{"status":"ok","message-type":"work","message-version":"1.0.0","message":{"indexed":{"date-parts":[[2026,6,2]],"date-time":"2026-06-02T23:30:11Z","timestamp":1780443011022,"version":"3.54.1"},"reference-count":23,"publisher":"Oxford University Press (OUP)","issue":"24","content-domain":{"domain":[],"crossmark-restriction":false},"short-container-title":[],"published-print":{"date-parts":[[2005,12,15]]},"abstract":"<jats:title>Abstract<\/jats:title>\n               <jats:p>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.<\/jats:p>\n               <jats:p>Results: Based on the structure of \u2018off-target\u2019 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\u2032-end subsequence that exceeds a threshold of duplex stability. We termed such a subsequence as a \u2018specificity-determining subsequence\u2019 (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\u2032-RACE experiments. Thus, the algorithm will be generally useful for improving various PCR-based applications.<\/jats:p>\n               <jats:p>Availability: The source code of the program is available upon request from the authors or can be obtained from<\/jats:p>\n               <jats:p>Supplementary information: Supplementary data are available at Bioinformatics online.<\/jats:p>\n               <jats:p>Contact: \u00a0ito@k.u-tokyo.ac.jp<\/jats:p>","DOI":"10.1093\/bioinformatics\/bti716","type":"journal-article","created":{"date-parts":[[2005,10,19]],"date-time":"2005-10-19T03:38:37Z","timestamp":1129693117000},"page":"4363-4370","source":"Crossref","is-referenced-by-count":36,"title":["A novel strategy to design highly specific PCR primers based on the stability and uniqueness of 3\u2032-end subsequences"],"prefix":"10.1093","volume":"21","author":[{"given":"Fumihito","family":"Miura","sequence":"first","affiliation":[{"name":"Department of Computational Biology, Graduate School of Frontier Sciences, University of Tokyo 1 \u00a0 1 \u00a0 \u00a0 Kashiwa, Japan"},{"name":"Institute for Bioinformatics Research and Development (BIRD), Japan Science and Technology Agency (JST) 2 \u00a0 2 \u00a0 \u00a0 Tokyo, Japan"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Chihiro","family":"Uematsu","sequence":"additional","affiliation":[{"name":"Central Research Laboratory, Hitachi Ltd 3 \u00a0 3 \u00a0 \u00a0 Tokyo, Japan"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Yoshiyuki","family":"Sakaki","sequence":"additional","affiliation":[{"name":"RIKEN Genomic Sciences Center 4 \u00a0 4 \u00a0 \u00a0 Yokohama, Japan"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Takashi","family":"Ito","sequence":"additional","affiliation":[{"name":"Department of Computational Biology, Graduate School of Frontier Sciences, University of Tokyo 1 \u00a0 1 \u00a0 \u00a0 Kashiwa, Japan"},{"name":"Institute for Bioinformatics Research and Development (BIRD), Japan Science and Technology Agency (JST) 2 \u00a0 2 \u00a0 \u00a0 Tokyo, Japan"}],"role":[{"vocabulary":"crossref","role":"author"}]}],"member":"286","published-online":{"date-parts":[[2005,10,18]]},"reference":[{"key":"2023061007215427300_b1","doi-asserted-by":"crossref","first-page":"10581","DOI":"10.1021\/bi962590c","article-title":"Thermodynamics and NMR of internal G\/T mismatches in DNA","volume":"36","author":"Allawi","year":"1997","journal-title":"Biochemistry"},{"key":"2023061007215427300_b2","doi-asserted-by":"crossref","first-page":"2170","DOI":"10.1021\/bi9724873","article-title":"Nearest neighbor thermodynamic parameters for internal G\/A mismatches in DNA","volume":"37","author":"Allawi","year":"1998","journal-title":"Biochemistry"},{"key":"2023061007215427300_b3","doi-asserted-by":"crossref","first-page":"9435","DOI":"10.1021\/bi9803729","article-title":"Nearest-neighbor thermodynamics of internal A\/C mismatches in DNA: sequence dependence and pH effects","volume":"37","author":"Allawi","year":"1998","journal-title":"Biochemistry"},{"key":"2023061007215427300_b4","doi-asserted-by":"crossref","first-page":"2694","DOI":"10.1093\/nar\/26.11.2694","article-title":"Thermodynamics of internal C\/T mismatches in DNA","volume":"26","author":"Allawi","year":"1998","journal-title":"Nucleic Acids Res."},{"key":"2023061007215427300_b5","doi-asserted-by":"crossref","first-page":"3751","DOI":"10.1093\/nar\/gkg560","article-title":"Primer design assistant (PDA): a web-based primer design tool","volume":"31","author":"Chen","year":"2003","journal-title":"Nucleic Acids Res."},{"key":"2023061007215427300_b6","doi-asserted-by":"crossref","first-page":"3801","DOI":"10.1093\/nar\/22.18.3801","article-title":"A simple procedure for optimizing the polymerase chain reaction (PCR) using modified Taguchi methods","volume":"22","author":"Cobb","year":"1994","journal-title":"Nucleic Acids Res."},{"key":"2023061007215427300_b7","doi-asserted-by":"crossref","first-page":"5227","DOI":"10.1093\/nar\/19.19.5227","article-title":"Oligodeoxyribonucleotide ligation to single-stranded cDNAs: a new tool for cloning 5\u2032 ends of mRNAs and for constructing cDNA libraries by in vitro amplification","volume":"11","author":"Edwards","year":"1991","journal-title":"Nucleic Acids Res."},{"key":"2023061007215427300_b8","doi-asserted-by":"crossref","first-page":"8998","DOI":"10.1073\/pnas.85.23.8998","article-title":"Rapid production of full-length cDNAs from rare transcripts: amplification using a single gene-specific oligonucleotide primer","volume":"85","author":"Frohman","year":"1988","journal-title":"Proc. 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