The orientation of DNA fragments in the agarose gels

Julian Borejdo, Kathryn DeFea

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Abstract

A microscopic method of measuring the orientation of nucleic acids in the agarose gels is described. A nucleic acid undergoing electrophoresis is stained with the dye ethidium bromide and is viewed under high magnification with a polarization microscope. A high-numerical-aperture microscope objective is used to illuminate and to collect the fluorescence signal, and therefore the orientation of the minute quantities of nucleic acid can be measured: in a typical experiment we can detect the orientation of one-tenth of a picogram (10-13 g) of DNA. Polarization properties of the fluorescent light emitted by the separate bands corresponding to different molecular weights of the DNA are examined. A linear dichroism equation relates the measured fluorescence to the mean orientation of the absorption dipole of the ethidium bromide (and therefore DNA) and to the extent to which it is disorganized. As an example, we measured the orientation of φX174 DNA RF HaeIII fragments undergoing electrophoresis in a field of 10 V/cm. Ethidium bromide bound to the fragments with an angle of the absorption dipole largely perpendicular to the direction of the electrophoretic current. The dichroism declined as the molecular weight of the fragments decreased which is interpreted as an increase in the degree of disorder for shorter DNA.

Original languageEnglish
Pages (from-to)393-398
Number of pages6
JournalAnalytical Biochemistry
Volume174
Issue number2
DOIs
Publication statusPublished - 1 Nov 1988

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Keywords

  • DNA orientation
  • agarose gels
  • electrophoresis
  • ethidium bromide
  • fluorescence microscopy
  • linear dichroism

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