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Related Experiment Videos

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
PubMed
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.

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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.

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  • 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.