It is claimed that the Tm value of Probe should be 10 degree higher than that of Primer, what is the reason of this?
Hello Samira
For a probe based qPCR system, fluorescence signal will generate only when the probe, which is equipped with a reporter and quencher on either ends will bind specifically to the ROI for which the probe is designed. Then, as soon the primers start to anneal at one end of this same probe-bound sequence, they start to amplify the strand through the DNA polymerase. So for a successful fluorescence generation, probe must remain bound with the ROI until the catalyzing DNA polymerase reaches its reporter end and performs hydrolytic cleavage there, which allows the reporter to get release and emit fluorescent signal.
In a qPCR system, the equipment performs ramping behavior ( means gradual increments or decrements in temperature rather than sudden changes), so lets say if the probe Tm is kept below or near equal to the set of primers, then as soon the Ta for the primers will reach the probe low Tm, the probe will automatically disintegrate from the sequence, which is absolutely undesirable, as in this case now the annealed primers of course will amplify the sequence including your ROI, but there will be no fluorescent signal at all as no probe exists anymore, so you can see what the qPCR machine will detect here, almost low-to-nil signal, which by no way represents the true amount of target (ROI) of your sample. Its a failure no doubt.
So overall, the strategy to keep probe Tm in high ranges also confers a sort of safeguarding for your qPCR, as in case of low Tm the probe percentage or chances bound to the target is very low. And vice versa with a probe Tm higher than the primer Ta there is no chance of any early displacement of bound probe by any DNA polymerase activity even at less than 72 C or just after the annealing of primers when the DNA polymerase enters into action of primer elongation.
Hello Samira
For a probe based qPCR system, fluorescence signal will generate only when the probe, which is equipped with a reporter and quencher on either ends will bind specifically to the ROI for which the probe is designed. Then, as soon the primers start to anneal at one end of this same probe-bound sequence, they start to amplify the strand through the DNA polymerase. So for a successful fluorescence generation, probe must remain bound with the ROI until the catalyzing DNA polymerase reaches its reporter end and performs hydrolytic cleavage there, which allows the reporter to get release and emit fluorescent signal.
In a qPCR system, the equipment performs ramping behavior ( means gradual increments or decrements in temperature rather than sudden changes), so lets say if the probe Tm is kept below or near equal to the set of primers, then as soon the Ta for the primers will reach the probe low Tm, the probe will automatically disintegrate from the sequence, which is absolutely undesirable, as in this case now the annealed primers of course will amplify the sequence including your ROI, but there will be no fluorescent signal at all as no probe exists anymore, so you can see what the qPCR machine will detect here, almost low-to-nil signal, which by no way represents the true amount of target (ROI) of your sample. Its a failure no doubt.
So overall, the strategy to keep probe Tm in high ranges also confers a sort of safeguarding for your qPCR, as in case of low Tm the probe percentage or chances bound to the target is very low. And vice versa with a probe Tm higher than the primer Ta there is no chance of any early displacement of bound probe by any DNA polymerase activity even at less than 72 C or just after the annealing of primers when the DNA polymerase enters into action of primer elongation.
Thank you for your full answer :) so the matter is to keep the probe stable on template till the DNA polymerase reaches it. i also thought like it is a matter of stringency, which could be same as what you say.
Thanks again,
Best regards
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