A single-molecule nanopore device detects DNA polymerase activity with single-nucleotide resolution
Journal
Journal of the American Chemical Society
Journal Volume
130
Journal Issue
3
Pages
818-820
Date Issued
2008
Author(s)
Abstract
The ability to monitor DNA polymerase activity with single-nucleotide resolution has been the cornerstone of a number of advanced single-molecule DNA sequencing concepts. Toward this goal, we report the first observation of the base-by-base DNA polymerase activity with single-base resolution at the single-molecule level. We describe the design and characterization of a supramolecular nanopore device capable of detecting up to nine consecutive DNA polymerase-catalyzed single-nucleotide primer extensions with high sensitivity and spatial resolution (≤2.4 Å). The device is assembled in a suspended lipid membrane by threading and mechanically capturing a single strand of DNA-PEG copolymer inside an α-hemolysin protein pore. Single-nucleotide primer extensions result in successive displacements of the template DNA strand within the protein pore, which can be monitored by the corresponding stepped changes in the ion current flowing through the pore under an applied transmembrane potential. The system described thus represents a promising advance toward nanopore-mediated single-molecule DNA sequencing concept and, in addition, might be applicable to studying a number of other biopolymer-protein interactions and dynamics. Copyright © 2008 American Chemical Society.
Other Subjects
alpha hemolysin; DNA; DNA polymerase; double stranded DNA; macrogol; single stranded DNA; article; device; DNA sequence; enzyme activity; lipid membrane; molecular dynamics; molecule; nanodevice; nanotechnology; nonhuman; nucleotide sequence; protein DNA interaction; Staphylococcus aureus; supramolecular chemistry; Bacterial Toxins; Catalysis; DNA Primers; DNA, Single-Stranded; DNA-Directed DNA Polymerase; Hemolysin Proteins; Nanotechnology; Nucleotides; Polyethylene Glycols
Type
journal article