Unless you’ve been stranded on a CpG island for the last 20 years, then you’ve probably heard of 5-methylcytosine (5mC) and its importance in epigenetics. Maybe you haven’t kept up with all the innovations to detect it though, since a new one seems to pop up every other month. 5mC is vital for controlling gene expression in mammals and it has been linked to countless developmental processes and diseases.
The diversity of 5mC techniques allow experiments of all scales and budgets to assess methylation. Chemical modifications, enzymatic analysis, antibodies combined with microarray and sequencing allow comprehensive analysis of 5mC.
5mC Analysis Tecniques
Bisulfite Conversion: Sodium bisulfite is the undisputed superstar of 5mC analysis, and is a chemical that alters the sequence of DNA allowing the discrimination of 5-methyl-C for C. The single-nucleotide resolution of this approach has secured bisulfite analyses as the gold-standard for 5mC studies.
WGSBS (Whole genome shotgun bisulfite sequencing): Shotgun sequencing is a processes for sequencing a lot DNA, such as a whole genome, and in WGSBS, the genomic DNA is treated with sodium bisulfite before sequencing.
RRBS (Reduced representation bisulfite sequencing):Provides the sensitivity of WGSBS without the cost by only sequencing a reduced, representative sample of the whole genome.
Enzymatic DNA Methylation Analysis: Relies on restriction enzymes that are methylation sensitive to analyze DNA methylation. This is the basis of techniques including HELP (HpaII tiny fragment Enrichment by Ligation-mediated PCR), RLGC (Restriction Landmark Genomic Scanning) and DNA methylation Restriction Enzyme Analysis (MSRE).
DNA Methylation Enrichment Methods: Enrich a sample for methylated DNA and compare the sequences obtained between experimental conditions. These methods include Methylated DNA immunoprecipitation (MeDIP), Methyl-CpG-binding domain (MBD) and others such as Agilent SureSelect that rely on sequence information to pull out specific regions, not methylation.
DNA Methylation Arrays: Designed to work with other techniques such as HELP, bisulfite, MeDIP and they can be targeted to just about any methylated or potentially methylated regions
5mC Additional Reading
Smith, Z.D., and Meissner, A. (2013). DNA methylation: roles in mammalian development. Nat. Rev. Genet. 14, 204-220.
This review gives a broad look at DNA methylation in the context of mammalian development. It gives a succinct summary of the enzymes governing 5mC catalysis and reading, genomic distribution, and various functions of 5mC.
Meaburn, E., and Schulz, R. (2012). Next generation sequencing in epigenetics: insights and challenges. Semin. Cell Dev. Biol. 23, 192-199.
This review introduces many of the techniques used in 5mC analysis via next-gen sequencing. Comparisons between the techniques as well as interesting results from various studies are reported.
Laird, P.W. (2010). Principles and challenges of genomewide DNA methylation analysis. Nat. Rev. Genet. 11, 191-203.
This review covers almost all DNA methylation techniques and compares the performance of each.
This textbook chapter provides a detailed protocol for various 5mC analyses. It provides a walk-through of several web-based tools and describes how to use the R programming language to determining meaningful differences in 5mC data between species and between disease and normal samples.