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Bisulfite Modification for Analysis of DNA Methylation

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Abstract
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Abstract

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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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 Materials

Sodium hydrogen sulfite (sodium bisulfite; NaHSO 3 ; Wako, Sigma)
Ammonium sulfite monohydrate [(NH 4 ) 2 SO 3 ⋅H 2 O; 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 2 O for a final volume of 10 mL) and/or 10 M H 2 SO 4 , for pH adjustment
64% to 66% ammonium bisulfite solution [(NH 4 ) 2 S 2 O 5 ], pH 4.1 (Daito Chemical; CAS no. 10192‐30‐0)
Sodium sulfite (Na 2 SO 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 Materials

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 Materials

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 Materials

Sodium bisulfite (NaHSO 3 ; Wako)
Ammonium sulfite monohydrate [(NH 4 ) 2 SO 3 .H 2 O; Wako]
50% ammonium hydrogen sulfite solution (NH 4 HSO 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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Figure 6.10.1 Determination of 5‐methylcytosine (Cm ) in DNA. Comparison of unmethylated and methylated genes by bisulfite modification. Bisulfite treatment converts C to U, which is then replaced with T by DNA polymerase during PCR amplification. Cm is inert to bisulfite treatment, and is thus maintained as C during amplification. Sequencing reveals the sites where C was replaced with T. Reproduced from Hayatsu () with permission from the Japanese Environmental Mutagen Society.

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Figure 6.10.2 Deamination of dC and 5‐methyl‐dC at pH 5.4. Circles: dC with 9 M bisulfite at 70°C. Diamonds: dC with 5.3 M sodium bisulfite at 70°C. Triangles: 5‐Me‐dC with 9 M bisulfite at 90°C. Squares: 5‐Me‐dC with 9 M bisulfite at 70°C. Reproduced from Hayatsu et al. () with permission from The Japan Academy.

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Figure 6.10.3 Bulk treatment of DNA by bisulfite. (A ) Untreated DNA and (B ) DNA treated with 9 M bisulfite for 10 min at 90°C was digested and the nucleosides were analyzed by HPLC, and monitored by absorbance at 260 nm. Elution times for 2′‐dC, 2′‐dU, 5‐Me‐2′‐dC, 2′‐dG, T, and 2′‐dA are 19, 22, 25, 26, 28, and 32 min, respectively. Reproduced from Hayatsu et al. () with permission from The Japan Academy.

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Figure 6.10.4 Conversion of cytosine to uracil. Reaction 1: The reversible addition of bisulfite across the 5,6‐double bond of cytosine is a rapid process that results in an equilibrium between C and C‐SO3 − . In acidic conditions, the equilibrium is shifted to the C‐SO3 − side. The lower the temperature, the more C‐SO3 − is formed. Reaction 2: Deamination of C‐SO3 − to U‐SO3 − is the rate‐determining step in the conversion from C to U. The effect of pH is remarkable; the optimum is pH 5 to 6. An unanticipated but important fact is that the velocity of reaction 2 increases in proportion to the bisulfite concentration. Reaction 3: Conversion of U‐SO3 − to U is an equilibrium process in the presence of bisulfite. In practice, however, after removal or dilution of bisulfite in the reaction mixture, U‐SO3 − is treated with an alkali to recover the 5,6‐double bond. With 5‐methylcytosine as reactant, both reactions 1 and 2 are much less efficient. Reproduced from Hayatsu () with permission from the Japanese Environmental Mutagen Society.

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Figure 6.10.5 pH‐Rate profile for deamination of 2′‐deoxycytidine in 7 M bisulfite. Reaction extents at 60°C and 5 min are plotted. Reproduced from Hayatsu et al.(2004) with permission from The Japan Academy.

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Figure 6.10.6 Relationship between the deamination rate and bisulfite concentration at pH 5.0 and 5.8. Solid lines: Calculated on the assumption that bisulfite participates in both reaction 1 and reaction 2 shown in Fig. . Dotted lines: Calculated on the assumption that bisulfite participates only in reaction 1. Reproduced from Hayatsu () with permission from the Japanese Environmental Mutagen Society.

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Figure 6.10.7 Two diastereomeric thymine‐bisulfite adducts. Reproduced from Hayatsu () with permission from the Japanese Environmental Mutagen Society.

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Figure 6.10.8 Efficiency of the conversion of cytosine to uracil by treatment with 9 M bisulfite (A ) at 90°C and (B ) at 70°C. Open circles indicate the ratio of conversion of nonmethylated cytosine to uracil per plasmid clone (e.g., the value 0.9 indicates 72 cytosine residues out of 80). Closed circles indicate the ratio of converted 5‐methylcytosine in the entire population of plasmid clones that were sequenced (e.g., the value 0.02 indicates a total of 1 out of 50 5‐methylcytosines). Similar experiments were performed again using independently treated samples and a similar efficiency of conversion was confirmed (data not shown). Reproduced from Shiraishi and Hayatsu () with permission from Oxford University Press.

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Literature Cited

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