Resolution of Quadruplex Polymorphism by Size‐Exclusion Chromatography

M. Clarke Miller1, John O. Trent1

1 James Graham Brown Cancer Center, University of Louisville, Louisville, Kentucky
Publication Name:  Current Protocols in Nucleic Acid Chemistry
Unit Number:  Unit 17.3
DOI:  10.1002/0471142700.nc1703s45
Online Posting Date:  June, 2011
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Abstract

This unit describes a method for separation of quadruplex species formed from the same sequence via size‐exclusion chromatography (SEC). Polymorphism is inherent to quadruplex formation, and even relatively simple quadruplex‐forming sequences, such as the human telomere sequence d(GGG(TTAGGG)3), can form a myriad of possible configurations. HPLC, especially using reversed‐phase and anion‐exchange methods, has been a mainstay of nucleic acids research and purification for many decades. These methods have been applied for separation of individual quadruplex species formed in a mixture from the same parent sequence. Curr. Protoc. Nucleic Acid Chem. 45:17.3.1‐17.3.18. © 2011 by John Wiley & Sons, Inc.

Keywords: SEC; quadruplex; size‐exclusion chromatography; telomere

     
 
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Table of Contents

  • Introduction
  • Basic Protocol 1: Size‐Exclusion Chromatography of G‐Quadruplexes
  • Support Protocol 1: Preparation of Quadruplex Sample
  • Commentary
  • Literature Cited
  • Figures
  • Tables
     
 
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Materials

Basic Protocol 1: Size‐Exclusion Chromatography of G‐Quadruplexes

  Materials
  • Distilled, deionized water
  • HPLC mobile phase: 100 mM KCl in 25 mM K 2HPO 4, pH 7.0 (prepare with K 2HPO 4 and titrate from pH ∼9.0 to pH 7.0 with HCl so that the total K+ concentration is known)
  • Gel filtration calibration kit LMW (GE Healthcare, cat. no. 28‐4038‐41)
  • Glycerol
  • Quadruplex sample (see protocol 2)
  • HPLC system with:
    • Waters 600 pump and controller
    • Waters 2998 UV‐vis photodiode array detector
    • Waters 2707 autosampler with 250‐µl sample loop
    • Waters fraction collector III (optional)
    • Computer with Waters Empower Software (optional)
  • Superdex 75 10/300 size‐exclusion column (GE Healthcare, cat. no. 17‐5174‐01)
  • 1.5‐mL microcentrifuge tubes (optional)

Support Protocol 1: Preparation of Quadruplex Sample

  Materials
  • DNA sample
  • Annealing buffer
  • Distilled, deionized water
  • 0.22‐µm filter
  • Dialysis unit
  • 1‐ to 2‐liter beakers
  • 100°C hotplate or water bath
  • Aluminum foil
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Figures

Videos

Literature Cited

Literature Cited
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