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Fluorescent labeling of DNA with ethidium homodimer without measurable decrease in DNA mobility: application to
S F Zakharov1, M M Garner, A Chrambach
1Section on Macromolecular Analysis, National Institute of Child Health and Human Development, National Institutes of Health, Bethesda, Maryland 20892-0001.
Analytical Biochemistry
|January 1, 1995
Summary
DNA mobility remains constant during electrophoresis when labeled with ethidium homodimer (EtD) at low densities. Below 1 EtD/40 bp, labeled DNA behaves like unlabeled DNA, simplifying electrophoretic analysis.
Area of Science:
- Molecular Biology
- Biophysics
- Analytical Chemistry
Background:
- Electrophoresis is a key technique for DNA analysis.
- Fluorescent intercalating dyes like ethidium homodimer (EtD) are used to visualize DNA.
- The binding of EtD to DNA can alter its electrophoretic mobility.
Purpose of the Study:
- To investigate the impact of ethidium homodimer (EtD) binding density on DNA electrophoretic mobility.
- To determine the threshold EtD binding density below which DNA mobility is unaffected.
- To assess the suitability of low-EtD-labeled DNA for automated electrophoresis with fluorescence detection.
Main Methods:
- Automated gel electrophoresis with fluorescence detection.
- Labeling of DNA with varying densities of ethidium homodimer (EtD).
- Normalization of DNA fragment mobilities to a 50-bp reference fragment.
Main Results:
- DNA mobilities, normalized to a 50-bp fragment, were constant over time.
- Increasing EtD binding density decreased DNA electrophoretic mobility.
- This effect became undetectable at binding densities below 1 EtD/40 bp.
Conclusions:
- DNA labeled with ethidium homodimer (EtD) at ratios less than 1/40 bp behaves similarly to unlabeled DNA in electrophoresis.
- Low-labeling ratios allow for analysis in automated systems with fluorescence detection.
- Reduced labeling sensitivity and increased loading requirements are necessary at low dye-DNA ratios.