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:

Single-molecule mechanical identification and sequencing

Nature Methods volume 9, pages 367–372 (2012)Cite this article

Subjects

Abstract

High-throughput, low-cost DNA sequencing has emerged as one of the challenges of the postgenomic era. Here we present the proof of concept for a single-molecule platform that allows DNA identification and sequencing. In contrast to most present methods, our scheme is not based on the detection of the fluorescent nucleotides but on DNA hairpin length. By pulling on magnetic beads tethered by a DNA hairpin to the surface, the molecule can be unzipped. In this open state it can hybridize with complementary oligonucleotides, which transiently block the hairpin rezipping when the pulling force is reduced. By measuring from the surface to the bead of a blocked hairpin, one can determine the position of the hybrid along the molecule with nearly single-base precision. Our approach can be used to identify a DNA fragment of known sequence in a mix of various fragments and to sequence an unknown DNA fragment by hybridization or ligation.

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

Access options

Buy this article

  • Purchase on Springer Link
  • Instant access to full article PDF
Buy now

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

Figure 1: Detection of oligonucleotide-induced blockages during rehybridization.
Figure 2: Sequence identification by hybridization.
Figure 3: Sequence identification by single ligation cycle.
Figure 4: Single-molecule sequencing by hybridization.
Figure 5: Single-molecule sequencing by ligation.

Similar content being viewed by others

References

  1. Sanger, F., Nicklen, S. & Coulson, A.R. DNA sequencing with chain-terminating inhibitors. Proc. Natl. Acad. Sci. USA 74, 5463–5467 (1977).

    Article  CAS  Google Scholar 

  2. Drmanac, R. et al. Human genome sequencing using unchained base reads on self-assembling DNA nanoarrays. Science 327, 78–81 (2010).

    Article  CAS  Google Scholar 

  3. Shendure, J. et al. Accurate multiplex polony sequencing of an evolved bacterial genome. Science 309, 1728–1732 (2005).

    Article  CAS  Google Scholar 

  4. Braslavsky, I., Hebert, B., Kartalov, E. & Quake, S.R. Sequence information can be obtained from single DNA molecules. Proc. Natl. Acad. Sci. USA 100, 3960–3964 (2003).

    Article  CAS  Google Scholar 

  5. Pushkarev, D., Neff, N.F. & Quake, S.R. Single-molecule sequencing of an individual human genome. Nat. Biotechnol. 27, 847–850 (2009).

    Article  CAS  Google Scholar 

  6. Pihlak, A. et al. Rapid genome sequencing with short universal tiling probes. Nat. Biotechnol. 26, 676–684 (2008).

    Article  CAS  Google Scholar 

  7. Shendure, J. & Ji, H. Next-generation DNA sequencing. Nat. Biotechnol. 26, 1135–1145 (2008).

    Article  CAS  Google Scholar 

  8. Shendure, J., Mitra, R.D., Varma, C. & Church, G.M. Advanced sequencing technologies: methods and goals. Nat. Rev. Genet. 5, 335–344 (2004).

    Article  CAS  Google Scholar 

  9. Metzker, M.L. Sequencing technologies—the next generation. Nat. Rev. Genet. 11, 31–46 (2010).

    Article  CAS  Google Scholar 

  10. Fuller, C.W. et al. The challenges of sequencing by synthesis. Nat. Biotechnol. 27, 1013–1023 (2009).

    Article  CAS  Google Scholar 

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

    Article  CAS  Google Scholar 

  12. Greenleaf, W.J. & Block, S.M. Single-molecule, motion-based DNA sequencing using RNA polymerase. Science 313, 801 (2006).

    Article  CAS  Google Scholar 

  13. Munroe, D.J. & Harris, T.J.R. Third-generation sequencing fireworks at marco island. Nat. Biotechnol. 28, 426–428 (2010).

    Article  CAS  Google Scholar 

  14. Clarke, J. et al. Continuous base identification for single-molecule nanopore DNA sequencing. Nat. Nanotechnol. 4, 265–270 (2009).

    Article  CAS  Google Scholar 

  15. Treffer, R. & Deckert, V. Recent advances in single-molecule sequencing. Curr. Opin. Biotechnol. 21, 4–11 (2010).

    Article  CAS  Google Scholar 

  16. Husale, S., Persson, H.H.J. & Sahin, O. DNA nanomechanics allows direct digital detection of complementary DNA and microRNA targets. Nature 462, 1075–1078 (2009).

    Article  CAS  Google Scholar 

  17. Clark, M.D. et al. An oligonucleotide fingerprint normalized and expressed sequence tag characterized zebrafish cDNA library. Genome Res. 11, 1594–1602 (2001).

    Article  Google Scholar 

  18. Herwig, R. et al. Information theoretical probe selection for hybridisation experiments. Bioinformatics 16, 890–898 (2000).

    Article  CAS  Google Scholar 

  19. Guerasimova, A. et al. New tools for oligonucleotide fingerprinting. Biotechniques 31, 490–495 (2001).

    Article  CAS  Google Scholar 

  20. Gosse, C. & Croquette, V. Magnetic tweezers: micromanipulation and force measurement at the molecular level. Biophys. J. 82, 3314–3329 (2002).

    Article  CAS  Google Scholar 

  21. Brower-Toland, B.D. et al. Mechanical disruption of individual nucleosomes reveals a reversible multistage release of DNA. Proc. Natl. Acad. Sci. USA 99, 1960–1965 (2002).

    Article  CAS  Google Scholar 

  22. Strick, T.R., Allemand, J., Bensimon, D., Bensimon, A. & Croquette, V. The elasticity of a single supercoiled DNA molecule. Science 271, 1835–1837 (1996).

    Article  CAS  Google Scholar 

  23. Essevaz-Roulet, B., Bockelmann, U. & Heslot, F. Mechanical separation of the complementary strands of DNA. Proc. Natl. Acad. Sci. USA 94, 11935–11940 (1997).

    Article  CAS  Google Scholar 

  24. McNally, B. et al. Optical recognition of converted DNA nucleotides for single-molecule DNA sequencing using nanopore arrays. Nano Lett. 10, 2237–2244 (2010).

    Article  CAS  Google Scholar 

  25. Mir, K.U., Qi, H., Salata, O. & Scozzafava, G. Sequencing by cyclic ligation and cleavage (CycLiC) directly on a microarray captured template. Nucleic Acids Res. 37, e5 (2009).

    Article  Google Scholar 

  26. Manosas, M., Spiering, M.M., Zhuang, Z., Benkovic, S.J. & Croquette, V. Coupling DNA unwinding activity with primer synthesis in the bacteriophage T4 primosome. Nat. Chem. Biol. 5, 904–912 (2009).

    Article  CAS  Google Scholar 

  27. Kim, K. & Saleh, O.A. A high-resolution magnetic tweezer for single-molecule measurements. Nucleic Acids Res. 37, e136 (2009).

    Article  Google Scholar 

  28. De Vlaminck, I. et al. Highly parallel magnetic tweezers by targeted DNA tethering. Nano Lett. 11, 5489–5493 (2011).

    Article  Google Scholar 

  29. Singh-Zocchi, M., Dixit, S., Ivanov, V. & Zocchi, G. Single-molecule detection of DNA hybridization. Proc. Natl. Acad. Sci. USA 100, 7605–7610 (2003).

    Article  CAS  Google Scholar 

  30. Melchior, W.B. & Von Hippel, P.H. Jr. Alteration of the relative stability of dA - dT and dG* dC base pairs in DNA. Proc. Natl. Acad. Sci. USA 70, 298–302 (1973).

    Article  CAS  Google Scholar 

Download references

Acknowledgements

We acknowledge useful suggestions by M. Volovitch, T. Lionnet and K. Neuman. This work was supported by an ERA-MolMachines grant (to D.B.), a Human Frontier Science Program grant (RGP003/2007 to V.C. and S.J.B.) and the European Research Council grant 'MagRepS' 267862 (to V.C., F.D., M.M. and S.J.B.). We thank J. Quintas for providing mechanical expertise on the instrument.

Author information

Authors and Affiliations

  1. Laboratoire de Physique Statistique, Ecole Normale Supérieure, Université Pierre et Marie Curie Université Paris 06, Université Paris Diderot, Centre National de la Recherche Scientifique, Paris, France

    Fangyuan Ding, Maria Manosas, David Bensimon, Jean-François Allemand & Vincent Croquette

  2. Institut de Biologie de l'Ecole Normale Supérieure, Paris, France

    Fangyuan Ding, David Bensimon, Jean-François Allemand & Vincent Croquette

  3. Departament de Física Fonamental, Facultat de Física, Universitat de Barcelona, Barcelona, Spain

    Maria Manosas

  4. Department of Chemistry, The Pennsylvania State University, University Park, Pennsylvania, USA

    Michelle M Spiering & Stephen J Benkovic

  5. Department of Chemistry and Biochemistry, University of California Los Angeles, Los Angeles, California, USA

    David Bensimon

Authors
  1. Fangyuan Ding
    View author publications

    You can also search for this author in PubMed Google Scholar

  2. Maria Manosas
    View author publications

    You can also search for this author in PubMed Google Scholar

  3. Michelle M Spiering
    View author publications

    You can also search for this author in PubMed Google Scholar

  4. Stephen J Benkovic
    View author publications

    You can also search for this author in PubMed Google Scholar

  5. David Bensimon
    View author publications

    You can also search for this author in PubMed Google Scholar

  6. Jean-François Allemand
    View author publications

    You can also search for this author in PubMed Google Scholar

  7. Vincent Croquette
    View author publications

    You can also search for this author in PubMed Google Scholar

Contributions

J.-F.A. and V.C. designed the apparatus; F.D., S.J.B., M.M., M.M.S. and V.C. discussed the T4 system that led to the concept of sequencing; F.D. and M.M. performed experiments on magnetic tweezers; F.D. and M.M.S. prepared DNA hairpins; F.D., D.B. and V.C. performed hybridization and ligation assays; F.D. and V.C. analyzed data; F.D., M.M., M.M.S., S.J.B., J.-F.A., D.B. and V.C. prepared the manuscript.

Corresponding author

Correspondence to Vincent Croquette.

Ethics declarations

Competing interests

D.B., J-F.A. and V.C. are collaborating with the company PicoTwist that manufactures magnetic tweezers. F.D., M.M., D.B., J-F.A. and V.C. have three patent applications on the work described here (EP 10305563.8, EP 10305564.6 and EP 11306743).

Supplementary information

Supplementary Text and Figures

Supplementary Figures 1–10, Supplementary Discussion, Supplementary Note 1 (PDF 1441 kb)

Rights and permissions

Reprints and permissions

About this article

Cite this article

Ding, F., Manosas, M., Spiering, M. et al. Single-molecule mechanical identification and sequencing. Nat Methods 9, 367–372 (2012). https://doi.org/10.1038/nmeth.1925

Download citation

  • Received:

  • Accepted:

  • Published:

  • Issue Date:

  • DOI: https://doi.org/10.1038/nmeth.1925

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