applications of electroporation of adherent cellsIn Situ,on a partly conductive slide

One of the most important factors affecting the quality of PCR is the choice of primers. In general, the longer the PCR product the more difficult it is to select efficient primers and set appropriate designing primers, and in general, the more DNA sequence information is available, the better the ch0ance of finding an optimal primer pair. Efficient primers can be designed by avoiding the following flaws: primer-dimer formation, self-complementarity, too lowT _m of the primers, and/or their incorrect internal stability profile. Tips on subcloning PCR products, calculating duplex stability (predicting dimer formation strength), and designing degenerate primers are given.     

Improved assay-dependent searching of nucleic acid sequence databases

Nucleic acid-based biochemical assays are crucial to modern biology. Key applications, such as detection of bacterial, viral and fungal pathogens, require detailed knowledge of assay sensitivity and specificity to obtain reliable results. Improved methods to predict assay performance are needed for exploiting the exponentially growing amount of DNA sequence data and for reducing the experimental effort required to develop robust detection assays. Toward this goal, we present an algorithm for the calculation of sequence similarity based on DNA thermodynamics. In our approach, search queries consist of one to three oligonucleotide sequences representing either a hybridization probe, a pair of Padlock probes or a pair of PCR primers with an optional TaqMantrade mark probe (i.e. in silico or 'virtual' PCR). Matches are reported if the query and target satisfy both the thermodynamics of the assay (binding at a specified hybridization temperature and/or change in free energy) and the relevant biological constraints (assay sequences binding to the correct target duplex strands in the required orientations). The sensitivity and specificity of our method is evaluated by comparing predicted to known sequence tagged sites in the human genome. Free energy is shown to be a more sensitive and specific match criterion than hybridization temperature.     

Molecular basis of action of HyBeacon fluorogenic probes: a spectroscopic and molecular dynamics study

HyBeacons, novel DNA probes for ultra-rapid detection of single nucleotide polymorphisms, contain a fluorophore covalently attached via a linker group to an internal nucleotide. As the probe does not require a quencher or self-complementarity to function, this study investigates the molecular-level mechanism underlying the increase of fluorescence intensity on hybridization of HyBeacons with target DNA. Spectroscopic ultraviolet-visible and fluorimetric studies, combined with molecular dynamics simulations, indicate projection of the fluorophore moiety away from the target-probe duplex into aqueous solution, although specific linker-DNA interactions are populated. Based on evidence from this study, we propose that for HyBeacons, the mechanism of increased fluorescence on hybridization is due to disruption of quenching interactions in the single-stranded probe DNA between the fluorophore and nucleobases. Hybridization leads to an extended linker conformation, removing the fluorophore from the immediate vicinity of the DNA bases.     

Hybridization of 2'-O-methyl and 2'-deoxy molecular beacons to RNA and DNA targets

Molecular beacons are stem-loop hairpin oligonucleotide probes labeled with a fluorescent dye at one end and a fluorescence quencher at the other end; they can differentiate between bound and unbound probes in homogeneous hybridization assays with a high signal-to-background ratio and enhanced specificity compared with linear oligonucleotide probes. However, in performing cellular imaging and quantification of gene expression, degradation of unmodified molecular beacons by endogenous nucleases can significantly limit the detection sensitivity, and results in fluorescence signals unrelated to probe/target hybridization. To substantially reduce nuclease degradation of molecular beacons, it is possible to protect the probe by substituting 2'-O-methyl RNA for DNA. Here we report the analysis of the thermodynamic and kinetic properties of 2'-O-methyl and 2'-deoxy molecular beacons in the presence of RNA and DNA targets. We found that in terms of molecular beacon/target duplex stability, 2'-O-methyl/RNA > 2'-deoxy/RNA > 2'-deoxy/DNA > 2'-O-methyl/DNA. The improved stability of the 2'-O-methyl/RNA duplex was accompanied by a slightly reduced specificity compared with the duplex of 2'-deoxy molecular beacons and RNA targets. However, the 2'-O-methyl molecular beacons hybridized to RNA more quickly than 2'-deoxy molecular beacons. For the pairs tested, the 2'-deoxy-beacon/DNA-target duplex showed the fastest hybridization kinetics. These findings have significant implications for the design and application of molecular beacons.     

Sulfur signaling: is the agent sulfide or sulfane?

Triplex-forming oligonucleotides (TFOs) are sequence-dependent DNA binders that may be useful for DNA targeting and detection. A sensitive and convenient method to monitor triplex formation by a TFO and its target DNA duplex is required for the application of TFO probes. Here we describe a novel design by which triplex formation can be monitored homogeneously without prelabeling the target duplex. The design uses a TFO probe tagged with a fluorophore that undergoes fluorescence resonance energy transfer with fluorescent dyes that intercalate into the target duplex. Through color compensation analysis, the specific emission of the TFO probe reveals the status of the triple helices. We used this method to show that triple helix formation with TFOs is magnesium dependent. We also demonstrated that the TFO probe can be used for detection of sequence variation in melting analysis and for DNA quantitation in real-time polymerase chain reaction.     

Stability, specificity and fluorescence brightness of multiply-labeled fluorescent DNA probes.

In this work, we studied the fluorescence and hybridization of multiply-labeled DNA probes which have the hydrophilic fluorophore 1-(straightepsilon-carboxypentynyl)-1'-ethyl- 3,3,3', 3'-tetramethylindocarbocyanine-5,5'-disulfonate (Cy3) attached via either a short or long linker at the C-5 position of deoxyuridine. We describe the effects of labeling density, fluorophore charge and linker length upon five properties of the probe: fluorescence intensity, the change in fluorescence upon duplex formation, the quantum yield of fluorescence (Phif), probe-target stability and specificity. For the hydrophilic dye Cy3, we have demonstrated that the fluorescence intensity andPhifare maximized when labeling every 6th base using the long linker. With a less hydrophilic dye, a labeling density this high could not be achieved without serious quenching of the fluorescence. The target specificity of multiply-labeled DNA probes was just as high as compared to the unmodified control probe, however, a less stable probe-target duplex is formed that exhibits a lower melting temperature. A mechanism that accounts for this destabilization is proposed which is consistent with our data. It involves dye-dye and dye-nucleotide interactions which appear to stabilize a single-stranded conformation of the probe.     

A label-free electrochemical DNA sensor based on exonuclease III-aided target recycling strategy for sequence-specific detection of femtomolar DNA

The present work demonstrates a rapid, single-step and ultrasensitive label-free and signal-off electrochemical sensor for specific DNA detection with excellent discrimination ability for single-nucleotide polymorphisms, taking advantage of Exonuclease III (Exo III)-aided target recycling strategy to achieve signal amplification. Exo III has a specifical exo-deoxyribonuclease activity for duplex DNAs in the direction from 3' to 5' terminus, however its activity on the duplex DNAs with 3'-overhang and single-strand DNA is limited. In response to the specific features of Exo III, the proposed E-DNA sensor is designed such that, in the presence of target DNA, the electrode self-assembled signaling probe hybridizes with the target DNA to form a duplex in the form of a 3'-blunt end at signaling probe and a 3'-overhang end at target DNA. In this way, Exo III specifically recognizes this structure and selectively digests the signaling probe. As a result, the target DNA dissociates from the duplex and recycles to hybridize with a new signaling probe, leading to the digestion of a large amount of signaling probes gradually. A redox mediator, Ru(NH(3))(6)(3+) (RuHex) is employed to electrostatically adsorbed onto signaling probes, which is directly related to the amount and the length of the signaling probes remaining in the electrode, and provides a quantitative measure of sequence-specific DNA with the experimentally measured (not extrapolated) detection limit as low as 20 fM. Moreover, this E-DNA sensor has an excellent differentiation ability for single mismatches with fairly good stability.     

Labeling of unique sequences in double-stranded DNA at sites of vicinal nicks generated by nicking endonucleases

We describe a new approach for labeling of unique sequences within dsDNA under nondenaturing conditions. The method is based on the site-specific formation of vicinal nicks, which are created by nicking endonucleases (NEases) at specified DNA sites on the same strand within dsDNA. The oligomeric segment flanked by both nicks is then substituted, in a strand displacement reaction, by an oligonucleotide probe that becomes covalently attached to the target site upon subsequent ligation. Monitoring probe hybridization and ligation reactions by electrophoretic mobility retardation assay, we show that selected target sites can be quantitatively labeled with excellent sequence specificity. In these experiments, predominantly probes carrying a target-independent 3′ terminal sequence were employed. At target labeling, thus a branched DNA structure known as 3′-flap DNA is obtained. The single-stranded terminus in 3′-flap DNA is then utilized to prime the replication of an externally supplied ssDNA circle in a rolling circle amplification (RCA) reaction. In model experiments with samples comprised of genomic λ-DNA and human herpes virus 6 type B (HHV-6B) DNA, we have used our labeling method in combination with surface RCA as reporter system to achieve both high sequence specificity of dsDNA targeting and high sensitivity of detection. The method can find applications in sensitive and specific detection of viral duplex DNA.     

致病性维罗纳气单胞菌检测方法的建立

发掘维罗纳气单胞菌特异性更强的检测靶点和毒力相关基因靶点,建立能够检测致病性维罗纳气单胞菌的PCR检测方法.通过序列比对分析气单胞菌的16S rRNA基因序列,筛选对维罗纳气单胞菌特异的引物,用于检测种特异性,利用气单胞菌气溶素基因保守引物,检测菌株的致病性,并进行反应条件和反应体系的优化,灵敏度试验和特异性试验.发掘并设计的维罗纳气单胞菌16S rRNA特异性引物结合气单胞菌气溶素基因保守引物建立的检测方法,对12株气单胞菌和10株非气单胞菌的检测结果显示,所有致病性维罗纳气单胞菌都能扩增到大小分别为343 bp和232 bp的特异性条带,而非维罗纳气单胞菌的致病性气单胞菌只能扩增到232 bp的气溶素基因特异性条带,其它菌株都不能扩增到目的条带.灵敏度试验表明,该反应体系的检测灵敏度为1.35×10-3 mg/L.我们建立的致病性维罗纳气单胞菌检测方法能特异地检测致病性维罗纳气单胞菌,并具有高度灵敏性.     

PNA openers as a tool for direct quantification of specific targets in duplex DNA

We report a new approach for target quantification directly within DNA duplex. Our assay is based on the formation of a new biomolecular structure, the PD-loop. The approach takes advantage of a selective hybridization of a probe to double-stranded DNA (dsDNA), which is locally opened by a pair of bis-PNA oligomers. To optimize the technique, several experimental formats are tested with the use of PNA and oligonucleotide probes. The highest sensitivity is achieved when the hybridized probe is extended and multiply labeled with 125I-dCTP by DNA polymerase via strand displacement in the presence of single-strand binding (SSB) protein. In this case, the PNA-assisted probe hybridization combined with the method of multiphoton detection (MPD) allows to monitor sub-attomolar amounts of the HIV-1 target on the background of unrelated DNA at sub-nCi level of radioactivity. The developed robust methodology is highly discriminative to single mutations, thus being of practical use for DNA analysis.     

Use of random amplified polymorphic DNA markers for the detection of Azospirillum strains in soil microcosms

Probes for the detection of Azospirillum strains were obtained from DNA fragments generated by random amplification of polymorphic DNA (RAPD) and tested to assess their specificity towards DNA extracted from pure cultures. The most specific probe, referred to as α4, produced a hybridization signal only with amplified DNA of A. lipoferum ATCC29731. This strain was inoculated, together with two other Azospirillum strains, in soil microcosms of different complexity and its presence tested with the probe α4. This probe confirmed its high specificity with amplified DNA extracted from the soil microcosm and in the presence of other A. lipoferum strains, indicating that the strategy for bacterial detection, based on RAPD markers, is useful for monitoring the presence of a particular strain under environment-like conditions. Other RAPD-derived probes, when tested on soil samples, did not show the same level of specificity as that shown on DNA from pure cultures. This result suggests that some precautions are necessary in the choice of a really specific RAPD marker. In a further development of this strategy, the α4 probe was sequenced and two pairs of “nested” primers were designed, which enabled a diagnostic polymerase chain reaction from soil samples that was specific for the A. lipoferum species. Received: 7 July 1997 / Accepted: 14 October 1997     

Exonuclease III-based and gold nanoparticle-assisted DNA detection with dual signal amplification

Herein we report a sensitive electrochemical biosensor for DNA detection by making use of exonuclease III and probe DNA functionalized gold nanoparticles. While probe DNA P1 modified on a gold electrode surface can self-hybridize into a stem-loop structure with an exonuclease III-resistant 3' overhang end, in the presence of target DNA, P1 may also hybridize with the target DNA to form a duplex region. Therefore, exonuclease III may selectively digest P1 from its 3'-hydroxyl termini until the duplex is fully consumed. Since a single target DNA can trigger exonuclease III digestion of numerous P1 strands, the first signal amplification is achieved. On the other hand, since the digested P1, exposing its complementary sequence to probe DNA P2, can further hybridize with P2 that has been previously modified on the surface of gold nanoparticles, many nanoparticles loaded with numerous DNA strands are immobilized onto the electrode surface. Consequently, large amount of electroactive molecules [Ru(NH(3))(6)](3+) can bind with the DNA strands to produce an intense electrochemical response as the second signal amplification. Based on the studies with cyclic voltammetry (CV) and chronocoulometry (CC) techniques, the proposed biosensor can sensitively detect specific target DNA at a picomolar level with high specificity.     

A novel strategy to design highly specific PCR primers based on the stability and uniqueness of 3'-end subsequences

MOTIVATION: In contrast with conventional PCR using a pair of specific primers, some applications utilize a single unique primer in combination with a common primer, thereby relying solely on the former for specificity. These applications include rapid amplification of cDNA ends (RACE), adaptor-tagged competitive PCR (ATAC-PCR), PCR-mediated genome walking and so forth. Since the primers designed by conventional methods often fail to work in these applications, an improved strategy is required, particularly, for a large-scale analysis. RESULTS: Based on the structure of 'off-target' products in the ATAC-PCR, we reasoned that the practical determinant of the specificity of primers may not be the uniqueness of entire sequence but that of the shortest 3'-end subsequence that exceeds a threshold of duplex stability. We termed such a subsequence as a 'specificity-determining subsequence' (SDSS) and developed a simple algorithm to predict the performance of the primer: the algorithm identifies the SDSS of each primer and examines its uniqueness in the target genome. The primers designed using this algorithm worked much better than those designed using a conventional method in both ATAC-PCR and 5'-RACE experiments. Thus, the algorithm will be generally useful for improving various PCR-based applications.     

Site-specific and photo-induced alkylation of DNA by a dimethylanthraquinone-oligodeoxynucleotide conjugate.

A dialkyl-substituted anthraquinone derivative was synthesized and ligated to a sequence-directing oligodeoxynucleotide to examine its efficiency and specificity for cross-linking to complementary sequences of DNA. The anthraquinone appendage stabilized spontaneous hybridization of the target and probe sequences through non-covalent interactions, as indicated by thermal denaturation studies. Covalent modification of the target was induced by exposure to near UV light (lambda > 335 nm) to generate cross-linked duplexes in yields as great as 45%. Reaction was dependent on the first unpaired nucleotide extended beyond the duplex formed by association of the target and probe. A specificity of C > T > A = G was determined for modification at this position. The overall site and nucleotide selectivity seems to originate from the chemical requirements of cross-linking and does not likely reflect the dominant solution structure of the complex prior to irradiation.     

产气荚膜梭菌实时荧光PCR方法的建立

目的:利用荧光定量PCR技术,建立快速敏感特异的检测产气荚膜梭菌的方法。方法:以产气荚膜梭菌基因为靶序列设计引物和探针,以自产气荚膜梭菌菌株中提取的DNA为模板,优化引物和探针的浓度比,同时验证方法的特异性、敏感性。结果:建立的反应体系在上游引物浓度为0.45μmol/L、下游引物浓度为0.15μmol/L、探针浓度为0.3μmol/L时,具有良好的特异性和敏感性,与创伤弧菌等12种相关细菌均无交叉反应;对纯菌检测的灵敏度低于10 CFU/反应体系。结论:建立的实时荧光PCR方法特异、灵敏、快速,能对战时气性坏疽做出快速准确的报告,实现对这种战时高发疾病的安全、快速和定量检测。     

Hybridization of 2'-O-methyl and 2'-deoxy molecular beacons to RNA and DNA targets

Molecular beacons are stem–loop hairpin oligonucleotide probes labeled with a fluorescent dye at one end and a fluorescence quencher at the other end; they can differentiate between bound and unbound probes in homogeneous hybridization assays with a high signal-to-background ratio and enhanced specificity compared with linear oligonucleotide probes. However, in performing cellular imaging and quantification of gene expression, degradation of unmodified molecular beacons by endogenous nucleases can significantly limit the detection sensitivity, and results in fluorescence signals unrelated to probe/target hybridization. To substantially reduce nuclease degradation of molecular beacons, it is possible to protect the probe by substituting 2′-O-methyl RNA for DNA. Here we report the analysis of the thermodynamic and kinetic properties of 2′-O-methyl and 2′-deoxy molecular beacons in the presence of RNA and DNA targets. We found that in terms of molecular beacon/target duplex stability, 2′-O-methyl/RNA > 2′-deoxy/RNA > 2′-deoxy/DNA > 2′-O-methyl/DNA. The improved stability of the 2′-O-methyl/RNA duplex was accompanied by a slightly reduced specificity compared with the duplex of 2′-deoxy molecular beacons and RNA targets. However, the 2′-O-methyl molecular beacons hybridized to RNA more quickly than 2′-deoxy molecular beacons. For the pairs tested, the 2′-deoxy-beacon/DNA-target duplex showed the fastest hybridization kinetics. These findings have significant implications for the design and application of molecular beacons.     

Strand-invading linear probe combined with unmodified PNA

Efficient strand invasion by a linear probe to fluorescently label double-stranded DNA has been implemented by employing a probe and unmodified PNA. As a fluorophore, we utilized ethynylperylene. Multiple ethynylperylene residues were incorporated into the DNA probe via a d-threoninol scaffold. The ethynylperylene did not significantly disrupt hybridization with complementary DNA. The linear probe self-quenched in the absence of target DNA and did not hybridize with PNA. A gel-shift assay revealed that linear probe and PNA combination invaded the central region of double-stranded DNA upon heat-shock treatment to form a double duplex. To further suppress the background emission and increase the stability of the probe/DNA duplex, a probe containing anthraquinones as well as ethynylperylene was synthesized. This probe and PNA invader pair detected an internal sequence in a double-stranded DNA with high sensitivity when heat shock treatment was used. The probe and PNA pair was able to invade at the terminus of a long double-stranded DNA at 40 °C at 100 mM NaCl concentration.     

A real-time PCR for the detection of Salmonella Enteritidis in poultry meat and consumption eggs

A robust duplex 5' nuclease (TaqMan) real-time PCR was developed and in-house validated for the specific detection of Salmonella enterica subspecies enterica serovar Enteritidis in whole chicken carcass rinses and consumption eggs. The assay uses specifically designed primers and a TaqMan probe to target the Prot6e gene located on the S. Enteritidis specific 60-kb virulence plasmid. As an internal amplification control to monitor Salmonella DNA in the sample, a second primer/TaqMan probe set detects simultaneously the Salmonella specific invA gene. The assay identified correctly 95% of the 79 Salmonella Enteritidis strains tested comprising 19 different phage types. None of the 119 non-Enteritidis strains comprising 54 serovars was positive for the Prot6e gene. The assay detection probability was for 10(2) or more genome equivalents 100% and for 10 equivalents 83%. A pre-PCR sample preparation protocol including a pre-enrichment step in buffered peptone water, followed by DNA extraction was applied on low levels of artificially contaminated whole chicken carcass rinses and eggs from hens as well as 25 potentially naturally contaminated chickens. The detection limit was less than three CFU per 50 ml carcass rinse or 10 ml egg. The sensitivity and specificity compared to the traditional culture-based detection method and serotyping were both 100%. Twenty-five potentially naturally contaminated chickens were compared by the real-time PCR and the traditional cultural isolation method resulting in four Salmonella positive samples of which two were positive for the Prot6e gene and serotyped as S. Enteritidis. We show also that Salmonella isolates which have a rough lipopolysaccharide structure could be assigned to the serovar Enteritidis by the real-time PCR. This methodology can contribute to meet the need of fast identification and detection methods for use in monitoring and control measures programmes.     

Enhanced photoelectrochemical method for linear DNA hybridization detection using Au-nanopaticle labeled DNA as probe onto titanium dioxide electrode

A photoelectrochemical method was proposed to detect DNA hybridization using Au nanoparticle modified DNA as one probe on TiO2 substrate, in which the TiO2 substrate was used not only as DNA anchors but also as the signal transducers. Hybridization between the probe and the target DNA oligonucleotides was confirmed by the decreased photocurrent of the TiO2 electrode. Compared with non-label probe, Au nanoparticles enhanced the photocurrent shifts after the hybridization. The photocurrent decreased with increasing the concentration of target DNA, indicating that this method could be used for quantitative measurements, and the discrimination of the complementary from mismatched DNA. Furthermore, the hybridization binding constant was obtained and photocurrent generation mechanism was discussed. The major advantages of this photochemical method are speed, simplicity and excellent specificity. This method provides a platform for studying a wide variety of biological processes using photoelectrochemical method.     

Annealing control primer system for improving specificity of PCR amplification

A novel primer designed to improve the specificity of PCR amplification, called the annealing control primer (ACP), comprises a tripartite structure with a polydeoxyinosine [poly(dI)] linker between the 3' end target core sequence and the 5' end nontarget universal sequence. We show that this ACP linker prevents annealing of the 5' end nontarget sequence to the template and facilitates primer hybridization at the 3' end to the target sequence at specific temperatures, resulting in a dramatic improvement of annealing specificity. The effect of this linker is demonstrated by the incorporation of ACP sequences as primers during the amplification of target nucleotide sequence and as hybridization probes in the genotyping of single nucleotide polymorphisms. This is the first report to show that a poly(dI) linker between two different sequences of ACP forms a bubble-like structure and disrupts or destabilizes DNA duplex formation at certain annealing temperatures.