Journal
SENSORS
Volume 8, Issue 1, Pages 193-210Publisher
MDPI
DOI: 10.3390/s8010193
Keywords
electrochemical detection; enzyme-linked assay; DNA hybridization; primer extension; PCR; gene expression
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Electrochemical enzyme-linked techniques for sequence-specific DNA sensing are presented. These techniques are based on attachment of streptavidin-alkaline phosphatase conjugate to biotin tags tethered to DNA immobilized at the surface of disposable screen-printed carbon electrodes (SPCE), followed by production and electrochemical determination of an electroactive indicator, 1-naphthol. Via hybridization of SPCE surface-confined target DNAs with end-biotinylated probes, highly specific discrimination between complementary and non-complementary nucleotide sequences was achieved. The enzyme-linked DNA hybridization assay has been successfully applied in analysis of PCR-amplified real genomic DNA sequences, as well as in monitoring of plant tissue-specific gene expression. In addition, we present an alternative approach involving sequence-specific incorporation of biotin-labeled nucleotides into DNA by primer extension. Introduction of multiple biotin tags per probe primer resulted in considerable enhancement of the signal intensity and improvement of the specificity of detection.
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