Dissolve the above solutions by gently inverting with a minimum amount of mixing. Keep the solutions cold and in the dark as much as possible.

Procedure

1. Dilute 1-2 µg (no more than 2 µg) DNA in 50 µl TE buffer or water. ^{[Note 2]}
2. Digest DNA overnight with a restriction enzyme that does not digest the DNA within the region of interest. ^{[Note 3]} Alternatively, pass the DNA through a narrow-gauge needle several times to shear the DNA. This step is optional. If restriction digestion is not used, proceed to step 4.
3. Extract the DNA with phenol:chloroform:isoamyl alcohol (PCI; 25:24:1), precipitate with 1/10 volume 5 M ammonium acetate and 2 volumes ethanol at -85°C for 15 min. Centrifuge (14,000 RPM) at room temperature, remove the supernatant and wash the pellet twice with 70% ethanol. Dry the pellet and resuspend in 50 µl TE (10 mM Tris-HCl, pH7.5, 1 mM EDTA).
4. Denature the DNA by adding 5 µl freshly prepared NaOH (3 M, final concentration 0.3 M). Incubate at 37-42°C for 15-30 min. ^{[Note 4]}
5. To a siliconized microcentrifuge tube add:

   510 µl 40.5% sodium bisulfite (final concentration: 3.3M)
   30 µl 10 mM hydroquinone ^{[Note 5]}
   55 µl denatured DNA from step 4
   add water to 610 µl
    

6. Gently mix. Cover the tube with aluminum foil to shield from the light.
7. Incubate at 55°C for 8-16 h. ^{[Note 6]}
8. Purify DNA using the Geneclean II kit (Intermountain Scientific Corporation) or any other methods of your choice. Both column based and silica based purification can be used.
9. After purification, resuspend DNA and add TE to a final volume of 50 µl.
10. Denature the sample with freshly prepared NaOH (as above) and incubate at 37°C for 15 min. ^{[Note 7]}
11. Neutralize by adding ammonium acetate (pH 7.0) to 3 M.
12. Precipitate the DNA with three volumes of ethanol, centrifuge for 10 min (14,000 RPM) at room temperature, wash twice with 70% ethanol and dry under a vacuum. Resuspend in 50 µl TE, and store at -20°C wrapped in foil. The treated DNA should be used within one month as degradation may occur in the cleaned and frozen sample.
13. The resulting DNA now can be used for PCR such as bisulfite genomic sequencing PCR (BSP), methylation specific PCR (MSP).

Bisulfite modification for nanogram quantities of DNA

        For modifications of smaller quantities of DNA such as DNA from microdissected tissues. The basic changes that are made to the above protocol include the method of extraction and the addition of mussel glycogen to precipitations.

The steps are the same as above and only changes are noted.

1. In Step 5, the bisulfite reaction is scaled down to accommodate the smaller volume of DNA:
   • 255 µl sodium bisulfate
   • 15 µl hydroquinone
   • 27.5 µl denatured DNA
   • 2.5 µl water
2. In Step 9, TE is added to a final volume of 25 µl.
3. In Step 12, the precipitation is performed in the presence of 40 µg mussel glycogen at -80°C for 30 min, followed by centrifugation (14,000 RPM) at room temperature for 30 min. The DNA is resuspended in 25 µl TE.

Note

[Note 2] To ensure complete denaturation, no more than 2 µg (or less) of starting material (DNA) should be used.

[Note 3] The purpose of digesting DNA with restriction endonuceleases is to facilitate DNA denaturation to create single stranded molecules because sodium bisulfite can only react with pyrimidines that are not involved in base-paring or cytosine residues in single stranded DNA. This step is not a must and can be omitted if other measures have been taken to ensure complete denaturation before and during bisulfite modification.

[Note 4] It is important that the DNA is completely denatured prior to and in the presence of the bisulfite solution or the modification will not be complete. We found prolonged incubation (30 min) at higher temperature (42°C) yields better results.

[Note 5] Hydroquinone is an alkalizing agent. Its purpose is to prevent DNA from strand breakage because of depurination.

[Note 7] This alkali treatment is to remove the bisulfite adduct from the uracil ring. It is important that this desulphonation step is complete.

[Note 8] This protocol was last modified on Aug. 10, 2004.

1. Hayatsu H, et al. Biochemistry 1970; 9:2858.
2. Tremblay, KD. 1998 Technical Tips Online.
3. Clark, SJ et al. Nucleic Acids Res 1994;22: 1827.
4. Frommer M, et al. PNAS 1992;89:1827.
5. Oakeley EJ Pharmacology & Therapeutics 1999;84:389.
6. Fraga MF and Esteller M. Biotechniques 2002;33:632.
7. Li, LC and Dahiya, R. Bioinformatics, 2002; 18(11):1427.