I'm wondering if anyone is aware of published literature that describes the PCR cloning technique I've just used. I'm aware of this paper, which is very similar but introduces a DNA fragment as an insertion without replacing an existing sequence in the plasmid: Article Overlap extension PCR cloning: A simple and reliable way to ...

I have an 11 Kb plasmid containing my 5.2 Kb GOI. I need to introduce 17 mutations in the C-terminal in the last 1.75 Kb region of coding sequence. By standard SDM procedures this would be laborious. So, I had a ds-DNA fragment synthesised containing 17 mutations flanked by restriction sites. I silently introduced RE sites into my WT GOI plasmid by SDM to insert the fragment by standard RE cloning but this failed. I tried a PCR approach that seemed to theoretically make sense in my head but I couldn't find any literature accurately describing the method.

I essentially used my 1.75 Kb DNA fragment with 17 mutations as a PCR primer in a 2-step PCR reaction with my WT GOI plasmid as template. The idea was that after melting, during annealing some products would form that would contain one strand of my plasmid containing WT GOI annealed to the 1.75 Kb DNA fragment that has complete homology across its length to the WT coding sequence except at the 17 mutation sites. Polymerase would extend from the 3' end all the way around the plasmid to meet the other 5' end of the fragment. This would lead to an 11 Kb product of a plasmid now containing my GOI with 17 mutations, which can be used as template in further PCR cycles. I dpn1 treat to remove parental plasmid and ligase to seal nicks then transform. I am awaiting sequencing results, but analysis of my PCR reaction on a gel shows an 11 Kb fragment that is not visible in the control reaction absent of the 1.75 Kb fragment.

This was a shot in the dark and I didn't expect you could use such a large DNA fragment as a megaprimer to completely replace an existing sequence with high homology.