A novel strategy to design highly specific PCR primers based on the stability and uniqueness of 3'-end subsequences.
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.
Miura
Fumihito
F
Department of Computational Biology, Graduate School of Frontier Sciences, University of Tokyo Kashiwa, Japan.
Uematsu
Chihiro
C
Sakaki
Yoshiyuki
Y
Ito
Takashi
T
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
D017931:DNA Primers; D004271:DNA, Fungal