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Single-molecule DNA digestion by lambda-exonuclease.

J Dapprich1

  • 1Praelux, Inc., Lawrenceville, New Jersey, USA. jdapprich@orchidbio.com

Cytometry
|July 15, 1999
PubMed
Summary

Lambda-exonuclease efficiently digests lambda-DNA molecules. This enzymatic shortening was measured using a bead displacement sensor, revealing digestion rates of 15-20 Hz.

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Area of Science:

  • Biophysics
  • Molecular Biology
  • Enzymology

Background:

  • Measuring enzymatic activity on single DNA molecules requires precise biophysical techniques.
  • Lambda-DNA is a well-characterized substrate for enzymatic studies.
  • Optically trapped beads can be used to monitor changes in molecular properties.

Purpose of the Study:

  • To determine the enzymatic shortening rate of individual lambda-DNA molecules.
  • To utilize a bead displacement sensor for real-time measurement of DNA digestion.

Main Methods:

  • Attachment of unstained lambda-DNA to optically trapped beads.
  • Measurement of bead displacement via viscous drag force changes.
  • Monitoring drag force alterations upon addition of lambda-exonuclease.

Main Results:

  • A single undigested lambda-DNA molecule on a bead exhibited stable drag force.
  • Addition of lambda-exonuclease caused a decrease in drag force over approximately 45 minutes.
  • The observed drag force reduction indicates enzymatic shortening of the DNA molecule.

Conclusions:

  • Lambda-exonuclease digests lambda-DNA at an average rate of 15-20 Hz.
  • Bead displacement sensing is a viable method for quantifying single-molecule enzymatic activity.
  • This technique allows for real-time monitoring of DNA processing by enzymes.

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