Amplifying plasmid - non specific binding of primer
Posted 15 March 2009 - 06:03 PM
I think my primer prefers to bind to the 9bp homologous region rather than the 11bp region I designed it to bind to... so I should have spotted the 9bp region before I ordered the primers, but I did not. However, since I got a 1kb band PCR product but was expecting a 3kb band product, I went back to investigate if there were other binding sites, and I found the 9bp one.
I know that primers should generally be >20bp, but as I had trouble with high Tm's (74.c) and as this was
amplification from a plasmid rather than genomic, I thought I could get away with a shorter primer.
So I designed the primer to be homologous to 11bp of the plasmid. But now when I do an alignment I see that it is also homologous to a 9bp region somewhere else on the plasmid. If it is binding here in preference to the 11bp region that I designed it for then I would get a 1kb pcr product out, which I do.
So my question is this... why might it be preferentially binding to the 9bp region over the 11bp region. I calculated the Tm or the 9bp region to be 43.c and the Tm of the 11bp region to be 51.c (using finnzymes Tm calculator). So shouldn't it bind preferentially to the 11bp region over the 9bp region? I'm annealing at 62.2 which is 3.c above my primer Tm of 59.2) so at this annealing temp, I'd expect the reaction to favor the longer 11bp strand over the shorter 9bp's. (higher reaction temperature means more energy, means tighter binding is more favorable, means longer strand has tighter binding as more bp available to bind, am I correct?)
I thought of increasing the annealing temperature, to increase the specificity, but I don't think that I have a problem with specificity, as I only get 1 band from the reaction, so changing the annealing temp shouldn't matter I think. My next option is just to redesign the primers, try and make them as short as possible to keep the Tm's down, but at the same time try and make them as long as possible to keep binding specificity high.
But I still don't see why it would prefer the 9bp over the 11bp...
Thanks in advance,
Posted 15 March 2009 - 09:22 PM
Posted 15 March 2009 - 10:15 PM
Sometimes you have no control on where your primer binds. Can you redesign your primer to let the 3' end of your primer to extend beyond the homologous region? Have you tried two step PCR with a 94C denaturing step and 68C-72C annealing and extension?
Sorry, but I don't see how I can extend the 3' end of the primer beyond the homologous region, doesn't the 3' end of the primer have to be homologous in order for it to bind at all?
I will try a two-step gradient PCR from 68-72.c tomorrow as you suggest.
So you said that I have no control over where my primers bind, but in general, am I correct in saying that in theory the higher Tm primer should bind preferentially? i.e a higher reaction temperature means more energy, which means a tighter binding is more favorable, which means longer strands have tighter binding as they have more bp available to bind with?
Posted 16 March 2009 - 07:10 AM
The longer the primer, the more chances that it actually has to misprime as well, so it's a two way street and you're talking about 2 nucleotides as well and more often then not, these effects are sequence and context specific.
Normally, you are right, and this sounds like one of those cases where normally doesn't work.
As far as complexity, your template is a plasmid, so Tm is less important in this case as you're searching for a discrepancy of 2nt in a single template versus hundreds of thousands of templates. Again, without the sequence, the 9nt homology may be more energetically favorable than the 11 and you may be forced to do some sort of nested PCR with the first being just a bit outside of your area of interest and then using that reaction as a template for the one you are wanting to do.