GC content of PCR primers - (Jan/22/2005 )
I am designing primers for PCR and not having great luck. I have to design one in a highly GC rich area with a Tm of ~60. I have successfully found an area where I can get a specific, 17nt forward primer with a Tm of 60, but my GC content is 70% and I was told to keep it lower (45-55%). Does the GC content really matter if my Tm is ideal? My reverse primer is 20nt long with the same Tm. Thanks in advance!
GC content isn't independent of the Tm. Aside from increasing Tms, The reason why they try to stipulate middle values for GC content is to avoid stretches of Gs or Cs (or As and Ts for that matter) that may cause mispriming.
I've had primers that have GC% above 60 and they still work, but a little optimization is required to get the best PCR environment.
I have been forced (by powers greater than little old me) to design primers that had 80% GC. Even IDT thought that they would never work. Well...they did work...badly...but they worked! I will admit that it took some time and some hairpulling to get them to work. A touchdown PCR rxn seemed to give the best results for those primers. In short....there is always hope.
Touchdown PCR is a very successful method for me to get a highly GC rich area with a Tm of ~60 Because I got them (17 fragments ) in two weeks.