Enzyme-linked electrochemical DNA ligation assay using magnetic beads

Stejskalová, Eva, Horáková, Petra, Vacek, Jan, Bowater, Richard P. ORCID: https://orcid.org/0000-0002-2745-7807 and Fojta, Miroslav (2014) Enzyme-linked electrochemical DNA ligation assay using magnetic beads. Analytical and Bioanalytical Chemistry, 406 (17). pp. 4129-4136. ISSN 1618-2642

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Abstract

DNA ligases are essential enzymes in all cells and have been proposed as targets for novel antibiotics. Efficient DNA ligase activity assays are thus required for applications in biomedical research. Here we present an enzyme-linked electrochemical assay based on two terminally tagged probes forming a nicked junction upon hybridization with a template DNA. Nicked DNA bearing a 5' biotin tag is immobilized on the surface of streptavidin-coated magnetic beads, and ligated product is detected via a 3' digoxigenin tag recognized by monoclonal antibody-alkaline phosphatase conjugate. Enzymatic conversion of napht-1-yl phosphate to napht-1-ol enables sensitive detection of the voltammetric signal on a pyrolytic graphite electrode. The technique was tested under optimal conditions and various situations limiting or precluding the ligation reaction (such as DNA substrates lacking 5′-phosphate or containing a base mismatch at the nick junction, or application of incompatible cofactor), and utilized for the analysis of the nick-joining activity of a range of recombinant Escherichia coli DNA ligase constructs. The novel technique provides a fast, versatile, specific, and sensitive electrochemical assay of DNA ligase activity.

Item Type:	Article
Uncontrolled Keywords:	electrochemistry,enzyme labeling,dna ligase,nicked dna,magnetic beads,immobilized assays
Faculty \ School:	Faculty of Science > School of Biological Sciences
UEA Research Groups:	Faculty of Science > Research Groups > Molecular Microbiology Faculty of Science > Research Centres > Centre for Molecular and Structural Biochemistry Faculty of Science > Research Groups > Biosciences Teaching and Education Research
Depositing User:	Pure Connector
Date Deposited:	11 Jun 2014 12:50
Last Modified:	18 Apr 2023 13:30
URI:	https://ueaeprints.uea.ac.uk/id/eprint/48703
DOI:	10.1007/s00216-014-7811-y

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