An Electrochemical Assay for Restriction Endonuclease Activity Using Graphene Monolayer

Abstract

Herein, we report a new electrochemical assay for the endonuclease activity using a graphene monolayer. The approach involves a methylene blue (MB) tagged double-stranded (ds) DNA as an enzyme substrate with a single-stranded (ss) segment at the end for anchoring on graphene through pi-pi stacking. The nuclease enzyme mediates DNA hydrolysis following the release of dsDNA-MB from the graphene surface, which reduces the electrochemical current signal. The protocol was validated for the HaeIII and EcoRV restriction endonuclease enzyme. We also assayed the activity of methyltransferase enzyme and inhibition of restriction by HaeIII. The method allows real-time measurement and quantitative assay for endonuclease activities in a simple manner. (c) 2014 The Electrochemical Society. All rights reserved.