Hello Rafika Nanda Putri

No, it is not okay. Preferably, the melting temperature (Tm) of the probe should be 6-8 °C higher than that of the primers. If the melting temperature is not as per the specification mentioned above, the percentage of probe bound to the target will be low. So, in your case, the primers may amplify a product, but sensitivity may be compromised as all target sites may not be saturated with the probe resulting in reduced fluorescence signal, which in turn will not provide the true amount of target present in your sample.

I hope this helps.

Best,

thank you Mr Malcolm Nobre

but i have done this experiment, and i just realized it now. Is the PCR result that I got is not the actual result? because the difference Tm is not as it should be? :(

can you recommend me some literature that discusses this? because I can't find some. Thanks before.

Hello Rafika Nanda Putri

Please refer to the link below.

https://sg.idtdna.com/pages/education/decoded/article/the-importance-of-tm-in-molecular-biology-applications

Selecting probe melting temperature

Designing qPCR assays with dual-labeled probes also requires careful coordination of primer Tm. When the reaction temperature is lowered from denaturing to annealing during cycling, the probe needs to anneal first to the target. If the probe binds to the target at the same time or after the primers bind, the polymerase may begin replication of target that does not contain bound probe. As a result, new DNA will be synthesized without associated probe degradation and, therefore, will not be detected as an increase in fluorescence. Such a situation leads to inaccurate data. For standard qPCR, IDT recommends a probe that has a Tm 5–10°C higher than the Tm of the primers.

Best.

Thank you so much Mr. Malcolm Nobre 🙏🏻