Modified dATP (2'-deoxyadenosine-5'-triphosphate) and dUTP (2'-deoxyuridine-5'-triphosphate) bearing ferrocene (Fc) labels linked via a conjugate acetylene spacer were prepared by single-step aqueous-phase cross-coupling reactions of 7-iodo-7-deaza-dATP or 5-iodo-dUTP with ethynylferrocene. The Fc-labeled dNTPs were good substrates for DNA polymerases and were efficiently incorporated to DNA by primer extension (PEX). Electrochemical analysis of the 2'-deoxyribonucleoside triphosphates (dNTPs) and PEX products revealed significant differences in redox potentials of the Fc label bound either to U or to 7-deazaA and between isolated dNTPs and conjugates incorporated to DNA. Prospective bioanalytical applications are outlined.
Primer extension is used to incorporate labeled nucleoside triphosphates into oligonucleotides (ONs). Aminophenyl and nitrophenyl modifications serve as electrochemical labels that are detectable by either oxidation or reduction, respectively (see representation of the ON double strand and redox curves). The redox potentials of the labels differ depending on the nucleobase and respond to incorporation into ONs.
Durch Primerverlängerung wurden markierte Nucleosidtriphosphate in Oligonucleotide (ONs) eingebaut. Aminophenyl‐ und Nitrophenylmodifikationen dienen als elektrochemische Markierungen, die oxidiert bzw. reduziert werden können (siehe Darstellung eines ON‐Doppelstrangs und Redoxkurven). Die Redoxpotentiale der Markierungen unterscheiden sich je nach Nucleobase und ändern sich beim Einbau in ONs.
Molecular diagnostics of inherited neurodegenerative disorders such as fragile X syndrome, myotonic dystrophy or Friedreich ataxia (FRDA) is based on analysis of the length of trinucleotide repetitive sequences in certain loci of genomic DNA. The current methods employ PCR and electrophoretic determination of the amplified DNA fragment size. We have recently shown that length of a triplet repetitive DNA sequence can be determined using a doublesurface electrochemical technique involving multiple hybridization of the expanded triplet repeat with short labeled reporter probe (spanning several trinucleotides). Here we propose a single-surface sensor employing an analogous principle. Target DNA (tDNA) is adsorbed onto surface of a carbon (pyrolytic graphite or screen-printed) electrode. Biotin-labeled reporter probe (RP) is hybridized with the immobilized tDNA followed by binding of streptavidinalkaline phosphatase (ALP) conjugate. The ALP catalyzes production of an electroactive indicator (1-naphthol) which is detected voltammetrically on the same electrode. Signal resulting from this electrochemical enzyme-linked DNA hybridization assay is normalized to the amount of tDNA immobilized at the transducer surface either by measuring intrinsic tDNA voltammetric response, or using electrochemical labeling of the tDNA with osmium tetroxide 2,2'-bipyridine complex. Detection of (GAA) n · (TTC) n triplet repeat expansion in nanogram quantities of PCR-amplified tDNAs, including amplicons of patients genomic DNA, is demonstrated. We show that our technique allow differentiation between normal and pathological alleles of X25 gene related to the FRDA.
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