Methylated DNA Analysis

Cloned DNA methyltransferases are used for in vivo footprinting to determine nucleosome positioning on chromatin (1). DamID is used to determine sequence specificity of DNA binding proteins – via methylation profiling, after expression of the methyltransferase-DNA binding fusion protein (2). DNA (cytosine-5) Methyltransferases (DNMTs) are also used for targeted gene silencing studies by expression as fusion proteins with a specific DNA-binding domain to direct methylation events to the promoters of target genes.

DNMT isoforms include DNMT1, DNMT1b, DNMT2, DNMT3a, and DNMT3b (and a host of DNMT3 splice variants). The activity of DNMTs can be quantitated via several methods (3). These include ELISA, radioactive filter-binding assay or a real time assay using a break light oligonucleotide in which the methylation of an unmethylated 5′-CG-3′ site is enzymatically coupled to the development of a fluorescent signal (4,5). The localization of DNMTs can be revealed by specific antibodies used in immunolocalization methods.

Methyltransferases are also used to block cleavage by restriction enzymes. In vitro methylation of genomic DNA, plasmids or purified PCR products is best accomplished by CpG Methyltransferase.

A new method for DNA methylation analysis at the single base level, NEBNext^® Enzymatic Methyl-seq (EM-seq™), is now available. This enzyme-based technology minimizes damage to DNA and produces high quality libraries that enable superior detection of 5mC and 5hmC from fewer sequencing reads. More information is available here.

1. Mehrnaz Fatemi et al. (2005) Nucl. Acids Res. 33(20): e176. PMID: 16314307
2. Bas van Steensel, Jeffrey Delrow & Steven Henikoff (2001) Nature Genetics 27:304. PMID: 11242113
3. Jurkowska RZ, (2011) Methods Mol Biol, 791:157-77. PMID: 21913079
4. Carina Frauer and Heinrich Leonhardt (2009) Nucl. Acids Res. 37 (3): e22. PMID: 19129216
5. Robert J. Wood et al. (2010) Nucl. Acids Res. 38(9): e107. PMID: 20139415