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.

• Klíčová slova
• Klenow (exo-) DNA polymerase, ferrocene-labeled nucleotides, nucleoside triphosphates, single-nucleotide polymorphism (SNP), single-point mutation, solid-phase primer elongation,
• MeSH
• jednonukleotidový polymorfismus MeSH
• metaloceny MeSH
• Mycobacterium tuberculosis * genetika   MeSH
• oxidace-redukce MeSH
• rifampin farmakologie   MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH
• Názvy látek
• metaloceny MeSH
• rifampin MeSH

A 153-mer target DNA was amplified using ethynyl ferrocene dATP and a tailed forward primer resulting in a duplex with a single-stranded DNA tail for hybridization to a surface-tethered probe. A thiolated probe containing the sequence complementary to the tail as well as a 15 polythimine vertical spacer with a (CH2)6 spacer was immobilized on the surface of a gold electrode and hybridized to the ferrocene-modified complementary strand. Potential step chronoamperometry and cyclic voltammetry were used to probe the potential of zero charge, PZC, and the rate of heterogeneous electron transfer between the electrode and the immobilized ferrocene moieties. Chronoamperometry gives three, well-resolved exponential current-time decays corresponding to ferrocene centers located within 13 Å (4 bases) along the duplex. Significantly, the apparent standard heterogeneous electron transfer rate constant, kappo, observed depends on the initial potential, i.e., the rate of electron transfer at zero driving force is not the same for oxidation and reduction of the ferrocene labels. Moreover, the presence of ions, such as Sr2+, that strongly ion pair with the negatively charged DNA backbone modulates the electron transfer rate significantly. Specifically, kappo = 246 ± 23.5 and 14 ± 1.2 s-1 for reduction and oxidation, respectively, where the Sr2+ concentration is 10 mM, but the corresponding values in 1 M Sr2+ are 8 ± 0.8 and 150 ± 12 s-1. While other factors may be involved, these results are consistent with a model in which a low Sr2+ concentration and an initial potential that is negative of the PZC lead to electrostatic repulsion of the negatively charged DNA backbone and the negatively charged electrode. This leads to the DNA adopting an extended configuration (concertina open), resulting in a slow rate of heterogeneous electron transfer. In contrast, for ferrocene reduction, the initial potential is positive of PZC and the negatively charged DNA is electrostatically attracted to the electrode (concertina closed), giving a shorter electron transfer distance and a higher rate of heterogeneous electron transfer. When the Sr2+ concentration is high, the charge on the DNA backbone is compensated by the electrolyte and the charge on the electrode dominates the electron transfer dynamics and the opposite potential dependence is observed. These results open up the possibility of electromechanical switching using DNA superstructures.

• MeSH
• DNA * genetika   MeSH
• elektrody MeSH
• elektrony * MeSH
• metaloceny MeSH
• statická elektřina MeSH
• transport elektronů MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH
• Názvy látek
• DNA * MeSH
• metaloceny MeSH

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