I want to mutate serine 409 and 410 into aspartic acid. For that two bases have to be changed for converting one serine into aspartic acid. So in my case four bases will be changed and between two unchanged bases one will be in the middle of the changed bases and one will be at the end of the changed bases. Is it possible to do these mutations with one primer or do I have to do it one by one with two different sets of primers???
You can only test it in an experiment, as success depends on primer selectivity to a particular target. One thing you should keep in mind that closer to 3' end of a primer, specificity of base matching is more important. While changes near 3' end may lead to fail of annealing, primer region near 5' can vary in some limits. So, for your goal you should design and test a set of primers varying distance between 3` end and your mutation sites, from the primer's middle to its 5' end.
Hi there,
four base changes via site directed mutagenesis (SDM) approach should be no problem. Personally, i allready mutated six bases within on mutagenesis PCR. For example, this is the forward primer i have used, where the lower letters represent bases changed.
5`-GCAATTGCAGgcATTGAAAgcGCAATTCATTAACTTCAACgcACTAAGAG-3`
Further i also mutated neighboring residues, similar to your goal, without significant problems. Again, my primer as an example:
5`-GCGCAATTGCAGgcATTGgcAgcGCAATTCATT-3´
As allready noted by Shamsunnahar, the SDM approach allows you to do this quite easily. If you are not familiar with the approach: You have two mutagenesis primer covering the region of interest. To avoid primer-primer annealing during the initial cycles of PCR, forward and reverse Primer are run seperately and joined together after a few cycles. Then you continue the PCR and digest the maternal, methylated DNA (i guess it´s plasmid based) via DpnI.
As a protocol i can recommend:
Edelheit O., Hanukoglu A., Hanukoglu I. 2009. Simple and efficient site-directed mutagenesis using two single-primer reactions in parallel to generate mutants for protein structure-function studies. BMC Biotech. 9: 61. https://doi.org/10.1186/1472-6750-9-61
If you run into trouble, i´d recommend playing with annealing temperature as well as the number of initial PCR cycles.
Best,
David
If the two mutations are so close You can certainly try to do it with a single primer set.
Give a look to the attached papers.
Ciao
Manuele
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