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What are the differences between the detection methods based on a fluorescent probe and an intercalating dye in qPCR?
Posted June 22, 2020
DNADNA and RNA QuantitationPCRPeptide and OligonucleotidePolymerase Chain Reaction (PCR)PolymerasesReal-Time PCR (qPCR)
Answer
qPCR is typically quantified in one of the two ways: via a fluorescent probe oligonucleotide that anneals to a specific sequence of the target DNA and fluoresces once the quencher on the probe is removed by the 5´ to 3´ nuclease activity of Taq DNA Polymerase, or via an intercalating dye that has increased quantum yield upon intercalating into double-stranded DNA. The main differences between these two methods are specificity and cost.
- Specificity: Fluorescent-probe-based detection has higher specificity than the one using an intercalating dye. Intercalating dyes cannot distinguish from target DNA products from by-products; therefore, non-specific products are also being measured. In contrast, fluorescent-probe-based method only detects the target DNA with complementary sequence of the probe, leading to higher specificity.
- Cost: The method based on intercalating dyes is generally more cost-effective, since only a pair of primers are needed to carry out the amplification. However, in fluorescent-probe-based method, in addition to the pair of primers, an oligonucleotide probe that is labeled with a fluorescent reporter at one end and a quencher at the opposite end is also needed, which increases the cost for experiment.
Additional resources
6-ROX glycine *25 uM fluorescence reference solution for PCR reactions*Postollec, F., Falentin, H., Pavan, S., Combrisson, J., & Sohier, D. (2011). Recent advances in quantitative PCR (qPCR) applications in food microbiology. Food microbiology, 28(5), 848-861.Valasek, M. A., & Repa, J. J. (2005). The power of real-time PCR. Advances in physiology education, 29(3), 151-159.