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

Fumihito Miura, Chihiro Uematsu, Yoshiyuki Sakaki, Takashi Ito

Research output: Contribution to journalArticlepeer-review

28 Citations (Scopus)


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 t he 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.

Original languageEnglish
Pages (from-to)4363-4370
Number of pages8
Issue number24
Publication statusPublished - Dec 2005
Externally publishedYes

All Science Journal Classification (ASJC) codes

  • Statistics and Probability
  • Biochemistry
  • Molecular Biology
  • Computer Science Applications
  • Computational Theory and Mathematics
  • Computational Mathematics


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