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.
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A PCR probe is a small DNA or RNA sequence labeled with a reporter molecule. The labeled sequence, or probe, is very specific and recognizes complementary sequences of RNA or DNA.
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.
Among the most popular PCR probes out there are hydrolysis probes, Molecular Beacon probes, dual hybridization probes, Eclipse 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 some form of fluorescence that binds to the target sequence, allowing its detection.
Figure 1. 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 a primer, a fluorescent reporter, and a quencher where the fluorescence emitted by the reporter is indicative of aligning to the target sequence.
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 a primer, 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.
In qPCR, the hydrolysis probe binds to the target sequence based on the primer.
Then, during the extension process, the DNA polymerase initiates the polymerization and executes 5' → 3' exonuclease activity on the primer.
The DNA polymerase cuts the fluorescent reporter from the primer and the quencher.
Then, the fluorescent reporter and quencher are separated, and the free fluorescent reporter fluoresces.
Figure 2. 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 components of a hydrolysis probe, the primer, the fluorescent reporter, and the quencher, are joined together, and their configuration is exact.
Think about an adjustable exercise weight. Two sets of barbells are separated by the metal bar.
Figure 3. Comparing a PCR hydrolysis probe to a dumbbell. The bar of the dumbbell represents the oligo of a probe. The two bells represent the fluorescent reporter and the quencher.
With a hydrolysis probe, the fluorescent reporter and the quencher is placed at specific sites of the oligo; therefore, the quencher is always quenching the fluorescence from the reporter, and the oligo acts as a bridge linking both molecules.
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.
You can use several primers and fluorescent reporters in the same reaction.
Some drawbacks of hydrolysis probes include the probe cost and complex experimental design
Figure 4. 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 bound to each end of the oligo, and the primer looping, the quencher and reporter are in close range.
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
Figure 5. 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 primer hybridizes to a 15–30 nucleotide section of the target sequence.
When the loop binds to the target sequence, it causes the stem to denature. After stem denaturation, the reporter and the quencher are separated, and then the free fluorescent reporter emits fluorescence.
Unlike the hydrolysis probes, the fluorescent reporter is separated from the quencher but not removed by the polymerase. Consequently, the DNA polymerase lacking 5' exonuclease activity is used.
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.
Figure 6. 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.
Two oligos, each one carrying one fluorophore, are used in dual hybridization.
These oligos and their fluorophores come into proximity with the target sequence. We could say that part of the complementary sequence to the target is in the donor and the other is in 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 molecular (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. The donor binds to the target sequence and when it comes close to the acceptor, it transfers its energy making the acceptor fluorophore fluoresce.
Figure 7. Dual hybridization probes require a donor and acceptor fluorophore. The two probes must come in close contact with each other. This proximity allows electronic transfer from the donor to the fluorophore, and then the acceptor fluoresces.
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.
Figure 8. Eclipse® probes comprise a minor-groove binder, a fluorescent reporter, a quencher and the complementary oligo.
An Eclipse® probe is a PCR probe used to target DNA sequences by complementation. Eclipse® probes have four components: a fluorescent reporter, a quencher, a minor-groove binder and the oligo. The aligning to the target sequence causes a physical distancing between the reporter and quencher favoring the fluorescence.
Minor-groove binder (MGB): A minor-groove binder is a small crescent-shaped molecule that interacts with DNA via minor grooves. The MGB can join the oligo sequence thanks to a hydrogen that binds to base pair edges.
The fluorescent reporter is localized at the 3' end of the oligo sequence and the quencher at 5' end in the Eclipse® probe.
The MGB is bound to the quencher. Because the Eclipse® probe has a loop conformation (like a "U" form), the quencher is close to the fluorescent reporter allowing it to quench the reporter.
The probe changes its conformation during the annealing stage and hybridizes to the target sequence, consequently separating the quencher from the fluorescent reporter.
Therefore, the reporter can fluoresce. The resulting fluorescence signal is proportional to the amplified product in the sample.
Figure 9. Eclipse® probe binding leads to a conformation change that separates the reporter from the quencher. Upon separation, the reporter fluoresces.
Figure 10. The Amplifluor® assay consists of a Z primer and Z sequence as well as the UniPrimer that holds the quencher and reporter.
In the Amplifluor® assay, a PCR probe hybridizes to the target sequence and the labeled probe changes its conformation to allow the fluorescence. It contains three components: a Z sequence, a Z primer and a UniPrimer.
The Z sequence: a sequence of 18 nucleotides used as a template.
The Z primer: a sequence that binds to the 5' extreme of the Z sequence.
The UniPrimer: a sequence in the form of a loop that harbors the fluorescent reporter and the quencher in close proximity.
In the Amplifluor® assay, the Z sequence is bound to the 5' extreme of the Z primer.
The Z primer hybridizes with the target sequence during the first amplification cycle and gets extended.
This Z sequence is the 3' extreme of the UniPrimer. During the second amplification cycle, this Z sequence hybridizes with the product of the first sequence. It serves as a template for the UniPrimer, which contains a sequence complementary to the Z sequence.
In the third amplification cycle, the extended UniPrimer acts like a template strand for the next amplification cycle.
During the fourth cycle, the opening of the UniPrimer causes the quencher and the fluorescent reporter to get separated, which then allows the reporter to fluoresce.
Here, the fluorescence signal is proportional to the amount of amplified product.
Figure 11. Shows the process of the Ampliflour® assay at each cycle.
The advantage of this probe is the specificity to the target sequence. The disadvantage is the design complexity of the oligo sequence.
Figure 12. 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. The hairpin-loop primer is modified during its annealing to the target sequence allowing fluorescence emission from the reporter.
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.
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.
Figure 13. The probe binds to the sequence, and the primer is extended. Upon denaturation, the probe opens. This causes the reporter and quencher to separate, and the reporter then fluoresces.
The Scorpion® probes are highly sensitive, sequence-specific, bi-labeled fluorescent probe/primer hybrids designed for qPCR. As with many of the advanced probes out there, a key disadvantage of this probe is the oligo design complexity.
Figure 14. The LUX™ probe consists of a reporter and primer.
LUX™ PCR probes are labeled probes used in qPCR to hybridize with target sequences and produce fluorescence to indicate the aligning. It contains a loop structure primer called LUX™ primer. It has a complementary sequence to the target and a fluorescence reporter.
Here the fluorescence emitted by the reporter is quenched by the secondary structure of the hairpin, so it does not need a quencher molecule.
The LUX™ PCR probe mechanism is relatively simple. Here, the LUX™ primer is opened, and the internal sequence is annealed to the target sequence. It causes the hairpin structure to be opened, which modifies the secondary structure of the probe. This modification causes the reporter to fluoresce.
Figure 15. Diagram shows how the LUX™ probe works. The probe binds to the target sequence and the primer is extended. Primer extension modifies the probe, freeing the reporter and allowing it to fluoresce. Note that the hairpin structure of the primer is what quenches the reporter in the first place.
Figure 16. QZyme™ probes consist of a universal oligonucleotide substrate, a target-specific reverse primer and a sequence for a catalytic DNA structure.
The QZyme™ is a labeled probe system used to target a specific sequence in qPCR. This probe uses a deoxyribozyme to cut the link between the reporter and the quencher and allow fluorescence once the probe is hybridized to the target sequence. It contains three components like a universal oligonucleotide substrate (UOS), a target-specific reverse primer (TRP), and a sequence for a catalytic DNA structure (SCD).
Universal oligonucleotide substrate (UOS): A sequence used for the catalytic DNA. It contains the fluorescent reporter and the quencher in close proximity quenching the fluorescence.
Target-specific reverse primer (TRP): A sequence that serves as a template for the catalytic DNA.
Sequence for a catalytic DNA structure (SCD): A sequence that is inactive and works in antisense. The catalytic DNA structure is a deoxyribozyme.
The Sequence for a catalytic DNA structure primer gets extended during the first amplification cycle and its products serve as a template in the second amplification cycle.
Target-specific reverse primer uses the Sequence for a catalytic DNA structure product as a template to be extended and generates a new product that contains the active catalytic DNA region.
Universal oligonucleotide substrate hybridizes to the Target-specific reverse primer product cleaving it in the next annealing step with the help of the DNAzyme.
This cleavage separates the quencher from the reporter, and the free reporter can fluoresce.
Figure 17. The QZyme™ probe binds to the target sequence. The antisense sequence gets extended which activates the universal oligonucleotide substrate. After the probe is cleaved, the reporter is free and fluoresces.
If you want to learn more about probe-based qPCR please visit our GoldBio article "All about probe based qPCR"
PCR probes, qPCR, fluorophores, reporter, quencher, fluorescence.
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