Bisulfite Modification for Analysis of DNA Methylation

Hikoya Hayatsu1, Masahiko Shiraishi2, Kazuo Negishi3

1 Faculty of Pharmaceutical Sciences, Okayama University, Tsushima, Okayama, Japan, 2 Department of Pharmaceutical Sciences, International University of Health and Welfare, Kitakanemaru, Otawara, Tochigi, Japan, 3 Kazuo Negishi, Nihon Pharmaceutical University, Komuro, Ina, Saitama, Japan
Publication Name:  Current Protocols in Nucleic Acid Chemistry
Unit Number:  Unit 6.10
DOI:  10.1002/0471142700.nc0610s33
Online Posting Date:  June, 2008
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Bisulfite is known to deaminate cytosine in nucleic acids, while 5‐methylcytosine resists this bisulfite action. For this reason, bisulfite treatment has been used for detecting 5‐methylcytosine in DNA, a minor component of eukaryotic DNA, presently recognized as playing an important role in the control of gene function. This procedure, called bisulfite genomic sequencing, is a principal method for the analysis of DNA methylation in various biological phenomena, including human diseases such as cancer. This unit describes an efficient procedure utilizing a newly developed high‐concentration bisulfite solution. Protocols for this methodology are supplemented with discussions focused on chemical aspects of the bisulfite treatment. Curr. Protoc. Nucleic Acid Chem. 33:6.10.1‐6.10.15. © 2008 by John Wiley & Sons, Inc.

Keywords: high‐concentration bisulfite; 5‐methylcytosine; cytosine deamination; bisulfite genomic sequencing; quantification of bisulfite

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Table of Contents

  • Introduction
  • Basic Protocol 1: Preparation of Bisulfite Solution
  • Basic Protocol 2: Bisulfite‐Mediated Deamination of Cytosine and 5‐Methylcytosine
  • Basic Protocol 3: Bisulfite Modification of Bulk DNA
  • Basic Protocol 4: Bisulfite Treatment for Genomic Sequencing of Methylated and Unmethylated DNA
  • Commentary
  • Literature Cited
  • Figures
  • Tables
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Basic Protocol 1: Preparation of Bisulfite Solution

  • Sodium hydrogen sulfite (sodium bisulfite; NaHSO 3; Wako, Sigma)
  • Ammonium sulfite monohydrate [(NH 4) 2SO 3⋅H 2O; Wako]
  • 50% ammonium bisulfite solution [(NH 4)HSO 3], pH 4.5 (Wako; CAS no. 10192‐30‐0)
  • NaOH (solid)
  • 10 M NaOH (dissolve 4 g solid NaOH in H 2O for a final volume of 10 mL) and/or 10 M H 2SO 4, for pH adjustment
  • 64% to 66% ammonium bisulfite solution [(NH 4) 2S 2O 5], pH 4.1 (Daito Chemical; CAS no. 10192‐30‐0)
  • Sodium sulfite (Na 2SO 3; Wako)
  • 0.1 M HCl
  • 15‐mL tubes with stoppers
  • 70°C water bath
  • pH meter equipped with electrode capable of measuring solutions in 1.5‐mL microcentrifuge tubes at a volume of ≥50 µL (e.g., Mettler Toledo InLab423)
  • Spectrophotometer

Basic Protocol 2: Bisulfite‐Mediated Deamination of Cytosine and 5‐Methylcytosine

  • 10 M bisulfite reagent (see protocol 1)
  • 2′‐Deoxycytidine
  • 0.2 M sodium phosphate buffer, pH 7.2 ( appendix 2A)
  • 5‐Methyl‐2′‐deoxycytidine
  • 1.5‐mL microcentrifuge tubes
  • 70°C water bath
  • UV spectrophotometer

Basic Protocol 3: Bisulfite Modification of Bulk DNA

  • 5 and 2 M NaOH
  • Salmon testis DNA (Sigma)
  • 10 M bisulfite solution, pH 5.4 (see protocol 1)
  • TE buffer, pH 8 ( appendix 2A)
  • 3 M NaOAc, pH 5.2
  • 99.5% ethanol, cold
  • 0.1 M MgCl 2/0.2 M Tris·Cl, pH 8 ( appendix 2A)
  • 1 mg/mL DNase I (Sigma)
  • 100 U/mL phosphodiesterase (PDase, Worthington)
  • 1000 U/mL alkaline phosphatase (calf intestine; Promega)
  • Buffer A: 100 mM potassium phosphate, pH 7 ( appendix 2A)
  • Buffer B: 90% methanol/1 mM potassium phosphate, pH 7
  • 37°C water bath or heating block
  • 0.2‐mL PCR tubes
  • Thermal cycler (Takara, BioRad)
  • NAP‐10 columns (GE Healthcare)
  • Spectrophotometer
  • Speedvac (Savant)
  • 4.6‐mm × 25‐cm HPLC column (Ultrasphere ODS, Beckman‐Coulter)
  • HPLC L‐7100 pump attached to UV detector and integrator (Hitachi)

Basic Protocol 4: Bisulfite Treatment for Genomic Sequencing of Methylated and Unmethylated DNA

  • Sodium bisulfite (NaHSO 3; Wako)
  • Ammonium sulfite monohydrate [(NH 4) 2SO 3.H 2O; Wako]
  • 50% ammonium hydrogen sulfite solution (NH 4HSO 3; Wako)
  • Restriction‐digested high‐molecular‐weight DNA
  • 5 M sodium hydroxide solution (NaOH), freshly prepared
  • Wizard DNA Clean‐Up System (Promega)
  • Ammonium acetate (Wako)
  • Yeast transfer RNA (yeast tRNA; Sigma)
  • 70% and 100% ethanol (Wako), cold
  • TE buffer, pH 8 ( appendix 2A)
  • 15‐mL tubes
  • 30° and 90°C water baths (Taitec, Thermo Minder SM‐05)
  • 1.5‐mL microcentrifuge tubes
  • Centrifuge (Sakuma M15‐IV)
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Literature Cited

Literature Cited
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