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Mutation detection and single-molecule counting using isothermal rolling-circle amplification


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 109 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.

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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.


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We are indebted to M. Salas for a generous gift of ø29 DNA polymerase, and for advice regarding the use of the enzyme. We also thank B. Moffett and Amersham for research samples of this enzyme. F. Barany kindly provided samples of T. thermophilus DNA ligase. C. 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).

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Correspondence to Paul M. Lizardi or David C. Ward.

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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).

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