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Detection of Single Nucleotide Polymorphisms Using Selective Incorporation of Biotin in DNA Strand and Subsequent Enzymatic Detection at Pencil Electrode
Plucnara, Medard
;
Ecsin, E.
;
Erdem, A.
;
Fojta, Miroslav
Enzyme-linked electrochemical assay using DNA labeling by biotin followed by binding of enzyme-streptavidin conjugates has been found to be a potent tool for DNA diagnostic. This approach brings a special advantage of signal amplification due to the fact, that only very small number of enzymatic labels can produce a number of molecules of an electrochemically detectable product from an inactive substrate to obtain sufficiently strong signal. A new way of using of this technique combined with selective primer extension reaction designed for the detection of single nucleotide polymorphism is presented here. The assay was combined with measurements at a pencil graphite electrode, which is a very practical tool for potential clinical applications due to its cheapness and disposability.
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Examples of Using of Electrochemical Detection at Pencil Graphite Electrode with Enzymatic Labeling for Analysis of Nucleotide Sequence
Plucnara, Medard
;
Haroniková, Lucia
;
Špaček, Jan
;
Havran, Luděk
;
Horáková, Petra
;
Pivoňková, Hana
;
Ecsin, E.
;
Erdem, A.
;
Fojta, Miroslav
Many examples of utilization of enzymatic labeling for DNA sequence analysis has been described in literature so far. Some of them involve hybridization with complementary biotinylated probe, while others use incorporation of biotinylated nucleotides into DNA strand by DNA polymerases. Common approach is then binding of streptavidine-enzyme conjugates to biotin tags and incubation with substrate, which is converted to detectable product. Here, two recent applications using this principle are described for the detection of PCR amplicons and for SNP typing. Both techniques are combined with detection at pencil graphite electrodes.
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