title: Chemistry of bisulfite genomic sequencing; advances and issues.
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 degrees 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.
authors.affiliation: Okayama University, Faculty of Pharmaceutical Sciences, Tsushima, Okayama 700-8530, Japan. hayatsuh@cc.okayam-u.ac.jp
authors.forename: Hikoya
authors.initials: H
authors.lastname: Hayatsu
authors.forename: Kazuo
authors.initials: K
authors.lastname: Negishi
authors.forename: Masahiko
authors.initials: M
authors.lastname: Shiraishi
authors.forename: Katsumi
authors.initials: K
authors.lastname: Tsuji
authors.forename: Kei
authors.initials: K
authors.lastname: Moriyama
mesh_terms: D044503:5-Methylcytosine / Q000737:chemistry*; D045744:Cell Line, Tumor; D003596:Cytosine / Q000737:chemistry; D019175:DNA Methylation; D003641:Deamination; D023281:Genomics; D006801:Humans; D000644:Quaternary Ammonium Compounds / Q000737:chemistry*; D017422:Sequence Analysis, DNA; D013447:Sulfites / Q000737:chemistry*; D014508:Urea / Q000737:chemistry
chemical_list: D000644:Quaternary Ammonium Compounds; D013447:Sulfites; D044503:5-Methylcytosine; D003596:Cytosine; C048169:ammonium sulfite; D014508:Urea