title: Reading DNA at single-nucleotide resolution with a mutant MspA nanopore and phi29 DNA polymerase.
abstract: Nanopore technologies are being developed for fast and direct sequencing of single DNA molecules through detection of ionic current modulations as DNA passes through a pore's constriction. Here we demonstrate the ability to resolve changes in current that correspond to a known DNA sequence by combining the high sensitivity of a mutated form of the protein pore Mycobacterium smegmatis porin A (MspA) with phi29 DNA polymerase (DNAP), which controls the rate of DNA translocation through the pore. As phi29 DNAP synthesizes DNA and functions like a motor to pull a single-stranded template through MspA, we observe well-resolved and reproducible ionic current levels with median durations of ∼28 ms and ionic current differences of up to 40 pA. Using six different DNA sequences with readable regions 42-53 nucleotides long, we record current traces that map to the known DNA sequences. With single-nucleotide resolution and DNA translocation control, this system integrates solutions to two long-standing hurdles to nanopore sequencing.
authors.affiliation: Department of Physics, University of Washington, Seattle, Washington, USA.
authors.forename: Elizabeth A
authors.initials: EA
authors.lastname: Manrao
authors.forename: Ian M
authors.initials: IM
authors.lastname: Derrington
authors.forename: Andrew H
authors.initials: AH
authors.lastname: Laszlo
authors.forename: Kyle W
authors.initials: KW
authors.lastname: Langford
authors.forename: Matthew K
authors.initials: MK
authors.lastname: Hopper
authors.forename: Nathaniel
authors.initials: N
authors.lastname: Gillgren
authors.forename: Mikhail
authors.initials: M
authors.lastname: Pavlenok
authors.forename: Michael
authors.initials: M
authors.lastname: Niederweis
authors.forename: Jens H
authors.initials: JH
authors.lastname: Gundlach
mesh_terms: D004259:DNA-Directed DNA Polymerase / Q000737:chemistry / Q000235:genetics; D059014:High-Throughput Nucleotide Sequencing / Q000295:instrumentation* / Q000379:methods*; D058608:Nanopores; D009711:Nucleotides / Q000737:chemistry / Q000235:genetics; D018272:Porins / Q000737:chemistry / Q000235:genetics
chemical_list: D009711:Nucleotides; D018272:Porins; C121838:mspA protein, Mycobacterium smegmatis; D004259:DNA-Directed DNA Polymerase