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A fluorometric assay for quantitating DNA strand breaks during apoptosis
1Center for Basic Research in Digestive Diseases, Mayo Clinic and Foundation, Rochester, Minnesota 55905, USA.
Analytical Biochemistry
|August 10, 1995
Summary
This study introduces a novel fluorescent end-labeling assay to quantify DNA fragmentation during apoptosis. The method accurately measures DNA strand breaks, offering high sensitivity for apoptosis research.
Area of Science:
- Molecular Biology
- Cell Biology
- Biochemistry
Background:
- Quantifying DNA fragmentation during apoptosis is challenging due to limited techniques.
- Apoptosis involves DNA cleavage, a critical hallmark of programmed cell death.
Purpose of the Study:
- To develop a quantitative assay for DNA fragmentation in apoptosis.
- To utilize enzymatic labeling with fluorescent dideoxynucleotides for DNA break detection.
Main Methods:
- Developed a fluorophore end-labeling assay using terminal deoxynucleotidyl transferase to label 3'-OH DNA ends.
- Validated the assay with DNase I-treated calf thymus DNA, comparing it to an isotopic method.
- Applied the assay to quantify DNA strand breaks in apoptotic hepatocytes using microscopy, flow cytometry, and fluorometry.
Main Results:
- Demonstrated direct proportionality between fluorescence intensity and the number of DNA strand breaks.
- Showed excellent correlation between fluorophore end-labeling and isotopic methods for quantifying 3'-OH ends.
- Detected DNA strand breaks earlier than morphological changes or hypodiploid DNA content in apoptotic hepatocytes.
Conclusions:
- Fluorophore end-labeling is a sensitive and quantitative method for assessing DNA fragmentation in apoptosis.
- The assay can detect DNA breaks preceding other indicators of apoptosis.
- This technique is valuable for investigating the molecular mechanisms of DNA cleavage during apoptosis.