I've designed a PCR reaction to amplify a region of the dog genome. One primer binds to a repetitive element in the dog genome. So, in order to gain specificity, it is imperative that my other primer be extremely specific to my region of interest.
Primer design tools, such as NCBI's, create primer pairs. The only way I've been able to test the specificity of my primer is to do an nBLAST. I always get a ton of his to regions that bind my primer non-specificaly - not good.
So I ask, is there a program that I can use to design a single, specific primer? When I use NCBI's tool, I utilize the most stringent parameters. However, when I then run the primer through nBLAST, it comes up with several hits to other locations....