Replication of individual DNA molecules under electronic control using a protein nanopore

Abstract

Nanopores can be used to analyse DNA by monitoring ion currents as individual strands are captured and driven through the pore in single file by an applied voltage. Here, we show that serial replication of individual DNA templates can be achieved by DNA polymerases held at the α-haemolysin nanopore orifice. Replication is blocked in the bulk phase, and is initiated only after the DNA is captured by the nanopore. We used this method, in concert with active voltage control, to observe DNA replication catalysed by bacteriophage T7 DNA polymerase (T7DNAP) and by the Klenow fragment of DNA polymerase I (KF). T7DNAP advanced on a DNA template against an 80-mV load applied across the nanopore, and single nucleotide additions were measured on the millisecond timescale for hundreds of individual DNA molecules in series. Replication by KF was not observed when this enzyme was held on top of the nanopore orifice at an applied potential of 80 mV. Sequential nucleotide additions by KF were observed upon applying controlled voltage reversals.

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Figure 1: The nanopore device.
Figure 2: Blocking oligomer inhibition of DNAP-catalysed DNA synthesis.
Figure 3: Blocking oligomer inhibition of bulk-phase T7DNAP binding and voltage-promoted deprotection of individual DNA substrate molecules.
Figure 4: Nucleotide addition may occur above the nanopore orifice (before the probing step) or at the nanopore orifice (during the probing step).
Figure 5: T7DNA replication of individual DNA substrate molecules deprotected and tethered in the nanopore.
Figure 6: KF replication of individual DNA substrate molecules deprotected and tethered in the nanopore.

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Acknowledgements

The authors are grateful to P. Walker at Stanford University PAN for expert oligonucleotide synthesis, Y. Kolodji for help with data analysis, B. Gyarfas for making the Supplementary movies and for assistance with FSM implementation, D. Garalde for advice on the use of T7DNAP, and W. Kibbe (Northwestern University) for modifications to OligoCalc. This work was supported by grants from Oxford Nanopore Technologies and from NHGRI (1RC2HG005553-01) to M.A. and D.D.

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F.O. designed experiments and performed data analysis. K.R.L. co-authored the manuscript, and designed and conducted experiments. S.B., G.M.C. and J.M.D. conducted nanopore experiments, including FSM implementation. D.W.D. conceived the idea of coupling polymerases to nanopores and helped design experiments. M.A. co-authored the manuscript, conceived the blocking oligomer strategy, designed experiments and is responsible for the overall quality of the work.

Corresponding author

Correspondence to Mark Akeson.

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Competing interests

M. Akeson and D. Deamer are consultants to Oxford Nanopore Technologies (Oxford, England). Oxford Nanopore Technologies has licensed rights to some of the inventions detailed in this manuscript and has sponsored some of the research presented here through a grant to the University of California.

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41565_2010_BFnnano2010177_MOESM232_ESM.avi

Supplementary movie 1 (AVI 5643 kb)

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Supplementary movie 1 (AVI 5643 kb)

41565_2010_BFnnano2010177_MOESM233_ESM.avi

Supplementary movie 2 (AVI 27245 kb)

Supplementary information

Supplementary movie 2 (AVI 27245 kb)

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Olasagasti, F., Lieberman, K., Benner, S. et al. Replication of individual DNA molecules under electronic control using a protein nanopore. Nature Nanotech 5, 798–806 (2010). https://doi.org/10.1038/nnano.2010.177

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