Designing Primers for Whole Genome PCR Scanning Using the Software Package GenoFrag: A Software Package for the Design of Primers Dedicated to Whole-G

Whole-genome polymerase chain reaction (PCR) scanning (WGPS) is based on the PCR amplification of small-sized chromosomes (e.g., bacterial chromosomes) by long-range PCR with a set of primers designed using a reference strain and applied to amplify several other strains. Such an approach of genome variability has specific requirements for the selection of primers and the design of primer pairs for the optimal coverage of the chromosome. To facilitate such analysis, we have developed GenoFrag, a software package for the design of primers optimized for whole-genome scanning by long-range PCR. GenoFrag works in a two-step procedure: first, a list of primers is selected according to the basic criteria, and second, the list of primer candidates is used for the coverage of the whole chromosome. These two steps are presented here with a part of the algorithm scripts developed for this software. Examples of what can be done using GenoFrag are illustrated by results obtained from the online version of the software. GenoFrag has already been validated in long-range (LR)-PCR experiment on several bacterial species. It is a robust and reliable tool for primer design for WGPS.