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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.

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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.

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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.

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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

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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.

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Correspondence to Hagan Bayley.

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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.

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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

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