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

WOS: 000353219700015In 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. [GRAPHICS] .Czech Science FoundationGrant Agency of the Czech Republic [P206/11/1638, P206/11/P739]; ASCRCzech Academy of Sciences [RVO 68081707]; Turkish Scientific and Technological Research CouncilTurkiye Bilimsel ve Teknolojik Arastirma Kurumu (TUBITAK) [111T050]; ASCR (TUBITAK)Turkiye Bilimsel ve Teknolojik Arastirma Kurumu (TUBITAK) [111T050]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)