primer tm with mismatch? - (Feb/27/2008 )
Does anyone know about some software that calculates primer Tm between sequences with mismatches?
I believe this amplification of mine for a target gene may also be amplifying another very similar gene.
The forward primer anneals almost completely with the other sequence (18 out of 20bp) and the reverse primer anneals only partially (11 out of 20bp). It's what you get when you don't BLAST your primer sequences before ordering them...
Both primers have a Tm of 60ºC, so I've been using 58ºC for the amplification. Do you think this is enough for the misannealing to occur? I think most probably not, but I've been having some weird results so I really want to have this sorted out.
Thanks in advance.
EDIT: I know I could clone the products and sequence them, but I'd like to avoid that if I could...
If there is another very similar gene where your primers can anneal, then they WILL anneal. And if this other gene is smaller than the one you want to amplify, then this will cause more problems, because PCR will give preference to the smaller product. Do you know the expected size of your product and the size of the other gene? If their sizes are different you can gel-extract the appropriate band. If not, then you really need to clone them or order new primers.
It's critical where the mismatches are located on the primer. 3' mismatches almost always fail to prime, while 5' mismatches are almost a don't care. A single base mismatch at the very 3' end of a primer is usually sufficient to prevent priming.
Luckuly enough there are matches at both ends of the primer, 3' and 5'...
The differences in amplification would be around 60bp... I'll try to run them properly on a gel and see if there's a possibility that 2 bands are there...
9 out of 20 is a big mismatch. trust me, it is best if you order your primers again instead of trying to interpret the strange results you are getting now, after all those could be only artifacts. it is also cheaper than sequencing.
As Phage said, it is the 3` end of your primers that are the most important. You want as many unique bases there as possible - the more the better. If you can, design your primers in a different region so that the primer sequence and the 3` end of your primers is more unique. For example, try a nested PCR and then isolate your sequence from that. Higher Tm (~70C) may also help increase specificity by allowing a higher annealing temperature but in your case the 3` end is the most important. Another reason for why the wrong sequence can be isolated is the abundance of the wrong sequence vs target. If you're trying to isolate your sequence from brain cDNA and a sequence similar to your target is 100 times more abundant then it will be more likely to amplify because there is more starting material. In this instance i might try a different cDNA in the hope that my target is more abundant in the new cDNA.
Thanks for your answers you guys.
I guess the best thing to do is designing new primers (and making sure they're unique before ordering )