Hello,

I would suggest a TM of 60°C for the primers and about 10°C higher for the probe. For designing primers and probes I would recommend this page:

http://bioinfo.ut.ee/primer3-0.4.0/primer3/

This page is really useful. Usually I only change the TM values and leave the rest as it is. Then I use BLAST to check the specificity of the primers.

Best wishes

Dirk

Dirk, thanks for your response. In a one-tube nested realtime PCR, there are two sets of primers (outer and inner) and one probe. I am asking which set primer's TM should be higher than the other set or it does not matter.

Sorry Zhenyu, I missed the word "nested". I guess that the probe should bind within your two inside primers. Maybe you can start with a lower TM for the first primer set for 20 cycles and then raise the TM for the second primer set and the probe for the next 25 cycles or so.

Thank you very much! This is also my thought.

I would suggest the reverse. Start with a high annealing temperature and high Tm for the outer primers, then drop the annealing temperature to allow the low-Tm inner primers and the probe to bind.

I would also recommend limiting the outer primer concentration so that they get used up before you drop the temperature and start the inner PCR. If not, then the second stage of the PCR will have four possible primer pairs (outer-outer; outer-inner; inner-outer; inner-inner) and this will reduce the specificity of the test. However, I'm not sure that it is possible to deplete a realistic amount of primers within 25 cycles unless your sample is very hot - in which case you wouldn't need to do nested PCR.

My own take on this is: don't do nested PCR unless you absolutely need to. It is entirely possible to achieve single-molecule sensitivity with one well-designed and well-placed primer pair, and there are lots of easily available programs to help you design your primers (e.g. Primer3). Nested PCR (potentially) increases the sensitivity at the cost of a complex and unpredictable loss of specificity and the loss of all quantitative information. I think that's a bad deal.