Nucleic Acids Research Advance Access first published online on June 15, 2009
This version published online on June 23, 2009
Nucleic Acids Research, doi:10.1093/nar/gkp443
| ||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
Methods online |
A thermodynamic approach to PCR primer design
Department of Genome Sciences, University of Washington, Seattle, WA, USA
*To whom correspondence should be addressed. Tel: (206) 543 8930; Email: william-noble{at}u.washington.edu Correspondence may also be addressed to John Stamatoyannopoulos. Tel: (206) 267 1098; Email: jstam{at}u.washington.edu
Received February 23, 2009. Revised May 11, 2009. Accepted May 11, 2009.
We developed a primer design method, Pythia, in which state of the art DNA binding affinity computations are directly integrated into the primer design process. We use chemical reaction equilibrium analysis to integrate multiple binding energy calculations into a conservative measure of polymerase chain reaction (PCR) efficiency, and a precomputed index on genomic sequences to evaluate primer specificity. We show that Pythia can design primers with success rates comparable with those of current methods, but yields much higher coverage in difficult genomic regions. For example, in RepeatMasked sequences in the human genome, Pythia achieved a median coverage of 89% as compared with a median coverage of 51% for Primer3. For parameter settings yielding sensitivities of 81%, our method has a recall of 97%, compared with the Primer3 recall of 48%. Because our primer design approach is based on the chemistry of DNA interactions, it has fewer and more physically meaningful parameters than current methods, and is therefore easier to adjust to specific experimental requirements. Our software is freely available at http://pythia.sourceforge.net.