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The free solution mobility of DNA

N C Stellwagen1, C Gelfi, P G Righetti

  • 1Department of Biochemistry, University of Iowa, Iowa City 52242, USA.

Biopolymers
|November 14, 1997
PubMed
Summary

DNA mobility differs between Tris-acetate-EDTA (TAE) and Tris-borate-EDTA (TBE) buffers. Borate complexation in TBE buffer increases DNA mobility by about 20% compared to TAE buffer.

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Area of Science:

  • Biophysical Chemistry
  • Molecular Biology
  • Analytical Chemistry

Background:

  • Accurate measurement of DNA free solution mobility is crucial for understanding DNA behavior in solution.
  • Standard DNA gel electrophoresis buffers like Tris-borate-EDTA (TBE) and Tris-acetate-EDTA (TAE) are widely used but their effects on DNA mobility are not fully elucidated.
  • Capillary electrophoresis (CE) offers a high-resolution technique for mobility measurements.

Purpose of the Study:

  • To measure the free solution mobility of DNA using capillary electrophoresis.
  • To compare DNA mobility in Tris-borate-EDTA (TBE) and Tris-acetate-EDTA (TAE) buffers.
  • To investigate the influence of novel capillary coatings on DNA mobility measurements.

Main Methods:

  • Capillary electrophoresis was employed to measure DNA free solution mobility.
  • Capillaries were coated with novel acrylamide monomers (N-acryloylaminoethoxyethanol or N-acryloylaminopropanol) to eliminate electroosmotic flow.
  • Measurements were conducted in Tris-borate-EDTA (TBE) and Tris-acetate-EDTA (TAE) buffers at 25 degrees C.

Main Results:

  • DNA free solution mobility in TAE buffer was measured as (3.75 +/- 0.04) x 10(-4) cm2 V-1 s-1, consistent with literature values.
  • DNA mobility in TAE buffer was independent of DNA concentration, sample size, electric field strength, and capillary coating for fragments from 400 bp to 48.5 kbp.
  • DNA mobility in TBE buffer was approximately 20% higher than in TAE buffer, measured at (4.5 +/- 0.1) x 10(-4) cm2 V-1 s-1.
  • A decrease in mobility was observed for DNA fragments smaller than 400 bp in TAE buffer.

Conclusions:

  • The choice of electrophoresis buffer significantly impacts DNA free solution mobility.
  • The higher DNA mobility in TBE buffer is likely due to the formation of nonspecific borate-deoxyribose complexes.
  • Novel capillary coatings effectively eliminate electroosmotic mobility, enabling accurate DNA mobility measurements.

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