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Quantification of nucleic acids on nitrocellulose membranes with time-resolved fluorometry
T K Christopoulos1, E P Diamandis, G Wilson
1Department of Clinical Biochemistry, Toronto Western Hospital, Ontario, Canada.
Nucleic Acids Research
|November 11, 1991
Summary
A novel streptavidin-based macromolecular complex (SBMC) offers sensitive, non-isotopic DNA detection. This method accurately stains biotinylated DNA on solid supports, with a detection limit of 10 pg.
Area of Science:
- Biochemistry
- Molecular Biology
- Analytical Chemistry
Background:
- Traditional DNA detection methods often involve isotopes, posing safety and disposal concerns.
- Non-isotopic methods are sought for safer and more versatile DNA analysis.
Purpose of the Study:
- To develop and validate a novel non-isotopic staining reagent for detecting biotinylated DNA on solid supports.
- To establish the sensitivity and applicability of this new method in molecular biology.
Main Methods:
- Utilized a streptavidin-based macromolecular complex (SBMC) labeled with a specific europium chelate (BCPDA).
- Applied the SBMC reagent for staining biotinylated DNA immobilized on nitrocellulose filters.
- Evaluated detection methods including UV illumination, instant photography, and time-resolved fluorometric scanning.
Main Results:
- Achieved a detection limit of approximately 10 pg of target DNA.
- Demonstrated successful staining of biotinylated DNA probes hybridized to Southern transferred targets.
- Confirmed the versatility of the method for DNA detection on various solid supports.
Conclusions:
- The developed SBMC-BCPDA reagent provides a sensitive and versatile non-isotopic methodology for DNA staining.
- This approach offers a viable alternative to isotopic methods for DNA analysis on solid supports.
- The method is suitable for applications requiring high-resolution DNA quantification and visualization.