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Nat Biotechnol. 2012 Feb 14;30(4):344-8. doi: 10.1038/nbt.2147.

Automated forward and reverse ratcheting of DNA in a nanopore at 5-Å precision.

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1
Nanopore Group, Department of Biomolecular Engineering, University of California Santa Cruz, Santa Cruz, California, USA.

Abstract

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 sensor. However, uncontrolled electrophoresis of DNA through these nanopores is too fast for accurate base reads. 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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PMID:
22334048
PMCID:
PMC3408072
DOI:
10.1038/nbt.2147
[Indexed for MEDLINE]
Free PMC Article

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