question about mutagenic PCR. - (Jan/27/2006 )

Howdy,

I got a question about mutagnenic PCR. I want to make a point mutation (Leucine to Glutamic acid) in a protein, which requires three-bases codon on DNA to be replaced by a codon that has totally different bases. For this reason, I have designed a 50-bp primer that contains three tandem mismatched bases. I was wondering if such kind of primer is going to work or not. Is three-base replacement possible?

thanks

I don't think it even needs to be 50bp long... I think it would work well with a smaller primer (about 25bp) just tilt it so most of the matching bases are 3' to the mutation say 10bp 3' of mutations and I think you could put even less... In my site directed mutagenesis, made one primer with only 2bp match after a 2bp mutation--did not work-- made one with 3bp match after 2bp mutation--worked great!!

It is most important to match the primer at the 3' end as this is what will be extended with the polymerase (5'-3' processivity so 3' end is where bases are added)

I would try should work as long as there isn't any crazy secondary structure or alot of inappropriate targets which are more likely with such a long primer... (you blasted to be sure it is fairly unique right?)

good luck...