Hi,
I am willing to mutate 5 specific consecutive amino acids to Alanine in a certain insert using site-directed mutagenesis in one PCR reaction only. I'm scanning a certain area of my protein to see wether it is involved in an interaction and I want to go 5 mutations, if not another 5 etc... Did anyone try it before and worked or are there better alternatives? I've read several publications but they're mostly for high throughput scanning and random mutations.
Thank you
I don't think there's a way to mutate 5 consecutive amino acids in one PCR reaction. The main reason being primers. You intend to mutate 5 consecutive amino acids, which means 15 consecutive nucleotides. The minimum requirement for good primer annealing is 20-22 nucleotides with more than 90% complementarity. In your case, if you want to introduce substitution mutations in 15 nucleotides, you decrease the chances of primer annealing at the site of your interest because the primers would not be entirely complementary. Also, you might increase the chances of non specific primer annealing at multiple sites in your plasmid resulting in non specific amplification.
There are kits that are available from NEB and Stratagene that allow multiple mutations at multiple sites but far from one another, but not next to each other. You can check their websites for further information on these kits.
The more reliable and sure shot method of SDM is sequential mutations at a single site. First round, mutate one amino acid, then mutate the second one on the already mutated clone and so on till you mutate all the five.
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