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.
- Mehrnaz Fatemi et al. (2005) Nucl. Acids Res. 33(20): e176. PMID: 16314307
- Bas van Steensel, Jeffrey Delrow & Steven Henikoff (2001) Nature Genetics 27:304. PMID: 11242113
- Jurkowska RZ, (2011) Methods Mol Biol, 791:157-77. PMID: 21913079
- Carina Frauer and Heinrich Leonhardt (2009) Nucl. Acids Res. 37 (3): e22. PMID: 19129216
- Robert J. Wood et al. (2010) Nucl. Acids Res. 38(9): e107. PMID: 20139415
FAQs for Methylated DNA Analysis
- What should be considered if the methylation using SssI Methyltransferase is not going to completion?
- How does Dnmt1 differ from SssI methylase?
- Is S-adenosylmethionine (SAM) supplied with the Methyltransferase?
- Can SssI methylated DNA be used to transform E. coli?
- Does SssI Methyltransferase require magnesium in the buffer?
- What is the activity of SssI Methyltransferase in other NEBuffers, including CutSmart?
- What is the specific activity of SssI Methyltransferase?
- Can SssI Methyltransferase single stranded DNA?
- Can SssI Methyltransferase be used for generating a positive control for methylation-specific PCR or bisulfate sequencing?
- What is the molecular weight of SssI (CpG) Methyltransferase?
- Will all the sites in the DNA become methylated by SssI Methyltransferase?
- Can DNA be radiolabeled with SssI Methyltransferase?
Protocols for Methylated DNA Analysis
- Capture and Elute Step for Control DNA (E2600)
- Capture Methylated CpG DNA (E2600)
- Cross-linking of IgG to Protein A or G Beads
- Cycling Protocol Using EpiMark™ Bisulfite Conversion Kit (E3318)
- DNA Fragmentation (E2600)
- Double Digest Protocol with Standard Restriction Enzymes
- Downstream Analysis (E2600)
- Elute Captured Methylated CpG DNA (E2600)
- End-point PCR Using EpiMark Bisulfite Conversion Kit (E3318)
- EpiMark® Hot Start Taq DNA Polymerase Guidelines for PCR (M0490)
- Immunoprecipitation using Protein A/G Magnetic Beads
- Loop-mediated Isothermal Amplification (LAMP)
- mRNA Isolation using Streptavidin Magnetic Beads (NEB #S1420 and NEB #S1421)
- Optimizing Restriction Endonuclease Reactions
- Phage Display: Solution-phase Panning with Affinity Bead Capture
- Prebind MBD2a-Fc to Protein A Magnetic Beads (E2600)
- Protocol for generating 32 bp fragments from modified CpG sites in genomic DNA using FspEI
- Protocol for generating 32 bp fragments from modified CpG sites in genomic DNA using LpnPI (R0663)
- Protocol for generating 32 bp fragments from modified CpG sites in genomic DNA using MspJI
- Protocol for Glucosylation and digestion of Genomic DNA using AbaSI (#R0665)
- Protocol for use with NEBNext ChIP-Seq Library Prep Reagent Set for Illumina (E6200)
- Purification of IgG using Protein A/G Magnetic Beads
- Reaction Protocol for EpiMark® 5-hmC and 5-mC Analysis Kit (E3317)
- Reagent Preparation Using EpiMark® Bisulfite Conversion Kit (E3318)
- Wash Off Unbound DNA (E2600)
If all cells are created from the same genetic material, why are there so many different cell types? Listen to Sriharsa Pradhan, Senior Scientist, RNA Biology at NEB, as he describes how DNA is methylated and how this affects the path of reading the DNA code the same way an obstruction would derail a train off its tracks.