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

Gökhan Tolun, Richard Myers

Research output: Contribution to journalArticle

31 Citations (Scopus)

Abstract

DNA nucleases (DNases) perform a wide variety of important cellular functions and are also very useful for research and in biotechnological applications. Due to the biological and technological importance of DNases and their use in a wide range of applications, DNase activity assays are essential. Traditional DNase assays employ radiolabeled DNA substrates and require separation of the products of the reaction from the unreacted substrate before quantification of enzyme activity. As a consequence, these methods are discontinuous. In this report, we describe a continuous DNase assay based on the differential fluorescence output of a DNA dye ligand called PicoGreen. The assay was developed to characterize a processive dsDNA exonuclease, lambda exonuclease. The assay appears to have general utility as it is also suitable for measuring the DNA digestion activities of a processive helicase/nuclease, RecBCD, a distributive exonuclease, T7 gene 6 exonuclease, and an endonuclease, DNaseI. The benefits of, and limitations to, the method are discussed.

Original languageEnglish (US)
JournalNucleic Acids Research
Volume31
Issue number18
StatePublished - Sep 15 2003

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Deoxyribonucleases
Fluorescence
Exonucleases
DNA
Endonucleases
Digestion
Coloring Agents
PicoGreen
Ligands
Enzymes
Research

ASJC Scopus subject areas

  • Genetics

Cite this

A real-time DNase assay (ReDA) based on PicoGreen fluorescence. / Tolun, Gökhan; Myers, Richard.

In: Nucleic Acids Research, Vol. 31, No. 18, 15.09.2003.

Research output: Contribution to journalArticle

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