Separation of Quadruplex Polymorphism in DNA Sequences by Reversed‐Phase Chromatography

M. Clarke Miller1, Carl J. Ohrenberg2, Ashani Kuttan1, John O. Trent3

1 James Graham Brown Cancer Center, University of Louisville, Louisville, Kentucky, 2 Department of Chemistry and Biochemistry, University of North Georgia, Oakwood, Georgia, 3 Department of Medicine, University of Louisville, Louisville, Kentucky
Publication Name:  Current Protocols in Nucleic Acid Chemistry
Unit Number:  Unit 17.7
DOI:  10.1002/0471142700.nc1707s61
Online Posting Date:  June, 2015
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Abstract

This unit describes a method for the separation of a mixture of quadruplex conformations formed from the same parent sequence via reversed‐phase chromatography (RPC). Polymorphism is inherent to quadruplex formation and even relatively simple quadruplex‐forming sequences can fold into a cornucopia of possible conformations and topologies. Isolation of a specific conformation for study can be problematic. This is especially true for conformations of the human telomere sequence d(GGG(TTAGGG)3). High performance liquid chromatography (HPLC), especially reversed‐phase chromatography, has been a mainstay of nucleic acid research and purification for many decades. We have successfully applied this method to the problem of separating individual quadruplex species in the ensemble from the same parent sequence. © 2015 by John Wiley & Sons, Inc.

Keywords: RPC; quadruplex; reversed‐phase chromatography; telomere

     
 
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Table of Contents

  • Introduction
  • Strategic Planning
  • Basic Protocol 1: Reversed‐Phase Chromatography of G‐Quadruplexes
  • Support Protocol 1: Preparation of Quadruplex Samples
  • Commentary
  • Literature Cited
  • Figures
  • Tables
     
 
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Materials

Basic Protocol 1: Reversed‐Phase Chromatography of G‐Quadruplexes

  Materials
  • Distilled, deionized water (18 MΩ or HPLC grade)
  • HPLC mobile phases:
    • Buffer A: water with 5% (v/v) acetonitrile, 50 mM triethylamine, 50 mM acetic acid, 35 mM KCl, pH 7.0
    • Buffer B: water with 85% (v/v) acetonitrile, 50 mM triethylamine, 50 mM acetic acid, 35 mM KCl, pH 7.0
  • Quadruplex sample (see protocol 2Support Protocol)
  • HPLC system equipped with:
    • Waters 600 pump and controller
    • Waters 2998 UV/vis photodiode array detector
    • Waters 2707 auto‐sampler with 250‐μL sample loop
    • Waters fraction collector III (optional)
    • Computer with Waters Empower Software (optional)
  • μRPC C2/C18 ST 4.6/100 column (GE Healthcare, cat. no. 17‐5057‐01)
  • Microcentrifuge (optional)
  • 0.22‐μm syringe filter (optional)
  • 1.5‐mL microcentrifuge tubes (optional)

Support Protocol 1: Preparation of Quadruplex Samples

  Materials
  • DNA sample
  • Annealing buffer: 100 mM KCl, 25 mM K 2HPO 4, pH 7.0
  • Distilled, deionized water
  • Dialysis unit
  • 100°C hotplate or water bath
  • Aluminum foil
  • 0.22‐μm syringe filter
  • 1.5‐ or 2.0‐mL microcentrifuge tube with a screw cap and rubber gasket
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Figures

Videos

Literature Cited

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