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Detection of Single Nucleotide Polymorphisms in p53 Mutation Hotspots and Expression of Mutant p53 in Human Cell Lines Using an Enzyme-Linked Electrochemical Assay
Authors: Horáková Petra | Šimková Eva | Vychodilová Zdenka | Brázdová Marie | Fojta Miroslav
Year: 2009
Type of publication: článek v odborném periodiku
Name of source: Electroanalysis
Publisher name: Wiley-VCH
Place: Weinheim
Page from-to: 1723-1729
Titles:
Language Name Abstract Keywords
cze V publikaci je představena enzymová elektrochemická metoda pro detekci jednonukleotidových polymorfismů (SNP) v nádorovém supresorovém genu p53. Tato metoda je založena na prodloužení primeru katalyzovaném DNA polymerázou o jeden nukleotid nesoucí biotin, navázání konjugátu streptavidinu s alkalickou fosfatázou a následné elektrochemické detekci produktu enzymové reakce. Kromě modelových studií se syntetickými oligonukleotidy byla provedena detekce exprese mutantního p53 v lidských buněčných liniích.
eng 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 suppressor gene is presented. The technique is based on a DNA polymerase-catalyzed extension of a primer hybridized to a target DNA strand upstream to the SNP site by one nucleotide bearing a biotin tag. Efficient incorporation of the biotinylated nucleotide occurs only in the case of complementarity between the first nucleotide in ss 5-overhang of the target strand. The 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 conjugate and enzymatic conversion of 1-naphthyl phosphate into 1-naphthol. 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. enzyme-linked electrochemical assay;SNP typing;p53;mutation;magnetic beads;polymorphism

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