A thermodynamic approach to PCR primer design

Tobias Mann, Rich Humbert, Michael Dorschner, John A. Stamatoyannopoulos and William Stafford Noble

Nucleic Acids Research. 37(13):e95, 2009.

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Abstract

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 PCR efficiency, and a precomputed index on genomic sequences to evaluate primer specificity. We show that Pythia can design primers with success rates comparable to 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 percent as compared to a median coverage of 51% for Primer3. For parameter settings yielding sensitivities of 81%, our method has a recall of 97%, compared to the Primer3 recall of 48%. Because our primer design approach 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.

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Nucleic Acids Research
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