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Denaturing reversed-phase HPLC using a mobile phase containing urea for oligodeoxynucleotide analysis.

Suntara Fueangfung1, Yinan Yuan, Shiyue Fang

  • 1a Department of Chemistry , Michigan Technological University , Houghton , Michigan , USA.

Nucleosides, Nucleotides & Nucleic Acids
|June 28, 2014
PubMed
Summary

This study introduces a novel denaturing method for reversed-phase high-performance liquid chromatography (RP-HPLC) using urea in the mobile phase at room temperature, enhancing oligonucleotide and double-stranded DNA analysis.

Keywords:
DNADenaturing HPLColigodeoxynucleotideurea

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Area of Science:

  • Biochemistry
  • Analytical Chemistry
  • Molecular Biology

Background:

  • Reversed-phase high-performance liquid chromatography (RP-HPLC) is a common technique for analyzing nucleic acids.
  • Denaturation of nucleic acids in RP-HPLC typically requires elevated column temperatures, which can be energy-intensive and potentially degrade samples.
  • There is a need for alternative, milder denaturation methods in RP-HPLC for oligonucleotide analysis.

Purpose of the Study:

  • To describe an alternative method for denaturing nucleic acids during RP-HPLC using urea in the mobile phase at room temperature.
  • To demonstrate the efficacy of this urea-based denaturation method for analyzing oligodeoxynucleotides (ODNs) and double-stranded (ds) ODNs.
  • To compare the chromatographic profiles of nucleic acids under normal and denaturing conditions using this new method.

Main Methods:

  • Utilized reversed-phase high-performance liquid chromatography (RP-HPLC).
  • Employed a mobile phase containing urea to achieve denaturation at room temperature.
  • Analyzed a 61-mer oligodeoxynucleotide (ODN) and double-stranded (ds) ODNs under both normal and denaturing conditions.

Main Results:

  • Under normal RP-HPLC conditions, the 61-mer ODN showed multiple peaks, which merged into a single peak under the urea-induced denaturing conditions.
  • Double-stranded ODNs (dsODNs) exhibited broad single peaks under normal conditions.
  • The urea-based denaturing method resulted in the splitting of broad dsODN peaks into two sharp, distinct peaks, indicating successful denaturation and separation.

Conclusions:

  • Urea in the mobile phase provides an effective alternative to elevated temperatures for denaturing nucleic acids in RP-HPLC.
  • This room-temperature denaturation method improves the resolution and peak shape for analyzing both single-stranded and double-stranded ODNs.
  • The described method offers a potentially more accessible and less energy-intensive approach for nucleic acid analysis via RP-HPLC.