Question: How do I find cross-reactive species given two primers?
gravatar for andytsaica
5.3 years ago by
United States
andytsaica0 wrote:

I have developed my own algorithm to find two 15-nt long primers for a specific gene. I now want to search NCBI primer-blast to see what organisms those two primers that I want to use will cross-react with. What search parameters do I use on primer-blast in order to do this?

primer blast bioinformatics • 2.5k views
ADD COMMENTlink modified 5.2 years ago by Biostar ♦♦ 20 • written 5.3 years ago by andytsaica0

What do you mean by cross-react? Do you mean amplify more than 1 unique region of the genome?
Typically cross-react is something you describe antibodies as doing, where as primers are just said to be non-specific. This is a kind of important distinction, because antibodies come into this world by biological means, to bind specifically to a given antigen.
Primers however are man-made with the intention of being specific, but even with the best will in the world we have to assume that they are not, to a degree. With the right salt concentration and the right temperature, you can push equilibrium in your favour, but its an important distinction to make in your head, especially if you are writing primer-designing software, because there are so so so many factors involved in designing good primers that no one can say 'this primer is specific' without actually testing it.

tl,dr: Primers are not born perfect but then naughtily cross-react. They want to bind to everything, and its your job to give them a soul-mate. Without a soulmate, and given enough rejection cycles, they will settle for lesser sequences.

Whatever - primer-blast finds primers for you - it is not for checking primers you already have. For that you'll want UCSC's "insilico pcr" which, 2 years ago, allowed you to specify the primers in the URL's GET request (making it really easy to automate in your tool). Since the biggest update to UCSC in my lifetime was a change of background, this is probably still the case.

ADD REPLYlink written 5.3 years ago by John12k
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