A single-molecule nanopore device detects DNA polymerase activity with single-nucleotide resolution.
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 (<or=2.4 A). The device is assembled in a suspended lipid membrane by threading and mechanically capturing a single strand of DNA-PEG copolymer inside an alpha-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.
Cockroft
Scott L
SL
Department of Chemistry, The Skaggs Institute for Chemical Biology, The Scripps Research Institute, 10550 North Torrey Pines Road, La Jolla, California 92037, USA.
Chu
John
J
Amorin
Manuel
M
Ghadiri
M Reza
MR
D001427:Bacterial Toxins / Q000737:chemistry; D002384:Catalysis; D017931:DNA Primers / Q000737:chemistry; D004277:DNA, Single-Stranded / Q000096:biosynthesis* / Q000737:chemistry; D004259:DNA-Directed DNA Polymerase / Q000737:chemistry*; D006460:Hemolysin Proteins / Q000737:chemistry; D036103:Nanotechnology / Q000295:instrumentation* / Q000379:methods*; D009711:Nucleotides / Q000737:chemistry*; D011092:Polyethylene Glycols / Q000737:chemistry
D001427:Bacterial Toxins; D017931:DNA Primers; D004277:DNA, Single-Stranded; D006460:Hemolysin Proteins; D009711:Nucleotides; C021091:staphylococcal alpha-toxin; D011092:Polyethylene Glycols; D004259:DNA-Directed DNA Polymerase