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Related Experiment Videos

A real-time DNase assay (ReDA) based on PicoGreen fluorescence.

Gökhan Tolun1, Richard S Myers

  • 1Department of Biochemistry and Molecular Biology, University of Miami School of Medicine, PO Box 016129, Miami, FL 33101-6129, USA.

Nucleic Acids Research
|September 5, 2003
PubMed
Summary

This study introduces a continuous DNA nuclease (DNase) activity assay using PicoGreen fluorescence. This novel method offers a simpler, more efficient alternative to traditional discontinuous assays for various DNase enzymes.

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Area of Science:

  • Molecular Biology
  • Enzymology

Background:

  • DNA nucleases (DNases) are crucial for cellular functions and biotechnological applications.
  • Existing DNase activity assays are discontinuous, often using radiolabeled DNA and requiring product separation.
  • There is a need for continuous, more efficient methods to measure DNase activity.

Purpose of the Study:

  • To develop and validate a continuous assay for measuring DNase activity.
  • To utilize the differential fluorescence of PicoGreen for real-time enzyme activity monitoring.
  • To assess the assay's utility across different types of DNases.

Main Methods:

  • A continuous assay was developed using the DNA-binding dye PicoGreen.
  • The assay measures changes in fluorescence intensity correlating with DNA digestion.

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  • The method was tested with lambda exonuclease, RecBCD, T7 gene 6 exonuclease, and DNase I.

    • Main Results:

    • The PicoGreen-based assay successfully monitored the activity of various DNases in real-time.
    • The continuous assay provides a simpler alternative to traditional discontinuous methods.
    • The assay demonstrated broad applicability for characterizing different classes of DNA nucleases.

    Conclusions:

    • A continuous DNase activity assay using PicoGreen fluorescence has been established.
    • This method offers advantages in simplicity and efficiency over traditional assays.
    • The assay is versatile and suitable for characterizing a wide range of DNA nucleases.