Electrochemical genosensor for the direct detection of tailed PCR amplicons incorporating ferrocene labelled dATP

An electrochemical genosensor for the detection and quantification of Karlodinium armiger is presented. The genosensor exploits tailed primers and ferrocene labelled dATP analogue to produce PCR products that can be directly hybridised on a gold electrode array and quantitatively measured using square wave voltammetry. Tailed primers consist of a sequence specific for the target, followed by a carbon spacer and a sequence specifically designed not to bind to genomic DNA, resulting in a duplex flanked by single stranded binding primers. The incorporation of the 7-(ferrocenylethynyl)-7-deaza-2′-deoxyadenosine triphosphate was optimised in terms of a compromise between maximum PCR efficiency and the limit of detection and sensitivity attainable using electrochemical detection via hybridisation of the tailed, ferrocene labelled PCR product. A limit of detection of 277aM with a linear range from 315aM to 10 fM starting DNA concentration and a sensitivity of 122 nA decade−1 was achieved. The system was successfully applied to the detection of genomic DNA in real seawater samples.info:eu-repo/semantics/acceptedVersio

Electrochemical Detection of Single-Nucleotide Polymorphism Associated with Rifampicin Resistance in Mycobacterium tuberculosis Using Solid-Phase Primer Elongation with Ferrocene-Linked Redox-Labeled Nucleotides.

Here, we report the electrochemical detection of single-point mutations using solid-phase isothermal primer elongation with redox-labeled oligonucleotides. A single-base mutation associated with resistance to rifampicin, an antibiotic commonly used for the treatment of Mycobacterium tuberculosis, was used as a model system to demonstrate a proof-of-concept of the approach. Four 5'-thiolated primers, designed to be complementary with the same fragment of the target sequence and differing only in the last base, addressing the polymorphic site, were self-assembled via chemisorption on individual gold electrodes of an array. Following hybridization with single-stranded DNA, Klenow (exo-) DNA polymerase-mediated primer extension with ferrocene-labeled 2'-deoxyribonucleoside triphosphates (dNFcTPs) was only observed to proceed at the electrode where there was full complementarity between the surface-tethered probe and the target DNA being interrogated. We tested all four ferrocenylethynyl-linked dNTPs and optimized the ratio of labeled/natural nucleotides to achieve maximum sensitivity. Following a 20 min hybridization step, Klenow (exo-) DNA polymerase-mediated primer elongation at 37 °C for 5 min was optimal for the enzymatic incorporation of a ferrocene-labeled nucleotide, achieving unequivocal electrochemical detection of a single-point mutation in 14 samples of genomic DNA extracted from Mycobacterium tuberculosis strains. The approach is rapid, cost-effective, facile, and can be extended to multiplexed electrochemical single-point mutation genotyping

Covalently linked purine dimers or trimers and purine-pyrimidine conjugates - analogues of nucleobase-pairs or triplets

Covalent analogues of pairs and triplets of nucleobases consisting of purine dimers and trimers and purine-pyrimidine conjugates connected by various types of carbon-linkers (acetylene, diacetylene, vinylene, ethylene, phenylene, benzenetriyl) are described

Regioselektivní cross-coupling reakce v syntéze multisubstituovaných purinových bází a nukleosidů

Regioselective cross-coupling reactions of di- and trihalopurines were studied and used in the synthesis of modified purine bases and nucleosides

Purinové báze a nukleosidy nesoucí funkcionalizované C-substituenty v pozici 6. Syntéza a biologická aktivita

Purine bases and nucleosides bearing diverse functionalized C-substituents have been prepared either via cross-coupling reactions of 6-halopurines with protected functionalized organometallic reagents or via conjugate additions of nucleophiles to 6-vinyl- or 6-ethynylpurines. Other types of substituents have been prepared by further functional group transformations of 6-(mesyloxymethyl)purines. Systematic biological activity screening revealed several new types of cytostatic and anti-HCV purine nucleosides

Chemie složek nukleových kyselin

This symposium covered latest developments in organic, medicinal and physical chemistry, biochemistry and chemical biology of nucleic acids and their components

Polymerase construction of base-modified DNA for chemical biology

An efficient two-step methodology for construction of base-modified DNA was developed based on aqueous-phase cross-coupling reactions of halogenated nucleoside triphosphates (dNTPs) followed by polymerase incorporation. A number of diverse chemical modifications have been successfully incorporated in this way for the use in chemical biology (modulation of cleavage by restriction enzymes, interactions with diverse DNA-binding proteins and bioconjugations)