Detection of Single Nucleotide Polymorphisms in p53 Mutation Hotspots and Expression of Mutant p53 in Human Cell Lines Using an Enzyme‐Linked Electrochemical Assay

Abstract

An enzyme-linked electrochemical technique for single nucleotide polymorphism (SNP) typing in the p53 tumor suppressor 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 1-naphthyl phosphate into 1-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.

Osmium Tetroxide Complexes as Versatile Tools for Structure Probing and Electrochemical Analysis of Biopolymers

“…Application of PCR to pre-amplify specific DNA fragments for the methylation analysis (as is routinely used e.g., in the case of single nucleotide polymorphisms typing [98,99]) is not possible because during the amplification reaction, the DNA methylation pattern is lost. Thus, to catch the methylation at the DNA sequence level, it seems to be necessary to modify with osmium directly the genomic DNA isolated from the biological material.…”

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

“…The PeGEs have been successfully applied in various modes of DNA sensing, using either label‐free detection (usually via electrooxidation signal of guanine, e. g. ) or employing enzyme‐linked techniques involving bioaffinity labeling . The latter techniques have utilized also other types of graphite‐based electrodes and some of them involved application of magnetic beads, using the “double‐surface” approach . Their applications included, for example, determination of lengths of repetitive DNA sequences , detection of point mutations , or simply monitoring of PCR amplification of specific DNA fragments .…”

“…The latter techniques have utilized also other types of graphite‐based electrodes and some of them involved application of magnetic beads, using the “double‐surface” approach . Their applications included, for example, determination of lengths of repetitive DNA sequences , detection of point mutations , or simply monitoring of PCR amplification of specific DNA fragments . The approach presented here involves application of bare (and used‐as‐received) PeGE and does not involve magnetic beads, which is inherently positively reflected in cost effectivity of the electroanalytical part.…”

“…Labeling of different probes by two different enzymes which convert their specific substrates to electrochemicaly well distinguishable, simultaneously detectable products was also described . Incorporation of biotinylated nucleotides by DNA polymerases was utilized for SNP typing or detection of PCR amplicons indicating presence of target sequence for annealing primers in a template DNA .…”

Sequence-specific electrochemical detection of nucleic acids in real samples

“…The same group proposed the detection of nucleotide polymorphisms in p53 mutation hotspots and expression of mutant p53 in human cell lines [77]. Combining the double-surface technique [70] with RT-PCR and PEX [76], they were able to detect single-nucleotide polymorphism in samples received from cell cultures, differentiating mutant and wild-type genotypes where mutant genotypes indicate the inability of p53 to act as a tumor suppressor.…”