). The reaction starts with denaturation, when the two DNA strands are separated. In the second step called annealing, primers anneal, attach to the DNA, and DNA polymerase is now capable of extending the DNA strand in the 5’-to-3’ direction during the third standard step called elongation or extension. The DNA-binding dye then binds to the newly synthesized DNA product, which as a result now emits fluorescent light.

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DNA-binding dye binds to the newly synthesized double-stranded DNA product, which as a result emits fluorescent light.
qPCR with DNA-binding dye

The second method uses a dye-containing probe (for example, TaqMan® probe). A probe is a short sequence of DNA that contains two important parts: a reporter that emits a fluorescent light and a quencher, which blocks the reporter. The quencher is very important as we want the reporter to emit fluorescent light once we get the amplified product, not at the beginning. The reaction is once again very similar to the standard PCR. First, the two strands are separated in denaturation, which is followed by annealing, when both the primers and the probes anneal to their complementary sequences. In extension, as DNA polymerase synthesises the new DNA strand, it cleaves the probe, which separates the quencher from the reporter and as a result, the reporter starts emitting fluorescent light.

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A dye-containing probe first anneals to the target DNA sequence and during extension, the quencher separates from the reporter, which then emits fluorescent light.
a dye-containing probe
qPCR with a dye-containing probe

As we already said, the key difference between standard PCR and quantitative PCR is that quantitative PCR gives the information on how much DNA there is after each cycle, from which the number of molecules in the original sample can be calculated. This is important, for example, in diagnostics, to determine the degree of infection by measuring how much viral DNA there is. qPCR can also be used to measure changes in gene expression by first transcribing RNA into DNA and then continuining with the reaction.

References:
Klug, W. S., Cummings, M. R., Spencer, C. A., Palladino, M. A., & Killian, D. (2019). Concepts of Genetics. Pearson.
Pierce, B. A. (2019). Genetics: A Conceptual Approach (Seventh ed.). W. H. Freeman.