RNA-templated single-base mutation detection based on T4 DNA ligase and reverse molecular beacon

A novel RNA-templated single-base mutation detection method based on T4 DNA ligase and reverse molecular beacon (rMB) has been developed and successfully applied to identification of single-base mutation in codon 273 of the p53 gene. The discrimination was carried out using allele-specific primers, which flanked the variable position in the target RNA and was ligated using T4 DNA ligase only when the primers perfectly matched the RNA template. The allele-specific primers also carried complementary stem structures with end-labels (fluorophore TAMRA, quencher DABCYL), which formed a molecular beacon after RNase H digestion. One-base mismatch can be discriminated by analyzing the change of fluorescence intensity before and after RNase H digestion. This method has several advantages for practical applications, such as direct discrimination of single-base mismatch of the RNA extracted from cell; no requirement of PCR amplification; performance of homogeneous detection; and easily design of detection probes.     

Characterization of DNA immobilization and subsequent hybridization using in situ quartz crystal microbalance, fluorescence spectroscopy, and surface plasmon resonance

We have characterized the immobilization of thiol-modified oligomers on Au surfaces and subsequent hybridization with a perfectly matched or single-base mismatched target using a quartz crystal microbalance (QCM) and fluorescence spectroscopy. The surface density of immobilized probe molecules and the hybridization efficiency depending on the type of buffer and salt concentration were investigated. We observed some ambiguities in surface coverage deduced from QCM measurement and adopted a complementary fluorescence displacement method. Direct comparison of surface coverage deduced from frequency change in QCM measurement and determined by the fluorescence exchange reaction revealed that QCM results are highly overestimated and the amount of overestimation strongly depends on the type of buffer and the structure of the film. Discrimination capability of the surface attached 15-mer probe was also examined using a single-base mismatched target at various hybridization temperatures. Hybridization efficiency depending on the type of single base mismatch was investigated using surface plasmon resonance (SPR).     

Genotyping by alkaline dehybridization using graphically encoded particles

This work describes a nonenzymatic, isothermal genotyping method based on the kinetic differences exhibited in the dehybridization of perfectly matched (PM) and single-base mismatched (MM) DNA duplexes in an alkaline solution. Multifunctional encoded hydrogel particles incorporating allele-specific oligonucleotide (ASO) probes in two distinct regions were fabricated by using microfluidic-based stop-flow lithography. Each particle contained two distinct ASO probe sequences differing at a single base position, and thus each particle was capable of simultaneously probing two distinct target alleles. Fluorescently labeled target alleles were annealed to both probe regions of a particle, and the rate of duplex dehybridization was monitored by using fluorescence microscopy. Duplex dehybridization was achieved through an alkaline stimulus using either a pH step function or a temporal pH gradient. When a single target probe sequence was used, the rate of mismatch duplex dehybridization could be discriminated from the rate of perfect match duplex dehybridization. In a more demanding application in which two distinct probe sequences were used, we found that the rate profiles provided a means to discriminate probe dehybridizations from both of the two mismatched duplexes as well as to distinguish at high certainty the dehybridization of the two perfectly matched duplexes. These results demonstrate an ability of alkaline dehybridization to correctly discriminate the rank hierarchy of thermodynamic stability among four sets of perfect match and single-base mismatch duplexes. We further demonstrate that these rate profiles are strongly temperature dependent and illustrate how the sensitivity can be compensated beneficially by the use of an actuating gradient pH field.     

Self-quenching smart probes as a platform for the detection of sequence-specific UV-induced DNA photodamage

Molecular beacons (MBs) are sensitive probes for many DNA sequence-specific applications, such as DNA damage detection, but suffer from technical and cost limitations. We have designed smart probes with self-quenching properties as an alternative to molecular beacons to monitor sequence-specific UV-induced photodamage of oligonucleotides. These probes have similar stem-loop structural characteristics as molecular beacons, but quenching is achieved instead via photoinduced intramolecular electron transfer by neighboring guanosine residues. Our results indicate that the probes are sensitive enough to detect nanomolar target concentrations and are specific enough to discriminate single-base damage. When the probes were used to monitor UV-induced photodamage in oligonucleotide sequences that differ by a single-base mismatch, the photodamage time constant was higher for the perfectly complementary target sequences than for the mismatch sequences, indicating that these probes are specific for each target sequence. In addition, time constants obtained for oligonucleotide target sequences with both stem and loop base mismatches are lower than those with only loop mismatches, suggesting that these sequences are also specifically distinguished by the smart probes. These probes thus constitute robust, sensitive, specific, and cheaper alternatives to MBs for sequence-specific DNA damage detection.     

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.     

A one-step highly sensitive method for DNA detection using dynamic light scattering

A one-step homogeneous DNA detection method with high sensitivity was developed using gold nanoparticles (AuNPs) coupled with dynamic light scattering (DLS) measurement. Citrate-protected AuNPs with a diameter of 30 nm were first functionalized with two sets of single-stranded DNA probes and then used as optical probes for DNA detection. In the presence of target DNA, the hybridization between target DNA and the two nanoparticle probes caused the formation of nanoparticle dimers, trimers, and oligomers. As a result, the nanoparticle aggregation increased the average diameter of the whole nanoparticle population, which can be monitored simply by DLS measurement. A quantitative correlation can be established between the average diameter of the nanoparticles and the target DNA concentration. This DLS-based assay is extremely easy to conduct and requires no additional separation and amplification steps. The detection limit is around 1 pM, which is 4 orders of magnitude better than that of light-absorption-based methods. Single base pair mismatched DNAs can be readily discriminated from perfectly matched target DNAs using this assay.     

Visual SNP genotyping using asymmetric PCR and split DNA enzymes

Harnessing and applying genomic technologies in resource limited environments demand a next generation of platforms, which are convenient, quick and easy to apply. We describe here a visual colour change assay that can be applied to SNP genotyping, which unlike traditional methods, does not adopt complicated procedures or expensive instrumentation, desirable features in bringing genetic capabilities outside the laboratory. Our strategy involved a two-step method that first enriched target genomic regions using asymmetric PCR, followed by direct in situ application of split DNA probes. In the presence of target sequences that perfectly matched the complementary probes, the split probes reassembled active DNAzymes, which catalysed a colour change reaction. A single-base mismatch (indicative of a polymorphism) prevented this reassembly and colour change, providing the means for accurate SNP calling. This is the first report, to our knowledge, that demonstrates successful visual SNP genotyping of actual human DNA samples using DNAzymes.     

基于锁核酸的高选择性新型分子信标

通过在分子信标的错配位点修饰锁核酸, 不仅可有效地改善其单碱基错配识别能力, 还可提高检测灵敏度. 因而有望发展成为一种通用的提高分子信标单碱基错配识别能力的方法.     

A MutS protein-immobilized au electrode for detecting single-base mismatch of DNA.

A novel electrochemical biosensor was developed to detect gene mutation by using a DNA-mismatch binding protein: MutS from Escherichia coli. The MutS protein was immobilized onto an Au-electrode surface via complex formation between a histidine tag of the MutS protein and a thiol-modified nitrilotriacetic acid chemically adsorbed on the Au-electrode surface. When a target double-stranded DNA having a single-base mismatch was captured by the MutS protein on the electrode, some electrostatic repulsion arose between polyanionic DNA strands and anionic redox couple ions. Consequently, their redox peak currents on a cyclic voltammogram with the Au electrode drastically decreased, depending on the concentration of the target DNA, according to the redox couple-mediated artificial ion-channel principle. By using this assay, one can detect all types of single-base mismatch and single-base deletion.     

A sensitive fluorescence anisotropy method for point mutation detection by using core-shell fluorescent nanoparticles and high-fidelity DNA ligase

The present study reports a proof-of-principle for a sensitive genotyping assay approach that can detect single nucleotide polymorphisms (SNPs) based on fluorescence anisotropy measurements through a core-shell fluorescent nanoparticles assembly and ligase reaction. By incorporating the core-shell fluorescent nanoparticles into fluorescence anisotropy measurements, this assay provided a convenient and sensitive detection assay that enabled straightforward single-base discrimination without the need of complicated operational steps. The assay was implemented via two steps: first, the hybridization reaction that allowed two nanoparticle-tagged probes to hybridize with the target DNA strand and the ligase reaction that generated the ligation between perfectly matched probes while no ligation occurred between mismatched ones were implemented synchronously in the same solution. Then, a thermal treatment at a relatively high temperature discriminated the ligation of probes. When the reaction mixture was heated to denature the duplex formed, the fluorescence anisotropy value of the perfect-match solution does not revert to the initial value, while that of the mismatch again comes back as the assembled fluorescent nanoparticles dispart. The present approach has been demonstrated with the discrimination of a single base mutation in codon 12 of a K-ras oncogene that is of significant value for colorectal cancers diagnosis, and the wild type and mutant type were successfully scored. Due to its ease of operation and high sensitivity, it was expected that the proposed detection approach might hold great promise in practical clinical diagnosis.     

Selective DNA detection at Zeptomole level based on coulometric measurement of gold nanoparticle-mediated electron transfer across a self-assembled monolayer

A selective DNA sensing with zeptomole detection level is developed based on coulometric measurement of gold nanoparticle (AuNPs)-mediated electron transfer (ET) across a self-assembled monolayer on the gold electrode. After immobilization of a thiolated hairpin-structured DNA probe, an alkanethiol monolayer was self-assembled on the resultant electrode to block [Fe(CN)6 ]-3-/4in a solution from accessing the electrode. In the presence of DNA target, hybridization between the DNA probe and the DNA target breaks the stem duplex of DNA probe. Consequently, stem moiety at the 3′-end of the DNA probes was removed from the electrode surface and made available for hybridization with the reporter DNA-AuNPs conjugates (reporter DNA-AuNPs). The thiolated reporter DNA matches the stem moiety at the 3′-end of the DNA probe. AuNPs were then enlarged by immersing the electrode in a growth solution containing HAuCl 4 and H2O2 after the reporter DNA-AuNPs bound onto the electrode surface. The enlarged AuNPs on the electrode restored the ET between the electrode and the [Fe(CN)6]3 -/4- , as a result, amplified signals were achieved for DNA target detection using the coulometric measurement of Fe(CN)6 3- electro-reduction by prolonging the electrolysis time. The quantities of ET on the DNA sensor increased with the increase in DNA target concentration through a linear range of 3.0 fM to 1.0 pM when electrolysis time was set to 300 s, and the detection limit was 1.0 fM. Correspondingly, thousands of DNA (zeptomole) copies were detected in 10L samples. Furthermore, the DNA sensor showed excellent differentiation ability for single-base mismatch.     

An Electrochemical Sensor Based on Gold Nanoparticles Incorporated in Mesoporous MFI Zeolite for Determination of Purine Bases in DNA

An electrochemical sensor was developed based on gold nanoparticles incorporated in mesoporous MFI zeolite for the determination of purine bases. Au nanoparticles (AuNPs) were incorporated into the mesoporous MFI zeolite (AuNPs/m‐MFI) by post‐grafting reaction. The composite materials were characterized by transmission electron microscopy (TEM), X‐ray photoelectron spectroscopy (XPS) and electrochemical methods. Au nanoparticles with a size of 5‐20 nm are uniformly dispersed in the pores of mesoporous MFI zeolite. And the morphology of MFI zeolite can be perfectly kept after pore expansion and Au nanoparticles incorporation. The electrocatalytic oxidation of purine bases (guanine and adenine in DNA) is investigated by cyclic voltammetry (CV) and differential pulse voltammetry (DPV). The surface‐confined Au nanoparticles provide the good catalytic activity for oxidation of purine bases. The simultaneous detection of guanine and adenine can be achieved at AuNPs/m‐MFI composites modified glassy carbon electrode (GCE). The electrochemical sensor based on AuNPs/m‐MFI exhibits wide linear range of 0.5–500 μM and 0.8–500 μM with detection limit of 0.25 and 0.29 μM for guanine and adenine, respectively. Moreover, the electrochemical sensor is applied to evaluation of guanine and adenine in herring sperm DNA samples with satisfactory results.     

Effect of oligonucleotide probes substituted by deoxyinosines on the specificity of SNP detection on the DNA microarray

One of the main factors that can affect the quality of microarray results is the microarray hybridization specificity. The key factor that affects hybridization specificity is the design of the probes. In this paper, we described a novel oligonucleotide probe containing deoxyinosines aimed at improving DNA hybridization specificity. We compared different probes to determine the distance between deoxyinosine base and SNPs site and the number of deoxyinosine bases. The new probe sequences contained two set of deoxyinosines (each set had two deoxyinosines), in which the interval between SNP site and each set of deoxyinosines was two bases. The new probes could obtain the highest hybridization specificity. The experimental results showed that probes containing deoxyinosines hybridized effectively to the perfectly matched target and improved the hybridization specificity of DNA microarray. By including a simple washing step after hybridization, these probes could distinguish matched targets from single‐base‐mismatched sequences perfectly. For the probes containing deoxyinosines, the fluorescence intensity of a match sequence was more than eight times stronger than that of a mismatch. However, the intensity ratio was only 1.3 times or less for the probes without deoxyinosines. Finally, using hybridization of the PCR product microarrays, we successfully genotyped SNP of 140 samples using these new labeled probes. Our results show that this is a useful new strategy for modifying oligonucleotide probes for use in DNA microarray analysis.     

Simultaneous identification of point mutations via DNA ligase-mediated gold nanoparticle assembly

Multiplex single nucleotide polymorphisms analysis has found a great demand in human genetics and pharmacogenetics. The present study reports a novel approach for a genotyping assay that could achieve simultaneous identification of multiple point mutations via a ligase-mediated gold nanoparticle assembly. Based on the allelic specificity of DNA ligase, gold nanoparticles modified by oligonucleotide probes perfectly matched to the DNA targets were assembled into a thermally-stable aggregate, while a single-base mismatch would result in the dissociation of the gold nanoparticle assembly at high temperature. Then, DNA targets and their point mutations could be differentiated using a multi-step temperature elevation analysis monitored by ultraviolet-visible measurements. This approach offered a direct colorimetric discrimination of multiple point mutations without stringent temperature control. The proposed approach is demonstrated using a model system for the identification of single-base mutations in codon 17 and position -28 of the beta-thalassemia gene. The results reveal that the wild and the mutant types could be simultaneously determined successfully. Owing to its ease of operation and high specificity, it was expected that the proposed procedure might hold great promise in both research-oriented and clinical genomic assays.     

基于金纳米粒子/聚阿魏酸/多壁碳纳米管修饰电极的DNA计时库仑法生物传感器的制备

构建了基于金纳米粒子/聚阿魏酸/多壁碳纳米管(AuNPs/PFA/MWCNTs)修饰电极的DNA计时库仑法生物传感器.利用循环伏安技术在多壁碳管修饰的玻碳电极表面上聚合一层阿魏酸,在恒电位条件下,在阿魏酸表面沉积金纳米粒子,巯基DNA作为探针通过金硫键固定在金纳米粒子表面.电化学交流阻抗技术(EIS)与扫描电镜(SEM...     

Quartz crystal microbalance detection of DNA single-base mutation based on monobase-coded cadmium tellurium nanoprobe

A new method for the detection of point mutation in DNA based on the monobase-coded cadmium tellurium nanoprobes and the quartz crystal microbalance (QCM) technique was reported. A point mutation (single-base, adenine, thymine, cytosine, and guanine, namely, A, T, C and G, mutation in DNA strand, respectively) DNA QCM sensor was fabricated by immobilizing single-base mutation DNA modified magnetic beads onto the electrode surface with an external magnetic field near the electrode. The DNA-modified magnetic beads were obtained from the biotin-avidin affinity reaction of biotinylated DNA and streptavidin-functionalized core/shell Fe(3)O(4)/Au magnetic nanoparticles, followed by a DNA hybridization reaction. Single-base coded CdTe nanoprobes (A-CdTe, T-CdTe, C-CdTe and G-CdTe, respectively) were used as the detection probes. The mutation site in DNA was distinguished by detecting the decreases of the resonance frequency of the piezoelectric quartz crystal when the coded nanoprobe was added to the test system. This proposed detection strategy for point mutation in DNA is proved to be sensitive, simple, repeatable and low-cost, consequently, it has a great potential for single nucleotide polymorphism (SNP) detection.     

纳米金颗粒增强信号的电化学生物传感器用于谷胱甘肽和半胱氨酸的检测

构建了一种高灵敏检测谷胱甘肽(GSH)和半胱氨酸(Cys)的新型电化学生物传感器. 先将富含T碱基的DNA1和DNA2探针分别修饰在金电极和纳米金颗粒(AuNPs)上, 再加入Hg²⁺, 通过形成T-Hg²⁺-T结构使AuNPs结合到金电极表面. 当加入GSH(或Cys)后, GSH(或Cys)可以竞争结合T-Hg²⁺-T结构中的Hg²⁺, 使AuNPs离开电极表面. 由于AuNPs上修饰的DNA探针能够静电吸附大量电活性物质六氨合钌(RuHex), 因此该过程可引起计时电量信号的显著变化, 据此实现了GSH(或Cys)的高灵敏检测. 该传感器的检出限达10 pmol/L, 比荧光法或比色法降低了2~3个数量级. 实验结果表明, 该传感器具有较好的选择性.     

Electrochemical biosensors for detection of point mutation based on surface ligation reaction and oligonucleotides modified gold nanoparticles

An electrochemical method for point mutation detection based on surface ligation reaction and oligonucleotides (ODNs) modified gold nanoparticles (AuNPs) was demonstrated. Point mutation identification was achieved using Escherichia coli DNA ligase. This system for point mutation detection relied on a sandwich assay comprising capture ODN immobilized on Au electrodes, target ODN and ligation ODN. Because of the sequence-specific surface reactions of E. coli DNA ligase, the ligation ODN covalently linked to the capture ODN only in the presence of a perfectly complementary target ODN. The presence of ligation products on Au electrode was detected using chronocoulometry through hybridization with reporter ODN modified AuNPs. The use of AuNPs improved the sensitivity of chronocoulometry in this approach, a detection limit of 0.9 pM complementary ODN was obtained. For single base mismatched ODN (smODN), a negligible signal was observed. Even if the concentration ratio of complementary ODN to smODN was decreased to 1:1000, a detectable signal was observed. This work may provide a specific, sensitive and cost-efficient approach for point mutant detection.     

Electronic detection of single-base mismatches in DNA with ferrocene-modified probes.

Genotyping and gene-expression monitoring is critical to the study of the association between genetics and drug response (pharmacogenomics) and the association of sequence variation with heritable phenotypes. Recently, we developed an entirely electronic method for the detection of DNA hybridization events by the site-specific incorporation of ferrocenyl derivatives into DNA oligonucleotides. To perform rapid and accurate point mutation detection employing this methodology, two types of metal-containing signaling probes with varying redox potentials are required. In this report we describe a new ferrocene-containing phosphoramidite 9 that provides a range of detectable redox potentials. Using automated DNA/RNA synthesis techniques the two ferrocenyl complexes were inserted at various positions along oligonucleotide probes. Thermal stability analysis of these metal-containing DNA oligonucleotides indicates that incorporation of 9 resulted in no destabilization of the duplex. A mixture of oligonucleotides containing compounds 9 and I was analyzed by alternating current voltammetry (ACV) monitored at the 1st harmonic. The data demonstrate that the two ferrocenyl oligonucleotide derivatives can be distinguished electrochemically. A CMS-DNA array was prepared on an array of gold electrodes on a printed circuit board substrate with a self-assembled mixed monolayer, coupled to an electronic detection system. Experiments for the detection of a single-base match utilizing two signaling probes were carried out. The results demonstrate that rapid and accurate detection of a single-base mismatch can be achieved by using these dual-signaling probes on CMS-DNA chips.     

Gold nanoparticle based signal enhancement liquid crystal biosensors for DNA hybridization assays

A novel signal enhanced liquid crystal biosensor based on using AuNPs for highly sensitive DNA detection has been developed. This biosensor not only significantly decreases the detection limit, but also offers a simple detection process and shows a good selectivity to distinguish perfectly matched target DNA from two-base mismatched DNA.