An emerging DNA sequencing technique uses protein or solid-state pores to analyze individual strands as they are driven in single-file order past a nanoscale sensor1,2,3. However, uncontrolled electrophoresis of DNA through these nanopores is too fast for accurate base reads4. Here, we describe forward and reverse ratcheting of DNA templates through the α-hemolysin nanopore controlled by phi29 DNA polymerase without the need for active voltage control. DNA strands were ratcheted through the pore at median rates of 2.5–40 nucleotides per second and were examined at one nucleotide spatial precision in real time. Up to 500 molecules were processed at ∼130 molecules per hour through one pore. The probability of a registry error (an insertion or deletion) at individual positions during one pass along the template strand ranged from 10% to 24.5% without optimization. This strategy facilitates multiple reads of individual strands and is transferable to other nanopore devices for implementation of DNA sequence analysis.
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The authors thank Oxford Nanopore Technologies (Oxford, UK) for supplying α-HL heptamers, P. Walker and Y. Tran (Stanford University Protein and Nucleic Acid Facility) for expert oligonucleotide synthesis, Enzymatics Corp. for supplying concentrated phi29 DNAP, and A. Mai for DNA purification. H. Wang, R. Abu-Shumays and H. Olsen commented on drafts of the manuscript. This work was supported by National Human Genome Research Institute grant HG006321.
M.A. is a consultant to Oxford Nanopore Technologies, Oxford, UK.
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Cherf, G., Lieberman, K., Rashid, H. et al. Automated forward and reverse ratcheting of DNA in a nanopore at 5-Å precision. Nat Biotechnol 30, 344–348 (2012). https://doi.org/10.1038/nbt.2147
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