Electrochemical identification of artificial oligonucleotides related to bovine species. Potential for identification of species based on mismatches in the mitochondrial cytochrome C1 oxidase gene

Our studies show that electrochemical impedance spectroscopy (EIS) and scanning electrochemical microscopy (SECM) of films of ds-DNA on gold allow us to distinguish between mitochondrial DNA fragments of the cytochrome c(1) oxidase (mt-Cox1) of three related species of the subfamily 'Bovinae' (Bos taurus, Bison bison, and Bison bonasus). In EIS, a perfectly matched DNA gives rise to a considerably larger charge transfer resistance R(ct) compared to mismatched pairings. Differences in charge transfer resistance, ΔR(ct), before and after the addition of Zn(2+) ions provide an additional tool for identification. In addition, all ds-DNA films were studied by SECM and their kinetic parameters were determined. Perfectly matched ds-DNAs are readily distinguished from mismatched duplexes by their lower rate constants. Our system can be used multiple times by dehybridization and rehybridization of capture strands up to the 250 pmole level.     

Scanning Electrochemical Microscopy for Electrochemical Detection of Single‐base Mismatches by Tagging Ferrocenecarboxylic Acid as a Redox Probe to DNA

Scanning electrochemical microscopy (SECM) was employed for sensitive detection of single base mismatches (SBMs) in a sandwiched dsDNA. Ferrocenecarboxylic acid (Fc), covalently conjugated to the dsDNA, was oxidized to Fc⁺ via the DNA‐mediated charge transfer from the underlying gold substrate, and reduced back to Fc by SECM tip generated ferrocyanide. The electrocatalytic oxidation of SECM tip‐generated ferrocyanide was sensitive to presence, as well as the type of SBMs. Apparent standard rate constants (k⁰_app) values for different SBMs, both near the electrode surface and far from it, were evaluated by SECM. The method can detect SBMs independent of their position in dsDNA.     

Insight into G[bond]T mismatch recognition using molecular dynamics with time-averaged restraints derived from NMR spectroscopy

Molecular dynamics (MD) simulations were conducted for a G[bond]T mismatch-containing DNA decamer, d(CCATGCGTGG)(2), and its Watson-Crick parent sequence, d(CCACGCGTGG)(2). Dynamics in unrestrained MD trajectories were in poor agreement with prior (13)C NMR studies. However, the accuracy of the trajectories was improved by the use of time-averaged interatomic distance restraints derived from (1)H NMR. Postprocess smoothing of the trajectories further improved accuracy. Comparison of restrained and smoothed trajectories of the two DNA molecules revealed distinct differences in dynamics. The major groove width of the mismatched oligomer was more variable over the course of the simulation compared to its parent sequence. Greater variability in helical parameters stretch and opening for the mismatches indicated less kinetically stable base pairing. Interbase helical parameters rise, roll, and tilt were also more variable in certain base steps involving mismatched bases. These dynamic differences between normal and G[bond]T mismatched DNA reflect differences in local flexibility that may play a role in mismatch recognition by the MutS. A potential alternate G[bond]T mismatch binding mode for MutS is also proposed.     

An Electrochemical DNA Biosensor for the Detection of the Apa I Polymorphism in the Vitamin D Receptor Gene Using Meldola's Blue as a Hybridization Indicator

Electrochemical detection of nucleic acid base mismatches related to Apa I single nucleotide polymorphism (SNP) in the vitamin D receptor gene was performed successfully using 7‐dimethyl‐amino‐1,2‐benzophenoxazinium salt (Meldola's blue, MDB) with 10.9 pmol/100 μL of detection limit. MDB reduction signals obtained from probe, mismatch(probe‐SNP containing target) and hybrid(probe‐target) modified pencil graphite electrode(PGE) increased respectively. The sensor was able to clearly distinguish perfect match from mismatch DNA in a 30 min. detection time. Several factors affecting on the hybridization and indicator response are studied to maximize sensitivity and selectivity. The advantages of the biosensor are discussed in comparison with previous electrochemical assays for DNA hybridization.     

High Sensitive Electrochemical Detection of Sequence‐Specific DNA Using Low Current Voltammetry

In this article, we report on efforts to construct a high sensitive electrochemical sensor with immobilized sandwich‐type DNA borne ferrocene (Fc) head for sequence‐specific DNA detection using ultramicroelectrode and low current voltammetry. Based on the difference in deformability between the bending rigid complementary DNA double helix and its anomalous flexile mismatches, the fully complementary target can be distinguished from mismatched targets including the single‐base mismatched target. Detection limit estimated as the amount of DNA is observed to be 100 fM via low current voltammetry. The method offers great promise of high sensitivity and selectivity simultaneously for effective gene identification.     

Impact of Single Basepair Mismatches on Electron‐Transfer Processes at Fc‐PNA⋅DNA Modified Gold Surfaces

Gold‐surface grafted peptide nucleic acid (PNA) strands, which carry a redox‐active ferrocene tag, present unique tools to electrochemically investigate their mechanical bending elasticity based on the kinetics of electron‐transfer (ET) processes. A comparative study of the mechanical bending properties and the thermodynamic stability of a series of 12‐mer Fc‐PNA?DNA duplexes was carried out. A single basepair mismatch was integrated at all possible strand positions to provide nanoscopic insights into the physicochemical changes provoked by the presence of a single basepair mismatch with regard to its position within the strand. The ET processes at single mismatch Fc‐PNA?DNA modified surfaces were found to proceed with increasing diffusion limitation and decreasing standard ET rate constants k⁰ when the single basepair mismatch was dislocated along the strand towards its free‐dangling Fc‐modified end. The observed ET characteristics are considered to be due to a punctual increase in the strand elasticity at the mismatch position. The kinetic mismatch discrimination with respect to the fully‐complementary duplex presents a basis for an electrochemical DNA sensing strategy based on the Fc‐PNA?DNA bending dynamics for loosely packed monolayers. In a general sense, the strand elasticity presents a further physicochemical property which is affected by a single basepair mismatch which may possibly be used as a basis for future DNA sensing concepts for the specific detection of single basepair mismatches.     

Scanning electrochemical microscopy imaging of DNA microarrays using methylene blue as a redox-active intercalator

Scanning electrochemical microscopy (SECM) has been employed in the imaging of DNA microarrays fabricated on gold substrates using methylene blue (MB) as a redox-active intercalator and ferrocyanide as the SECM mediator in solution. MB intercalated between base pairs of immobilized ds-DNA is electrochemically reduced via electron transfer from the underlying gold substrate, and the product is reoxidized in solution by SECM tip-generated ferricyanide. The resulting feedback current allows a heterogeneous electron-transfer rate constant for the MB-intercalated DNA to be deduced. Moreover, DNA microarray spots can be imaged at a detection level of 14 fmol/spot for ds-DNA consisting of 15 base pairs. Microarrays prepared using 20 microM DNA solutions are easily visualized, and the feasibility of detecting base pair mismatches is also demonstrated.     

Reactivity of ferrocenylcarbodiimide with DNA duplex containing single-mismatched base pairs.

Ferrocenylcarbodiimide (1), which is known to react with a guanine (G) or thymine (T) base of single stranded DNA, was allowed to react with DNA duplex having a single mismatched base pair of G-T, T-T, or T-cytosine (C). Electrophoreograms of the reaction mixture showed that 1 could react with G or T base of the mismatched sites on the DNA duplex. However, 1 also reacted with the G base of the terminal site on the DNA duplex. This showed that 1 can react with an unpaired base or unstable base pair such as a terminal or mismatched base on the DNA duplex. Electrochemical mismatch detection could be achieved after hybridization of the ferrocenylated mismatched DNA duplex with a selected DNA probe-immobilized electrode. These results revealed that 1 has a potentiality of serving as a labeling reagent of mismatched bases on the DNA duplex, which is important in the search for heterozygous single nucleotide polymorphisms (SNPs).     

Design of allele-specific primers and detection of the human ABO genotyping to avoid the pseudopositive problem

PCR experiments using DNA primers forming mismatch pairing with template lambda DNA at the 3' end were carried out in order to develop allele-specific primers capable of detecting SNP in genomes without generating pseudopositive amplification products, and thus avoiding the so-called pseudopositive problem. Detectable amounts of PCR products were obtained when primers forming a single or two mismatch pairings at the 3' end were used. In particular, 3' terminal A/C or T/C (primer/template) mismatches tended to allow PCR amplification to proceed, resulting in pseudopositive results in many cases. While less PCR product was observed for primers forming three terminal mismatch pairings, target DNA sequences were efficiently amplified by primers forming two mismatch pairings next to the terminal G/C base pairing. These results indicate that selecting a primer having a 3' terminal nucleotide that recognizes the SNP nucleotide and the next two nucleotides that form mismatch pairings with the template sequence can be used as an allele-specific primer that eliminates the pseudopositive problem. Trials with the human ABO genes demonstrated that this primer design is also useful for detecting a single base pair difference in gene sequences with a signal-to-noise ratio of at least 45.     

Protein-DNA interaction: impedance study of MutS binding to a DNA mismatch

MutS binding to a double-stranded DNA containing a single nucleotide mismatch can be conveniently monitored by impedance spectroscopy and represents the first step in developing an electrochemical binding essay for single nucleotide mismatch detection.     

Thermodynamics of translesion synthesis across a major DNA adduct of antitumor oxaliplatin: differential scanning calorimetric study

Differential scanning calorimetry (DSC) was used to measure the thermodynamic changes associated with translesion synthesis across major lesion induced in DNA by antitumor oxaliplatin [1,2-d(GG) intrastrand cross-link]. Insertion of matched nucleotides dC at the primer terminus (across unique 3'- or 5'-dG in the unplatinated template) and subsequent extensions resulted in an incremental increase in thermodynamic parameters. In contrast, incorporation of dC opposite either platinated dG in the intrastrand cross-link formed in the template strand and subsequent extensions by one nucleotide resulted only in little changes in thermodynamics. A similar thermodynamic delay was observed for a control template primer containing a dG:dT mismatch across 3'- or 5'-dG in the template and subsequent Watson-Crick primer extensions. The thermodynamic scarcity generated by either the lesion or mismatches was not localized but extended to the 5'-downstream sites, which may be connected with the phenomenon termed "short-term memory" of replication errors retained by some DNA polymerases responding to DNA damages or mismatches. Interestingly, formation of the 1,2-d(GG) intrastrand cross-link of oxaliplatin altered the overall DSC profiles of the dG:dT mismatch template/primers only in a very small extent. While addition of matched nucleotide dC across either dG in the template strand was thermodynamically favored over the presence of a mismatched dT (ΔΔG(0)(310) was 7.6 or 6.8 kJ mol(-1), ΔΔH was 14 or 49 kJ mol(-1)), no such thermodynamic advantage was observed with the 1,2-d(GG) intrastrand cross-link of oxaliplatin at these positions (ΔΔG(0)(310) was 2.8 or -0.3 kJ mol(-1), ΔΔH was 4 or 9 kJ mol(-1)). The equilibrium thermodynamic data also provide insight into the processes associated with misincorporation of incorrect nucleotides during replication bypass across major cross-links of antitumor oxaliplatin. On the other hand, besides thermodynamic effects also kinetic factors play an important role in the processing of the cross-links of antitumor platinum drugs. The impact of the two effects in overall processing DNA adducts by a particular DNA polymerase will depend on its nature.     

The detection of DNA deamination by electrocatalysis at DNA-modified electrodes

The method of electrocatalysis based on using a methylene blue (MB) as an electrochemical indicator and ferricyanide ions [Fe(CN)6]3- as an electron acceptor was applied in screening DNA for lesions caused by deamination of nucleobases. The damaged DNA was modeled by short 18-mer oligonucleotides containing the different number of mismatched target bases (uracil instead of cytosine residues). The hybridization capacity of these oligomers with complementary probes (immobilized on gold electrodes or free) was investigated by both electrochemical methods and UV spectroscopy. We have shown that the amplitude of the reduction signal corresponding to ferricyanide ions considerably increases in the presence of MB. This electrocatalytic effect allowed us to detect the changes in electrochemical properties of DNA caused by dU.dG mismatches. Using differential pulse voltammetry and cyclic voltammetry, we showed that the electron transport from the electrode through the double-stranded DNA to MB and then to ferricyanide ions is suppressed by the mismatches in duplex structure. According to UV-monitored melting data, single or multiple wobble dU.dG base pairs destabilize 18-mer DNA duplex by 9-27 degrees C.     

SNPs Feasibility of Nonlabeled Oligonucletides on Using Electrochemical Sensing

We have demonstrated an electrochemical gene chip protocol for the SNPs detection of nonlabeled DNA. Using an array consisting of streptavidin‐modified gold electrodes, probe DNA were attached through the application of a direct electric field. Electrochemical response changes originating from the hybridization of nucleic acids to protein‐bound nucleic acids using soluble mediators in K₃Fe(CN)₆ solution could then be observed. The electrochemical protocol developed showed high sensitivity and good reproducibility in the detection of DNA hybridization. Significant changes in electrochemical signals were also observed when using target DNA with a single base mismatch, indicating the applicability of this method to single nucleotide polymorphisms (SNPs) detection.     

Progress in Scanning Electrochemical Microscopy by Coupling with Electrochemical Impedance and Quartz Crystal Microbalance

Scanning electrochemical microscopy (SECM) is a powerful technique for performing quantitative measurements at a local scale. This paper covers the development of combinations of SECM with electrochemical impedance spectroscopy (EIS) and electrochemical quartz crystal microbalance (EQCM). Basic aspects are described and potential applications reported by several research groups are covered. The unique advantages of the coupled techniques—with additional information being obtained from each coupling—are also discussed.     

Real Time Electrochemical Monitoring of DNA/PNA Dissociation by Melting Curve Analysis

An immobilization‐free electrochemical method is reported for real‐time monitoring of the DNA hybrid dissociation between a ferrocene labeled peptide nucleic acid (PNA) and a fully‐complementary or single‐base‐mismatched DNA. This method takes advantages of electrostatic charge characteristics and interactions among the neutrally charged PNA, the negatively charged DNA and the negatively charged electrode surface made of indium tin oxide (ITO). When a ferrocene labeled PNA (Fc‐PNA) sequence is hybridized to a complementary DNA strand, electrostatic repulsion between the negatively charged PNA/DNA hybrid and the negative ITO surface retards the diffusion of the electroactive Fc to the electrode, resulting in a much reduced electrochemical signal. On the other hand, when the Fc‐PNA is dissociated from the hybrid at elevated temperatures, the neutrally charged Fc‐PNA easily diffuses to the electrode with an enhanced electrochemical signal. Therefore, an electrochemical melting curve of the Fc‐PNA/DNA hybrid can be obtained by measuring the Fc signal with the increasing temperature. This strategy allows monitoring of the dissociation of the DNA hybrid in real time, which might lead to a simple detection method for single nucleotide polymorphism (SNP) analysis.     

寡聚酰胺为探针的电化学DNA生物传感器

DNA分子中的碱基对可以长程传递电荷, DNA分子中的碱基π堆积结构为电荷的长程传递提供了良好的通道. 电荷在DNA分子中的传递受碱基序列的影响, 利用这种性质可以构建DNA碱基错配检测的电化学传感器. 寡聚酰胺能和DNA以小沟绑定方式高亲和力地结合, 并且具有序列识别功能, 本文以带有硝基官能团的寡聚酰胺分子为电化学探针, 设计了电化学DNA生物传感器. 结果显示, 寡聚酰胺与DNA修饰电极作用后, 电化学响应显著增强, 并且可以作为检测DNA碱基错配的电化学探针分子.     

In situ hybridization of PNA/DNA studied label-free by electrochemical impedance spectroscopy

The in situ hybridization kinetics of label-free DNA on mixed monolayers of peptide nucleic acid (PNA) and 6-mercapto-1-hexanol (MCH) on Au electrodes was investigated by electrochemical impedance spectroscopy (EIS) and used to discriminate the fully complementary DNA from the single-base mismatched hybrids.     

Method for detection of single-base mismatches using bimolecular beacons

This paper describes a method for the detection of single-base mismatches using DNA microarrays in a format that does not require labeling of the sample ("target") DNA. The method is based on disrupting fluorescence energy transfer (FRET) between a fluorophore attached to an immobilized DNA strand ("probe") and a quencher-containing sequence that is complementary except for an artificial mismatch (e.g. 5-nitroindole, 3-nitropyrole, or abasic site) at the site of interrogation. As the displacement of the FRET acceptor and hybridization of the unlabeled probe are bimolecular, the term "bimolecular beacons" is used to describe this approach. The analysis of a mismatch was based on differences in the amount of disruption in FRET upon hybridization of perfectly matched DNA targets and those containing single-base mismatches. Using this method and an oligonucleotide model system, A/C single-base mismatches were successfully discriminated at levels greater than that observed using surface-immobilized molecular beacons. The amount of discrimination was dependent on the identity of the artificial mismatch; greater discrimination was observed with 5-nitroindole (a "universal" base) than with an abasic site. G/T mismatches, considered to be particularly difficult to detect, were also successfully discriminated when quencher sequences containing 5-nitroindole were used.     

Tuning the DNA reactivity of cis-platinum: conjugation to a mismatch-specific metallointercalator

A novel bimetallic conjugate combining a rhodium intercalator that selectively binds DNA mismatches and a reactive cis-platinum analogue that targets DNA by coordination has been prepared. The site-selectivity of the bimetallic complex in forming coordination adducts is examined using mismatched and well-matched oligonucleotides of different sequences. The results indicate that through the bifunctional complex, the platinum center can be targeted near mismatched sites. Interestingly, with mismatched, DNA both intrastrand and the less common interstrand cross-linked adducts are formed. The recognition of a DNA mismatch by the bulky Rh intercalator appears to direct the Pt unit, depending upon steric contraints, to react preferentially with mismatched DNA at a site that may or may not be the preferred site of Pt coordination. Thus, the presence of a permanent link to a site-specific intercalator is able to tune the reactivity of the cis-platinum analogue.     

Electrochemical Determination of the p53 Tumor Suppressor Gene Using a Gold Nanoparticle-Graphene Nanocomposite Modified Glassy Carbon Electrode

A novel sensitive and simple electrochemical DNA sensor is reported for the determination of p53 tumor suppressor gene. A gold nanoparticle/graphene nanocomposite-modified glassy carbon electrode was prepared and methylene blue was used as the hybridization redox indicator. Scanning electron microscopic and electrochemical characterization demonstrated that the gold nanoparticles and graphene were present on the electrode. The resulting sensor provided suitable electrochemical response to the p53 tumor suppressor gene with a linear dynamic range from 0.1 to 1000?nM. The limit of detection was 0.012?nM. The sensor was able to differentiate a complete complementary DNA sequence, single-base mismatched DNA sequence, and a three-base mismatched DNA sequence. The precision of the device was satisfactory, with a relative standard deviation of 4.1% for 11 measurements. The combination of gold nanoparticles and a graphene nanocomposite provided enhanced capabilities for the determination of DNA for clinical applications.