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

Abstract

AbstractA novel method of SNP typing in human mitochondrial DNA utilizing enzymatic labeling and electrochemical detection at disposable pencil graphite electrodes is described. The procedure is based on amplification of DNA stretches by cyclic primer extension (PEx) of SNP‐specific diagnostic primers in a mixture of biotinylated and natural nucleotides. The diagnostic primers are designed to recognize, by its 3'‐terminal nucleotide, the SNP‐site in target template. Under optimized conditions of the PEx reaction, efficient polymerase synthesis of biotin‐labeled strands takes place only in the case of full complementarity between the diagnostic primer and the target SNP site. There is also benefit from introducing many biotin molecules per extended DNA strand, resulting in another level of signal amplification. After adsorption of biotinylated PEx products at the electrode surface, streptavidin‐alkaline phosphatase conjugate was bound to the biotin tags, 1‐naphthol was enzymatically produced and electrochemically detected. Several critical steps and parameters of the assay, including termination of 3’‐OH ends of residual amplification primers, temperature for annealing of diagnostic primers, relative amount of biotinylated deoxynucleoside triphosphate in the PEx mixture and number of PEx cycles were optimized in this study to attain best SNP resolution, and reduction of time needed for the analysis.