DNA methylation occurs by the covalent addition of a methyl group (CH3) at the 5-carbon of the cytosine ring by DNA methyltransferases (DNMT), resulting in 5-methylcytosine (5-mC). When a CpG island is methylated in the promoter region of a gene, expression of the gene is repressed. Several human diseases, such as cancer, occur when DNA methylation is not properly established and / or maintained.
DNA methylation is a dynamic and reversible process that can result in various forms of intermediate modification. 5-mC converted from cytosine by DNMT can be hydroxylated to 5-hydroxymethylcytosine (5-hmC) by TET enzymes. 5-hmC is further modified to 5-formylcytosine (5-fC) and 5-carboxytosine (5-caC) by the same TET enzymes. DNA methylation analysis is critical to understanding and targeting changes associated with DNA methylation, which would help identify DNA methylation markers and their biological regulation.
There are several methods available to profile DNA methylation throughout the genome and in a specific region. Although cost, minimum sample input requirements, precision, speed, and throughput are important considerations, choosing the correct method is most important to the success of your analysis.
Before you begin, we suggest that you review any literature that exists related to your proposed experiment. If other researchers have already completed the groundwork, you can also use this available information as a basis for building your study. Then you and your team can employ unique variations and angles to make the study different and interesting, carefully reviewing previously completed work to identify potential obstacles or problems that previous researchers encountered.
If you are unsure which method will work for your study, we suggest starting with a simple preselection of the overall DNA methylation and hydroxymethylation status in your samples. 1) Using a rapid global DNA methylation or hydroxymethylation assay, you can assess whether and how much your sample contains 5-mC or 5-hmC.
Then you can determine the next step and which method is appropriate to use. 2) If you find that your sample contains both 5-mC and 5-hmC, bisulfite oxidative sequencing could be used. This method can identify and distinguish 5-mC from 5-hmC on a genome-wide scale with single-base resolution.
If only 5-mC is detected with little or no 5-hmC, conventional WGBS or RRBS can be performed. (3) If the sample quantity is limited or single-cell level analysis is desired, post-bisulfite sequencing can meet the requirement. (4) To analyze the methylation / hydroxymethylation of specific gene panels, such as a cancer gene panel or multiple genomic regions, directed bisulfite sequencing or oxidative directed bisulfite sequencing will work.