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Fluorometric assay using dimeric dyes for double- and single-stranded DNA and RNA with picogram sensitivity
H S Rye1, J M Dabora, M A Quesada
1Department of Molecular and Cell Biology, University of California, Berkeley 94720.
Analytical Biochemistry
|January 1, 1993
Summary
Thiazole orange homodimer (TOTO) and oxazole yellow homodimer (YOYO) are fluorescent dyes that bind strongly to nucleic acids. These dyes enable sensitive and rapid detection of DNA and RNA using fluorometry and array scanning methods.
Area of Science:
- Biochemistry
- Molecular Biology
- Analytical Chemistry
Background:
- Thiazole orange homodimer (TOTO) and oxazole yellow homodimer (YOYO) are high-affinity nucleic acid binding dyes.
- These dyes exhibit significant fluorescence enhancement upon binding to nucleic acids.
Purpose of the Study:
- To develop a sensitive method for DNA quantitation using TOTO and YOYO.
- To explore the utility of these dyes for high-throughput nucleic acid detection.
Main Methods:
- Utilized TOTO and YOYO dyes for fluorescence-based nucleic acid detection.
- Employed a conventional fluorometer for DNA quantitation over a range of 0.5-100 ng/ml.
- Implemented a laser-excited confocal fluorescence scanner with a 25-microliter well array for high-throughput analysis.
Main Results:
- Demonstrated a linear relationship between fluorescence intensity and double-stranded DNA concentration.
- Achieved sensitive detection of as little as 20 pg of double-stranded DNA per well using array scanning.
- Showcased the method's speed, high throughput, and minimal sample requirement.
- Extended quantitation capabilities to single-stranded DNA and RNA.
Conclusions:
- TOTO and YOYO provide a sensitive and efficient means for nucleic acid quantitation.
- The array scanning method offers a rapid, high-throughput approach for detecting small amounts of DNA and RNA.
- These fluorescent dyes are valuable tools for molecular biology and biochemical analyses.