DNA Methylation is a naturally occurring event that happens in both prokaryotic and eukaryotic organisms. In prokaryotes DNA methylation provides a way to protect host DNA from digestion by their own restriction enzymes that are designed to eliminate foreign DNA. In higher eukaryotes DNA methylation acts as another level of gene regulation. It has been clearly demonstrated that aberrant methylation is a widespread phenomenon in cancer and may be among the earliest changes during oncogenesis. DNA methylation has also been shown to play a central role in gene imprinting, embryonic development, X-Chromosome gene silencing and cell cycle regulation. In many plants and animals, including mammals, DNA methylation consists of the addition of a methyl group to the fifth carbon position of the cytosine pyrimidine ring via a methyltransferase enzyme. The majority of the DNA methylation in mammals is found in 5’-CpG-3’ dinucleotides, but other methylation patterns do exist. In fact, about 80 percent of all 5’-CpG-3’ dinucleotides in mammalian genomes are methylated, and the majority of the 20 percent that remain unmethylated are found within promoters or in the first exons of genes. It is obvious that the ability to quantify and detect DNA methylation efficiently and accurately is essential for the study of cancer, gene expression, genetic diseases, and many other important aspects of biology. To date, a number of methods have been developed to quantify DNA methylation such as high performance capillary electrophoresis and methylation-sensitive arbitrarily primed PCR, but currently the most commonly used technique is the bisulfite method. This technique consists of treating DNA with bisulfite, which causes unmethylated cytosines to be converted into uracil while methylated cytosines remain unchanged. In this protocol the bisulfite modified DNA is amplified by PCR and the resulting PCR products are either analyzed by DNA sequencing or restriction endonuclease digestion. The methylation status of the DNA segment is then determined by comparing the bisulfite treated DNA to the untreated DNA.

The EZ DNA Methylation™ Kit uses a simplified procedure and streamlines the bisulfite method for DNA methylation analysis. The kit is based on the three step reaction that takes place between cytosine and sodium bisulfite where cytosine is converted into uracil. The EZ DNA Methylation Kit’s innovative in-column desulphonation reaction eliminates several precipitation steps and provides researchers with consistent reaction conditions. The kit is designed to reduce template degradation, minimize DNA loss during treatment and clean-up, and result in the complete conversion of unmethylated cytosines. The recovered, treated DNA template is ideal for Methylation Specific PCR (MSP) followed by methylation analysis using restriction endonucleases, sequencing, microarrays, etc.