ICON Probes: Synthesis and DNA Methylation Typing

Kazuki Tainaka1, Akimitsu Okamoto2

1 Laboratory for Synthetic Biology, Quantitative Biology Center (QBiC), RIKEN, Kobe, Hyogo, Japan, 2 PRESTO, Japan Science and Technology Agency, Kawaguchi, Saitama, Japan
Publication Name:  Current Protocols in Nucleic Acid Chemistry
Unit Number:  Unit 8.7
DOI:  10.1002/0471142700.nc0807s47
Online Posting Date:  December, 2011
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DNA methylation and demethylation significantly affect the deactivation and activation processes of gene expression, respectively. The determination of the location and frequency of DNA methylation is important for the elucidation of the mechanisms of cell differentiation and carcinogenesis and may be a useful and effective index for cancer diagnosis. We have developed an artificial DNA probe that induces a methylation detection reaction of a target cytosine in a long DNA sequence (ICON probe). This artificial DNA allows the rapid detection of a methyl group attached at the C5 position of the target cytosine. In addition, there is no nonspecific cleavage of genomic DNA in this reaction. The ICON probe also facilitates the quantification of methylation at the target cytosine using a small amount of genomic DNA sample. This unit provides a procedure for synthesizing bipyridine‐modified adenosine phosphoramidite and preparation of ICON probes. Additionally, the protocol for the methylation quantification experiments by quantitative PCR utilizing ICON probes is also presented. Curr. Protoc. Nucleic Acid Chem. 47:8.7.1‐8.7.17. © 2011 by John Wiley & Sons, Inc.

Keywords: ICON probe; DNA methylation; osmium; artificial DNA

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Table of Contents

  • Introduction
  • Basic Protocol 1: Synthesis of N‐(2‐Aminoethyl) (4′‐Methyl‐2,2′‐Bipyridine‐4‐yl)Hexanamide
  • Basic Protocol 2: Synthesis of a Bipyridine‐Modified Adenosine Phosphoramidite
  • Basic Protocol 3: Synthesis, Isolation, and Characterization of the Icon Probe
  • Basic Protocol 4: Methylation Quantification Experiments Using Icon Probes and Quantitative PCR
  • Reagents and Solutions
  • Commentary
  • Literature Cited
  • Figures
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Basic Protocol 1: Synthesis of N‐(2‐Aminoethyl) (4′‐Methyl‐2,2′‐Bipyridine‐4‐yl)Hexanamide

  • Diisopropylamine (DIPA; Wako)
  • Dry nitrogen (N 2) source
  • Tetrahydrofuran (THF; Wako), anhydrous
  • Acetone (Wako)
  • 1.6 M n‐butyl lithium solution in hexane (n‐BuLi; Wako)
  • 4,4′‐dimethyl‐2,2′‐dipyridyl ( S.1; Aldrich)
  • 5‐bromovaleronitrile (TCI)
  • 1 N hydrochloric acid (HCl)
  • Ethyl acetate (EtOAc; Wako)
  • Saturated aqueous sodium chloride (NaCl)
  • Magnesium sulfate (MgSO 4), anhydrous
  • Sodium hydroxide (NaOH)
  • Chloroform (CHCl 3; Wako)
  • Benzotriazol‐1‐yl‐oxytripyrrolidinophosphonium hexafluorophosphate (PyBOP; Novabiochem)
  • N,N‐Dimethylformamide (DMF; Wako), anhydrous
  • Ethylenediamine (Wako)
  • Round‐bottom flasks (1 L, 500 mL, 200 mL, and 50 mL), oven dried
  • Teflon‐coated magnetic stir bars
  • Rubber septa
  • Magnetic stir plate
  • Dewar flask
  • Syringe
  • Separatory funnels (1 L and 300 mL)
  • Rotary evaporator with diaphragm pump
  • Vacuum oil pump
  • Reflux condenser
  • Silicone oil bath

Basic Protocol 2: Synthesis of a Bipyridine‐Modified Adenosine Phosphoramidite

  • 6‐Chloropurine‐2′‐deoxyriboside ( S.5; Carbon Scientific)
  • 4,4′‐Dimethoxytrityl chloride (DMTrCl; TCI)
  • Dry nitrogen (N 2) source
  • Pyridine (Wako), anhydrous
  • Ethyl acetate (EtOAc)
  • Saturated aqueous sodium bicarbonate (NaHCO 3)
  • Saturated aqueous sodium chloride (NaCl)
  • Magnesium sulfate (MgSO 4), anhydrous
  • Silica gel (Wako; Wakogel C‐200)
  • Hexane (Wako)
  • 1:1 ethyl acetate/hexane
  • Methanol (MeOH; Wako)
  • 10:1 chloroform (CHCl 3)/methanol
  • Dimethylformamide (DMF), anhydrous
  • N,N‐diisopropylethylamine (DIPEA; Wako)
  • Sodium sulfate (Na 2SO 4), anhydrous
  • 28% ammonium hydroxide (NH 4OH)
  • 1H‐tetrazole (Dojindo, http://www.dojindo.com)
  • Dichloromethane (CH 2Cl 2; Wako), anhydrous
  • 2‐cyanoethyl tetraisopropylphosphorodiamidite (Aldrich)
  • Acetonitrile (CH 3CN; Wako), anhydrous
  • Round‐bottom flasks (2 L, 500 L ,300 mL, 100 mL)
  • Teflon‐coated magnetic stir bars
  • Rubber septa
  • Magnetic stir plate
  • Separatory funnels (2 L, 500 mL, and 200 mL)
  • Rotary evaporator with diaphragm pump
  • Chromatography columns (diameter, 7 cm; length, 50 cm)
  • TLC sheets (Merck, silica gel 60 F254 aluminum sheets)
  • UV lamp (254 nm)
  • Silicone oil bath
  • Vacuum oil pump
  • Additional reagents and equipment for TLC (see appendix 3D)

Basic Protocol 3: Synthesis, Isolation, and Characterization of the Icon Probe

  • Bipyridine‐modified adenosine phosphoramidite ( S.8; see protocol 2)
  • Dry CH 3CN (DNA‐synthesis grade; Wako)
  • Dry CH 2Cl 2 (Wako)
  • Controlled Pore Glass (CPG) for the synthesis of 3′‐phosphorylated oligonucleotides (1 µmol; Glen Research)
  • 2′‐Deoxyribonucleoside phosphoramidites (Glen Research)
  • Activator (0.45 M sublimed 1H‐tetrazole in CH 3CN; Glen Research)
  • Cap Mix A (1:1 THF/acetic anhydride; Glen Research)
  • Cap Mix B (10% 1‐methylimidazole in 8:1 THF/pyridine; Glen Research)
  • Oxidizing solution (0.02 M iodine in THF/pyridine/water; Glen Research)
  • Deblocking Mix (3% trichloroacetic acid/CH 2Cl 2; Glen Research)
  • 28% ammonium hydroxide (NH 4OH)
  • Mobile phase A: 0.1 M triethylammonium acetate (TEAA), pH 7.0
  • Mobile phase B: 100% CH 3CN
  • Liquid nitrogen
  • Calf intestine alkaline phosphatase (Nippon Gene, http://nippongene.com)
  • Snake venom phosphodiesterase (Boehringer Ingelheim)
  • P1 nuclease (Wako)
  • Automated DNA synthesizer (ABI 392 DNA/RNA synthesizer, Applied Biosystems; also see appendix 3C)
  • Vials and bottles for attachment of the phosphoramidites and reagents to the synthesizer
  • Screw‐capped tube (Assist)
  • SpeedVac evaporator
  • Membrane filter (0.45 µm; Cosmonice filter W 13 mm; Nacalai Tesque; http://www.nacalai.co.jp/)
  • Reversed‐phase HPLC column (10 × 150 mm or 4.6 × 150 mm Chemco CHEMCOBOND 5‐ODS‐H column; also see unit 10.5)
  • Centrifuge tubes (Falcon)
  • Dewar flask
  • Freeze dryer
  • Additional reagents and equipment for automated DNA synthesis ( appendix 3C), oligoribonucleotide purification by HPLC (unit 10.5), and MALDI‐TOF mass spectrometry (unit 10.1)

Basic Protocol 4: Methylation Quantification Experiments Using Icon Probes and Quantitative PCR

  • Genomic DNA sample (20 ng) and standard samples
  • ICON probes (see protocol 3)
  • 10× ferrate [1 M K 3Fe(CN) 6 aqueous solution; Wako]
  • 2× buffer mix (see recipe)
  • Potassium osmate(VI) dihydrate (K 2OsO 4; Aldrich)
  • 5× osmate (25 mM K 2OsO 4 aqueous solution)
  • Takara Ex Taq kit
  • 20 µL each of 10 µm forward and reverse PCR primers (GeneDesign)
  • SYBR Green I (Cambrex)
  • Reaction tubes (BMBio, http://www.bmbio.com)
  • Micro Bio‐Spin columns (Bio‐Rad)
  • High‐speed refrigerated microcentrifuge (Kubota)
  • PCR tubes (BMBio, cat. no. PCR‐02F)
  • Real‐time rotary analyzer (Rotor‐Gene; Corbett Life Science, http://www.corbettlifescience.com)
  • Spreadsheet software (e.g., Microsoft Excel)
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Literature Cited

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