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Quantitation of supercoiled circular content in plasmid DNA solutions using a fluorescence-based method
M S Levy1, P Lotfian, R O'Kennedy
1The Advanced Centre for Biochemical Engineering, Department of Biochemical Engineering, University College London, Torrington Place, London WC1E 7JE, UK. myriom.levy@ucl.ac.uk
Nucleic Acids Research
|June 28, 2000
Summary
A new method, SCFluo, quantifies supercoiled circular (SC) DNA. This simple, rapid technique measures SC DNA proportion by analyzing fluorescence changes after heat treatment, offering an alternative to gel electrophoresis.
Area of Science:
- Molecular Biology
- Biochemistry
- Analytical Chemistry
Background:
- Supercoiled circular (SC) DNA is crucial for gene therapy and DNA vaccination.
- Current methods for quantifying SC DNA can be time-consuming or less accurate.
- DNA degradation during processing can significantly reduce SC DNA content.
Purpose of the Study:
- To develop and validate a novel, rapid, and quantitative method for determining the proportion of SC DNA in solutions.
- To offer an alternative to existing methods for SC DNA quantification.
Main Methods:
- The SCFluo method utilizes the reversible denaturation property of SC DNA.
- PicoGreen fluorochrome quantifies double-stranded DNA (dsDNA).
- Fluorescence is measured after a standardized heating and cooling step and normalized to total dsDNA.
Main Results:
- The SCFluo method showed good correlation with densitometric scanning for SC content >20-30%.
- The method accurately quantified SC DNA in degraded plasmid preparations (6.9, 13, and 20 kb).
- SCFluo is demonstrated as a simple, rapid, and quantitative technique.
Conclusions:
- SCFluo provides a reliable and efficient alternative for SC DNA quantification.
- This method is suitable for assessing DNA quality in large-scale preparations and during storage.
- Accurate SC DNA quantification is essential for pharmaceutical applications like gene therapy and DNA vaccination.