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Enzyme-linked electrochemical detection of DNA fragments amplified by PCR in the presence of a biotinylated deoxynucleoside triphosphate using disposable pencil graphite electrodes

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Abstract

In this report, we present a simple electrochemical detection protocol for the detection of specific PCR-amplified DNA fragments, based on incorporation of biotin tags into DNA amplicons during PCR run in the presence of a biotinylated nucleoside triphosphate. For detection, an enzyme-linked electrochemical system involving streptavidin–alkaline phosphatase conjugate attached to the biotinylated DNA, adsorbed at the surface of a disposable pencil graphite electrode, is used. The enzyme converts an inactive indicator, 1-naphthyl phosphate, into electrochemically oxidizable indicator 1-naphthol that is subsequently detected. Excellent selectivity of this fast, facile, and inexpensive analysis not requiring any sophisticated electrode modification and its applicability for off-line monitoring of DNA amplification is demonstrated. Applications of the technique include detection of the presence of specific nucleotide sequences in biological samples, such as sequences related to pathogenic microorganism or transgenes.

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Acknowledgments

This work was supported by the Czech Science Foundation (Grant P206/11/1638 to M. F. and P206/11/P739 to P. H.) and by the ASCR (RVO 68081707). A. E and M. F acknowledges to the grant of international joint project through between Turkish Scientific and Technological Research Council and the ASCR (TUBITAK Project No. 111T050).

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Authors and Affiliations

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

    Lucia Hároníková, Jan Špaček, Medard Plucnara, Petra Horáková, Hana Pivoňková, Luděk Havran & Miroslav Fojta

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

    Jan Špaček & Miroslav Fojta

  3. Analytical Chemistry Department, Faculty of Pharmacy, Ege University, Bornova, 35100, Izmir, Turkey

    Arzum Erdem

Authors
  1. Lucia Hároníková
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  2. Jan Špaček
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  3. Medard Plucnara
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  4. Petra Horáková
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  5. Hana Pivoňková
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  6. Luděk Havran
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  7. Arzum Erdem
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  8. Miroslav Fojta
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Corresponding author

Correspondence to Miroslav Fojta.

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Hároníková, L., Špaček, J., Plucnara, M. et al. Enzyme-linked electrochemical detection of DNA fragments amplified by PCR in the presence of a biotinylated deoxynucleoside triphosphate using disposable pencil graphite electrodes. Monatsh Chem 146, 849–855 (2015). https://doi.org/10.1007/s00706-015-1436-5

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  • DOI: https://doi.org/10.1007/s00706-015-1436-5

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