These tools will no longer be maintained as of December 31, 2024. Archived website can be found here. PubMed4Hh GitHub repository can be found here. Contact NLM Customer Service if you have questions.


Pubmed for Handhelds

PUBMED FOR HANDHELDS

Search MEDLINE/PubMed

  • Title: Highly sensitive electrochemical methyltransferase activity assay.
    Author: Deng H, Yang X, Yeo SP, Gao Z.
    Journal: Anal Chem; 2014 Feb 18; 86(4):2117-23. PubMed ID: 24471506.
    Abstract:
    A simple and highly sensitive electrochemical DNA methyltransferase (MTase) activity assay is presented in this report. The assay employs the electrocatalytic oxidation of ascorbic acid (AA) by a threading intercalator (N,N'-bis(3-propylimidazole)-1,4,5,8-naphthalene diimide (PIND) functionalized with electrocatalytic redox Os(bpy)2Cl(+) moieties (PIND-Os)). Briefly, a double-stranded DNA (ds-DNA) containing the symmetric sequence of 5'-CCGG-3' is first immobilized on a gold electrode. The electrode is then incubated with M.SssI CpG methyltransferase (M.SssI MTase) which catalyzes the methylation of the specific CpG dinucleotides, and the electrode is subsequently treated with a restriction endonuclease HpaII which recognizes the 5'-CCGG-3' sequence. Once the CpG site in the 5'-CCGG-3' is methylated, HpaII recognition is blocked. Higher M.SssI MTase activity leads to more CpG sites being methylated and consequently impedes more the restriction endonuclease HpaII digestion process. Thus, a larger amount of ds-DNA remains on the electrode surface after the HpaII treatment. Thereafter, the electrode is incubated with PIND-Os during which PIND-Os specifically inserts itself between base pairs of ds-DNA and catalyzes the electrooxidation of AA. The methylation event corresponding to the MTase activity can therefore be monitored and amplified by the electrocatalytic oxidation of AA. A linear correlation between the catalytic oxidation current of AA and the activity of M.SssI MTase ranged from 0 to 120 U/mL with a current sensitivity of 0.046 μA mL U(-1) is obtained. The inhibitor screening ability of the developed MTase activity assay is also demonstrated.
    [Abstract] [Full Text] [Related] [New Search]