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Sequence-specific detection of individual DNA polymerase complexes in real time using a nanopore

Abstract

Nanoscale pores have potential to be used as biosensors and are an established tool for analysing the structure and composition of single DNA or RNA molecules1,2,3. Recently, nanopores have been used to measure the binding of enzymes to their DNA substrates4,5. In this technique, a polynucleotide bound to an enzyme is drawn into the nanopore by an applied voltage. The force exerted on the charged backbone of the polynucleotide by the electric field is used to examine the enzyme–polynucleotide interactions. Here we show that a nanopore sensor can accurately identify DNA templates bound in the catalytic site of individual DNA polymerase molecules. Discrimination among unbound DNA, binary DNA/polymerase complexes, and ternary DNA/polymerase/deoxynucleotide triphosphate complexes was achieved in real time using finite state machine logic. This technique is applicable to numerous enzymes that bind or modify DNA or RNA including exonucleases, kinases and other polymerases.

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Figure 1: Detection of DNA translocation events with the nanopore device.
Figure 2: Distinguishing DNA, DNA/KF complexes or DNA/KF/dNTP complexes in the nanopore device.
Figure 3: Detection of correct dNTP binding to the KF/primer-template complex.
Figure 4: Proposed mechanism for voltage-facilitated dissociation of DNA from KF/DNA or KF/DNA/dNTP complexes.
Figure 5: Recognition and control of DNA complexes in real time using FPGA.

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Acknowledgements

We wish to thank D. Branton, V. Tabard-Cossa, M. Wiggin, D. Krapf and R. Mathies for reading early versions of this manuscript. We also thank A. Kottas for guidance on statistical analysis. This work was supported by National Institutes of Health grants GM073617-01A1 (M.A.), HG003703-01 (M.A., D.D.) and HG004035-01 (W.D.).

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Correspondence to William B. Dunbar or Mark Akeson.

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Supplementary figures S1, S2 and supplementary table 1 (PDF 125 kb)

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Benner, S., Chen, R., Wilson, N. et al. Sequence-specific detection of individual DNA polymerase complexes in real time using a nanopore. Nature Nanotech 2, 718–724 (2007). https://doi.org/10.1038/nnano.2007.344

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