Enzymatic analysis are tried and true method for cost effective, reliable characterization of DNA methylation. Enzymatic analysis of DNA methylation relies on restriction enzymes that are methylation sensitive such as HpaI.
HpaII and its isoschizomer MspI are the original odd couple: HpaII is very particular and won’t cut its CCGG recognition site if it’s methylated. MspI on the other hand is totally chilled out and will cut CCGG regardless of methylation. Though different, together these enzymes form the perfect pair to study DNA methylation. When examining a region with a CCGG site, if the fragment is cut by HpaII then it was unmethylated, if uncut then it was methylated. The sample is also digested with MspI as a control for proper digestion.
Several closely related techniques use these enzymes. In the HELP (HpaII tiny fragment Enrichment by Ligation-mediated PCR) assay, adapters are ligated to the HpaII and MspI digested DNA followed by and PCR amplification (Oda and Greally, 2009). The fragments are then identified using microarray (HELP-chip) or sequencing (HELP-seq).
RLGC (Restriction Landmark Genomic Scanning) involves running the fragments on a two-dimensional gel to detect methylation of many regions simultaneously. RLGC is low cost, semi-quantitative, interrogates many regions, and can be used on any organism (Ando and Hayashizaki, 2006). Finally DNA methylation Restriction Enzyme Analysis (MSRE) is very similar to HELP but often uses real-time PCR. Zymo has developed an all-in-one reaction approach to this technique.
Enzymatic DNA Methylation Analysis Additional Reading
This paper examines the details of the HELP assay, its pros and cons and how it can be combined with next-gene sequencing techniques.
This paper describes a virtual-gel approach to analysing RLGC data. It also gives a good description of the RLGC method itself.
Reference List
- Ando, Y., and Hayashizaki, Y. (2006). Restriction landmark genomic scanning. Nat. Protoc. 1, 2774-2783.
- Oda, M., and Greally, J.M. (2009). The HELP assay. Methods Mol. Biol. 507, 77-87.