Three sets of 7-deazaadenine and cytosine nucleosides and nucleoside triphosphates bearing either unsubstituted ferrocene, octamethylferrocene and ferrocenecarboxamide linked through an alkyne tether to position 7 or 5, respectively, were designed and synthesized. The modified dNFcX TPs were good substrates for KOD XL DNA polymerase in primer extension and were used for enzymatic synthesis of redox-labelled DNA probes. Square-wave voltammetry showed that the octamethylferrocene oxidation potential was shifted to lower values, whilst the ferrocenecarboxamide was shifted to higher potentials, as compared to ferrocene. Tailed PEX products containing different ratios of Fc-labelled A (dAFc ) and FcPa-labelled C (dCFcPa ) were synthesized and hybridized with capture oligonucleotides immobilized on gold electrodes to study the electrochemistry of the redox-labelled DNA. Clearly distinguishable, fully orthogonal and ratiometric peaks were observed for the dAFc and dCFcPa bases in DNA, demonstrating their potential for use in redox coding of nucleobases and for the direct electrochemical measurement of the relative ratio of nucleobases in an unknown sequence of DNA.

• Klíčová slova
• DNA, electrochemistry, ferrocenes, nucleobases, redox labelling,
• MeSH
• barvení a značení metody   MeSH
• cytidintrifosfát chemie   MeSH
• DNA sondy chemická syntéza   chemie   MeSH
• DNA-dependentní DNA-polymerasy metabolismus   MeSH
• DNA chemie   metabolismus   MeSH
• elektrochemické techniky MeSH
• metaloceny chemie   MeSH
• nukleotidy chemie   MeSH
• oxidace-redukce MeSH
• substrátová specifita MeSH
• železnaté sloučeniny chemie   MeSH
• Publikační typ
• časopisecké články MeSH
• Názvy látek
• cytidintrifosfát MeSH
• DNA sondy MeSH
• DNA-dependentní DNA-polymerasy MeSH
• DNA MeSH
• ferrocene MeSH Prohlížeč
• metaloceny MeSH
• nukleotidy MeSH
• železnaté sloučeniny MeSH

We report the duplex amplification of two plasmid DNA markers involved in the virulence of Bacillus anthracis, CAP and PAG, and the direct electrochemical detection of these amplicons. The method consists of the simultaneous amplification of the two targets in a single-pot reaction via polymerase chain reaction (PCR) using tailed primers and ferrocene-labeled dATP. Following amplification, the PCR products hybridize to probes immobilized on electrodes in a microfabricated electrode array chip. The incorporated ferrocene labeled dATP is then detected using square wave voltammetry. We evaluated the effect of electrolyte cations, anions, and concentration to condense, bend, and shrink double-stranded DNA and their effect on the intensity of the ferrocene signal. We obtained detection limits of 0.8 and 3.4 fM for CAP and PAG targets, respectively. We successfully developed a method to detect the presence of both targets in genomic DNA extracted from real samples.

• Publikační typ
• časopisecké články MeSH

Microribonucleic acids (miRNAs) have been linked with various regulatory functions and disorders, such as cancers and heart diseases. They, therefore, present an important target for detection technologies for future medical diagnostics. We report here a novel method for rapid and sensitive miRNA detection and quantitation using surface plasmon resonance (SPR) sensor technology and a DNA*RNA antibody-based assay. The approach takes advantage of a novel high-performance portable SPR sensor instrument for spectroscopy of surface plasmons based on a special diffraction grating called a surface plasmon coupler and disperser (SPRCD). The surface of the grating is functionalized with thiolated DNA oligonucleotides which specifically capture miRNA from a liquid sample without amplification. Subsequently, an antibody that recognizes DNA*RNA hybrids is introduced to bind to the DNA*RNA complex and enhance sensor response to the captured miRNA. This approach allows detection of miRNA in less than 30 min at concentrations down to 2 pM with an absolute amount at high attomoles. The methodology is evaluated for analysis of miRNA from mouse liver tissues and is found to yield results which agree well with those provided by the quantitative polymerase chain reaction (qPCR).

• MeSH
• biosenzitivní techniky metody   MeSH
• DNA MeSH
• hybridizace nukleových kyselin imunologie   MeSH
• játra chemie   MeSH
• limita detekce MeSH
• metody MeSH
• mikro RNA analýza   MeSH
• myši MeSH
• povrchová plasmonová rezonance metody   MeSH
• protilátky MeSH
• RNA MeSH
• zvířata MeSH
• Check Tag
• myši MeSH
• zvířata MeSH
• Publikační typ
• časopisecké články MeSH
• hodnotící studie MeSH
• práce podpořená grantem MeSH
• Research Support, N.I.H., Extramural MeSH
• Názvy látek
• DNA MeSH
• mikro RNA MeSH
• protilátky MeSH
• RNA MeSH