Overview: Types of PCR Probes
by Adriana Gallego, Ph.D.
When performing a qPCR or RT-qPCR, a PCR probe is usually used to detect the target sequence using fluorescence.
However, there are several PCR probes commercially available.
This article will provide a deeper look into the different types of PCR probes and their applications.
In this article
How does a hydrolysis probe work?
How does a molecular beacon probe work?
How does a dual hybridization probe work?
How does a Scorpion probe work?
What is a PCR probe?
A PCR probe is a short, sequence-specific oligonucleotide, usually DNA, labeled with one or more fluorescent detection elements such as a reporter dye and, in many probe types, a quencher. The labeled sequence, or probe, is highly specific and recognizes a complementary target sequence. In PCR and qPCR applications, this target is typically DNA, including cDNA generated during RT-qPCR.
Dyes, by comparison, intercalate with DNA nonspecifically.
The reason probes are so important is because they allow you to study your target gene among many.
For instance, imagine you are interested in a gene that produces a specific metabolite in a plant species or a gene that causes disease in an animal.
With all the genes present in an organism the question becomes, how do you study your target gene? This is why you’ll need a probe.
Because of the specificity, PCR probes bind to your complementary target sequences among the whole cocktail of sequences.
Types of PCR Probes
Among the most popular PCR probes out there are hydrolysis probes, Molecular Beacon probes, dual hybridization probes, Scorpion probes, and a few others.
There are different types of PCR probes commercially available. In general, these PCR probes share common characteristics, such as using fluorescent labels attached to probes that bind to the target sequence, allowing its detection.
Hydrolysis Probes
The components of a hydrolysis probe consist of a complementary oligonucleotide, or oligo, sequence (small nucleotide sequence that can hybridize with the target DNA/RNA sequence), a fluorescent reporter and a quencher.
Hydrolysis probes are qPCR labeled probes used to target specific sequences by complementation. Hydrolysis probes are comprised of an oligonucleotide probe, a fluorescent reporter, and a quencher. During amplification, polymerase-mediated cleavage of the probe separates the reporter from the quencher, producing fluorescence that is proportional to target amplification.
Oligonucleotide: The oligonucleotide, or oligo, for PCR probes is a DNA sequence complementary to the target sequence.
Fluorescent reporter: The fluorescent reporter is a 5'-fluorescent dye. This molecule fluoresces once the target sequence is detected and it is separated from the quencher. Fluorescein (FAM), which emits green fluorescence, is commonly used as a fluorescent reporter.
Quencher: A quencher is a molecule that absorbs excitation energy from a fluorophore (eats the reporter’s fluorescence). In other words, a quencher is attached to the oligonucleotide probe, and from there, it quenches the fluorescence emitted by a fluorophore when they are in close proximity. Commonly used quenchers are Black Hole Quencher 1 dye and TAMRA.
How does a hydrolysis probe work?
In qPCR, the hydrolysis probe hybridizes to a complementary sequence within the target amplicon, typically between the forward and reverse primer binding sites.
Then, during extension, DNA polymerase synthesizes the new strand. When it reaches the bound probe, its 5′ nuclease activity cleaves the probe.
Then, the fluorescent reporter and quencher are separated, allowing fluorescence to be detected.
The hydrolysis probe binds to the complementary sequence. The polymerase (purple arrow) separates the fluorescent reporter from the probe, which separates it from the quencher as well. Separation from the quencher leads to reporter fluorescence.
The three main components of a hydrolysis probe are the oligonucleotide probe, the fluorescent reporter, and the quencher. The reporter and quencher are attached to specific positions on the probe so fluorescence remains quenched until the probe is cleaved.
With a hydrolysis probe, the fluorescent reporter and quencher are attached to specific sites on the oligonucleotide. While the probe is intact, their proximity allows the quencher to suppress reporter fluorescence.
These hydrolysis probes present advantages such as high specificity because the oligo binds to your target sequence by complementary hybridization and the ability to perform multiplex reactions.
Multiplex reactions can use multiple primer pairs and probes labeled with different fluorescent reporters, allowing more than one target sequence to be detected in the same reaction.
Some drawbacks of hydrolysis probes include the probe cost and complex experimental design
Molecular Beacon Probes
A molecular beacon probe consists of a complementary oligo, a fluorescent reporter and a quencher. However, the structure differs in that the oligo forms a stem and loop, bringing the reporter and quencher closer together.
A molecular beacon probe is designed so the oligonucleotide has complementary sequences that leads to the formation of a stem and loop. With the quencher and reporter attached to opposite ends of the stem-loop probe, the folded probe brings them into close proximity and suppresses fluorescence.
Like a hydrolysis probe, molecular beacon probes contain the same elements. It has an oligo (oligonucleotide), a fluorescent reporter, and a quencher.
Molecular Beacon Probes
Shows how the molecular beacon probe works. Once the probe binds to the complementary sequence, the stem denatures, causing the reporter and quencher to become too far apart for the quencher to work. As a result, the reporter fluoresces.
In a qPCR, the loop section of the probe, which is typically 15–30 nucleotides in length, hybridizes to the complementary target sequence.
When the loop binds to the target sequence, it causes the stem to denature. After stem denaturation, the reporter and quencher are separated far enough that the reporter can emit fluorescence while still attached to the probe.
Unlike hydrolysis probes, molecular beacons generate signal through hybridization-induced separation of the reporter and quencher, not through enzymatic probe cleavage.
Some of the advantages of a molecular beacon probe are the high specificity and multiplexing ability (identifying multiple target sequences in the same run).
A significant disadvantage involves the complexity of designing the probes. For instance, the oligo sequence (forming the stem) must be strong enough to avoid reporter and the quencher separation leading to false fluorescence.
Dual Hybridization Probes
Dual hybridization probes work on the basis of donor and acceptor fluorophores instead of quenching a reporter.
In dual hybridization probes, there are no quenchers. Instead, there are two oligos, one carrying a donor fluorophore, and the other carrying an acceptor fluorophore.
Donor fluorophore: A dye excited by a specific wavelength. This dye donates its energy to an acceptor fluorophore.
Acceptor fluorophore: An acceptor fluorophore is a dye that receives energy from another fluorophore (or donor fluorophore), and with that energy, the acceptor fluorophore emits fluorescence. Both the donor and acceptor fluorophores must come close for this to happen.
How does a dual hybridization probe work?
Two oligos, each one carrying one fluorophore, are used in dual hybridization.
These oligos and their fluorophores come into proximity with the target sequence. One probe hybridizes to one region of the target and carries the donor fluorophore, while the second probe hybridizes to an adjacent region and carries the acceptor fluorophore.
Then, a process called fluorescence resonance energy transfer (or FRET) occurs. FRET is a distance-dependent physical process that transfers energy from an excited molecule (or donor fluorophore) to another molecule (the acceptor fluorophore) through a dipole–dipole coupling process.
Here the donor fluorophore is excited and transfers its energy to the acceptor fluorophore, making it fluoresce. Both oligos bind to adjacent regions of the same target sequence. This co-localization brings the donor and acceptor fluorophores into close proximity, enabling FRET: the excited donor transfers its energy to the acceptor, causing it to fluoresce.
Dual hybridization probes require a donor and acceptor fluorophore. The two probes must come in close contact with each other. This proximity allows energy transfer from the excited donor fluorophore to the acceptor fluorophore, causing the acceptor to emit fluorescence.
The advantage of this probe is the specificity to the target sequence. The disadvantage is the complex design of the oligo sequence for both the donor and acceptor fluorophores.
Scorpion® Probes
Scorpion® probes consist of a PCR blocker and the Scorpion® primer linked to a probe.
Scorpion® probes are a PCR probe containing a hairpin-loop primer and a PCR blocker. After primer extension, the probe sequence within the Scorpion® primer hybridizes intramolecularly to its complementary sequence in the newly synthesized amplicon, opening the hairpin and separating the reporter from the quencher.
Scorpion® primer: a hairpin loop primer in the form of "U,” containing a quencher and a fluorescent reporter. Due to this loop structure, the quencher is close to the fluorescent reporter quenching its fluorescence. It also includes the sequence complementary to the target sequence.
PCR blocker: a hexethylene glycol (HEG) monomer used to prevent read-through during the extension of the opposite strand.
How does a Scorpion® probe work?
During the amplification cycles, the loop structure in the Scorpion® primer is opened. This allows the hybridization of the internal sequence to the target sequence and separates the quencher from the fluorescent reporter.
Finally, this separation allows the reporter to fluoresce.
The PCR blocker is located downstream of the quencher in the 3' direction. It is used to prevent read-through during the extension of the opposite strand. If the opposite strand would be read, the quencher could be separated from the reporter and this will lead to a false fluorescence.
The probe binds to the sequence, and the primer is extended. Following primer extension, the probe sequence hybridizes intramolecularly to its complementary target sequence within the newly synthesized amplicon. This opens the hairpin structure, separates the reporter from the quencher, and allows fluorescence to occur.
Scorpion® probes are highly sensitive, sequence-specific, bi-labeled self-probing primers designed for qPCR, in which the detection probe element is covalently linked to the 5' end of the amplification primer.
If you want to learn more about probe-based qPCR please visit our GoldBio article "All about probe based qPCR"
References
Bio-Rad. (2022). Introduction to PCR Primer & Probe Chemistries Real-time. DNA Binding Dyes. 04 of May 2022
Gudnason, H., Dufva, M., Bang, D. D., & Wolff, A. (2007). Comparison of multiple DNA dyes for real-time PCR: Effects of dye concentration and sequence composition on DNA amplification and melting temperature. Nucleic Acids Research, 35(19), 1–8. https://doi.org/10.1093/nar/gkm671
Murray, J. L., Hu, P., & Shafer, D. A. (2014). Seven novel probe systems for real-time PCR provide absolute single-base discrimination, higher signaling, and generic components. Journal of Molecular Diagnostics, 16(6), 627–638. https://doi.org/10.1016/j.jmoldx.2014.06.008
Nagy, A., Vitásková, E., Černíková, L., Křivda, V., Jiřincová, H., Sedlák, K., Horníčková, J., & Havlíčková, M. (2017). Evaluation of TaqMan qPCR system integrating two identically labelled hydrolysis probes in single assay. Scientific Reports, 7, 1–10. https://doi.org/10.1038/srep41392
Wong, M. L., & Medrano, J. F. (2005). One-Step Versus Two-Step Real- Time PCR. BioTechniques, 39(1), 75–85. https://doi.org/10.2144/05391RV01
Wong, W., Farr, R., Joglekar, M., Januszewski, A., & Hardikar, A. (2015). Probe-based real-time PCR approaches for quantitative measurement of microRNAs. Journal of Visualized Experiments, 2015(98), 16–18. https://doi.org/10.3791/52586
Zhang, H., Yan, Z., Wang, X., Gaňová, M., Chang, H., Laššáková, S., Korabecna, M., & Neuzil, P. (2021). Determination of Advantages and Limitations of qPCR Duplexing in a Single Fluorescent Channel. ACS Omega, 6(34), 22292–22300. https://doi.org/10.1021/acsomega.1c02971
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