Abstract
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 language | English |
|---|---|
| Pages (from-to) | 4363-4370 |
| Number of pages | 8 |
| Journal | Bioinformatics |
| Volume | 21 |
| Issue number | 24 |
| DOIs | |
| Publication status | Published - Dec 1 2005 |
| Externally published | Yes |
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All Science Journal Classification (ASJC) codes
- Statistics and Probability
- Biochemistry
- Molecular Biology
- Computer Science Applications
- Computational Theory and Mathematics
- Computational Mathematics
Cite this
A novel strategy to design highly specific PCR primers based on the stability and uniqueness of 3′-end subsequences. / Miura, Fumihito; Uematsu, Chihiro; Sakaki, Yoshiyuki; Ito, Takashi.
In: Bioinformatics, Vol. 21, No. 24, 01.12.2005, p. 4363-4370.Research output: Contribution to journal › Article
}
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
PY - 2005/12/1
Y1 - 2005/12/1
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.
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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 -