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Reading DNA at single-nucleotide resolution with a mutant MspA nanopore and phi29 DNA polymerase

Nature Biotechnology volume 30, pages 349–353 (2012)Cite this article

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Abstract

Nanopore technologies are being developed for fast and direct sequencing of single DNA molecules through detection of ionic current modulations as DNA passes through a pore's constriction1,2. Here we demonstrate the ability to resolve changes in current that correspond to a known DNA sequence by combining the high sensitivity of a mutated form of the protein pore Mycobacterium smegmatis porin A (MspA)3 with phi29 DNA polymerase (DNAP)4, which controls the rate of DNA translocation through the pore. As phi29 DNAP synthesizes DNA and functions like a motor to pull a single-stranded template through MspA, we observe well-resolved and reproducible ionic current levels with median durations of ∼28 ms and ionic current differences of up to 40 pA. Using six different DNA sequences with readable regions 42–53 nucleotides long, we record current traces that map to the known DNA sequences. With single-nucleotide resolution and DNA translocation control, this system integrates solutions to two long-standing hurdles to nanopore sequencing2.

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Figure 1: Event structure.
Figure 2: Current trace for polymerase synthesis.
Figure 3: Reading a repetitive DNA template.
Figure 4: Reading heteromic DNA.

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

    Article  CAS  Google Scholar 

  3. Manrao, E.A., Derrington, I.M., Pavlenok, M., Niederweis, M. & Gundlach, J.H. Nucleotide discrimination with DNA immobilized in the MspA nanopore. PLoS ONE 6, e25723 (2011).

    Article  CAS  Google Scholar 

  4. Cherf, G.M. et al. Automated forward and reverse ratcheting of DNA in a nanopore at 5-Å precision. Nat. Biotechnol. advance online publication, doi:10.1038/nbt.2147 (14 February 2012).

  5. Wallace, E.V.B. et al. Identification of epigenetic DNA modifications with a protein nanopore. Chem. Commun. (Camb.) 46, 8195–8197 (2010).

    Article  CAS  Google Scholar 

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

    Article  CAS  Google Scholar 

  7. Stoddart, D., Heron, A.J., Mikhailova, E., Maglia, G. & Bayley, H. Single-nucleotide discrimination in immobilized DNA oligonucleotides with a biological nanopore. Proc. Natl. Acad. Sci. USA 106, 7702–7707 (2009).

    Article  CAS  Google Scholar 

  8. Purnell, R.F., Mehta, K.K. & Schmidt, J.J. Nucleotide identification and orientation discrimination of DNA homopolymers immobilized in a protein nanopore. Nano Lett. 8, 3029–3034 (2008).

    Article  CAS  Google Scholar 

  9. Purnell, R.F. & Schmidt, J.J. Discrimination of single base substitutions in a DNA strand immobilized in a biological nanopore. ACS Nano 3, 2533–2538 (2009).

    Article  CAS  Google Scholar 

  10. 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 

  11. Niederweis, M. et al. Cloning of the MspA gene encoding a porin from Mycobacterium smegmatis. Mol. Microbiol. 33, 933–945 (1999).

    Article  CAS  Google Scholar 

  12. Faller, M., Niederweis, M. & Schulz, G.E. The structure of a mycobacterial outer-membrane channel. Science 303, 1189–1192 (2004).

    Article  CAS  Google Scholar 

  13. Butler, T.Z., Pavlenok, M., Derrington, I.M., Niederweis, M. & Gundlach, J.H. Single-molecule DNA detection with an engineered MspA protein nanopore. Proc. Natl. Acad. Sci. USA 105, 20647–20652 (2008).

    Article  CAS  Google Scholar 

  14. 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 

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

    Article  CAS  Google Scholar 

  16. 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 

  17. Hurt, N., Wang, H.Y., 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 

  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. Blanco, L. & Salas, M. Relating structure to function in phi29 DNA polymerase. J. Biol. Chem. 271, 8509–8512 (1996).

    Article  CAS  Google Scholar 

  20. Salas, M., Blanco, L., Lazaro, J.M. & de Vega, M. The bacteriophage phi29 DNA polymerase. IUBMB Life 60, 82–85 (2008).

    Article  CAS  Google Scholar 

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

    Article  CAS  Google Scholar 

  22. Blanco, L. et al. Highly efficient DNA Synthesis by phage phi29 DNA polymerase. J. Biol. Chem. 264, 8935–8940 (1989).

    CAS  PubMed  Google Scholar 

  23. Durbin, R., Eddy, S., Krogh, A. & Mitchison, G. Biological Sequence Analysis, ed. 11 (Cambridge University Press, Cambridge, UK, 2006).

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Acknowledgements

We thank M. Akeson and G.M. Cherf for getting us started with the blocking oligomer phi29 DNAP technique, sharing their CAT DNA and reading the manuscript. We thank J. Bartlett and G. Nayler for their help running experiments and D. Feldman for writing data acquisition code. This work was supported by the US National Institutes of Health, National Human Genome Research Institute $1000 Genome Program Grants R21HG004145, R01HG005115 and R01HG006321.

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Authors and Affiliations

  1. Department of Physics, University of Washington, Seattle, Washington, USA

    Elizabeth A Manrao, Ian M Derrington, Andrew H Laszlo, Kyle W Langford, Matthew K Hopper, Nathaniel Gillgren & Jens H Gundlach

  2. Department of Microbiology, University of Alabama at Birmingham, Birmingham, Alabama, USA

    Mikhail Pavlenok & Michael Niederweis

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  1. Elizabeth A Manrao
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  2. Ian M Derrington
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  6. Nathaniel Gillgren
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  7. Mikhail Pavlenok
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  9. Jens H Gundlach
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Contributions

E.A.M., I.M.D. and J.H.G. conceptualized the project. E.A.M., I.M.D., A.H.L. and J.H.G. designed the experiments, wrote the paper and contributed equally. E.A.M., I.M.D., A.H.L., K.W.L., M.K.H., N.G. and J.H.G. analyzed the data. K.W.L., M.K.H. and N.G. collected data. M.P. and M.N. produced the MspA mutants. J.H.G. supervised the project.

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Correspondence to Jens H Gundlach.

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The authors declare no competing financial interests.

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Supplementary Table 1 and Supplementary Figures 1–16 (PDF 2049 kb)

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Manrao, E., Derrington, I., Laszlo, A. et al. Reading DNA at single-nucleotide resolution with a mutant MspA nanopore and phi29 DNA polymerase. Nat Biotechnol 30, 349–353 (2012). https://doi.org/10.1038/nbt.2171

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