TY - JOUR
T1 - A novel strategy to design highly specific PCR primers based on the stability and uniqueness of 3′-end subsequences
AU - Miura, Fumihito
AU - Uematsu, Chihiro
AU - Sakaki, Yoshiyuki
AU - Ito, Takashi
N1 - Funding Information:
This work was supported by the Bioinformatics Research and Development (BIRD) project of the Japan Science and Technology Agency (JST), Grants-in-Aid for Scientific Research from the Ministry of Education, Culture, Sports, Science and Technology (MEXT) and the Industrial Science and Technology Program of the New Energy and Industrial Technology Development Organization (NEDO), Japan.
PY - 2005/12
Y1 - 2005/12
N2 - 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.
AB - 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.
UR - http://www.scopus.com/inward/record.url?scp=28944444909&partnerID=8YFLogxK
UR - http://www.scopus.com/inward/citedby.url?scp=28944444909&partnerID=8YFLogxK
U2 - 10.1093/bioinformatics/bti716
DO - 10.1093/bioinformatics/bti716
M3 - Article
C2 - 16234322
AN - SCOPUS:28944444909
VL - 21
SP - 4363
EP - 4370
JO - Bioinformatics
JF - Bioinformatics
SN - 1367-4803
IS - 24
ER -