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Bioinformatics Advance Access published online on October 18, 2005
Bioinformatics, doi:10.1093/bioinformatics/bti716
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© The Author (2005). Published by Oxford University Press. All rights reserved. For Permissions, please email: journals.permissions@oxfordjournals.org
Received July 27, 2005
Revised October 6, 2005
Accepted October 12, 2005

Article

A novel strategy to design highly specific PCR primers based on the stability and uniqueness of 3'-end subsequences

Fumihito Miura 1, Chihiro Uematsu 2, Yoshiyuki Sakaki 3, and Takashi Ito 1*

1 Department of Computational Biology, Graduate School of Frontier Sciences, University of Tokyo, Kashiwa, Japan; Institute for Bioinformatics Research and Development (BIRD), Japan Science and Technology Agency (JST), Tokyo, Japan
2 Central Research Laboratory, Hitachi Ltd., Tokyo, Japan
3 RIKEN Genomic Sciences Center, Yokohama, Japan

* To whom correspondence should be addressed.
Takashi Ito, E-mail: ito{at}k.u-tokyo.ac.jp


  Abstract

Motivation: In contrast with conventional PCR using a pair of specific primers, some applications utilize a single unique primer in combination with a common primer, thereby relying solely on the former for specificity. These applications include rapid amplification of cDNA ends (RACE), adaptor-tagged competitive PCR (ATAC-PCR), PCR-mediated genome walking, and so forth. Since the primers designed by conventional methods often fail to work in these applications, an improved strategy is required, particularly, for a large-scale analysis.

Results: Based on the structure of "off-target" products in the ATAC-PCR, we reasoned that the practical determinant of the specificity of primers may not be the uniqueness of entire sequence but that of the shortest 3'-end subsequence that exceeds a threshold of duplex stability. We termed such a subsequence as a "specificity-determining subsequence" (SDSS) and developed a simple algorithm to predict the performance of the primer: the algorithm identifies the SDSS of each primer and examines its uniqueness in the target genome. The primers designed using this algorithm worked much better than those designed using a conventional method in both ATAC-PCR and 5'-RACE experiments. Thus, the algorithm will be generally useful for improving various PCR-based applications.

Availability: The source code of the program is available upon request from the authors or can be obtained from http://itolab.cb.k.u-tokyo.ac.jp/GATC/.


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