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.


  1. 1

    Nilsson, M. et al. Padlock probes: Circularizing oligonucleotides for localized DNA detection . Science 265, 2085–2088 (1994).

  2. 2

    Nilsson, M. et al. Padlock probes reveal single-nucleotide differences, parent of origin and in situ distribution of centromeric sequences in human chromosomes 13 and 21. Nature Genet. 16, 252– 255 (1997).

  3. 3

    Fire, A. & Xu, S.Q. Rolling replication of short DNA circles . Proc. Natl Acad. Sci. USA 92, 4641– 4645 (1995).

  4. 4

    Liu, D., Daubendiek, S.L., Zillman, M.A., Ryan, K. & Kool, E.T. Rolling circle DNA synthesis: Small circular oligonucleotides as efficient templates for DNA polymerases. J. Am. Chem. Soc. 118, 1587–1594 (1996).

  5. 5

    Blanco, L. & Salas, M. Characterization and purification of a phage ø29-encoded DNA polymerase required for the initiation of replication . Proc. Natl Acad. Sci. USA 81, 5325– 5329 (1984).

  6. 6

    Blanco, L. et al. Highly efficient DNA synthesis by the phage ø29 DNA polymerase. J. Biol. Chem. 264, 8935–8940 (1989).

  7. 7

    Abravaya, K., Carrino, J.J., Muldoon, S. & Lee, H. Detection of point mutations with a modified ligase chain reaction. Nucleic Acids Res. 23, 675–682 ( 1995).

  8. 8

    Schwarz, K. Improved yields of long PCR products using gene 32 protein. Nucleic Acids Res. 18, 1079 (1990).

  9. 9

    Newton, C.R. et al. Analysis of any point mutation in DNA. The amplification refractory mutation system (ARMS). Nucleic Acids Res. 17, 2503 –2516 (1989).

  10. 10

    Luo, J., Bergstrom, D.E. & Barany, F. Improving the fidelity of Thermus thermophilus DNA ligase. Nucleic Acids Res. 24, 3071–3078 (1996).

  11. 11

    Vogelstein, B., Pardoll, D.M. & Coffey, D.S. Supercoiled loops and eucaryotic DNA replication. Cell 22, 79–85 ( 1980).

  12. 12

    Gerdes, M. G, Carter, K.C., Moen, P.T. Jr & Lawrence, J.B. Dynamic changes in the higher-level chromatin organization of specific sequences revealed by in situ hybridization in nuclear halos. J. Cell Biol. 126, 289–304 ( 1994).

  13. 13

    Aliotta, J.M. et al. Thermostable Bst DNA polymerase lacks a 3´-5´ proofreading exonuclease activity. Genet. Anal. 12, 185– 195 (1996).

  14. 14

    Kong, H., Kucera, R. & Jack, W. Characterization of a DNA polymerase from the hyperthermophile Archea Thermococcus litoralis. J. Biol. Chem. 268, 1965– 1975 (1993).

  15. 15

    Speicher, M.R., Ballard, S.G. & Ward, D.C. Karyotyping human chromosomes by combinatorial multicolor FISH. Nature Genet. 12, 368– 375 (1996).

  16. 16

    Guo, Z., Guifoyle, R.A., Thiel, A.J., Wang, R. & Smith, L.M. Direct fluorescence analysis of genetic polymorphisms by hybridization with oligonucleotide arrays on glass supports. Nucleic Acids Res. 22, 5456–5465 (1994).

  17. 17

    Heinoven, P., Itia, A., Torresani, T. & Lovgren, T. Simple triple-label detection of seven cystic fibrosis mutations by time-resolved fluorometry . Clin. Chem. 43, 1142– 1150 (1997).

  18. 18

    Schena, M., Shalon, D., Davis, R.W. & Brown, P. Quantitative monitoring of gene expression patterns with a complementary DNA microarray. Science 270, 467–470 ( 1995).

  19. 19

    Rychlik, W. & Rhoads, R.E. A computer program for choosing optimal oligonucleotides for filter hybridization, sequencing, and in vitro amplification of DNA. Nucleic Acids Res. 17, 8543–8551 (1989).

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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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