Can qPCR primer with several mismatch works?

Go to the profile of imnani.mbio
May 07, 2017

I am designing primers for qPCR from conserved regions of a few different fish species.This is because gene sequence for my fish species are yet unavailable in NCBI database.

Is it OK (can the primer anneal to the target sequence) if the primers have several mismatch at the interior and towards it 5’ end? At the 3’ end, I can only design primer with maximum 5 conserved bases.

Thank you.


Go to the profile of OldCloner
OldCloner 9 months ago

Well, you are correct that it is important for the 3’ end of the primer to match the template exactly. A few mismatches in the middle might be tolerated, and at the 5’ end you can have tails that don’t match at all- which is the basis of adding restriction sites to amplified fragments for cloning. I’m sorry I can’t define “a few;” this has to be worked out by trial and error. You can also consider “degenerate primers,” which can have a mixture of bases at a particular position. If at position X there could be an A or a T for instance, you use the code “W” in that position when you order the primer, and they will synthesize the oligo with both A’s and T’s at that position (About half will have an A, and the others will have a T). Look up “nucleotide ambiguity code” if you are not familiar with all the possible codes.

If you have no idea what nt is at position X, use the code “N” for any nucleotide. They will put all 4 bases at that position. If your primer design has several degenerate positions, they will synthesize a mixed primer in which all combinations are represented, and some subpopulation of the primer should stick to each template. I’d limit degenerate positions to maybe 2 or 3 if possible, and keep them away from that 3’ end.

To validate, test your primers on a number of control samples (different species) and sequence the products to confirm that you are getting the correct product. Good luck!