Biophysical Analysis of Triple‐Helix Formation

Alexandre S. Boutorine1, Christophe Escudé1

1 Muséum National d'Histoire Naturelle, Paris
Publication Name:  Current Protocols in Nucleic Acid Chemistry
Unit Number:  Unit 7.12
DOI:  10.1002/0471142700.nc0712s29
Online Posting Date:  June, 2007
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Two methods for DNA triple‐helix analysis are described in this unit: a gel‐shift assay based on the slower electrophoretic migration of a triplex in a polyacrylamide gel under nondenaturing conditions, and an optical method in which the thermal denaturation of the triple helix is followed by UV spectrophotometry. Both methods give valuable information on the characteristics of DNA triple‐helix formation and triplex stability under different conditions.

Keywords: DNA triple helix; nondenaturing gel electrophoresis; gel‐shift assay; thermal denaturation; UV spectrophotometry; melting point

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Table of Contents

  • Strategic Planning
  • Basic Protocol 1: Nondenaturing Gel‐Shift Studies of Triple‐Helix Formation
  • Basic Protocol 2: Thermal Denaturation Studies of Triple‐Helix Formation
  • Reagents and Solutions
  • Commentary
  • Literature Cited
  • Figures
  • Tables
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Basic Protocol 1: Nondenaturing Gel‐Shift Studies of Triple‐Helix Formation

  • 50 to 100 µM fluorescently or radioactively labeled duplex (target DNA) in H 2O (two separate strands or a single hairpin oligonucleotide)
  • 50 to 100 µM triplex‐forming oligonucleotide (TFO) in H 2O
  • 10× MES buffer (see recipe) or HEPES buffer (see recipe)
  • 30% (w/v) sucrose with 0.025% (w/v) bromphenol blue and 0.025% (w/v) xylene cyanol FF
  • 40% acrylamide/N,N′‐methylene‐bis‐acrylamide (19:1)
  • N,N,NN′‐Tetramethylethylenediamine (TEMED)
  • 10% (w/v) ammonium persulfate
  • 0.5‐ to 1.5‐mL polypropylene microcentrifuge tubes (e.g., Eppendorf)
  • 90°C water bath or dry heating block
  • 50‐ to 100‐mL side‐arm flask and rubber or silicone stopper
  • Sintered‐glass funnel filter, no. 3 or 4
  • Vertical electrophoresis system with glass plates (e.g., 16.5 × 14–cm plates with 0.3‐ to 0.6‐mm spacers and 12‐ to 16‐well comb with 0.5‐ to 0.8‐cm‐long teeth)
  • High‐voltage electrophoresis power supply with power regulation
  • Whatmann 3MM chromatographic paper
  • Gel dryer
  • Fluorescence gel scanner or radioactive phosphor imager (e.g., Phosphor Imager or Typhoon instrument from GE Healthcare)
  • Storage phosphor screens and cassettes for exposure to radioactive gels ()
  • Scanning image analysis software (e.g., ImageQuant v. 5, Molecular Dynamics)
  • Additional reagents and equipment for gel electrophoresis ( appendix 3B)
CAUTION: Radioactive labeling of oligonucleotides must be done in specially equipped laboratories that have permission to work with isotopes such as 32P or 33P.CAUTION: Acrylamide is toxic and must be handled using gloves and appropriate eye protection.

Basic Protocol 2: Thermal Denaturation Studies of Triple‐Helix Formation

  • 10× cacodylate buffer (see recipe)
  • 130 µM target DNA in H 2O (oligonucleotide duplex in the form of two complementary strands in equimolar concentrations)
  • 130 µM triplex‐forming oligonucleotide (TFO) in H 2O
  • Mineral oil
  • 0.5‐ to 1.5‐mL polypropylene microcentrifuge tubes (e.g., Eppendorf)
  • UV‐Vis spectrophotometer with thermostatted cells (e.g., UVIKON XL), linked to a computer with data‐acquisition software
  • Programmed thermostat (e.g., ThermoElectron, Lauda, Haake, Heto, Neslab)
  • Vacuum desiccator
  • Quartz spectrophotometer cells with black side walls and hermetic Teflon caps (e.g., Hellma QS114B quartz cells)
  • Graphing software: e.g., Microsoft Excel or KalaidaGraph (Synergy Software;
CAUTION: Cacodylic acid and its salts are highly toxic. Observe all precautions for handling and disposal of this material, including the use of gloves and appropriate eye protection.
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Literature Cited

Literature Cited
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