When using molecular beacon probes in realtime PCR, why the Taq polymerase won’t cleave the beacon probes like what happens in traditional realtime PCR using Taqman probes?
This question is as old as beacons and Taqman probes themselves. When the polymerase arrives at the site where either kind of probe is bound, one of the two things will happen, the probe will get displaced, or after displacing the probe for a few nucleotides, a cleavege event will occur in the probe. Both beacon and Taqman probes are displaced in some of the passages and cleaved in some others. The probability of cleavage is lower for beacons because they have an alternative thermodynamically stable state, the hairpin. They are also designed to be bound during the annealing stage (lower probe-target hybrid Tm). As a part of probe bound to the target is ploughed off by the polymerase, the arms of the beacon snap back forming the hairpin. You can do a successful real-time PCR with beacons using a polymerase that is deficient in the endonucleolytic activity or using a beacon made from an uncleavable backbone. Also, Taqman probes are more fluorescent when bound to the target then they are free in solution.
They are not expected to be cleaved. When hybridization occurs, the quencher and the fluorophore are separated apart and we can detect fluorescence. No need for cleavage.
Hi Eric,
Most likely, the probe is designed in such a way that the flourophore is quenched (often due to proximity of the probe and the quencher at different ends of the probe). As Taq comes through, it chews up the nucleosides and places new bases behind it in the polymerase reaction. During this the flourophore is released into the solution where it is no longer quenched (often because it is no longer in proximity to the quencher).
There are other ways of quenching (eg. minor groove binding) but in each of these cases the action of Taq releases the flourophore to fluoresce and allows detection.
-Matt
Thanks Iker and Matt.
I understand how molecular beacon and Taqman probe work. But just wondering why the Taq polymerase won't cleave the molecular beacon probes during extension considering its 3'-5' exonuclease activity.
Ordinary molecular beacon probe cleaves during in realtime PCR. and acts as TaqMan.
There are no refenences Eric. Its obvious. If you will run PCR products in the proper gel, you will see probe degradationg products because there is no principal difference in structure between beacon and TaqMan.
According to the information from Thermofisher (https://www.thermofisher.com/us/en/home/references/ambion-tech-support/rtpcr-analysis/tech-notes/real-time-pcr-goes-prime-time.html), " Unlike TaqMan probes, molecular beacons are designed to remain intact during the amplification reaction, and must rebind to target in every cycle for signal measurement. "
I suspect the reason that it won't be digested by Taq polymerase is because the short stretches of DNA at the two ends of the molecular beacon, which used to form the stem when in solution, do not hybridize to the PCR product.
With this being said, I still don't understand how dual hybridization probes, such as the one in the following paper, won't be digested by Taq polymerase during PCR process:
https://www.nature.com/articles/6604925.pdf
Or maybe the sensor probe is digested but won't matter since it is in excess and the digested one won't give any signal anyway?
The simple answer based on several resources is "molecular beacons are displaced but not destroyed during amplification because a DNA polymerase lacking 5' exonuclease activity is used."
http://www.bio-rad.com/en-us/applications-technologies/introduction-pcr-primer-probe-chemistries?ID=LUSOJW3Q3
https://www.idtdna.com/pages/products/qpcr-and-pcr/custom-probes/molecular-beacons
This might not be true as I found several papers use Taq polymerase, which has 5' exonuclease activity.
i.e., this one use Platinum Taq DNA polymerase
http://www.archivesofpathology.org/doi/pdf/10.1043/1543-2165%282003%291272.0.CO%3B2
This question is as old as beacons and Taqman probes themselves. When the polymerase arrives at the site where either kind of probe is bound, one of the two things will happen, the probe will get displaced, or after displacing the probe for a few nucleotides, a cleavege event will occur in the probe. Both beacon and Taqman probes are displaced in some of the passages and cleaved in some others. The probability of cleavage is lower for beacons because they have an alternative thermodynamically stable state, the hairpin. They are also designed to be bound during the annealing stage (lower probe-target hybrid Tm). As a part of probe bound to the target is ploughed off by the polymerase, the arms of the beacon snap back forming the hairpin. You can do a successful real-time PCR with beacons using a polymerase that is deficient in the endonucleolytic activity or using a beacon made from an uncleavable backbone. Also, Taqman probes are more fluorescent when bound to the target then they are free in solution.
The phase or step in which the fluorescence is measured is different in each case.
With Taq-Man probe the fluorescence is measured during extension.
With Hairpin probe and dual hybridization probe the fluorescence is measured during annealing step. During the extension step these probe are no longer bound to the target.
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