I want to change only one amino acid in my insert DNA for this I chose Quick change site-directed mutagenesis and designed primer for the same. Now I want to know another type of site-directed mutagenesis for the same purpose (change only 1 amino acid). kindly suggest me the name of another type of site-directed mutagenesis and primer designing strategy which is more efficient ?
Hi Monika,
There are lots of commercially available SDM (site-directed mutagenesis) kits out there, so if you're struggling with the QuikChange system they might be worth a try.
However, if you're trying to avoid SDM entirely, it might be a little more difficult. Perhaps the easiest option is to check your insert DNA sequence for single-cutting restriction enzyme sites; if you can find two that are relatively close to your mutation site, you can always order two complementary primers that contain your mutation site and have restriction enzyme cleavage-complementary overhangs; once you anneal these primers, you can just ligate them into your insert DNA like a cloning, without having to do any synthesis. This approach is obviously dependent on suitable RE sites etc., however.
Hope that helps!
Dan
Thank you, Scott, but I need SDM strategy which does not involve restriction site. I need a forward primer is in the region of mutation and a reverse primer is bit upstream from the mutation site. How to design primer for this?
I need to change only one amino acid in my insert DNA.
Maybe I am not getting the problem correctly. Do you want two amino acid changes?
If you cannot get both amino acid changes in one step, you might have to do it twice. Do the SDM with the first primer and confirm the first amino acid change by restriction digestion and sequencing. The second SDM can be done on this mutated plasmid using the second mutagenic primer followed by confirmation as before.
Hope you are also creating restriction sites for screening clones and will eventually sequence the entire gene as SDM PCR can sometimes introduce random mutation.
Hello Monika,
You can amplify the Insert from your plasmid specific primers and then you can design a megaprimer (80mer) which includes mutation region at the centre and Reverse primer upstream to the mutation site.
Including terminal Restriction enzyme sites will be helpful to clone it back the mutated insert, and also you can avoid secondary mutations after sequence is confirmed through your PCR product.
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