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Fluorometric determination of DNA in epidermis and cultured fibroblasts, using 4'-6-diamidino-2-phenylindole (DAPI)
Abstract:
4'-6-diamidino-phenylindole (DAPI) which is used for the detection of mycoplasmic infections of cell cultures or cytofluorometric investigations proved to be a sensitive agent for the fluorometric assay of alkali extracted DNA in cultured cells and human epidermis. Various experiments to optimize assay conditions led to the following procedure: 1 part of freeze dried tissue (1 mg/ml) and 1 part of 0.2 mol/l NaOH(w/v) were heated at 95 degrees for 40 min. After cooling for 1 h to room temperature, 0.1 ml alkaline extract was mixed with 2.9 ml DAPI-solution (0.2 microgram/ml in McIlvaine buffer containing 1 mmol/l MgCl2). After 10 min in the dark the emitted fluorescence was estimated at 453 nm with an excitation at 360 nm. This assay procedure allowed a detection limit of 25 ng DNA.
Insights
This study demonstrates that 4
Area of Science:
- Biochemistry
- Molecular Biology
- Cell Biology
Background:
- 4'-6-diamidino-phenylindole (DAPI) is a fluorescent stain used in cell biology.
- DAPI is commonly used for detecting mycoplasmic infections and in cytofluorometric analysis.
- Accurate DNA quantification is crucial for various biological and medical applications.
Purpose of the Study:
- To establish and optimize a sensitive fluorometric assay for DNA quantification using DAPI.
- To determine the applicability of the DAPI assay for DNA extracted from cultured cells and human epidermis.
- To establish the detection limit of the optimized DAPI-based DNA assay.
Main Methods:
- Alkali extraction of DNA from freeze-dried tissue samples (1 mg/ml) using 0.2 mol/l NaOH.
- Incubation of the alkaline extract at 95°C for 40 minutes, followed by cooling.
- Fluorometric measurement of DNA using DAPI solution (0.2 µg/ml in McIlvaine buffer with 1 mmol/l MgCl2), with excitation at 360 nm and emission at 453 nm.
Main Results:
- An optimized protocol for alkali extraction and DAPI-based DNA quantification was developed.
- The assay demonstrated sensitivity for DNA extracted from cultured cells and human epidermis.
- A detection limit of 25 ng DNA was achieved with the described fluorometric method.
Conclusions:
- The DAPI fluorometric assay provides a sensitive method for quantifying DNA from biological samples.
- This optimized assay is suitable for analyzing DNA from cultured cells and human tissues.
- The established protocol offers a reliable tool for DNA detection in various research settings.