PCR on template with trinucleotide repeats - PCR tips for complexe DNA template (Apr/15/2008 )
I'm trying to clone a sequence containing a trinucleotide repeat into a pGL3 vector. The triplet repeats is a chain of 7, 8, 9 or 10 GAG within a 5'UTR sequence. I'm trying to clone the various forms of the repeat by doing this:
1) PCR the 5'UTR (500bp)
2) PCR purification
4) Ligation into pGL3
My problem is that the PCR isn't working that much.
I have enough PCR product for cloning if I'm using a Taq polymerase, but not with a proofreading enzyme such as Pfu. I already tried different primers (they must have a restriction site overhang), I also tried to add some buffer (DMSO/Q-Buffer) to the PCR or to change the annealing temperature . It looks like I have a lot of unspecific products as well... I think that the problem comes from the GAG repeats (when I try to sequence this region, sequence stop at the GAG)
Is there a specific PCR programm that I could use for this kind of PCR?
What I'm doing:
95°C 2 min
94°C 30 sec |
58°C 30 sec | 35x
72°C 1 min |
72°C 10 min
Any other advice?
I do lots of PCRs with trinucleotide repeats. I've used normal Taq as well as a Taq+proofreading mix. I also had lots of unspecific products, but I do PCR on genomic DNA (don't know if that's your case).
You could try a touchdown protocol to decrease the number of unspecific products and increase the expected product (usually works), use less template or less primers (always an idea to keep in mind).
You could also use nested PCR (worked very well for me, goodbye unspecific products).
I would try the Phusion enzyme, and also try lowering the extension temperature (64-66) with corresponding increase in extension time.
Try a Pfu/Taq cocktail. We used to use 1:9 Pfu:Taq. That gave enough proofreading to ensure fidelity, and the robustness of taq to give good yields.
Thanks a lot for your help. I will start by trying touchdown PCR or perhaps nested PCR. Mixed Pfu/Taq is a good idea also.