Electrochemical Detection of SNP in Human Mitochondrial DNA using Cyclic Primer Extension with Biotinylated Nucletides and Enzymatic Labeling at Disposable Pencil Graphite Electrodes

Abstrakt

A novel method of SNP typing in humanmitochondrial DNA utilizing enzymatic labeling andelectrochemical detection at disposable pencil graphiteelectrodes is described. The procedure is based onamplification of DNA stretches by cyclic primer extension(PEx) of SNP-specific diagnostic primers in a mixture ofbiotinylated and natural nucleotides. The diagnosticprimers are designed to recognize, by its 3’-terminalnucleotide, the SNP-site in target template. Under opti-mized conditions of the PEx reaction, efficient polymerasesynthesis of biotin-labeled strands takes place only in thecase of full complementarity between the diagnosticprimer and the target SNP site. There is also benefit from introducing many biotin molecules per extended DNAstrand, resulting in another level of signal amplification.After adsorption of biotinylated PEx products at theelectrode surface, streptavidin-alkaline phosphatase con-jugate was bound to the biotin tags, 1-naphthol wasenzymatically produced and electrochemically detected.Several critical steps and parameters of the assay,including termination of 3’-OH ends of residual amplifica-tion primers, temperature for annealing of diagnosticprimers, relative amount of biotinylated deoxynucleosidetriphosphate in the PEx mixture and number of PExcycles were optimized in this study to attain best SNPresolution, and reduction of time needed for the analysis.