Multiplexed single-molecule assay for enzymatic activity on flow-stretched DNA

Nat Methods. 2007 May;4(5):397-9. doi: 10.1038/nmeth1037. Epub 2007 Apr 15.

Abstract

We report a single-molecule assay for nucleic-acid enzymes on flow-stretched DNA templates. To facilitate the detection of slow or intermittent enzymatic activities, we developed the assay with 15-nm spatial resolution at a frame rate of 1 Hz and approximately 10 nm mechanical stability over the timescale of hours. With multiplexed data collection, we applied the assay to phi29 DNA polymerase, HIV-1 reverse transcriptase, lambda exonuclease and Escherichia coli RNA polymerase.

Publication types

  • Research Support, N.I.H., Extramural
  • Research Support, Non-U.S. Gov't

MeSH terms

  • Bacillus Phages / enzymology
  • DNA / chemistry
  • DNA, Single-Stranded / chemistry
  • DNA, Viral / metabolism*
  • DNA-Directed DNA Polymerase / metabolism*
  • DNA-Directed RNA Polymerases / metabolism*
  • Elasticity
  • Escherichia coli / enzymology
  • Exodeoxyribonucleases / metabolism*
  • HIV Reverse Transcriptase / metabolism*
  • Microscopy / methods
  • Viral Proteins / metabolism*

Substances

  • DNA, Single-Stranded
  • DNA, Viral
  • Viral Proteins
  • DNA
  • HIV Reverse Transcriptase
  • DNA-Directed RNA Polymerases
  • DNA-Directed DNA Polymerase
  • Exodeoxyribonucleases
  • exo protein, Bacteriophage lambda