Fluorometric quantification of DNA in cells and tissue

Area of Science:

• Biochemistry
• Molecular Biology
• Analytical Chemistry

Background:

• Accurate DNA quantification is crucial for molecular biology applications.
• Traditional DNA extraction methods can be time-consuming and require harsh chemicals or enzymes.
• Existing methods may not be compatible with sensitive downstream assays.

Purpose of the Study:

• To validate a simple and rapid DNA solubilization procedure.
• To enable direct use of DNA extracts in fluorescence-enhancement assays.
• To provide a reliable method for quantitative DNA estimation in cell and tissue extracts.

Main Methods:

• Cell lysis or tissue homogenization using an ammonium hydroxide-Triton X-100 solution.
• Direct application of the DNA extract to a fluorescence-enhancement assay using bisbenzimidazole (Hoechst 33258) reagent.
• Comparison of results with the standard diphenylamine assay.

Main Results:

• Quantitative extraction of intact, polymerized DNA was achieved.
• The procedure inactivates endogenous DNAase and enhances fluorescence activity, eliminating the need for enzyme treatment or high salt solutions.
• DNA estimates were linear, parallel to a bovine DNA standard, and unaffected by sample volume, with an optimal range of 5-150 ng.
• Results showed excellent agreement with the standard diphenylamine assay.

Conclusions:

• The described one-step DNA extraction procedure is simple, rapid, and effective for quantitative DNA estimation.
• This method is compatible with Hoechst 33258 reagent for sensitive fluorescence-based DNA quantification.
• The procedure offers a reliable alternative to traditional methods for analyzing DNA levels in biological samples.