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Magnesium-dependent thermostability of DNase I
S W Bickler1, M C Heinrich, G C Bagby
1Department of Surgery, Oregon Health Sciences Univ., Portland.
Biotechniques
|July 1, 1992
Summary
Heat inactivation of DNase I (Deoxyribonuclease I) for RNA purification is unreliable. Enzyme activity can be restored by magnesium (Mg2+) concentration changes, impacting downstream RNA analysis.
Area of Science:
- Molecular Biology
- Biochemistry
- RNA Purification
Background:
- DNase I is crucial for removing DNA contamination from RNA samples.
- Heat treatment is a common method to inactivate DNase I before RNA analysis.
- Inactivated DNase I allows for subsequent reverse transcription and PCR amplification.
Purpose of the Study:
- To investigate the efficacy of heat treatment for DNase I inactivation.
- To determine factors affecting DNase I inactivation by heat.
- To assess the reliability of heat treatment for preserving RNA integrity.
Main Methods:
- Enzymatic assays to measure DNase I activity.
- Heat treatment protocols at varying magnesium (Mg2+) concentrations.
- Analysis of DNase I deoxyribonucleolytic activity post-heat treatment and Mg2+ manipulation.
Main Results:
- DNase I heat inactivation is highly dependent on magnesium (Mg2+) concentration.
- Deoxyribonucleolytic activity of heat-treated DNase I can be restored by altering Mg2+ levels.
- Heat treatment alone is insufficient for complete and permanent DNase I inactivation.
Conclusions:
- Caution is advised when using heat treatment solely to inactivate DNase I.
- Magnesium ion concentration significantly influences the stability of heat-inactivated DNase I.
- Alternative or supplementary methods may be necessary for reliable DNase I inactivation in RNA sample preparation.