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. 2009 Jun 15;37(13):e95. doi: 10.1093/nar/gkp443

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© 2009 The Author(s)

This is an Open Access article distributed under the terms of the Creative Commons Attribution Non-Commercial License (http://creativecommons.org/licenses/by-nc/2.0/uk/) which permits unrestricted non-commercial use, distribution, and reproduction in any medium, provided the original work is properly cited.

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Figure 2. — Flowchart of the Pythia algorithm. Inputs are the genomic sequence, locus coordinates and user-specified parameters. In Step 1, Pythia identifies all primer pairs meeting the user-specified requirements and sorts these primer pairs by the sum of the differences between the computed and target primer melting temperatures. In Step 2, Pythia computes the thermodynamic quality metric for the top ranked candidate. If this candidate meets a user-specified metric threshold, then Pythia proceeds to Step 3. If not, the top ranked candidate is removed from the list and Pythia returns to Step 2. In Step 3, Pythia performs a specificity check. If the primer passes the specificity check, it is given to the user, and the program terminates. If not, the top ranked candidate is removed from the list and Pythia returns to Step 2.