title: Automated forward and reverse ratcheting of DNA in a nanopore at 5-Å precision.
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.
authors.affiliation: Nanopore Group, Department of Biomolecular Engineering, University of California Santa Cruz, Santa Cruz, California, USA.
authors.forename: Gerald M
authors.initials: GM
authors.lastname: Cherf
authors.forename: Kate R
authors.initials: KR
authors.lastname: Lieberman
authors.forename: Hytham
authors.initials: H
authors.lastname: Rashid
authors.forename: Christopher E
authors.initials: CE
authors.lastname: Lam
authors.forename: Kevin
authors.initials: K
authors.lastname: Karplus
authors.forename: Mark
authors.initials: M
authors.lastname: Akeson
mesh_terms: D004261:DNA Replication / Q000235:genetics; D004259:DNA-Directed DNA Polymerase / Q000737:chemistry / Q000235:genetics; D006460:Hemolysin Proteins / Q000737:chemistry; D059014:High-Throughput Nucleotide Sequencing / Q000295:instrumentation* / Q000379:methods*; D058608:Nanopores; D009711:Nucleotides / Q000737:chemistry / Q000235:genetics
chemical_list: D006460:Hemolysin Proteins; D009711:Nucleotides; D004259:DNA-Directed DNA Polymerase