DNA Methylation Analysis    

One of the fastest growing fields in biology and cancer research is epigenetics. While the underlying genetic code defines which proteins and gene products are synthesized, it is epigenetic control that defines when and where they are expressed.

Epigenetic control is often mediated by methylation of cytosine to 5-methylcytosine (5-mC) in CpG islands. Methylation and / or hydroxymethylation of CpGs near promoters is associated with gene silencing, and has important consequences for gene expression by contributing to the re-modeling of chromatin via recruitment of methyl-CpG-binding domain (MBD) protein complexes and subsequent chromatin modifiers. Disease phenotypes have been shown to arise when these activities are perturbed, resulting in undesired gene expression.

How it works

DNA methylation status can be evaluated in two basic ways:

• Whole Genome Bisulfite Conversion. Chemical treatment with bisulfite converts non-methylated cytosines into uracils that are converted to thymidines after one round of PCR. 5-methylcytosines remain unaffected. After mapping, this can be used to identify the positions of all 5-mC’s in the genome. The disadvantage of this approach is the enormous amount of sequencing that is required for large vertebrate and plant genomes (see Table 1 and Important Considerations). Currently, ACGT, Inc. is not able to do WGBC&S for genomes larger that 500 Mb.
• Affinity-based isolation of methylated DNA. The application of the affinity-based technologies appears to be the best way to differentially and specifically enrich 5-mC sequences from complex sources such as genomic DNA. In brief, genomic DNA from cultured cells or tissues is prepared, sheared, and then denatured. Then, immunoselection and immunoprecipitation can take place using the antibody directed against 5 meC and antibody binding beads, or a bead-linked MBD protein. After isolation and purification is performed, the IP’d methylated DNA is ready for any subsequent analysis by next generation sequencing in a manner similar to ChIP-Seq samples (see Sample Submission Guidelines). The output is the comparative abundance of various sequences, suggesting their level of methylation in the sample relative to other samples.

Table 1. Genome Size vs Fold Coverage

* 7 lanes is maximum per 1 flow cell