title: Enzyme-Linked Electrochemical Detection of PCR-Amplified Nucleotide Sequences Using Disposable Screen-Printed Sensors. Applications in Gene Expression Monitoring.
abstract: Electrochemical enzyme-linked techniques for sequence-specific DNA sensingare presented. These techniques are based on attachment of streptavidin-alkalinephosphatase conjugate to biotin tags tethered to DNA immobilized at the surface ofdisposable screen-printed carbon electrodes (SPCE), followed by production andelectrochemical determination of an electroactive indicator, 1-naphthol. Via hybridizationof SPCE surface-confined target DNAs with end-biotinylated probes, highly specificdiscrimination between complementary and non-complementary nucleotide sequences wasachieved. The enzyme-linked DNA hybridization assay has been successfully applied inanalysis of PCR-amplified real genomic DNA sequences, as well as in monitoring of planttissue-specific gene expression. In addition, we present an alternative approach involvingsequence-specific incorporation of biotin-labeled nucleotides into DNA by primerextension. Introduction of multiple biotin tags per probe primer resulted in considerableenhancement of the signal intensity and improvement of the specificity of detection.
authors.affiliation: Institute of Biophysics, v.v.i., Academy of Sciences of the Czech Republic, Kralovopolska 135, CZ-612 65 Brno, Czech Republic. petrahor@ibp.cz.
authors.forename: Petra
authors.initials: P
authors.lastname: Horaková-Brazdilova
authors.affiliation: Institute of Biophysics, v.v.i., Academy of Sciences of the Czech Republic, Kralovopolska 135, CZ-612 65 Brno, Czech Republic. fojtova@ibp.cz.
authors.forename: Miloslava
authors.initials: M
authors.lastname: Fojtova
authors.affiliation: Department of Analytical Chemistry, University of Pardubice, Nam. Cs. Legii 565, CZ-53210, Czech Republic. karel.vytras@upce.cz.
authors.forename: Karel
authors.initials: K
authors.lastname: Vytras
authors.affiliation: Institute of Biophysics, v.v.i., Academy of Sciences of the Czech Republic, Kralovopolska 135, CZ-612 65 Brno, Czech Republic. fojta@ibp.cz.
authors.forename: Miroslav
authors.initials: M
authors.lastname: Fojta
keywords: DNA hybridization; PCR; electrochemical detection; enzyme-linked assay; gene expression; primer extension