Overlapping sequence PCR primers - (Feb/13/2013 )
a quick question: I have sequence A and sequence B, which i would like to fuse together, so that i get a product A-B (fragment A is aroudn 500bp and fragment B around 1200bp), which i will then clone. I have been told that the easiest thing to do is overlapping sequence PCR. My question is, how many nucleotides of overlap are sufficient for both fragments to be fused? If I design "overlapping" primers of around 20nt of length, can I intrtoduce 5nt sequence on the 5' end of each of these primers, which would be complementary to a sequence of the other region? So overall, the overlap will be 10nucleotides. Will this be enough for the fusion of the two fragments to occu? Or should the overlap region be bigger? I am not really sure how many non-complementary nucleotides i can introduce to the 5'end of a primer without seriously affecting its template annealing capability.
No, 10 bp is not enough. Think of each of the fragments as a "megaprimer" for the other. You need 20-24 bp of overlap, just as you need that much for a good primer.