Automated amplicon design suitable for analysis of DNA variants by melting techniques

Per Olaf Ekstrøm, Sigve Nakken, Morten Johansen, Eivind Hovig

The technological development of DNA analysis has had tremendous development in recent years, and the present deep sequencing techniques present unprecedented opportunities for detailed and high-throughput DNA variant detection. Although DNA sequencing has had an exponential decrease in cost per base pair analyzed, focused and target-specific methods are however still much in use for analysis of DNA variants. With increasing capacity in the analytical procedures, an equal demand in automated amplicon and primer design has emerged. We have constructed a web-based tool that is able to batch design DNA variant assay suitable for analysis by denaturing gel/capillary electrophoresis and high resolution melting. The tool is developed as a computational workflow that implements one of the most widely used primer design tools, followed by validation of primer specificity, as well as calculation and visualization of the melting properties of the resulting amplicon, with or without an artificial high melting domain attached. The tool will be useful for scientists applying DNA melting techniques in analysis of DNA variations. The tool is freely available at http://meltprimer.ous-research.no/ . Herein, we demonstrate a novel tool with respect to covering the whole amplicon design workflow necessary for groups that use melting equilibrium techniques to separate DNA variants.