title: A novel strategy to design highly specific PCR primers based on the stability and uniqueness of 3'-end subsequences.
abstract: BACKGROUND
  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.
authors.affiliation: Department of Computational Biology, Graduate School of Frontier Sciences, University of Tokyo Kashiwa, Japan.
authors.forename: Fumihito
authors.initials: F
authors.lastname: Miura
authors.forename: Chihiro
authors.initials: C
authors.lastname: Uematsu
authors.forename: Yoshiyuki
authors.initials: Y
authors.lastname: Sakaki
authors.forename: Takashi
authors.initials: T
authors.lastname: Ito
mesh_terms: D000465:Algorithms; D001483:Base Sequence; D019295:Computational Biology; D017931:DNA Primers / Q000235:genetics*; D004271:DNA, Fungal / Q000235:genetics; D015195:Drug Design; D016133:Polymerase Chain Reaction / Q000379:methods* / Q000706:statistics & numerical data; D012441:Saccharomyces cerevisiae / Q000235:genetics
chemical_list: D017931:DNA Primers; D004271:DNA, Fungal