Background: A large number of PCR primer-design softwares are available online. However, only very few of them can be used for the design of primers to amplify bisulfite-treated DNA templates, necessary to determine genomic DNA methylation profiles. Indeed, the number of studies on bisulfite-treated templates exponentially increases as determining DNA methylation becomes more important in the diagnosis of cancers. Bisulfite-treated DNA is difficult to amplify since undesired PCR products are often amplified due to the increased sequence redundancy after the chemical conversion. In order to increase the efficiency of PCR primer-design, we have developed BiSearch web server, an online primer-design tool for both bisulfite-treated and native DNA templates.
Results: The web tool is composed of a primer-design and an electronic PCR (ePCR) algorithm. The completely reformulated ePCR module detects potential mispriming sites as well as undesired PCR products on both cDNA and native or bisulfite-treated genomic DNA libraries. Due to the new algorithm of the current version, the ePCR module became approximately hundred times faster than the previous one and gave the best performance when compared to other web based tools. This high-speed ePCR analysis made possible the development of the new option of high-throughput primer screening. BiSearch web server can be used for academic researchers at the http://bisearch.enzim.hu site.
Conclusion: BiSearch web server is a useful tool for primer-design for any DNA template and especially for bisulfite-treated genomes. The ePCR tool for fast detection of mispriming sites and alternative PCR products in cDNA libraries and native or bisulfite-treated genomes are the unique features of the new version of BiSearch software.