"Ligate" two overlapping pcr products with PCR fusion? - (Apr/23/2007 )

I recently found the term "PCR fusion" and tries to find information about it, and would like to have your opinions about that. Have it worked well for you? Is it better to cut and paste with restriction enzymes? It sounds easy in theory but is it?

How would you have done if you had two fragments at about 1000 bp that you want to join together with this method? I have seven fragments at about 1000 bp that I want to join together, they are overlapping with at least 80 bp, but some of them do not share a good restriction site.

Suggestion from me:
1) first amplify the two products from their plasmids (where they are now) separately with primers 1F and 1R (product 1), and 2F and 2R (product 2). Product 1 and 2 overlaps
2) add the produkts together (about 10-100 ng DNA) and primer 1F and 2R and do the fusion PCR.

I have read that it is good to have a separate step first without the primers for some cycles?(pcrproduct 1 and primer 1F in one vial and pcr-product2 together with primer 2R in another to make single stranded DNA - 10 cycles, and then mix them 1:1 and continue with 15 cycles more) I planned to use Pfu Ultra polymerase.

I have never used primer 1F and 2R together but they have similar Tm. I planned to use an annealing temperature at Tm-5 degrees. Could I take all parameters from the Pfu Ultra protocol directly?

Would be happy for help and hints!

I am slo working on fusion PCR. I fused two fragments 1Kb and 2Kb final product 3kb with 30bp overlap. Now in am trying to fuse two other 1kb fragments.
you have to do PCR in two steps.
1. Determin the Tm for overlaping region.
then run ten PCR cylcles with out adding primers.
2. after 10 PCR cycles add the 1F and 2R primers and then 35 to 40 cycles.(I also add some more Taq at this step).
The Tm for second PCR should be according to the Tm of 1F and 2R.
hope it will work.

wish u best of luck.