© 2007 Oxford University Press
Chemistry of Bisulfite Genomic Sequencing; Advances and Issues
1Okayama University, Faculty of Pharmaceutical Sciences, Tsushima, Okayama 700-8530, Japan.
2Nihon Pharmaceutical University, Department of Health and Medicine, Inacho, Saitama 362-0816, Japan.
3International University of Health and Welfare, Department of Pharmaceutical Sciences, Otawara, Tochigi 324-8501, Japan.
4Toyobo Co. Ltd., Biotechnology Frontier Project, Tsuruga, Fukui 914-0047, Japan.
5Shujitsu University School of Pharmacy, Nishigawara, Okayama 703-8516, Japan.
*Corresponding author. hayatsuh{at}cc.okayama-u.ac.jp
Abstract
Methylation at position 5 of cytosine in DNA plays a major role in epigenetic gene control. The methylation analysis can be performed by bisulfite genomic sequencing. Conventional procedures in this analysis include a treatment of single stranded DNA with 3–5 M sodium bisulfite at pH 5 and at 50–55° for 4–20 hr. This will convert cytosine into uracil, while 5-methylcytosine resists this deamination. Amplification by PCR of the bisulfite-treated DNA followed by sequencing reveals the positions of 5-methylcytosine in the gene. We reported recently that the whole procedure can be speeded up by use of a highly concentrated bisulfite solution, 10 M ammonium bisulfite. We also reported that urea, which has been often added to the reaction mixture with the purpose of facilitating the reaction, may not work as anticipated. This time, we would like to address the need for further investigating the chemistry of the bisulfite modification of DNA. Particularly important is to study side reactions that may occur due to the exhaustive bisulfite treatment required for achieving complete deamination of all the cytosine residues in a given sample of DNA.