Homogeneous time-resolved fluorescence DNA hybridization assay by DNA-mediated formation of an EDTA-Eu(III)-beta-diketonate ternary complex.

A sensitive homogenous time-resolved fluorescence DNA hybridization assay method based on the formation of an EDTA-Eu(3+)-beta-diketonate ternary complex in the DNA hybrid was developed. The new approach combined the use of two DNA probes whose sequences compose the whole complementary strand to the target DNA, in which one probe was labeled with an EDTA-Eu(3+) complex on the 5'-terminus and the other, labeled with a bidentate beta-diketone on the 3'-terminus. After hybridization of two DNA probes with target DNA, EDTA-Eu(3+) and beta-diketone come close to each other, and an EDTA-Eu(3+)-beta-diketonate ternary complex with a strong and long-lived fluorescence was formed; thus the target DNA was detected sensitively with a detection limit of 6 pM (0.6 fmol per assay) by time-resolved fluorescence measurement. In the absence of the target DNA, due to the poor stability of bidentate beta-diketonate-Eu(3+) complex in very diluted solution, only a small amount of ternary fluorescence complex was formed.     

Electrospray ionisation-cleavable tandem nucleic acid mass tag–peptide nucleic acid conjugates: synthesis and applications to quantitative genomic analysis using electrospray ionisation-MS/MS

The synthesis and characterization of isotopomer tandem nucleic acid mass tag–peptide nucleic acid (TNT–PNA) conjugates is described along with their use as electrospray ionisation-cleavable (ESI-Cleavable) hybridization probes for the detection and quantification of target DNA sequences by electrospray ionisation tandem mass spectrometry (ESI-MS/MS). ESI-cleavable peptide TNT isotopomers were introduced into PNA oligonucleotide sequences in a total synthesis approach. These conjugates were evaluated as hybridization probes for the detection and quantification of immobilized synthetic target DNAs using ESI-MS/MS. In these experiments, the PNA portion of the conjugate acts as a hybridization probe, whereas the peptide TNT is released in a collision-based process during the ionization of the probe conjugate in the electrospray ion source. The cleaved TNT acts as a uniquely resolvable marker to identify and quantify a unique target DNA sequence. The method should be applicable to a wide variety of assays requiring highly multiplexed, quantitative DNA/RNA analysis, including gene expression monitoring, genetic profiling and the detection of pathogens.     

Electrospray ionisation-cleavable tandem nucleic acid mass tag-peptide nucleic acid conjugates: synthesis and applications to quantitative genomic analysis using electrospray ionisation-MS/MS

The synthesis and characterization of isotopomer tandem nucleic acid mass tag-peptide nucleic acid (TNT-PNA) conjugates is described along with their use as electrospray ionisation-cleavable (ESI-Cleavable) hybridization probes for the detection and quantification of target DNA sequences by electrospray ionisation tandem mass spectrometry (ESI-MS/MS). ESI-cleavable peptide TNT isotopomers were introduced into PNA oligonucleotide sequences in a total synthesis approach. These conjugates were evaluated as hybridization probes for the detection and quantification of immobilized synthetic target DNAs using ESI-MS/MS. In these experiments, the PNA portion of the conjugate acts as a hybridization probe, whereas the peptide TNT is released in a collision-based process during the ionization of the probe conjugate in the electrospray ion source. The cleaved TNT acts as a uniquely resolvable marker to identify and quantify a unique target DNA sequence. The method should be applicable to a wide variety of assays requiring highly multiplexed, quantitative DNA/RNA analysis, including gene expression monitoring, genetic profiling and the detection of pathogens.     

Nonradioactive nucleic acid detection by enhanced chemiluminescence using probes directly labeled with horseradish peroxidase

The use of nucleic acid probes directly labeled with horseradish peroxidase for detection of single copy sequences on Southern blots of human genomic DNA by enhanced chemiluminescence is described. Of the target sequences, 6 x 10(5) molecules (1 amol) have been detected on blue sensitive film using exposures of up to 60 min and probes of 0.3-5.1 kb. The chemiluminescent signal quantified using a cooled charge coupled device (CCD) camera is proportional to probe length for DNA probes in the range 50-3571 bases. The enzyme has no significant effect on the stability of a DNA/DNA hybrid formed with a 3571-base probe and target as determined by increasing the stringency of posthybridization washes by decreasing the concentration of a monovalent cation (NaCl) and by a Tm analysis. The kinetics of DNA hybridization have been analyzed by a cooled CCD camera to provide quantitative data. Ten nanograms per milliliter of probe may be used for an overnight hybridization. Southern blots can be reprobed using a DNA probe for the same or a different sequence without the necessity of stripping off the previously bound probe.     

Hairpin DNA probe based electrochemical biosensor using methylene blue as hybridization indicator

In this paper, a label-free, rapid and simple method was proposed to study the hybridization specificity of hairpin DNA probe using methylene blue (MB) as a hybridization indicator. Thiolated hairpin DNA probe was immobilized on the gold electrode by self-assembly. The voltammetric signals of MB were investigated at these modified electrodes by means of cyclic voltammetry (CV) detection. Single-base mutation oligonucleotide and random oligonucleotide can be easily discriminated from complementary target DNA. The effect of mismatch position in target DNA was investigated. Experimental results showed that mutation in the center of target DNA had greatest effect on the hybridization with hairpin DNA probe. The relationship between electrochemical responses and DNA target concentration was also studied. The reduction current of MB intercalation decreased with increasing the concentration of target DNA. Taken together, these experiments demonstrate that the hybridization indicator MB provides great promise for rapid and specific measurement of target DNA.     

Computer simulation study of molecular recognition in model DNA microarrays

DNA microarrays have been widely adopted by the scientific community for a variety of applications. To improve the performance of microarrays there is a need for a fundamental understanding of the interplay between the various factors that affect microarray sensitivity and specificity. We use lattice Monte Carlo simulations to study the thermodynamics and kinetics of hybridization of single-stranded target genes in solution with complementary probe DNA molecules immobilized on a microarray surface. The target molecules in our system contain 48 segments and the probes tethered on a hard surface contain 8-24 segments. The segments on the probe and target are distinct and each segment represents a sequence of nucleotides ( approximately 11 nucleotides). Each probe segment interacts exclusively with its unique complementary target segment with a single hybridization energy; all other interactions are zero. We examine how the probe length, temperature, or hybridization energy, and the stretch along the target that the probe segments complement, affect the extent of hybridization. For systems containing single probe and single target molecules, we observe that as the probe length increases, the probability of binding all probe segments to the target decreases, implying that the specificity decreases. We observe that probes 12-16 segments ( approximately 132-176 nucleotides) long gave the highest specificity and sensitivity. This agrees with the experimental results obtained by another research group, who found an optimal probe length of 150 nucleotides. As the hybridization energy increases, the longer probes are able to bind all their segments to the target, thus improving their specificity. The hybridization kinetics reveals that the segments at the ends of the probe are most likely to start the hybridization. The segments toward the center of the probe remain bound to the target for a longer time than the segments at the ends of the probe.     

Mechanistic approach to the problem of hybridization efficiency in fluorescent in situ hybridization

In fluorescent in situ hybridization (FISH), the efficiency of hybridization between the DNA probe and the rRNA has been related to the accessibility of the rRNA when ribosome content and cell permeability are not limiting. Published rRNA accessibility maps show that probe brightness is sensitive to the organism being hybridized and the exact location of the target site and, hence, it is highly unpredictable based on accessibility only. In this study, a model of FISH based on the thermodynamics of nucleic acid hybridization was developed. The model provides a mechanistic approach to calculate the affinity of the probe to the target site, which is defined as the overall Gibbs free energy change (DeltaG degrees overall) for a reaction scheme involving the DNA-rRNA and intramolecular DNA and rRNA interactions that take place during FISH. Probe data sets for the published accessibility maps and experiments targeting localized regions in the 16S rRNA of Escherichia coli were used to demonstrate that DeltaG degrees overall is a strong predictor of hybridization efficiency and superior to conventional estimates based on the dissociation temperature of the DNA/rRNA duplex. The use of the proposed model also allowed the development of mechanistic approaches to increase probe brightness, even in seemingly inaccessible regions of the 16S rRNA. Finally, a threshold DeltaG degrees overall of -13.0 kcal/mol was proposed as a goal in the design of FISH probes to maximize hybridization efficiency without compromising specificity.     

时间分辨荧光分析法在DNA探针检测中的初步应用

应用时间分辨荧光技术进行核酸杂交分析,选用自制整合剂异硫氰酸苯基-EDTA将铕离子标记连接于链霉亲和素分子中,通过光化学反应制备生物素标记pUC118DNA探针,与固定在聚苯乙烯微滴板中的靶DNA杂交后,以铕离子Eu(3+)标记的链霉亲和素为检测物,检测靶DNA的含量,可检测到30pg的靶DNA．     

Evaluation of non-radioactive labelling and detection of deoxyribonucleic acids: Part one: chemiluminescent methods

The growth of analytical methods for the detection of nucleic acid from various biological samples reflects recent advances in biotechnology development especially in the areas of genetic, infections and cancer diagnosis. The target DNA is detected by hybridization techniques derived from Southern's blotting. However such assays, based on the use of ³²P labelled DNA probes, bring with them the associated problems of handling radioactive materials. In order to overcome these difficulties, a number of chemiluminescent detection methods have recently been developed.These new, alternative probe labelling procedures and chemiluminescent detection methods are easy to use in routine assays performed in research laboratories as well as for medical applications, and can reach the level of sensitivity found in classical radiolabelling techniques.The techniques investigated include peroxydase, biotin 16-dUTP or digoxigenin 11-dUTP probe labelling. The target DNAs are transferred onto nitrocellulose or nylon membranes and further fixed by heat or UV crosslinking. Specific hybridization on the target DNA is finally revealed by the use of chemiluminescent substrates. For all these techniques the detection limit is 10 aM (attomol) of a 561 bp target DNA. However for the probes labelled with peroxydase and with digoxigenin the detection limit drops to 1.0 aM of the target DNA. In the present paper we shall compare several of these DNA labelling and detection procedures and show that the detection threshold can vary by as much as a factor of 20 from method to method. This is the first time that various chemiluminescent methods for label and detection of DNA are compared and evaluated in order to determine the best protocol.     

Introduction of hematoxylin as an electroactive label for DNA biosensors and its employment in detection of target DNA sequence and single-base mismatch in human papilloma virus corresponding to oligonucleotide

For the detection of DNA hybridization, a new electrochemical biosensor was developed on the basis of the interaction of hematoxylin with 20-mer deoxyoligonucleotides (from human papilloma virus, HPV). The study was performed based on the interaction of hematoxylin with an alkanethiol DNA probe self-assembled gold electrode (ss-DNA/AuE) and its hybridization form (ds-DNA/AuE). The optimum conditions were found for the immobilization of HPV probe on the gold electrode (AuE) surface and its hybridization with the target DNA. Electrochemical detection of the self-assembled DNA and the hybridization process were performed by cyclic voltammetry (CV) and differential pulse voltammetry (DPV) over the potential range where the accumulated hematoxylin at the modified electrode was electroactive. Observing a remarkable difference between the voltammetric signals of the hematoxylin obtained from different hybridization samples (non-complementary, mismatch and complementary DNAs), we confirmed the potential of the developed biosensor in detecting and discriminating the target complementary DNA from non-complementary and mismatch oligonucleotides. Under optimum conditions, the electrochemical signal had a linear relationship with the concentration of the target DNA ranging from 12.5 nM to 350.0 nM, and the detection limit was 3.8 nM.     

Electrochemical DNA biosensor for the detection of interaction between di[azino-di(5,6-azafluorene)-kappa2-NN'] dibromicoppers and DNA

A new Cu(II) complex CuL(2)Br(2) (L = azino-di(5,6-azafluorene)-kappa(2)-NN') was synthesized, and a new method of electrochemical probe has been proposed for the determination of hepatitis B virus (HBV) based on its interaction with [CuL(2)](2+). This ligand, containing functional groups, as well as planar aromatic domains, is capable of binding to double-stranded DNA (dsDNA) more efficiently than to single-stranded DNA (ssDNA). Emphasis has been placed on the elucidation of the nature of the interaction by electrochemical techniques. The electroactive [CuL(2)](2+) could be employed as an electrochemical indicator to detect hybridization events in DNA biosensors. These biosensors have been constructed by immobilization of a probe DNA sequence from HBV onto glassy carbon electrode (GCE). After hybridization with the complementary target sequence, [CuL(2)](2+) was accumulated within the dsDNA layer. Electrochemical detection was performed by differential pulse voltammetry over the potential range. Using this approach, complementary target sequences of HBV can be quantified over the range of 1.74 x 10(-9) to 3.45 x 10(-7) M, with a detection limit of 8.32 x 10(-10) M and a linear correlation coefficient of 0.9936.In addition, this approach is capable of detecting hybridization of complementary sequences containing one or three mismatched bases.     

Label-free electrochemical detection of short sequences related to the hepatitis B virus using 4,4'-diaminoazobenzene based on multiwalled carbon nanotube-modified GCE

A novel label-free electrochemical DNA biosensor based on 4,4'-diaminoazobenzene (4,4'-DAAB) and multiwalled carbon nanotube (MWNT)-modified glassy carbon electrode (GCE) for short DNA sequences related to the hepatitis B virus (HBV) hybridization detection was presented. Differential pulse voltammetry (DPV) was used to investigate hybridization event. The decrease in the peak current of 4,4'-DAAB was observed on hybridization of probe with the target. This electrochemical approach was sequence specific as indicated by the control experiments, in which no peak current change was observed when a noncomplementary DNA sequence was used. Numerous factors affecting the target hybridization were optimized to maximize the sensitivity. Under optimal conditions, this sensor showed a good calibration range between 7.94 x 10(-8) M and 1.58 x 10(-6) M, with HBV DNA sequence detection limit of 1.1 x 10(-8) M.     

Quantitative DNA hybridization in solution using magnetic/luminescent core-shell nanoparticles

Nanoscale magnetic/luminescent core-shell particles were used for DNA quantification in a hybridization-in-solution approach. We demonstrated a rapid, simple, and non-polymerase chain reaction-based DNA hybridization-in-solution assay for quantifying bacteria capable of biodegrading methyl tertiary-butyl ether. Fe3O4/Eu:Gd2O3 core-shell nanoparticles synthesized by spray pyrolysis were biofunctionalized with NeutrAvidin. Following immobilization of a biotinylated probe DNA on the particles' surfaces via passive adsorption, target DNA labeled with fluorescein isothiocyanate was hybridized with probe DNA. The hybridized DNA complex was separated from solution with a magnet, while nonhybridized DNA remained in solution. The normalized fluorescence (fluorescein isothiocyanate/nanoparticles) measured with a spectrofluorometer indicated a linear quantification (R(2)=0.98) of the target bacterial 16 S rDNA. The rate of hybridization increased concurrently with the target DNA concentration. In addition, this approach differentiated between the signal outputs from perfectly complementary target and two-base mismatched target DNA in a range of concentrations, showing the specificity of the assay and the possibility for environmental applications.     

A nucleic acid biosensor for the detection of a short sequence related to the hepatitis B virus using bis(benzimidazole)cadmium(II) dinitrate as an electrochemical indicator

A novel hybridization indicator, bis(benzimidazole)cadmium(II) dinitrate (Cd(bzim)(2)(NO(3))(2)), was utilized to develop an electrochemical DNA biosensor for the detection of a short DNA sequence related to the hepatitis B virus (HBV). The sensor relies on the immobilization and hybridization of the 21-mer single-stranded oligonucleotide from the HBV long repeat at the glassy carbon electrode (GCE). The hybridization between the probe and its complementary sequence as the target was studied by enhancement of the peak of the Cd(bzim)(2)(2+) indicator using cyclic voltammetry (CV) and differential pulse voltammetry (DPV). Numerous factors affecting the probe immobilization, target hybridization, and indicator binding reactions were optimized to maximize the sensitivity and speed of the assay time. With this approach, a sequence of the HBV could be quantified over the range from 1.49x10(-7)M to 1.06x10(-6)M, with a linear correlation of r=0.9973 and a detection limit of 8.4x10(-8)M. The Cd(bzim)(2)(2+) signal observed from the probe sequence before and after hybridization with a four-base mismatch containing sequence was lower than that observed after hybridization with a complementary sequence, showing good selectivity. These results demonstrate that the Cd(bzim)(2)(2+) indicator provides great promise for the rapid and specific measurement of the target DNA.     

Trans-diamminedichlorplatinum (II)-modified probes for detection of picogram quantities of DNA.

A novel simple nonradioactive method for detection of specific nucleotide sequences has been developed. This method consists of the hybridization of a target DNA with a DNA probe modified with trans-diamminedichlorplatinum(II) (trans-DDP) followed by detection of DNA/DNA hybrids with affinity-isolated anti-DNA-trans-DDP antibodies and poly-horseradish peroxidase-protein A conjugate. Major advantages of this approach are the low cost and the extreme simplicity of the labeling procedure, which involves only mixing of the reagents. The sensitivity of the proposed technique is sufficient to detect 0.8 pg of DNA in Southern blot hybridization and 25 fg in dot hybridization and permits colony screening.     

G‐rich sequence‐functionalized polystyrene microsphere‐based instantaneous derivatization for the chemiluminescent amplified detection of DNA

Herein, we develop a novel chemiluminescence (CL) approach with high sensitivity and excellent selectivity, by taking advantage of magnetic beads as preconcentration carriers and polystyrene microspheres as an amplification platform. Briefly, a ‘sandwich‐type’ detection strategy is employed in our design, which involves capture probe DNA immobilized on the surface of carboxyl‐terminated magnetic beads and multiple biotinylated reporter DNA self‐assembled on the surface of streptavidin‐modified polystyrene microspheres. The reporter DNA includes a guanine nucleobase‐rich (G‐rich) sequence domain for the generation of light and an additional tethered nucleic acid domain complementary with the target DNA. The CL signal is obtained via a novel instantaneous derivatization reaction between a specific CL reagent and the guanine nucleo­bases rich in the target and reporter DNA. As a result, we demonstrate that this DNA assay is reproducible, stable, easy to use, and can sensitively detect femtomolar target DNA related to anthrax lethal factors with excellent differentiation ability for single‐base mismatched sequences. Overall, this new CL protocol couples the high sensitivity of CL analysis with effective magnetic separation for discriminating against unwanted constituents such as mismatched sequences, and hence, offers great promise for DNA hybridization analysis. Copyright © 2009 John Wiley & Sons, Ltd.     

A gold nanoparticle-based chronocoulometric DNA sensor for amplified detection of DNA

We report a protocol for the amplified detection of target DNA by using a chronocoulometric DNA sensor (CDS). Electrochemistry is known to be rapid, sensitive and cost-effective; it thus offers a promising approach for DNA detection. Our CDS protocol is based on a 'sandwich' detection strategy, involving a capture probe DNA immobilized on a gold electrode and a reporter probe DNA loaded on gold nanoparticles (AuNPs). Each probe flanks one of two fragments of the target sequence. A single DNA hybridization event brings AuNPs, along with hundreds of reporter probes, in the proximity of the electrode. We then employ chronocoulometry to interrogate [Ru(NH3)6]3+ electrostatically bound to the captured DNA strands. This AuNP-amplified DNA sensor can selectively detect as low as femtomolar (zeptomoles) concentrations of DNA targets and conveniently analyze a breast cancer-associated BRCA-1 mutant DNA. The time range for the entire protocol is approximately 3 d, whereas the DNA sensing takes less than 2 h to complete.     

Efficient detection of secondary structure folded nucleic acids related to Alzheimer's disease based on junction probes

Single stranded DNA often forms stable secondary structures under physiological conditions. These DNA secondary structures play important physiological roles. However, the analysis of such secondary structure folded DNA is often complicated because of its high thermodynamic stability and slow hybridization kinetics. In this article, we demonstrate that Y-shaped junction probes could be used for rapid and highly efficient detection of secondary structure folded DNA. Our approach contained a molecular beacon (MB) probe and an assistant probe. In the absence of target, the MB probe failed to hybridize with the assistant probe. Whereas, the MB probe and the assistant probe could cooperatively unwind the secondary structure folded DNA target to form a ternary Y-shaped junction structure. In this condition, the MB probe was also opened, resulting in separating the fluorophores from the quenching moiety and emitting the fluorescence signal. This approach allowed for the highly sensitive detection of secondary structure folded DNA target, such as a tau specific DNA fragment related to Alzheimer's disease in this case. Additionally, this approach showed strong SNPs identifying capability. Furthermore, it was noteworthy that this newly proposed approach was capable of detecting secondary structure folded DNA target in cell lysate samples.     

Label-free and homogeneous DNA hybridization detection using gold nanoparticles-based chemiluiminescence system

We find that the catalytic activity of gold nanoparticles (GNPs) on luminol-H₂O₂ chemiluminescence (CL) system is greatly enhanced after it is aggregated by 0.5 M NaCl. We use this observation to design a CL detection of DNA hybridization. It is based on that the single- and double-stranded oligonucleotides have different propensities to adsorb on GNPs in colloidal solution, and the hybridization occurred between the probe DNA and target DNA can result in aggregation of the GNPs, producing strong CL emission. In the assay, no covalent functionalization of the GNPs, the probe, or the target DNA is required. The assay, including hybridization and detection, occurs in homogenous solution. The detection limit of target DNA (3σ) was estimated to be as low as 1.1 fM. The sensitivity was increased more than 6 orders of magnitude over that of GNPs-based colorimetric method. The present CL method for DNA hybridization detection offers the advantages of being simple, cheap, rapid and sensitive.