PCRTiler: automated design of tiled and specific PCR primer pairs

Abstract

Efficiency and specificity of PCR amplification is dependent on several parameters, such as amplicon length, as well as hybridization specificity and melting temperature of primer oligonucleotides. Primer design is thus of critical importance for the success of PCR experiments, but can be a time-consuming and repetitive task, for example when large genomic regions are to be scanned for the presence of a protein of interest by chromatin immunoprecipitation experiments. We present here a webserver that allows the automated design of tiled primer pairs for any number of genomic loci. PCRTiler splits the target DNA sequences into smaller regions, and identifies candidate primers for each sub-region by running the well-known program Primer3 followed by the elimination of primers with a high cross-hybridization potential via BLAST. Tiling density and primer characteristics are specified by the user via a simple and user-friendly interface. The webserver can be accessed at http://pcrtiler.alaingervais.org:8080/PCRTiler. Additionally, users may download a standalone Java-based implementation of this software. Experimental validation of PCRTiler has demonstrated that it produces correct results. We have tiled a region of the human genome, in which 96 of 123 primer pairs worked in the first attempt, and 105 of 123 (85%) could be made to work by optimizing the conditions of the PCR assay.