ICON Probes: Synthesis and DNA Methylation Typing

互联网2013-12-31

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Abstract
Table of Contents
Materials
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Literature Cited

Abstract

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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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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Materials

Basic Protocol 1: Synthesis of N‐(2‐Aminoethyl) (4′‐Methyl‐2,2′‐Bipyridine‐4‐yl)Hexanamide

Materials

Diisopropylamine (DIPA; Wako)
Dry nitrogen (N₂ ) 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 (MgSO₄ ), anhydrous
Sodium hydroxide (NaOH)
Chloroform (CHCl₃ ; 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

Materials

6‐Chloropurine‐2′‐deoxyriboside ( S.5 ; Carbon Scientific)
4,4′‐Dimethoxytrityl chloride (DMTrCl; TCI)
Dry nitrogen (N₂ ) source
Pyridine (Wako), anhydrous
Ethyl acetate (EtOAc)
Saturated aqueous sodium bicarbonate (NaHCO₃ )
Saturated aqueous sodium chloride (NaCl)
Magnesium sulfate (MgSO₄ ), anhydrous
Silica gel (Wako; Wakogel C‐200)
Hexane (Wako)
1:1 ethyl acetate/hexane
Methanol (MeOH; Wako)
10:1 chloroform (CHCl₃ )/methanol
Dimethylformamide (DMF), anhydrous
N ,N ‐diisopropylethylamine (DIPEA; Wako)
Sodium sulfate (Na₂ SO₄ ), anhydrous
28% ammonium hydroxide (NH₄ OH)
1H ‐tetrazole (Dojindo, http://www.dojindo.com)
Dichloromethane (CH₂ Cl₂ ; Wako), anhydrous
2‐cyanoethyl tetraisopropylphosphorodiamidite (Aldrich)
Acetonitrile (CH₃ CN; 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

Materials

Bipyridine‐modified adenosine phosphoramidite ( S.8 ; see protocol 2 )
Dry CH₃ CN (DNA‐synthesis grade; Wako)
Dry CH₂ Cl₂ (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 CH₃ CN; 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/CH₂ Cl₂ ; Glen Research)
28% ammonium hydroxide (NH₄ OH)
Mobile phase A: 0.1 M triethylammonium acetate (TEAA), pH 7.0
Mobile phase B: 100% CH₃ CN
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

Materials

Genomic DNA sample (20 ng) and standard samples
ICON probes (see protocol 3 )
10× ferrate [1 M K₃ Fe(CN)₆ aqueous solution; Wako]
2× buffer mix (see recipe )
Potassium osmate(VI) dihydrate (K₂ OsO₄ ; Aldrich)
5× osmate (25 mM K₂ OsO₄ 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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Figures

Figure 8.7.1 Synthesis of N ‐(2‐aminoethyl) (4′‐methyl‐2,2′‐bipyridin‐4‐yl)hexanamide. Abbreviations: n ‐BuLi, n ‐butyl lithium; DIPA, diisopropylamine; THF, tetrahydrofuran; PyBOP, benzotriazol‐1‐yl‐oxytripyrrolidinophosphonium hexafluorophosphate; DMF, N,N ‐dimethylformamide.

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Figure 8.7.2 Synthesis of a bipyridine‐modified adenosine phosphoramidite. Abbreviations: DMTrCl, 4,4′‐dimethoxytrityl chloride; DIPEA, N,N ‐diisopropylethylamine; DMF, N,N ‐dimethylformamide.

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Figure 8.7.3 An example of methylation quantification using the calibration curve. The methylation status was calculated from the cycle number showing the maximum based on the second differential of the curve (32.6). The calibration curve was drawn based on the data obtained from the standard samples (100%, 50% and 25% methylation).

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Figure 8.7.4 Osmium complex formation. A stable methylcytosine glycol‐osmate‐bipyridine complex was formed after the reaction.

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Figure 8.7.5 Interstrand cross‐link formation by ICON probe in the presence of osmate.

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Videos

Literature Cited

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