Single Codon Deletion - SOE PCR - (Jan/23/2007 )

I am trying to delete a single codon from a 1.7kb piece of cDNA. I have successfully used SOE PCR to delete up to 40 amino acids in the same cDNA, but I am having trouble with the single amino acid.

My primers flank the codon with ~10 nucleotides on each side. The initial reaction to create hybrid primers works well, but the hybrid primers seem to have trouble annealing to each other, so the mutated cDNA without the codon is not formed. I have tried various reagent conditions and thermal cycling settings, but to no avail.

Does anyone have any other ideas regarding the deletion of a single codon from cDNA? I not exactly an expert on these matters, so I thought there might be an easier approach?

Thanks

Two sugestions. Firstly, add more nucleotides onto the 5` side of the primers so that a great overlap will exist. Because your initial PCRs work well, there is no need to add bases to the 3` end, and due to the nature of this technique adding bases to the 3` end of the primers will not create a great overlap. Secondly, try using as much of each half in the splice-overlap PCR as possible, and use as concentrated solution as possible. Sometimes I find that the more DNA I use the better the yield of the final product I get. Generally, I find that some SOE PCRs work better than others for reasons I don't know. For that reason, I think the first suggestion may be the best option.

On an aside, are you sure you want to delete the codon of interest? Are you sure you wouldn't be better off just replacing the codon with a similar amino acid to avoid any major structural change a deletion may cause? That's usually what is done. I may be out of line but just in case you didn't know about this idea it's something that may make your experiments more relevant.

Thanks for the suggestions killerkoz17. I will design new primers with longer 5' extensions and try again.

As for your second post, I am trying to mimick, in-vitro, a human mutation which codes for a single AA deletion (i.e. I want the major structural change that the deletion causes). I don't think that studying a protein with a conservative AA mutation would have the same effect.

Thanks again! I'll let you know how it turns out.