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Bioinformatics Advance Access published online on March 22, 2007
Bioinformatics, doi:10.1093/bioinformatics/btm091
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© 2007 The Author(s)
This is an Open Access article distributed under the terms of the Creative Commons Attribution Non-Commercial License (http://creativecommons.org/licenses/by-nc/2.0/uk/) which permits unrestricted non-commercial use, distribution, and reproduction in any medium, provided the original work is properly cited.

Enhancements and modifications of primer design program Primer3

Triinu Koressaar and Maido Remm *

Department of Bioinformatics, Institute of Molecular and Cell Biology, University of Tartu, Riia str. 23, Tartu 51010, Estonia; Tel: +372 7375001; Fax: +372 7420286.

*To whom correspondence should be addressed. Maido Remm, E-mail: maido.remm{at}ut.ee


  Abstract

Summary: The determination of annealing temperature is a critical step in PCR design. This parameter is typically derived from the melting temperature of the PCR primers, so for successful PCR work it is important to determine the melting temperature of primer accurately. We introduced several enhancements in the widely-used primer design program Primer3. The improvements include a formula for calculating melting temperature and a salt correction formula. Also, the new version can take into account the effects of divalent cations, which are included in most PCR buffers. Another modification enables using lowercase masked template sequences for primer design.

Availability: Features described in this paper have been implemented into the development code of Primer3 and will be available in future versions (version 1.1 and newer) of Primer3. Also, a modified version is compiled under the name of mPrimer3 which is distributed independently. The web-based version of mPrimer3 is available at http://bioinfo.ebc.ee/mprimer3/ and the binary code is freely downloadable from the URL http://bioinfo.ebc.ee/download/.

Associate Editor: Dr. Limsoon Wong

Received on January 31, 2007; revised on March 5, 2007; accepted on March 5, 2007

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