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A quantitative microtiter plate nuclease assay based on ethidium/DNA fluorescence
P Friedhoff1, S E Matzen, G Meiss
1Institut für Biochemie, Justus-Liebig-Universität, Heinrich-Buff-Ring 58, Giessen, D-35392, Germany.
Analytical Biochemistry
|September 5, 1996
Summary
A new microtiter plate assay accurately quantifies Serratia marcescens endonuclease activity. This rapid method precisely measures nuclease activity using fluorescence changes, applicable to DNA and RNA substrates.
Area of Science:
- Biochemistry
- Molecular Biology
- Enzymology
Background:
- Extracellular nucleases play roles in various biological processes.
- Accurate quantification of nuclease activity is crucial for biochemical studies.
- Serratia marcescens endonuclease is an enzyme with potential biotechnological applications.
Purpose of the Study:
- To develop and validate a microtiter plate assay for quantifying extracellular Serratia marcescens endonuclease activity.
- To assess the assay's performance under various buffer conditions.
- To establish the assay's dynamic range and efficiency.
Main Methods:
- Development of a microtiter plate assay.
- Monitoring ethidium/DNA fluorescence decrease to track substrate cleavage.
- Utilizing a uv-transilluminator and video documentation system.
- Recording time courses of DNA cleavage and determining cleavage rates.
Main Results:
- The assay precisely quantifies nuclease activity within a factor of 1.2.
- A linear dynamic range covering three orders of magnitude of nuclease activity was achieved.
- The assay is rapid, completed within minutes.
- The assay is effective for both DNA and RNA substrates.
Conclusions:
- A rapid, precise, and sensitive microtiter plate assay for quantifying Serratia marcescens endonuclease activity was successfully developed.
- The assay demonstrates a wide dynamic range and can be adapted for other enzymatic reactions.
- This method provides a valuable tool for biochemical and molecular biology research involving nucleases.