DNA Methylation Analysis by Bisulfite Sequencing (BS) (PROT34)

Introduction 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 (Figure 1). After denaturation and bisulfite modification, double-stranded DNA is obtained by primer extension and the fragment of interest is amplified by PCR. Procedure DNA Extraction from Frozen Tissue Sections