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Nucleic Acids Research Advance Access published online on March 5, 2009
Nucleic Acids Research, doi:10.1093/nar/gkp073
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© 2009 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.

Computational Biology

PrimerHunter: a primer design tool for PCR-based virus subtype identification

Jorge Duitama1, Dipu Mohan Kumar2, Edward Hemphill3, Mazhar Khan2, Ion I. Mandoiu1,* and Craig E. Nelson3,*

1Department of Computer Science & Engineering, University of Connecticut, Unit 2155, Storrs, CT 06269-2155, 2Department of Pathobiology & Veterinary Science, University of Connecticut, Unit 3089, Storrs, CT 06269-3089, 3Genetics & Genomics Program, Department of Molecular & Cell Biology, University of Connecticut, Unit 2131, Storrs, CT 06269-2131, USA

*To whom correspondence should be addressed. Tel: +1 860 486 3784; Fax: +1 815 301 8557; Email: ion{at}engr.uconn.edu

Received September 19, 2008. Revised January 19, 2009. Accepted January 27, 2009.

Rapid and reliable virus subtype identification is critical for accurate diagnosis of human infections, effective response to epidemic outbreaks and global-scale surveillance of highly pathogenic viral subtypes such as avian influenza H5N1. The polymerase chain reaction (PCR) has become the method of choice for virus subtype identification. However, designing subtype-specific PCR primer pairs is a very challenging task: on one hand, selected primer pairs must result in robust amplification in the presence of a significant degree of sequence heterogeneity within subtypes, on the other, they must discriminate between the subtype of interest and closely related subtypes. In this article, we present a new tool, called PrimerHunter, that can be used to select highly sensitive and specific primers for virus subtyping. Our tool takes as input sets of both target and nontarget sequences. Primers are selected such that they efficiently amplify any one of the target sequences, and none of the nontarget sequences. PrimerHunter ensures the desired amplification properties by using accurate estimates of melting temperature with mismatches, computed based on the nearest neighbor model via an efficient fractional programming algorithm. Validation experiments with three avian influenza HA subtypes confirm that primers selected by PrimerHunter have high sensitivity and specificity for target sequences.

Correspondence may also be addressed to Craig E. Nelson. Tel: +1 860 486 5617; Fax: +1 860 486 1936; Email: craig.nelson{at}uconn.edu


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