Figure 2: Validation of the 5mC-labelling approach using model dsDNA and genomic DNA.
From: Tet-mediated covalent labelling of 5-methylcytosine for its genome-wide detection and sequencing
(a) Mass spectrometry characterization of the model reaction with a 5mC containing a 5hmC-containing 12mer DNA, annealed to a 5mC-containing 16mer DNA. The reactions were monitored by matrix-assisted laser desorption/ionization–time of flight with the calculated molecular weight and observed molecular weight indicated. (b) Comparison of the 5mC conversion and labelling by using the one-pot mTet1/β-GT method (black curve) and sequential mTet1/β-GT method (red curve). 32mer dsDNA (50 pmol) containing a single 5mC was used as the substrate, and various concentrations of recombinant mTet1 were used as indicated. The conversion ratio was evaluated by dot blot assay of the attached biotin. As shown in the figure, the one-pot mTet1/β-GT method labelled over 90% of 5mC in the model DNA. Error bars represent data with s.d. from triplicate experiments. (c) 5mC labelling on mESC genomic sample by using the one-pot mTet1/β-GT method. Various concentrations of recombinant mTet1 were used as indicated. mTet1 protein (80–120 pmol) labelled most 5mC in 1 μg genomic DNA. Error bars represent data from triplicate experiments. (d) The labelled genomic DNA (mESC) products were validated by using antibodies against 5mC (1 μg), 5fC (150 ng) and 5caC (150 ng), and dot blot assay for 5hmC (150 ng). The one-pot mTet1/β-GT method converted most 5mC to 5hmC for subsequent labelling, with no overoxidation products detected.
