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.

  • Article
  • Published:

Continuous base identification for single-molecule nanopore DNA sequencing

Nature Nanotechnology volume 4, pages 265–270 (2009)Cite this article

Abstract

A single-molecule method for sequencing DNA that does not require fluorescent labelling could reduce costs and increase sequencing speeds. An exonuclease enzyme might be used to cleave individual nucleotide molecules from the DNA, and when coupled to an appropriate detection system, these nucleotides could be identified in the correct order. Here, we show that a protein nanopore with a covalently attached adapter molecule can continuously identify unlabelled nucleoside 5'-monophosphate molecules with accuracies averaging 99.8%. Methylated cytosine can also be distinguished from the four standard DNA bases: guanine, adenine, thymine and cytosine. The operating conditions are compatible with the exonuclease, and the kinetic data show that the nucleotides have a high probability of translocation through the nanopore and, therefore, of not being registered twice. This highly accurate tool is suitable for integration into a system for sequencing nucleic acids and for analysing epigenetic modifications.

This is a preview of subscription content, access via your institution

Access options

Buy this article

  • Purchase on SpringerLink
  • Instant access to full article PDF
Buy now

Prices may be subject to local taxes which are calculated during checkout

Figure 1: Structures of haemolysin mutants.
Figure 2: Single-channel recordings comparing permanent and transient adapters.
Figure 3: Nucleotide event distributions with the permanent adapter.
Figure 4: Nucleotide dwell times and kinetics with a permanent adapter.
Figure 5: Detection of methyl-dCMP.
Figure 6: Detection of nucleotides cleaved from ssDNA by exonuclease I.

Similar content being viewed by others

References

  1. Bayley, H. Sequencing single molecules of DNA. Curr. Opin. Chem. Biol. 10, 628–637 (2006).

    Article  CAS  Google Scholar 

  2. Branton, D. et al. The potential and challenges of nanopore sequencing. Nature Biotechnol. 26, 1146–1153 (2008).

    Article  CAS  Google Scholar 

  3. Kasianowicz, J. J., Brandin, E., Branton, D. & Deamer, D. W. Characterization of individual polynucleotide molecules using a membrane channel. Proc. Natl Acad. Sci. USA 93, 13770–13773 (1996).

    Article  CAS  Google Scholar 

  4. Deamer, D. W. & Branton, D. Characterization of nucleic acids by nanopore analysis. Acc. Chem. Res. 35, 817–825 (2002).

    Article  CAS  Google Scholar 

  5. Ashkenasy, N., Sanchez-Quesada, J., Bayley, H. & Ghadiri, M. R. Recognizing a single base in an individual DNA strand: a step toward DNA sequencing in nanopores. Angew Chem. Int. Ed. Engl. 44, 1401–1404 (2005).

    Article  CAS  Google Scholar 

  6. Mitchell, N. & Howorka, S. Chemical tags facilitate the sensing of individual DNA strands with nanopores. Angew Chem. Int. Ed. Engl. 47, 5565–5568 (2008).

    Article  CAS  Google Scholar 

  7. Astier, Y., Kainov, D. E., Bayley, H., Tuma, R. & Howorka, S. Stochastic detection of motor protein–RNA complexes by single-channel current recording. Chem. Phys. Chem. 8, 2189–2194 (2007).

    Article  CAS  Google Scholar 

  8. Hornblower, B. et al. Single-molecule analysis of DNA–protein complexes using nanopores. Nature Methods 4, 315–317 (2007).

    Article  CAS  Google Scholar 

  9. Cockroft, S. L., Chu, J., Amorin, M. & Ghadiri, M. R. A single-molecule nanopore device detects DNA polymerase activity with single-nucleotide resolution. J. Am. Chem. Soc. 130, 818–820 (2008).

    Article  CAS  Google Scholar 

  10. Maglia, G., Restrepo, M. R., Mikhailova, E. & Bayley, H. Enhanced translocation of single DNA molecules through alpha-hemolysin nanopores by manipulation of internal charge. Proc. Natl Acad. Sci. USA 105, 19720–19725 (2008).

    Article  CAS  Google Scholar 

  11. Jett, J. H. et al. High-speed DNA sequencing: an approach based upon fluorescence detection of single molecules. J. Biomol. Struct. Dyn. 7, 301–309 (1989).

    Article  CAS  Google Scholar 

  12. Sauer, M. et al. Single molecule DNA sequencing in submicrometer channels: state of the art and future prospects. J. Biotechnol. 86, 181–201 (2001).

    Article  CAS  Google Scholar 

  13. Stephan, J. et al. Towards a general procedure for sequencing single DNA molecules. J. Biotechnol. 86, 255–267 (2001).

    Article  CAS  Google Scholar 

  14. Werner, J. H. et al. Progress towards single-molecule DNA sequencing: a one color demonstration. J. Biotechnol. 102, 1–14 (2003).

    Article  CAS  Google Scholar 

  15. Werner, J. H., Cai, H., Keller, R. A. & Goodwin, P. M. Exonuclease I hydrolyzes DNA with a distribution of rates. Biophys. J. 88, 1403–1412 (2005).

    Article  CAS  Google Scholar 

  16. Bayley, H. & Cremer, P. S. Stochastic sensors inspired by biology. Nature 413, 226–230 (2001).

    Article  CAS  Google Scholar 

  17. Cheley, S., Gu, L. Q. & Bayley, H. Stochastic sensing of nanomolar inositol 1,4,5-trisphosphate with an engineered pore. Chem. Biol. 9, 829–838 (2002).

    Article  CAS  Google Scholar 

  18. Shin, S. H., Luchian, T., Cheley, S., Braha, O. & Bayley, H. Kinetics of a reversible covalent-bond-forming reaction observed at the single-molecule level. Angew Chem. Int. Ed. Engl. 41, 3707–3709, 3523 (2002).

    Article  CAS  Google Scholar 

  19. Gu, L. Q., Braha, O., Conlan, S., Cheley, S. & Bayley, H. Stochastic sensing of organic analytes by a pore-forming protein containing a molecular adapter. Nature 398, 686–690 (1999).

    Article  CAS  Google Scholar 

  20. Astier, Y., Braha, O. & Bayley, H. Toward single molecule DNA sequencing: direct identification of ribonucleoside and deoxyribonucleoside 5′-monophosphates by using an engineered protein nanopore equipped with a molecular adapter. J. Am. Chem. Soc. 128, 1705–1710 (2006).

    Article  CAS  Google Scholar 

  21. Brody, R. S., Doherty, K. G. & Zimmerman, P. D. Processivity and kinetics of the reaction of exonuclease I from Escherichia coli with polydeoxyribonucleotides. J. Biol. Chem. 261, 7136–7143 (1986).

    CAS  Google Scholar 

  22. Enderlein, J. Nucleotide specificity versus complex heterogeneity in exonuclease activity measurements. Biophys. J. 92, 1556–1558 (2007).

    Article  CAS  Google Scholar 

  23. Cheley, S., Braha, O., Lu, X., Conlan, S. & Bayley, H. A functional protein pore with a ‘retro’ transmembrane domain. Protein Sci. 8, 1257–1267 (1999).

    Article  CAS  Google Scholar 

  24. Song, L. et al. Structure of staphylococcal alpha-hemolysin, a heptameric transmembrane pore. Science 274, 1859–1866 (1996).

    Article  CAS  Google Scholar 

  25. Gu, L. Q., Cheley, S. & Bayley, H. Capture of a single molecule in a nanocavity. Science 291, 636–640 (2001).

    Article  CAS  Google Scholar 

  26. Wu, H. C., Astier, Y., Maglia, G., Mikhailova, E. & Bayley, H. Protein nanopores with covalently attached molecular adapters. J. Am. Chem. Soc. 129, 16142–16148 (2007).

    Article  CAS  Google Scholar 

  27. Sanchez-Quesada, J., Ghadiri, M. R., Bayley, H. & Braha, O. Cyclic peptides as molecular adapters for a pore-forming protein. J. Am. Chem. Soc. 122, 11757–11766 (2000).

    Article  CAS  Google Scholar 

  28. Brena, R. M., Huang, T. H. & Plass, C. Toward a human epigenome. Nature Genet. 38, 1359–1360 (2006).

    Article  CAS  Google Scholar 

  29. Schaefer, C. B., Ooi, S. K., Bestor, T. H. & Bourc'his, D. Epigenetic decisions in mammalian germ cells. Science 316, 398–399 (2007).

    Article  CAS  Google Scholar 

  30. Ooi, S. K. & Bestor, T. H. The colorful history of active DNA demethylation. Cell 133, 1145–1148 (2008).

    Article  CAS  Google Scholar 

  31. Hayatsu, H. The bisulfite genomic sequencing used in the analysis of epigenetic states, a technique in the emerging environmental genotoxicology research. Mutat. Res. 659, 77–82 (2008).

    Article  CAS  Google Scholar 

  32. Howorka, S., Cheley, S. & Bayley, H. Sequence-specific detection of individual DNA strands using engineered nanopores. Nature Biotechnol. 19, 636–639 (2001).

    Article  CAS  Google Scholar 

Download references

Acknowledgements

The authors would like to thank O. Braha, S. Cheley, T. Reid and G. Sanghera for valuable discussion, S. Lewis for cyclodextrin preparation and analysis, M. Crawford, G. Hall and L. Woodward for nanopore testing, J. Kilgour and J. White for protein production, Z. McDougall for assistance with the manuscript, and all the staff at ONT. This work was supported by grants from the NIH, the European Commission's seventh Framework Programme (FP7) READNA Consortium and Oxford Nanopore Technologies. H.B. is the holder of a Royal Society Wolfson Research Merit Award.

Author information

Authors and Affiliations

  1. Oxford Nanopore Technologies Ltd, Begbroke Science Park, Sandy Lane, Oxford, OX5 1PF, UK

    James Clarke, Lakmal Jayasinghe, Alpesh Patel & Stuart Reid

  2. Department of Chemistry, University of Oxford, Chemistry Research Laboratory, Mansfield Road, Oxford, OX1 3TA, UK

    Hai-Chen Wu, Lakmal Jayasinghe & Hagan Bayley

Authors
  1. James Clarke
    View author publications

    You can also search for this author in PubMed Google Scholar

  2. Hai-Chen Wu
    View author publications

    You can also search for this author in PubMed Google Scholar

  3. Lakmal Jayasinghe
    View author publications

    You can also search for this author in PubMed Google Scholar

  4. Alpesh Patel
    View author publications

    You can also search for this author in PubMed Google Scholar

  5. Stuart Reid
    View author publications

    You can also search for this author in PubMed Google Scholar

  6. Hagan Bayley
    View author publications

    You can also search for this author in PubMed Google Scholar

Contributions

J.C. and H.B. conceived the experiments and wrote the manuscript. J.C. designed the mutant constructs and analysed the data. H.W. designed and synthesized the cyclodextrin. L.J. engineered the proteins. A.P. performed the single-channel recordings. S.R. wrote data analysis algorithms and software.

Corresponding author

Correspondence to Hagan Bayley.

Ethics declarations

Competing interests

H.B. is the founder, a director and a shareholder of Oxford Nanopore Technologies Ltd, a company engaged in the development of nanopore sequencing technology. J.C., L.J., A.P. and S.R. are employees of Oxford Nanopore Technologies Ltd.

Supplementary information

Supplementary Information

Supplementary Information (PDF 1892 kb)

Rights and permissions

Reprints and permissions

About this article

Cite this article

Clarke, J., Wu, HC., Jayasinghe, L. et al. Continuous base identification for single-molecule nanopore DNA sequencing. Nature Nanotech 4, 265–270 (2009). https://doi.org/10.1038/nnano.2009.12

Download citation

  • Received:

  • Accepted:

  • Published:

  • Issue Date:

  • DOI: https://doi.org/10.1038/nnano.2009.12

This article is cited by

Search

Advanced search

Quick links

Nature Briefing

Sign up for the Nature Briefing newsletter — what matters in science, free to your inbox daily.

Get the most important science stories of the day, free in your inbox. Sign up for Nature Briefing