Bioinformatics Advance Access originally published online on October 18, 2005
Bioinformatics 2005 21(24):4363-4370; doi:10.1093/bioinformatics/bti716
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A novel strategy to design highly specific PCR primers based on the stability and uniqueness of 3'-end subsequences
1Department of Computational Biology, Graduate School of Frontier Sciences, University of Tokyo Kashiwa, Japan
2Institute for Bioinformatics Research and Development (BIRD), Japan Science and Technology Agency (JST) Tokyo, Japan
3Central Research Laboratory, Hitachi Ltd Tokyo, Japan
4RIKEN Genomic Sciences Center Yokohama, Japan
*To whom correspondence should be addressed.
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/
Supplementary information: Supplementary data are available at Bioinformatics online.
Contact: ito{at}k.u-tokyo.ac.jp
Received on July 27, 2005; revised on October 6, 2005; accepted on October 12, 2005
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