While designing the mutagenic primers for substitution, my final primer sequences have two mismatches with the template for the necessary mutation to be present in the final sequence. For e.g., For a serine to cysteine mutation (TCA-->TGC), I have to change two bases in one of the primer sequences. Will this drastically affect the annealing efficiency of the primer to the template? I will be using Pfu poly for the mutagenesis steps.
Any suggestions will be helpful.
Thanks
Check this: https://pubs.acs.org/doi/10.1021/jacs.0c02243?goto=supporting-info
In this paper, they have mutated 3nt at the same time. In Supplementary Table 6 you can see the primer design and in page S3 you have the protocol followed to create the mutations in the coding sequence.
Good luck.
If possible, make the change at the 5' end of the primer. As long as the rest of the primer anneals well, you can add in as many novel bases as you like on the 5' end. That's the basis of adding barcoding tags or universal cloning sites, etc to a PCR product.
If you have trouble coming up with primers, there is also a web server by Agilent. You paste in the sequence, select whether you want to mutate or delete any residues, tell it which mutations you want to introduce on which residues, and it spits out the primer sequences for you to copy and order from whatever supplier is available to you.
https://www.agilent.com/store/primerDesignProgram.jsp
Hope this helps.
I'm also working on site-direct mutagenesis, and I'm following the primer-design and mutagenesis method of this article: Article An efficient one-step site-directed deletion, insertion, sin...
Two mismatches should be fine if the rest of the primer anneals well with the template as Burnette mentioned. You can try to make the primers with C or G at their 3' end. Hope this help and good luck!
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