PCR fusion issue

[Themos Vasilopoulos]

Hello. I am currently working on creating mutations on a specific site of mitochondrial genome in order to use it for EMSA assay. I have already created a deletion using the well known PCR spicing technique and everything went well. On the first step I created two fragments (right and left of the sequence I wanted to delete) using chimeric primers that were complementary and then in a second step PCR I used the fragments as templates and I amplified them to produce the final product. What I want to do now is to replace this sequence, so I created new primers with flanking regions that are complementary and contain the new sequence. The first step of PCR works fine and it’s exactly the same with what I did to produce the deletion (although now I have a bigger flanking regions) but on the second step I have many issues. I never get the product I want. In fact I get nothing… It is like the hybridization of the flanking regions never happens… The fragments I mix have a long string of Gs (about 27 nt) which is the replacement I want to do (the original sequence that I need to replace was a string of As). I have read many things about the problems that are caused by an increased GC content but I tried many PCRprograms. I have already tried putting the external primers in the beginning or after the first 15 cycles, I have used long denaturation period at about 98oC and so forth…I would be very grateful if someone can help me with an alteration or with a specific strategy to follow. For any information feel free to aks me. Thanks in advance!

[Maxwell Peterson]

I was once stuck for days on a tough PCR, but it worked when we varied the temperature during the annealing stage of PCR - if I remember right, we were getting absolutely no product, increased temp at annealing by like 3 C and suddenly we had all kind of product. Maybe you've already tried this.

Max