taq man probes non specific? (View forum version)


Posted 16 September 2010 - 11:52 AM

Hi All

I have doing quite a few experiments examining expressing of a novel gene in a specific tissue type and I am getting postive readings from my taq man probes. I was just wondering what are the chances of the taq man probes being a false positive or more importantly has anybody ever experience getting a false positive with the taq man probes. I have also ran the resulting product on a gel and i get an expected band however i also get another unexpected band which is of a lesser intensity so that has just made me start to wonder.



Posted 17 September 2010 - 02:53 AM

Specifity of the probe (and primers) depends on its sequence. You can check the probe (and primer) sequence in BLAST. Usually you would need both the primers and the probe to be binded to the same unspecific sequence to get a false positive read. What more, the probes has a higher melting temperature, so it's more unlikely to bind unspecifically. So when you design your primers and probe well, it's very improbable that you get false positives. But there is no real way to check it, unless you have a template in which you're sure your target gene is not expressed.

If you have multiple products it means your primers are not specific or form primer-dimers (if the sequence is very small, like < 100bp, it's more likely it is a dimer). That doesn't mean the probe binds to that. So you only get fluorescent reads from specific sequence.

Anyway it's usually not so good to have unspecifities or dimers in your real-time reactions, you should increase the annealing temperature or redesign the primers.