DNA Methylation Analysis by Bisulfite Sequencing (Prot 34)
Genomic DNA methylation is one of the most important epigenetic modifications in eukaryotes. It is essential for life and its alteration is often associated with disease. In animals, most of the methylation occurs at the 5´ position of the pyrimidine ring of the cytosine. The resulting methylcytosine (mC) is mainly found in cytosine-guanine (CpG) dinucleotides. The presence of 5-mC in the promoter of specific genes alters the binding of transcriptional factors and other proteins to DNA and recruits methyl-DNA-binding proteins and histone deacetylases that compact the chromatin around the gene-transcription start site. Both mechanisms block transcription and cause gene silencing. Methylation of cytosine residues in genomic DNA plays a key role in the regulation of gene expression. There is an extensive range of methods based on the sodium bisulfite treatment for quantifying the methylation status of cytosines located in specific DNA regions. Bisulfite modification converts unmethylated cytosine to uracil, while methylated cytosine does not react. After denaturation and bisulfite modification, double-stranded DNA is obtained by primer extension and the fragment of interest is amplified by PCR.
Cancer Epigenetics Laboratory
Spanish National Cancer Centre (CNIO) - 3 Melchor Fernandez Almagro - 28029 Madrid - Madrid, Spain
Methylated DNA Immunoprecipitation (MeDIP) (Prot 33)
Methylation of cytosines can mediate epigenetic gene silencing and is the most prominent DNA modification in eukaryotes. MeDIP is an immunocapturing approach to enrich DNA that is methylated. The principle is that genomic DNA is randomly sheared by sonication and immunoprecipitated with an antibody that specifically recognizes 5-methylcytidine (5mC). This protocol has been used to generate comprehensive DNA methylation profiles on a genome scale in mammals and plants, and to identify abnormally methylated genes in cancer cells.
Institute of Molecular Genetics
CNRS UMR 5535 - 1919 Route de Mende - 34293 Montpellier, France
Plant DNA methylation analysis by "bisulfite genomic sequencing" (Prot 14)
The following "bisulfite genomic sequencing" protocol has been optimized for the determination of DNA cytosine methylation at transgene loci in plants. The conditions used are based on the method given by Pelissier and Wassenegger (2000).
Research Group Epigenetics
Department of Cytogenetics
Institute of Plant Genetics and Crop Plant Research
Corrensstraße 3 - 06466 Gatersleben, Germany