MFEprimer: multiple factor evaluation of the specificity of PCR primers.

Summary: We developed a program named MFEprimer for evaluating the specificity of PCR primers based on multiple factors, including sequence similarity, stability at the 3 � -end of the primer, melting temperature, GC content and number of binding sites between the primer and DNA templates. MFEprimer can help the user to select more suitable primers before running either standard or multiplex PCR reactions. The cDNA and genomic DNA databases of 10 widely used species, as well as user custom databases, were used as DNA templates for analyzing primers specificity. Furthermore, we maintained a Primer3Plus server with a modified Primer3Manager for one-stop primer design and specificity checking. Availability: The web service of MFEprimer is freely available at http://biocompute.bmi.ac.cn/MFEprimer/. A stand-alone version of MFEprimer can be downloaded at http://biocompute.bmi.ac.cn/ MFEprimer/download/. Primer3Plus on our server is located at http:// biocompute.bmi.ac.cn/MFEprimer/primer3plus.cgi. All programs are licensed under the General Public License (Free Software Foundation, 1991).