Analysis of DNA methylation using bisulphite sequencing - METHODS.info choice!
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Bisulphite treatment by Dr. Ushijima (Modified by Alexei Gratchev)

This protocol for bisulphite treatment was kindly sent to me by Dr. Toshikazu Ushijima. I was intrigued, since in this protocol you can use relatively high amounts of DNA and there is no need of generating agarose beads – the most critical step in our old protocol. Unfortunately I was not able to test it myself yet, but my colleague, Dr. Natalia Kisseljova from the Russian Cancer Research Centre tested it. She was really impressed by the results, since this protocol leads to almost 100% conversion of nonmethylated cytosines. Even the region of DNA Dr. Kisseljova considers the most difficult, was converted the best when bisulphite treatment protocol Dr. Ushijima suggested. The usage of up to 2 mkg of DNA makes it much easier to amplify your target sequence.

Solutions required

  • 6N NaOH: dissolve 6g of NaOH (pellets) in 24.2 ml H2O.
  • 4.04M NaHSO3: dissolve 1.92g of Sodium bisulphite (Na2S2O5 Sigma S9000) in 4.4 ml H2O.
  • 10 mM Hydroquinone: dissolve 11 mg of hydroquinone (Sigma H9003) in 10 ml H2O.


  1. Digest your DNA with appropriate restriction enzyme and purify using your method of choice.
  2. Dissolve up to 2 µg DNA in 19 µl H2O.
  3. For one sample prepare bisulphite conversion solution by mixing 107 µl 4.04M NaHSO3, 7µl 10 mM Hydroquinone  and 6µl 6N NaOH. Total volume – 120 µl, pH 5.00.
  4. To DNA solution add 1µl of 6N NaOH. Incubate for 15 min at 37°C.
  5. Add 120 µl conversion solution.
  6. Perform 15 cycles: denaturing for 30 sec at 95°C, bisulphite treatment for 15 min at 50°C.
  7. Desalt DNA using Wizard DNA clean up (Promega). Elute in 50µl TE buffer.
  8. Add 2.5 µl of 6N NaOH, incubate 5 minuter at RT.
  9. Precipitate DNA or purify it with the method of your choice.


  1. Kaneda A, Kaminishi M, Sugimura T, Ushijima T. Decreased expression of the seven ARP2/3 complex genes in human gastric cancers. Cancer Lett. 2004;212:203-210.
  2. Kaneda A, Tsukamoto T, Takamura-Enya T, Watanabe N, Kaminishi M, Sugimura T, Tatematsu M, Ushijima T. Frequent hypomethylation in multiple promoter CpG islands is associated with global hypomethylation, but not with frequent promoter hypermethylation. Cancer Sci. 2004;95:58-64.

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\METHODS\DNA methylation analysis\Analysis of methylation using bisulphite sequencing