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Enzyme-linked electrochemical DNA ligation assay using magnetic beads

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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.

Enzyme-linked electrochemical detection of a ligated DNA strand using magnetic beads. Anti-digoxigenin antibody conjugate with alkaline phosphatase (ALP) is bound to digoxigenin label of the ligated product immobilized at streptavidin-coated magnetic beads via biotin tag on its opposite end. Then substrate for ALP (napht-1-yl phosphate) is added and enzymatically converted to napht-1-ol, an electroactive indicator, which is subsequently detected electrochemically at a carbon electrode

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Acknowledgments

This work was supported by the Czech Science Foundation (grant P206/11/P739 to P.H. and P206/11/1638 to M.F.) and by the Grant Agency of the ASCR (grant IAA400040901), and by the Academy of Sciences of the Czech Republic (RVO 68081707).

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Author notes

  1. Eva Stejskalová

    Present address: Institute of Molecular Genetics, v.v.i., Academy of Sciences of the Czech Republic, Vídeňská 1083, 142 20, Prague, Czech Republic

Authors and Affiliations

  1. Institute of Biophysics, v.v.i., Academy of Sciences of the Czech Republic, Královopolská 135, 612 65, Brno, Czech Republic

    Eva Stejskalová, Petra Horáková & Miroslav Fojta

  2. Department of Medical Chemistry and Biochemistry, Faculty of Medicine and Dentistry, Palacký University, Hněvotínská 3, 775 15, Olomouc, Czech Republic

    Jan Vacek

  3. School of Biological Sciences, University of East Anglia, Norwich, NR4 7TJ, UK

    Richard P. Bowater

  4. Central European Institute of Technology, Masaryk University, Kamenice 753/5, 625 00, Brno, Czech Republic

    Miroslav Fojta

Authors
  1. Eva Stejskalová
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  2. Petra Horáková
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  3. Jan Vacek
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  4. Richard P. Bowater
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  5. Miroslav Fojta
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Correspondence to Miroslav Fojta.

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Stejskalová, E., Horáková, P., Vacek, J. et al. Enzyme-linked electrochemical DNA ligation assay using magnetic beads. Anal Bioanal Chem 406, 4129–4136 (2014). https://doi.org/10.1007/s00216-014-7811-y

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  • DOI: https://doi.org/10.1007/s00216-014-7811-y

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