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Are there different types of fluorescent probes used in qPCR?
Posted June 22, 2020
Peptide and Oligonucleotide LabelingDNA and RNA QuantitationDNA and RNAPCRPhysiological ProbesPolymerase Chain Reaction (PCR)Real-Time PCR (qPCR)
Answer
Yes! The hydrolysis (e.g., TaqMan) probe is the most common but just one of the probes being used in qPCR. The hydrolysis probe is a short oligonucleotide labeled with a fluorescent reporter and a quencher at each end, which is later degraded by the 5' ? 3' exonuclease activity of polymerase and fluoresce in accordance with the concentration of amplified target DNA. Apart from the hydrolysis probe, there are also other types of probes available in market for qPCR. Common examples are:
- Molecular beacon probes: Molecular beacon probes are also short oligonucleotides labeled with a fluorescent reporter at one end and a quencher at the opposite end, which form a stem-loop structure in the absence of target DNA, allowing the quencher prevents fluorescence FRET. When molecular beacon probes hybridize to the target DNA, the fluorescent reporter and quencher are separated, resulting in increased fluorescence.
- Scorpion probes: Scorpion probes serve as both the primer and the probe in qPCR. A stem-loop structure carrying a fluorescent reporter and a quencher is linked to the PCR primer. The loop of the Scorpion probe has a sequence that is complementary to the newly synthesized strand, downstream of the primer binding site. After an amplification cycle, a copy of the complementary target sequence is created, and the loop of the Scorpion probe can then hybridize to the recently synthesized DNA strand. This hybridization separates the fluorescent reporter from the quencher, resulting in increased fluorescence signal.
- Dual hybridization probes: Dual hybridization probes involve the usage of two sequence-specific oligonucleotide probes, which rely on FRET to produce, instead of quench, a detectable fluorescence signal. The two probes are designed to bind to adjacent sequence in the target DNA. The donor dye is labeled to the 3’ end of the first probe, while the acceptor dye is labeled to the 5’ end of the second probe. Once both probes bind to the target DNA, the increased fluorescence signal from the acceptor can be used to monitor the amplified target sequence.
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.Helixyte™ Green *10,000X Aqueous PCR Solution*