Please see the picture below.
As shown one base in my primer does not match the template sequence.
Is there a rule of thumb for how many bases should match the template 5' (upstream) of the mismatched base?
Do there have to be any matching bases 5' of the mismatched base?
Dear Neal Edward Beeman
i do not think that there is a rigid rule.
However in mutagenesis is essential that there is an overlap between for and reverse primes in the region that contai the mismatch and i my experience if the overlap is too short is possible that you do not obtain many colonies because the primer annealing region is too short.
Generally i'm considering a total overlapping region of at least 15bp that means that for each primer i'm adding at least 7-8 bp upstream to the mismatch.
good luck
Manuele
Hi Neal,
I agree with Manuele Martinelli - the length of the 5' part depends only on the amount of overlap you want to achieve with your reverse primer.
The 5' end does not have to match at all, this is how you can add restriction enzyme recognition sites to PCR primers and still have them work for amplification. You will need a good 18-20 bp of match on the 3' side.
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