Primer Tm - How low can you go? - (Jul/08/2005 )
Hi,
I'm designing primers for bisulfite treated DNA, which converts unmethylated cytosines to uracil (read as thymine by taq). So the problem that I am having is that because my GC content is so low, the primers which I have come up with so far all have Tms in the low 40's. I don't believe that using these sets will be acceptable for product amplfication using PCR. So whats the lowest Tm that I should be shooting for? The primers I'm designing are between 21-24 nt long.
-CW
I'm designing primers for bisulfite treated DNA, which converts unmethylated cytosines to uracil (read as thymine by taq). So the problem that I am having is that because my GC content is so low, the primers which I have come up with so far all have Tms in the low 40's. I don't believe that using these sets will be acceptable for product amplfication using PCR. So whats the lowest Tm that I should be shooting for? The primers I'm designing are between 21-24 nt long.
-CW
I've found that I can get away with low Tms by cycling substantailly above the primer Tm but allowing relatively long annealing times (up to 5 minutes). My guess is you can anneal around 50oC for a few minutes. If available, do a PCR in a gradient block thermocycler setting the gradient from just below your primers calculated Tms to as high as the machine will allow. Anneal for 2-5 minutes depending upon your patience or if you're just going to go home.
Question for you: we've looked at a couple of primer design tools and they leave Gs in the primer. If all the template Cs are converted to U then why are Gs in the primer?
Why not simply make the primers longer?
The "rules" about primer design are not absolute. PCR will work just fine with longer primers...
LeserattePD
I'm designing primers for bisulfite treated DNA, which converts unmethylated cytosines to uracil (read as thymine by taq). So the problem that I am having is that because my GC content is so low, the primers which I have come up with so far all have Tms in the low 40's. I don't believe that using these sets will be acceptable for product amplfication using PCR. So whats the lowest Tm that I should be shooting for? The primers I'm designing are between 21-24 nt long.
-CW
You definitely should be using longer primers. You should also use a low extension temperature in your PCR (I use 65). Ideally your primer should contain several modified bases, so that you selectively amplify only one of the two converted strands (they are no longer complementary after bisulfite treatment).