Bisulfite Sequencing of DNA

Russell P. Darst1, Carolina E. Pardo1, Lingbao Ai1, Kevin D. Brown1, Michael P. Kladde1

1 University of Florida College of Medicine, Gainesville, Florida
Publication Name:  Current Protocols in Molecular Biology
Unit Number:  Unit 7.9
DOI:  10.1002/0471142727.mb0709s91
Online Posting Date:  July, 2010
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Exact positions of 5‐methylcytosine (m5C) on a single strand of DNA can be determined by bisulfite genomic sequencing (BGS). Treatment with bisulfite ion preferentially deaminates unmethylated cytosines, which are then converted to uracil upon desulfonation. Amplifying regions of interest from deaminated DNA and sequencing products cloned from amplicons permits determination of methylation at single‐nucleotide resolution along single DNA molecules, which is not possible with other methylation analysis techniques. This unit describes a BGS technique suitable for most DNA sources, including formaldehyde‐fixed tissue. Considerations for experimental design and common sources of error are discussed. Curr. Protoc. Mol. Biol. 91:7.9.1‐7.9.17. © 2010 by John Wiley & Sons, Inc.

Keywords: DNA methylation; epigenetics; chromatin

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Table of Contents

  • Introduction
  • Basic Protocol 1: Bisulfite Conversion of DNA
  • Alternate Protocol 1: Rapid Bisulfite Conversion of DNA
  • Alternate Protocol 2: Bisulfite Genomic Sequencing of DNA from Formalin‐Fixed Paraffin‐Embedded Tissue
  • Basic Protocol 2: Molecular Amplification and Cloning of Deaminated DNA
  • Support Protocol 1: Primer Design for PCR of Deaminated DNA
  • Reagents and Solutions
  • Commentary
  • Literature Cited
  • Figures
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Basic Protocol 1: Bisulfite Conversion of DNA

  • DNA of interest: up to 2 µg genomic DNA or 1 ng purified DNA fragment in 20 µl total volume (see protocol 3)
  • 0.5 M Na 2EDTA, pH 8.0 (store indefinitely at room temperature)
  • 3 N NaOH (see recipe)
  • Glycogen, molecular biology grade (optional; store indefinitely at −20°C)
  • Degassed, distilled H 2O (degassed dH 2O; see recipe)
  • 100 mM hydroquinone, freshly prepared (see recipe)
  • Sodium bisulfite/sodium metabisulfite: unopened, single‐use 5‐g vial (Sigma catalog no. 243973)
  • 80% (v/v) ethanol
  • TE buffer, pH 8.0 ( appendix 22), filter sterilized
  • 20‐ml glass scintillation vial
  • 5‐ml serological pipet and pipetting device
  • 50°C water bath
  • Thermal cycler with heated lid
  • Minicolumn‐based DNA purification kit (e.g., Zymo Research, EZ Bisulfite DNA Clean‐Up Kit, catalog no. D5026)
CAUTION: Bisulfite‐ and sulfite‐based reagents are harmful if swallowed or inhaled; they cause irritation to skin, respiratory tract, and eyes; pose risk of serious eye damage; may cause allergic respiratory reactions; and react with acids and water to release toxic sulfur dioxide gas. Use in a well‐ventilated chemical fume hood and wear protective gear, including gloves and dust and face masks.NOTE: Use only molecular‐biology‐grade water (i.e., DNase and nucleic acid free) in all steps and solutions.

Alternate Protocol 1: Rapid Bisulfite Conversion of DNA

  • 45% ammonium bisulfite solution (Spectrum catalog no. A1145; aliquot to the rim in 5‐g glass vials with Teflon PTFE‐lined caps so there is minimal excess volume and cap tightly; store at room temperature in a vessel containing Drierite)
  • Sodium bisulfite (≥90%)
  • Ammonium sulfite monohydrate (≥92%)
  • 70°C water bath
CAUTION: The bisulfite‐ and sulfite‐based reagents are harmful if swallowed or inhaled; they cause irritation to skin, respiratory tract, and eyes; pose risk of serious eye damage; may cause allergic respiratory reactions; and react with acids and water to release toxic sulfur dioxide gas. Use in a well‐ventilated chemical fume hood and wear protective gear, including gloves and dust and face masks.

Alternate Protocol 2: Bisulfite Genomic Sequencing of DNA from Formalin‐Fixed Paraffin‐Embedded Tissue

  • Tissue blocks or microdissected tissue
  • Xylene
  • Ethanol
  • Lysis buffer (see recipe)
  • TE buffer, pH 8.0 ( appendix 22), filter sterilized
  • Microtome
CAUTION: Xylene is harmful when swallowed or in contact with skin. Vapor has a narcotic effect and can induce unconsciousness which may be fatal. May form a flammable/explosive vapor‐air mixture during use. Use in a well‐ventilated chemical fume hood and wear protective gear, including gloves and face mask. Discard according to institutional guidelines.

Basic Protocol 2: Molecular Amplification and Cloning of Deaminated DNA

  • Deaminated DNA as template (see protocol 1 or protocol 2 or protocol 32)
  • Distilled, molecular biology–grade water (dH 2O)
  • 25 mM and 1 M MgCl 2
  • 2.5 mM dNTP mix: 2.5 mM each dATP, dCTP, dGTP, and dTTP in water (store 100‐µl aliquots indefinitely at –20°C, discarding after five freeze‐thaw cycles)
  • 20 µM primers (a1 and a2, or b1 and b2; see protocol 5 and Fig. )
  • Hot‐start Taq DNA polymerase (HotStarTaq, Qiagen catalog no. 203203) with supplied buffer
  • 5 mM trimethylammonium chloride (TMAC; optional)
  • Commercial kit for DNA purification (e.g., Qiaex II gel extraction kit, Qiagen catalog no. 20051)
  • Vector plasmid
  • Restriction endonucleases with supplied buffer (optional, depending on primer design or if genomic DNA needs to be digested)
  • T4 DNA ligase and supplied buffer
  • Competent DH5α, DH10B, or Top10 E. coli
  • SOC (see recipe)
  • LB medium with appropriate selective antibiotic (unit 1.1)
  • LB X‐gal plates (see recipe)
  • 20 µM primers that anneal upstream and downstream of clone insertion site in the vector
  • Generic Taq polymerase with supplied buffer
  • Thermal cycler
  • Additional reagents and equipment for agarose gel electrophoresis (unit 2.5) and PCR optimization (unit 15.1)
CAUTION: Ethidium bromide is a suspected mutagen, is harmful if swallowed, is very toxic if inhaled, and is irritating to eyes, respiratory system, and skin. There is a possible risk of irreversible effects. Wear protective gear, including gloves and dust and face masks.
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Key Reference
   Frommer et al., 1992. See above.
  First demonstration of the utility of bisulfite conversion in sequencing of 5‐methylcytosine.
Internet Resources
  CpGviewer at the University of Leeds.
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