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High-performance membrane chromatography of supercoiled plasmid DNA
R Giovannini1, R Freitag, T B Tennikova
1Laboratoire de Biotechnologie Cellulaire, Ecole Polytechnique Federale de Lausanne, Switzerland.
Analytical Chemistry
|September 3, 1998
Summary
This study explores polynucleotides using membrane adsorbers, finding isocratic elution is possible for DNA chromatography. Optimized conditions separate plasmid DNA into three forms: supercoiled, nicked, and open circular.
Area of Science:
- Biochemistry
- Chromatography
- Molecular Biology
Background:
- Membrane adsorbers are established for protein chromatography.
- The behavior of polynucleotides on these phases was previously uninvestigated.
Purpose of the Study:
- To investigate polynucleotide behavior on membrane adsorbers.
- To compare high-performance membrane chromatography (HPMC) with conventional methods for DNA separation.
Main Methods:
- Utilized a 7.2-kb supercoiled plasmid as a model polynucleotide.
- Performed gradient and isocratic elution experiments.
- Compared HPMC with packed-bed and monolithic anion-exchange columns.
Main Results:
- Isocratic elution is feasible for DNA chromatography, unlike protein HPMC.
- Optimized conditions resolved the plasmid into three distinct peaks: supercoiled, nicked, and open circular.
- Linearized plasmid eluted earlier, indicating reduced adsorption energy.
Conclusions:
- Membrane adsorbers are effective for separating different plasmid DNA forms.
- HPMC offers distinct advantages for DNA chromatography, including higher salt tolerance in gradient elution.
- Results align with a new theory of HPMC.