Mutation detection and single-molecule counting using isothermal rolling-circle amplification | Nature Genetics Skip to main content Thank you for visiting nature.com. You are using a browser version with limited support for CSS. To obtain the best experience, we recommend you use a more up to date browser (or turn off compatibility mode in Internet Explorer). In the meantime, to ensure continued support, we are displaying the site without styles and JavaScript. Advertisement Abstract Rolling-circle amplification (RCA) driven by DNA polymerase can replicate circularized oligonucleotide probes with either linear or geometric kinetics under isothermal conditions. In the presence of two primers, one hybridizing to the + strand, and the other, to the – strand of DNA, a complex pattern of DNA strand displacement ensues that generates 10 9 or more copies of each circle in 90 minutes, enabling detection of point mutations in human genomic DNA. Using a single primer, RCA generates hundreds of tandemly linked copies of a covalently closed circle in a few minutes. If matrix-associated, the DNA product remains bound at the site of synthesis, where it may be tagged, condensed and imaged as a point light source. Linear oligonucleotide probes bound covalently on a glass surface can generate RCA signals, the colour of which indicates the allele status of the target, depending on the outcome of specific, target-directed ligation events. As RCA permits millions of individual probe molecules to be counted and sorted using colour codes, it is particularly amenable for the analysis of rare somatic mutations. RCA also shows promise for the detection of padlock probes bound to single-copy genes in cytological preparations. This is a preview of subscription content, access via your institution Access options Access through your institution Subscription info for Chinese customers We have a dedicated website for our Chinese customers. Please go to naturechina.com to subscribe to this journal. Go to naturechina.com Buy this article Purchase on SpringerLink Instant access to the full article PDF. 39,95 € Prices may be subject to local taxes which are calculated during checkout Figure 1: Probe circularization by ligation, and amplification by a rolling circle-reaction. Figure 2: Ligation of circularizable probes by Ampligase, a thermostable DNA ligase. Figure 3: Analysis of RCA products. Figure 4: Rolling-circle amplification of a circularized probe using two primers. Figure 5: HRCA of circularized DNA and its use in allele detection. Figure 6: Design of the RCA-CACHET ligation-dependent assay using immobilized DNA probes. Figure 7: Detection of individual ligated probe molecules on glass slides by RCA-CACHET. Figure 8: Detection of padlock probes amplified by RCA on cytological preparations. References Nilsson, M. et al. Padlock probes: Circularizing oligonucleotides for localized DNA detection . 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Radding and his group kindly provided us with E. coli SSB. We thank E. Winn-Deen for critical reading of the manuscript. This work was supported in part by a research grant from ONCOR, Inc., to P.M.L. and a grant from the National Institutes of Health to D.C.W. (HG00272). Author information Authors and Affiliations Department of Pathology, Yale University School of Medicine, 333 Cedar St., New Haven, 06520, Connecticut, USA Paul M. Lizardi Department of Genetics, Yale University School of Medicine, 333 Cedar St., New Haven, 06520, Connecticut, USA Xiaohua Huang, Zhengrong Zhu, Patricia Bray-Ward & David C. Ward Oncor, Inc., 209 Perry Parkway, Gaithersburg, 20877, Maryland, USA David C. Thomas Authors Paul M. Lizardi View author publications Search author on: PubMed Google Scholar Xiaohua Huang View author publications Search author on: PubMed Google Scholar Zhengrong Zhu View author publications Search author on: PubMed Google Scholar Patricia Bray-Ward View author publications Search author on: PubMed Google Scholar David C. Thomas View author publications Search author on: PubMed Google Scholar David C. Ward View author publications Search author on: PubMed Google Scholar Corresponding authors Correspondence to Paul M. Lizardi or David C. Ward . Rights and permissions Reprints and permissions About this article Cite this article Lizardi, P., Huang, X., Zhu, Z. et al. Mutation detection and single-molecule counting using isothermal rolling-circle amplification. Nat Genet 19 , 225–232 (1998). https://doi.org/10.1038/898 Download citation Issue date : July 1998 DOI : https://doi.org/10.1038/898 Share this article Anyone you share the following link with will be able to read this content: Get shareable link Sorry, a shareable link is not currently available for this article. Copy shareable link to clipboard Provided by the Springer Nature SharedIt content-sharing initiative Access through your institution Buy or subscribe Advertisement Search Search articles by subject, keyword or author Show results from All journals This journal Search Advanced search Quick links Explore articles by subject Find a job Guide to authors Editorial policies Close banner Close Sign up for the Nature Briefing newsletter — what matters in science, free to your inbox daily. Email address Sign up I agree my information will be processed in accordance with the Nature and Springer Nature Limited Privacy Policy . 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