title: DNA methylation: bisulphite modification and analysis.
abstract: DNA methylation is an important epigenetic modification of DNA in mammalian genomes. DNA methylation patterns are established early in development, modulated during tissue-specific differentiation and disrupted in many disease states, including cancer. To understand further the biological functions of these changes, accurate and reproducible methods are required to fully analyze the DNA methylation sequence. Here, we describe the 'gold-standard' bisulphite conversion protocol that can be used to re-sequence DNA from mammalian cells in order to determine and quantify the methylation state of a gene or genomic region at single-nucleotide resolution. The process of bisulphite treatment exploits the different sensitivities of cytosine and 5-methylcytosine (5-MeC) to deamination by bisulphite under acidic conditions--in which cytosine undergoes conversion to uracil, whereas 5-MeC remains unreactive. Bisulphite conversion of DNA, in either single tubes or in a 96-well format, can be performed in a minimum of 8 h and a maximum of 18 h, depending on the amount and quality of starting DNA.
authors.affiliation: Cancer Program, Garvan Institute of Medical Research, 384 Victoria St, Darlinghurst, Sydney, NSW 2010, Australia. s.clark@garvan.org.au
authors.forename: Susan J
authors.initials: SJ
authors.lastname: Clark
authors.forename: Aaron
authors.initials: A
authors.lastname: Statham
authors.forename: Clare
authors.initials: C
authors.lastname: Stirzaker
authors.forename: Peter L
authors.initials: PL
authors.lastname: Molloy
authors.forename: Marianne
authors.initials: M
authors.lastname: Frommer
mesh_terms: D044503:5-Methylcytosine / Q000032:analysis*; D000818:Animals; D019175:DNA Methylation; D015894:Genome, Human; D006801:Humans; D017422:Sequence Analysis, DNA; D013447:Sulfites
chemical_list: D013447:Sulfites; C005200:sodium metabisulfite; D044503:5-Methylcytosine