qPCR primer design - possible off-target (Apr/30/2011 )

Hi all,

I'm designing primers for qPCR.
I've tried to check the specificity of the primers by blasting them. However, one of the potential off-targets given by Primer-BLAST is:

product length = 196
Forward primer 1 AAGCAGAAAACCAGCAGCTC 20
Template 3134 C..G..C.C.G......... 3153

Forward primer 1 AAGCAGAAAACCAGCAGCTC 20
Template 3329 C......C.C.AG....... 3310

Anyone how would like to share their thoughts on this potential "forward-forward" problem.

Kind regards

Dr_D on Sat Apr 30 14:23:30 2011 said:

It is essentially telling you that your forward primer could potentially anneal to 2 sites on the same off-target gene: one site from 3134 to 3153 and the other from 3329 to 3310. There are 4 mismatches in each site and if you choose your annealing temperature properly, it should not cause you any problems.