Automated forward and reverse ratcheting of DNA in a nanopore at 5-Å precision
Open Access
- 14 February 2012
- journal article
- research article
- Published by Springer Science and Business Media LLC in Nature Biotechnology
- Vol. 30 (4), 344-348
- https://doi.org/10.1038/nbt.2147
Abstract
A key obstacle to sequencing DNA as it passes through a nanopore is that the translocation rate is too fast to resolve individual bases. Cherf et al. solve this problem with an improved method for ratcheting DNA forward and backward through the nanopore using a DNA polymerase. 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.Keywords
This publication has 21 references indexed in Scilit:
- Origins and Consequences of Velocity Fluctuations during DNA Passage through a NanoporeBiophysical Journal, 2011
- Processive Replication of Single DNA Molecules in a Nanopore Catalyzed by phi29 DNA PolymeraseJournal of the American Chemical Society, 2010
- Nanopore DNA sequencing with MspAProceedings of the National Academy of Sciences of the United States of America, 2010
- Nucleobase Recognition in ssDNA at the Central Constriction of the α-Hemolysin PoreNano Letters, 2010
- Proofreading dynamics of a processive DNA polymeraseThe EMBO Journal, 2009
- Electronic Control of DNA Polymerase Binding and Unbinding to Single DNA MoleculesACS Nano, 2009
- Specific Nucleotide Binding and Rebinding to Individual DNA Polymerase Complexes Captured on a NanoporeJournal of the American Chemical Society, 2009
- The potential and challenges of nanopore sequencingNature Biotechnology, 2008
- Single-Molecule DNA Sequencing of a Viral GenomeScience, 2008
- Sequence-specific detection of individual DNA polymerase complexes in real time using a nanoporeNature Nanotechnology, 2007