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Related Concept Videos

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DNA isolation protocols can be fast and straightforward or complex and time-consuming depending on the type and quality of DNA required for further processing. For example, plasmid DNA extraction is a bit more complicated than genomic DNA extraction because of the need for an appropriate lysis method to separate plasmid DNA from gDNA during isolation. However, for specific applications, such as long-range DNA sequencing that require a good yield of high- quality DNA samples, we need to follow...
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Related Experiment Video

Updated: Jun 13, 2026

Single-Molecule Dwell-Time Analysis of Restriction Endonuclease-Mediated DNA Cleavage
09:53

Single-Molecule Dwell-Time Analysis of Restriction Endonuclease-Mediated DNA Cleavage

Published on: February 7, 2021

Digestion of individual DNA molecules by lambda-exonuclease at liquid-solid interface.

Seong Ho Kang1, Seungah Lee, Edward S Yeung

  • 1Ames Laboratory-USDOE and Department of Chemistry, Iowa State University, Ames, Iowa 50011, USA.

The Analyst
|May 4, 2010
PubMed
Summary

Single DNA molecules were observed undergoing enzyme digestion in real time using total internal reflection fluorescence microscopy (TIRFM). This study revealed distinct digestion rates for lambda-exonuclease, indicating varied enzyme molecule dynamics.

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Last Updated: Jun 13, 2026

Single-Molecule Dwell-Time Analysis of Restriction Endonuclease-Mediated DNA Cleavage
09:53

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Published on: February 7, 2021

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08:23

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Area of Science:

  • Molecular Biology
  • Biophysics
  • Microscopy

Background:

  • Enzyme digestion is crucial for DNA manipulation.
  • Real-time observation of single-molecule enzymatic activity provides detailed insights.
  • Total Internal Reflection Fluorescence Microscopy (TIRFM) allows high-resolution imaging of surface-bound molecules.

Purpose of the Study:

  • To directly observe and quantify the real-time enzyme digestion of single DNA molecules.
  • To investigate the digestion dynamics of lambda-exonuclease at the single-molecule level.
  • To compare length-based and fluorescence-intensity-based measurements of DNA digestion.

Main Methods:

  • Utilizing dual-color TIRFM to visualize individual lambda-DNA molecules.
  • Immobilizing stretched DNA on a fused-silica surface via hydrophobic and electrostatic interactions.
  • Initiating lambda-exonuclease digestion and monitoring DNA shortening in real time.

Main Results:

  • DNA digestion was successfully monitored in real time at a dye:base pair ratio of 1:50.
  • Lambda-exonuclease exhibited 3 distinct length-based digestion rates (0.173–0.462 µm/s) at 37°C.
  • Length-based measurements were more accurate than fluorescence intensity-based measurements due to TIRF mode limitations.

Conclusions:

  • Single-molecule TIRFM enables real-time observation of DNA enzyme digestion.
  • Lambda-exonuclease displays heterogeneous digestion dynamics.
  • Accurate kinetic measurements require careful optimization of dye labeling and imaging parameters.