Primer design and tolerances - (Jun/26/2008 )
Time for another possibly dumb question from a PCR newbie. I've been trying to help a colleague design RT-PCR primers for a rat gene (Beta actin) for SYBR green and we have been having a hell of a time trying to find a pair that is specific yet free of dimers. We've been at it for a day using Primer3 and OligoAnalyzer from IDT, and still haven't been able to find one. Are there certain requirements for primer design that I could safely relax (i.e. a deltaG of less than -6 for homodimers)? Also, the more I think about it, I wonder, does it matter if the primers are nonspecific as long as they are nonspecific for different genes? I mean, yes, it would decrease the primer concentration in the reaction if they were binding nonspecifically, but as long as they aren't on the same gene, the nonspecific product wouldn't be amplified exponentially like the target gene, so wouldn't the nonspecific products cause a negligible effect in results? Or am I just going to have to bite the bullet and go with a probe-based reaction?
I would recommend a commercially available design software for RT primers (go for Applied Biosystems or Roche Applied Science)...
A couple of points.
1. Primer dimers are only really a problem if you have 3' end binding. Unless the 3' end of one primer binds to the other primer or to itself, you won't get extension; in any case the temperature during the annealing stage of the reaction will probably be too high to allow primer dimerisation, unless most of the primer is involved. Look at the regions that lead to the dimerisation alert in your software: unless you have, say, 10 nt continuous binding, it'll probably not be stable. Take-home message: try it and see.
2. Non-specific binding. Unless it is 100% complementary, you shouldn't have too much of a problem, especially if you do a touchdown PCR. Touchdown PCR starts with an annealing temp that is too high for anything to bind, then drops by 1 degree every two cycles until you get to 8-10 degrees below the Tms of the primers; after that, you do 'n' cycles to a total of 30-35 cycles at that lower temperature. Take-home message: try it and see.
remember this: PCR is really a fairly straightforward reaction, so don't sweat it too hard. Nowadays, primers are really pretty cheap to manufacture, so even if you make a mistake or two, the total cost won't be too much.
I second Swanny's points, the best thing is to try them. On the other hand, you should be able to find plenty of primers for beta-actin in the literature.
Regarding the software, I'm pretty happy with primer3, and it's free!!