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.

Automated forward and reverse ratcheting of DNA in a nanopore at 5-Å precision

Nature Biotechnology volume 30, pages 344–348 (2012)Cite this article

Subjects

Abstract

An emerging DNA sequencing technique uses protein or solid-state pores to analyze individual strands as they are driven in single-file order past a nanoscale sensor1,2,3. However, uncontrolled electrophoresis of DNA through these nanopores is too fast for accurate base reads4. Here, we describe forward and reverse ratcheting of DNA templates through the α-hemolysin nanopore controlled by phi29 DNA polymerase without the need for active voltage control. DNA strands were ratcheted through the pore at median rates of 2.5–40 nucleotides per second and were examined at one nucleotide spatial precision in real time. Up to 500 molecules were processed at ∼130 molecules per hour through one pore. The probability of a registry error (an insertion or deletion) at individual positions during one pass along the template strand ranged from 10% to 24.5% without optimization. This strategy facilitates multiple reads of individual strands and is transferable to other nanopore devices for implementation of DNA sequence analysis.

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

Relevant articles

Open Access articles citing this article.

Access options

Rent or buy this article

Prices vary by article type

from$1.95

to$39.95

Learn more

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

Figure 1: Experimental set-up.
Figure 2: Forward and reverse ratcheting of DNA through the nanopore.
Figure 3: Reproducible ionic current states as DNA is ratcheted through the nanopore.
Figure 4: Estimating DNA template registry errors in the nanopore during phi29 DNAP–controlled translocation.

References

  1. 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 

  2. Akeson, M., Branton, D., Kasianowicz, J.J., Brandin, E. & Deamer, D.W. Microsecond time-scale discrimination among polycytidylic acid, polyadenylic acid, and polyuridylic acid as homopolymers or as segments within single RNA molecules. Biophys. J. 77, 3227–3233 (1999).

    Article  CAS  Google Scholar 

  3. Meller, A., Nivon, L., Brandin, E., Golovchenko, J. & Branton, D. Rapid nanopore discrimination between single polynucleotide molecules. Proc. Natl. Acad. Sci. USA 97, 1079–1084 (2000).

    Article  CAS  Google Scholar 

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

    Article  CAS  Google Scholar 

  5. Eid, J. et al. Real-time DNA sequencing from single polymerase molecules. Science 323, 133–138 (2009).

    Article  CAS  Google Scholar 

  6. Harris, T.D. et al. Single-molecule DNA sequencing of a viral genome. Science 320, 106–109 (2008).

    Article  CAS  Google Scholar 

  7. Movileanu, L., Cheley, S. & Bayley, H. Partitioning of individual flexible polymers into a nanoscopic protein pore. Biophys. J. 85, 897–910 (2003).

    Article  CAS  Google Scholar 

  8. Church, G.M., Deamer, D.W., Branton, D., Baldarelli, R. & Kasianowicz, J. Characterization of individual polymer molecules based on monomer-interface interaction. US patent 5,795,782 (1998).

  9. Blanco, L. et al. Highly efficient DNA synthesis by the phage phi 29 DNA polymerase. Symmetrical mode of DNA replication. J. Biol. Chem. 264, 8935–8940 (1989).

    CAS  PubMed  Google Scholar 

  10. Lu, B., Albertorio, F., Hoogerheide, D.P. & Golovchenko, J.A. Origins and consequences of velocity fluctuations during DNA passage through a nanopore. Biophys. J. 101, 70–79 (2011).

    Article  CAS  Google Scholar 

  11. Olasagasti, F. et al. Replication of individual DNA molecules under electronic control using a protein nanopore. Nat. Nanotechnol. 5, 798–806 (2010).

    Article  CAS  Google Scholar 

  12. Lieberman, K.R. et al. Processive replication of single DNA molecules in a nanopore catalyzed by phi29 DNA polymerase. J. Am. Chem. Soc. 132, 17961–17972 (2010).

    Article  CAS  Google Scholar 

  13. Soengas, M.S., Gutierrez, C. & Salas, M. Helix-destabilizing activity of phi 29 single-stranded DNA binding protein: effect on the elongation rate during strand displacement DNA replication. J. Mol. Biol. 253, 517–529 (1995).

    Article  CAS  Google Scholar 

  14. Ibarra, B. et al. Proofreading dynamics of a processive DNA polymerase. EMBO J. 28, 2794–2802 (2009).

    Article  CAS  Google Scholar 

  15. Wilson, N.A. et al. Electronic control of DNA polymerase binding and unbinding to single DNA molecules. ACS Nano 3, 995–1003 (2009).

    Article  CAS  Google Scholar 

  16. Hurt, N., Wang, H., Akeson, M. & Lieberman, K.R. Specific nucleotide binding and rebinding to individual DNA polymerase complexes captured on a nanopore. J. Am. Chem. Soc. 131, 3772–3778 (2009).

    Article  CAS  Google Scholar 

  17. Gyarfas, B. et al. Mapping the position of DNA polymerase-bound DNA templates in a nanopore at 5 A resolution. ACS Nano 3, 1457–1466 (2009).

    Article  CAS  Google Scholar 

  18. Benner, S. et al. Sequence-specific detection of individual DNA polymerase complexes in real time using a nanopore. Nat. Nanotechnol. 2, 718–724 (2007).

    Article  CAS  Google Scholar 

  19. Derrington, I.M. et al. Nanopore DNA sequencing with MspA. Proc. Natl. Acad. Sci. USA 107, 16060–16065 (2010).

    Article  CAS  Google Scholar 

  20. Stoddart, D. et al. Nucleobase recognition in ssDNA at the central constriction of the alpha-hemolysin pore. Nano Lett. 10, 3633–3637 (2010).

    Article  CAS  Google Scholar 

  21. Tsutsui, M. & Taniguchi, M. Yokota, K., Kawai, T. Identifying single nucleotides by tunnelling current. Nat. Nanotechnol. 5, 286–290 (2010).

    Article  CAS  Google Scholar 

Download references

Acknowledgements

The authors thank Oxford Nanopore Technologies (Oxford, UK) for supplying α-HL heptamers, P. Walker and Y. Tran (Stanford University Protein and Nucleic Acid Facility) for expert oligonucleotide synthesis, Enzymatics Corp. for supplying concentrated phi29 DNAP, and A. Mai for DNA purification. H. Wang, R. Abu-Shumays and H. Olsen commented on drafts of the manuscript. This work was supported by National Human Genome Research Institute grant HG006321.

Author information

Authors and Affiliations

  1. Department of Biomolecular Engineering, Nanopore Group, University of California Santa Cruz, Santa Cruz, California, USA

    Gerald M Cherf, Kate R Lieberman, Hytham Rashid, Christopher E Lam, Kevin Karplus & Mark Akeson

Authors
  1. Gerald M Cherf
    View author publications

    You can also search for this author in PubMed Google Scholar

  2. Kate R Lieberman
    View author publications

    You can also search for this author in PubMed Google Scholar

  3. Hytham Rashid
    View author publications

    You can also search for this author in PubMed Google Scholar

  4. Christopher E Lam
    View author publications

    You can also search for this author in PubMed Google Scholar

  5. Kevin Karplus
    View author publications

    You can also search for this author in PubMed Google Scholar

  6. Mark Akeson
    View author publications

    You can also search for this author in PubMed Google Scholar

Contributions

G.M.C. co-wrote the manuscript and performed and conceived experiments, K.R.L. conceived experiments and edited the final draft, H.R. designed and performed PAGE assays, C.E.L. performed nanopore experiments, K.K. articulated the indel error problem in the context of nanopore sequence analysis and co-wrote the paper, and M.A. co-wrote the manuscript and directed the project.

Corresponding author

Correspondence to Mark Akeson.

Ethics declarations

Competing interests

M.A. is a consultant to Oxford Nanopore Technologies, Oxford, UK.

Supplementary information

Rights and permissions

Reprints and Permissions

About this article

Cite this article

Cherf, G., Lieberman, K., Rashid, H. et al. Automated forward and reverse ratcheting of DNA in a nanopore at 5-Å precision. Nat Biotechnol 30, 344–348 (2012). https://doi.org/10.1038/nbt.2147

Download citation

  • Received:

  • Accepted:

  • Published:

  • Issue Date:

  • DOI: https://doi.org/10.1038/nbt.2147

This article is cited by

Search

Advanced search

Quick links

Nature Briefing: Translational Research

Sign up for the Nature Briefing: Translational Research newsletter — top stories in biotechnology, drug discovery and pharma.

Get what matters in translational research, free to your inbox weekly. Sign up for Nature Briefing: Translational Research