Detection of Single Nucleotide Polymorphisms in p53 Mutation Hotspots and Expression of Mutant p53 in Human Cell Lines Using an Enzyme-Linked Electrochemical Assay
An enzyme-linked electrochemical technique for single nucleotide polymorphism (SNP) typing in the p53 tumor suppres or gene is presented. The technique is based on a DNA polymerase-catalyzed extension of a primer hybridized to a target DNA strand upstream (5'->3') to the SNP site by one nucleotide bearing a biotin tag. Under optimized conditions, efficient incorporation of the biotinylated nucleotide occurs only in the case of complementarity between the first nucleotide in single-stranded 5'-overhang of the target strand. The introduced biotin tag is detected after capture of the primer extension products at magnetic beads bearing oligoT strands via oligoA adaptors at 5'-ends of the primer, binding of streptavidin-alkaline phosphatase conjugate and enzymatic conversion of I-naphthyl phosphate into I-naphthol which is determined electrochemically at carbon electrodes. In addition to model studies with synthetic oligonucleotides, we report on detection of mutant p53 expression in human cell lines using reverse transcription-PCR technique combined with amplified primer extension and the magnetic beads-based electrochemical assay.