Related Experiment Video
Updated: Jan 28, 2026
Continuous Fluorescence-Based Endonuclease-Coupled DNA Methylation Assay to Screen for DNA Methyltransferase Inhibitors
Published on: August 5, 2022
Electrochemical determination of the activity of DNA methyltransferase based on the methyl binding domain protein and
Lingsong Lu1, Bei Liu2, Jianhang Leng3
1Department of Central Laboratory, Affiliated Hangzhou First People's Hospital, Zhejiang University School of Medicine, Hangzhou, Zhejiang Province, 310006, People's Republic of China. lls0917@163.com.
Abstract:
An electrochemical method is described for the determination of the activity of the DNA methyltransferase (MTase). The assay was based on the use of a commercially available customized electromagnetic modular detector, which consisted of a magnetic switch, electrical connectors and a screen-printed electrode modified with graphene oxide. The biotinylated single-strand DNA (ss-DNA) S1 was absorbed by streptavidin-modified magnetic beads (MBs) via streptavidin-biotin interaction. The biotinylated ss-DNA S1 was hybridized with the complementary ss-DNA S2. After the symmetrical sequences 5'-CCGG-3' of the duplex DNA (ds-DNA) were methylated by M. SssI CpG methyltransferase (M. SssI MTase), the symmetrical sequences 5'-CCGG-3' in the ds-DNA were recognized by glutathione S-transferase (GST) tagged methyl CpG binding protein 2 (MeCP2). The unmethylated 5'-CCGG-3' sequences were specifically cleaved by HpaII restriction endonuclease. After magnetic separation and washing, HRP-labeled GST tag monoclonal antibody and H2O2 were used as a tracer label and enzyme substrate, respectively. Electrochemical measurement was carried out at pH 7.4 in the presence of 50 μM thionine and 0.5 mM H2O2. Stepwise changes in the microscopic features of the SPE surface upon the formation of each layer were studied by scanning electron microscopy. Cyclic voltammetry and differential pulse voltammetry were used to characterize the electrochemical behavior of the different modified electrodes. Under the optimal conditions, the activity of M. SssI MTase can be determined in the activity range of 0.5-125 unit·mL-1 with a detection limit of 0.2 unit·mL-1 (at an S/N ratio of 3). The sensitivity of the immunoassay is 0.489 μA·μM-1·cm-2. Graphical abstract Schematic presentation of the electrochemical immunosensor for the determination of the activity of M. SssI CpG methyltransferase (M. SssI MTase). It is based on an electromagnetic modular detector and the use of glutathione S-transferase tagged methyl CpG binding protein 2 (GST-MeCP2).
More Related Videos
12:36In vitro tRNA Methylation Assay with the Entamoeba histolytica DNA and tRNA Methyltransferase Dnmt2 Ehmeth Enzyme
Published on: October 19, 2010
13:21Comprehensive DNA Methylation Analysis Using a Methyl-CpG-binding Domain Capture-based Method in Chronic Lymphocytic Leukemia Patients
Published on: June 16, 2017
Related Concept Videos
02:26Conservation of Protein Domains Over Different Proteins
A limited set of protein domains often duplicate and recombine during evolution. These domains can be organized in different combinations to...
01:22Protein-Drug Binding: Determination Methods
Indirect methods involve isolating the bound drug from its free form in biological samples such as blood, serum, or plasma. These techniques aim to measure the percentage of drugs bound to proteins. Equilibrium dialysis is a commonly used method where the free drug concentration at equilibrium is measured by separating the bound...
01:03Single-Strand DNA Binding Proteins
03:06From DNA to Protein
02:36RNA Polymerase II Accessory Proteins
01:26What is an Electrochemical Gradient?
The chemical gradient relies on differences in the abundance of a substance on the outside versus the inside of a cell and flows from areas of high to low ion concentration. In contrast, the electrical gradient revolves around an...