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Nucleic acid quantification by delta pH measurement.

F U Gast1, A Pingoud

  • 1Institut für Biochemie, Justus-Liebig-Universität Giessen, Heinrich-Buff-Ring 58, D-35392, Giessen, Germany. frank-ulrich.gast@gmd.de

Journal of Biotechnology
|June 14, 2001
PubMed
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This study presents a new enzymatic method for quantifying nucleic acids using pH electrodes and nucleases. This robust technique measures cleaved phosphodiester bonds, offering an alternative to optical assays for nucleic acid and nuclease activity determination.

Area of Science:

  • Biochemistry
  • Molecular Biology
  • Analytical Chemistry

Background:

  • Nucleic acid quantification is crucial in molecular biology.
  • Current methods like UV absorbance, fluorimetry, and hybridization have limitations.
  • Spectroscopic methods are sensitive to contaminants, while hybridization is time-consuming.

Purpose of the Study:

  • To develop a simple, robust, and accurate method for quantifying microgram quantities of nucleic acids.
  • To offer an alternative to existing optical assays for nucleic acid and nuclease activity determination.

Main Methods:

  • Utilized highly active nonspecific nucleases for nucleic acid cleavage.
  • Employed a temperature-controlled mixing chamber with miniaturized pH electrodes.
  • Measured the acidification resulting from the cleavage of phosphodiester bonds.

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Main Results:

  • Successfully quantified microgram nucleic acid quantities.
  • The method is independent of nucleic acid composition and secondary structure.
  • Demonstrated a simple and robust alternative to optical assays.

Conclusions:

  • The developed enzymatic assay provides a reliable method for nucleic acid quantitation.
  • This approach is suitable for determining total nucleic acid concentration and nuclease activity.
  • The method overcomes limitations of traditional spectroscopic and hybridization techniques.