DNA hybridization sensor based on aurothiomalate electroactive label on glassy carbon electrodes

In this work, a gold complex is used as electroactive label for monitoring hybridization assays on glassy carbon electrodes. Ionic gold is bound to a 30-mer sequence of the SARS (severe acute respiratory syndrome) virus, responsible for the atypical pneumonia, using sodium aurothiomalate. In order to label this single strand, a mixture of sodium aurothiomalate and the strand is prepared. Then, it is incubated for 24 h at 37 degrees C and, finally, free gold is separated from the labeled strand by a dialysis against a 0.15M NaCl solution (pH 7.5). The DNA hybridization sensor is designed immobilizing the complementary probe on the pre-treated electrode surface and, then, the hybridization reaction takes place with the gold labeled strand. The electrochemical determination is based on the catalytic effect of electrodeposited gold on the reduction of silver ions. In non-stringent experimental conditions, a limit of detection of 15 fmol (30 microL) is obtained, and discrimination between a complementary oligonucleotide and a three-based mismatch complementary oligonucleotide is achieved. For the discrimination of a single-base mismatch, is needed to use stringent conditions (50% of formamide in the hybridization buffer).     

Solution-phase detection of polynucleotides using interacting fluorescent labels and competitive hybridization

DNA was assayed in a homogeneous format using DNA probes containing hybridization-sensitive labels. The DNA probes were prepared from complementary DNA strands in which one strand was covalently labeled on the 5'-terminus with fluorescein and the complementary strand was covalently labeled on the 3'-terminus with a quencher of fluorescein emission, either pyrenebutyrate or sulforhodamine 101. Probes prepared in this manner were able to detect unlabeled target DNA by competitive hybridization producing fluorescence signals which increased with increasing target DNA concentration. A single pair of complementary probes detected target DNA at a concentration of approximately 0.1 nM in 10 min or about 10 pM in 20-30 min. Detection of a 4 pM concentration of target DNA was demonstrated in 6 h using multiple probe pairs. The major limiting factors were background fluorescence and hybridization rates. Continuous monitoring of fluorescence during competitive hybridization allowed correction for variable sample backgrounds at probe concentrations down to 20 pM; however, the time required for complete hybridization increased to greater than 1 h at probe concentrations below 0.1 nM. A promising application for this technology is the rapid detection of amplified polynucleotides. Detection of 96,000 target DNA molecules in a 50-microliters sample was demonstrated following in vitro amplification using the polymerase chain reaction technique.     

Digital chemiluminescence imaging of DNA sequencing blots using a charge-coupled device camera.

Digital chemiluminescence imaging with a cryogenically cooled charge-coupled device (CCD) camera is used to visualize DNA sequencing fragments covalently bound to a blotting membrane. The detection is based on DNA hybridization with an alkaline phosphatase(AP) labeled oligodeoxyribonucleotide probe and AP triggered chemiluminescence of the substrate 3-(2'-spiro-adamantane)-4-methoxy-4-(3"-phosphoryloxy)phenyl- 1,2-dioxetane (AMPPD). The detection using a direct AP-oligonucleotide conjugate is compared to the secondary detection of biotinylated oligonucleotides with respect to their sensitivity and nonspecific binding to the nylon membrane by quantitative imaging. Using the direct oligonucleotide-AP conjugate as a hybridization probe, sub-attomol (0.5 pg of 2.7 kb pUC plasmid DNA) quantities of membrane bound DNA are detectable with 30 min CCD exposures. Detection using the biotinylated probe in combination with streptavidin-AP was found to be background limited by nonspecific binding of streptavidin-AP and the oligo(biotin-11-dUTP) label in equal proportions. In contrast, the nonspecific background of AP-labeled oligonucleotide is indistinguishable from that seen with 5'-32P-label, in that respect making AP an ideal enzymatic label. The effect of hybridization time, probe concentration, and presence of luminescence enhancers on the detection of plasmid DNA were investigated.     

Oligonucleotide microchips as genosensors for determinative and environmental studies in microbiology.

The utility of parallel hybridization of environmental nucleic acids to many oligonucleotides immobilized in a matrix of polyacrylamide gel pads on a glass slide (oligonucleotide microchip) was evaluated. Oligonucleotides complementary to small-subunit rRNA sequences of selected microbial groups, encompassing key genera of nitrifying bacteria, were shown to selectively retain labeled target nucleic acid derived from either DNA or RNA forms of the target sequences. The utility of varying the probe concentration to normalize hybridization signals and the use of multicolor detection for simultaneous quantitation of multiple probe-target populations were demonstrated.     

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.     

A novel endonuclease IV post-PCR genotyping system

Here we describe a novel endonuclease IV (Endo IV) based assay utilizing a substrate that mimics the abasic lesions that normally occur in double-stranded DNA. The three component substrate is characterized by single-stranded DNA target, an oligonucleotide probe, separated from a helper oligonucleotide by a one base gap. The oligonucleotide probe contains a non-fluorescent quencher at the 5′ end and fluorophore attached to the 3′ end through a special rigid linker. Fluorescence of the oligonucleotide probe is efficiently quenched by the interaction of terminal dye and quencher when not hybridized. Upon hybridization of the oligonucleotide probe and helper probe to their complementary target, the phosphodiester linkage between the rigid linker and the 3′ end of the probe is efficiently cleaved, generating a fluorescent signal. In this study, the use of the Endo IV assay as a post-PCR amplification detection system is demonstrated. High sensitivity and specificity are illustrated using single nucleotide polymorphism detection.     

Hybridization of glass-tethered oligonucleotide probes to target strands preannealed with labeled auxiliary oligonucleotides

In this article we introduce a strategy of preanncaling labeled auxiliary oligonucleotides to single-stranded target DNA, prior to hybridization of the DNA target to oligonucleotide arrays (genosensors) formed on glass slides for the purpose of mutation analysis. Human genomic DNA samples from normal individuals and cystic fibrosis (CF) patients (including homozygous δF508 and heterozygous δF508/wild type (wt) in the region examined) were used. A PCR fragment of length 138 bp (wt) or 135 bp (mutant) was produced from exon l0 in the cystic fibrosis transmembrane conductance regulator (CFTR) gene, using a new pair of polymerase chain reaction (PCR) primers. This fragment contains four of the most frequent mutation sites causing the disease (Q493X, δI507, δF508, and V520F). Each of these mutations was tested using a pair of nonamer (9-mer) probes covalently attached to glass slides, representing the normal (wt) and the mutant allcles. Single-stranded target DNA was isolated from the PCR fragment using one PCR primer labeled with biotin and a streptavidin minicolumn to capture the biotin-labeled strand. Prior to hybridization to the 9-mer array on a glass slide, the unlabeled target strand was preannealed with one, three, or four auxiliary oligonucleotides, at least one being labeled with³²P. As observed previously in several laboratories, the discrimination between normal (wt) and mutant alleles at each site using oligonucleotide array hybridization ranged from very good to poor, depending on the number and location of mismatches between probe and target. Terminal mismatches along the probe were difficult to discriminate, internal mismatches were more easily discriminated, and multiple mismatches were very well discriminated. An exceptionally intense hybridization signal was obtained with a 9-mer probe that hybridized contiguously (in tandem) with one auxiliary oligonucleotide preannealed to the target DNA. The increased stability is apparently caused by strong base slacking interactions between the “capture probe” and the auxiliary oligonucleotide. The presence of the δF508 mutation was delected with this system, including discrimination between homozygous and heterozygous conditions. Base mismatch discrimination using the arrayed 9-mcr probes was improved by increasing the temperature of hybridization from 15 to 25‡C. Auxiliary oligonucleotides, preannealed to the single-stranded template, may serve several purposes to enable a more robust genosensor-based DNA sequence analysis:

Streptavidin conjugates with gold nanoparticles for visualization of single DNA interactions on the silicon surface

Applicability of scanning electron microscopy (SEM) for visualization of individual acts of DNA hybridization with oligonucleotide probes has been investigated using gold nanoparticles as a label. DNA or oligonucleotides were labeled with biotin molecules, which were then detected in DNA duplexes using a streptavidin conjugate with gold nanoparticles. Effective imaging of DNA duplexes was possible using the conjugate prepared by covalent binding. The detection limit of the model oligonucleotide of 19 bases was 20 pg.     

Oligonucleotide quartz crystal microbalance sensor for the microalgae Alexandrium minutum (Dinophyceae)

We report the immobilization on a gold surface of a 20-base DNA probe labeled with disulfide group and on the selective hybridization with the complementary 20-base DNA strand. The oligonucleotide probe is the complementary strand of a partial sequence of the gene encoding for a large ribosomal RNA sub-unit which is a coding sequence of Alexandrium minutum DNA, a microalgae that produces neurotoxins responsible for paralytic shellfish poisoning on European and Asian coasts. The kinetics of DNA probe immobilization and hybridization were monitored in situ by using a 27 MHz quartz crystal microbalance under controlled hydrodynamic conditions. The frequency of the setup is stable to within a few hertz, corresponding to the nanogram scale, for 3h and makes it possible to follow frequency change from immobilization of the probe to hybridization of the complementary DNA target. This setup constitutes a biosensor, which is sensitive and selective, and the hybridization ratio between hybridized complementary DNA and immobilized DNA probes is 47%.     

Oligozymes

The simple use of nonisotopic hybridization probes to detect complementary sequences provides valuable information in a large number of research and commercial applications. In hybridization assays, the four ‘S’s (speed, simplicity, sensitivity, and specificity) are important criteria for determining the choice of probe and label. The direct chemical combination of synthetic oligonucleotide probes and enzyme labels offer advantages unmatched by other approaches, with the oligonucleotide providing rapid hybridization and high specificity, and the direct enzyme label providing simple and sensitive detection. Such oligonucleotide-enzyme conjugates (“oligozymes”) can be used in a variety of hybridization and detection formats, including dot blots, Southern/northern blots,in situ, and solution hybridization/capture schemes. The practical synthesis and use of such oligozymes are summarized.     

Improved hybridization assays employing tailed oligonucleotide probes: a direct comparison with 5'-end-labeled oligonucleotide probes and nick-translated plasmid probes

Terminal deoxynucleotidyl transferase was used to add labeled dAMP residues to the 3' end of oligonucleotide probes that hybridize to the 5' end of the neomycin phosphotransferase II gene. Southern hybridization conditions were described in which the sensitivity per unit of exposure time was about 30-fold greater for the tailed probe as compared to the 5'-end-labeled probe. The tailed oligonucleotide probe had the sensitivity per unit of exposure time comparable to that of a nick-translated probe of high specific activity: in 3 h of autoradiographic exposure both easily detected an amount of target equivalent to a single-copy gene in 10 micrograms of human DNA. The thermal dissociation profiles of 5'-end-labeled and tailed oligonucleotide probes were virtually identical and the tailed oligonucleotide probe was as allele specific as the 5'-end-labeled oligonucleotide probe. The useful lifetime of a 32P-tailed probe was about 1-2 weeks. Finally, by adding 50 35S-labeled nucleotides to the 3' end, we prepared a stable oligonucleotide probe with a sensitivity per unit of exposure time comparable to that of the unstable 5'-32P-labeled oligonucleotide probe.     

Simultaneous detection of several oligonucleotides by time-resolved fluorometry: the use of a mixture of categorized microparticles in a sandwich type mixed-phase hybridization assay.

Porous, uniformly sized (50 micrometer) glycidyl methacrylate/ethylene dimethacrylate particles (SINTEF) were used as a solid phase to construct a sandwich type hybridization assay that allowed simultaneous detection of up to six oligonucleotides from a single sample. The assay was based on categorization of the particles by two organic prompt fluorophores, viz. fluorescein and dansyl, and quantification of the oligonucleotide hybridization by time-resolved fluorometry. Accordingly, allele-specific oligodeoxyribonucleotide probes were assembled on the particles by conventional phosphoramidite strategy using a non-cleavable linker, and the category defining fluorescein and/or dansyl tagged building blocks were inserted in the 3'-terminal sequence. An oligonucleotide bearing a photoluminescent europium(III) chelate was hybridized to the complementary 3'-terminal sequence of the target oligonucleotide, and the resulting duplex was further hybridized to the particle-bound allele-specific probes via the 5'-terminal sequence of the target. After hybridization each individual particle was subjected to three different fluorescence intensity measurements. The intensity of the prompt fluorescence signals of fluorescein and dansyl defined the particle category, while the europium(III) chelate emission quantified the hybridization. The length of the complementary region between the target oligonucleotide and the particle-bound probe was optimized to achieve maximal selectivity. Furthermore, the kinetics of hybridization and the effect of the concentration of the target oligomer on the efficiency of hybridization were evaluated. By this approach the possible presence of a three base deletion (DeltaF508), point mutation (G542X) and point deletion (1078delT) related to cystic fibrosis could unequivocally be detected from a single sample.     

Enhancement of DNA immobilization and hybridization on gold electrode modified by nanogold aggregates

Gold electrodes modified by nanogold aggregates (nanogold electrode) were obtained by the electrodeposition of gold nanoparticles onto planar gold electrode. The Electrochemical response of single-stranded DNA (ssDNA) probe immobilization and hybridization with target DNA was measured by cyclic voltammograms (CV) using methylene blue (MB) as an electroactive indicator. An improving method using long sequence target DNA, which greatly enhanced the response signal during hybridization, was studied. Nanogold electrodes could largely increase the immobilization amount of ssDNA probe. The hybridization amount of target DNA could be increased several times for the manifold nanogold electrodes. The detection limit of nanogold electrode for the complementary 16-mer oligonucleotide (target DNA1) and long sequence 55-mer oligonucleotide (target DNA2) could reach the concentration of 10(-9) mol/L and 10(-11) mol/L, respectively, which are far more sensitive than that of the planar electrode.     

DNA bioassays based on the fluorescence ‘turn off’ of silver nanocluster beacon

Recognition and quantification of oligonucleotide sequences play important roles in medical diagnosis. In this study, a new fluorescent oligonucleotide‐stabilized silver nanocluster beacon (NCB) probe was designed for sensitive detection of oligonucleotide sequence targets. This probe contained two tailored DNA strands. One strand was a signal probe strand containing a cytosine‐rich strand template for fluorescent silver nanocluster (Ag NC) synthesis and a detection sections at each end. The other strand was a fluorescence enhancing strand containing a guanine‐rich section for signal enhancement at one end and a linker section complementary to one end of the signal probe strand. After synthesis of the Ag NCs and hybridization of the two strands, the fluorescence intensity of the as‐prepared silver NCB was enhanced 200‐fold compared with the Ag NCs. Two NCBs were designed to detect two disease‐related oligonucleotide sequences, and results indicated that the two target oligonucleotide sequences in the range 50.0–600.0 and 50.0–200.0 nM could be linearly detected with detection limits of 20 and 25 nM, respectively. The developed fluorescence method using NCBs for oligonucleotide sequence detection was sensitive, facile and had potential for use in bioanalysis and diagnosis.     

Mutation detection by stacking hybridization on genosensor arrays

A new strategy for analysis of point mutations using oligonucleotide array (genosensor) hybridization was investigated. In the new approach, a single-stranded target strand is preannealed with a labeled "stacking oligonucleotide," and then the partially duplex labeled target molecule is hybridized to an array of glass-tethered oligonucleotide probes, targeted to the region on the target immediately adjacent to the stacking oligomer. In this configuration, the base-stacking interactions between the "capture probe" and the contiguously stacking oligomer stabilize the binding of the target molecule to its complementary probe on the genosensor array. The temperature of hybridization can be adjusted so that the target molecule will bind to the glass-tethered probe only in the presence of the stacking oligomer, and a single mismatch at or near the terminal position ol the capture probe disrupts the stacking interactions and thereby eliminates or greatly reduces the hybridization. This stacking hybridization approach was investigated using a collection of synthetic targets, probes, and stacking oligonucleotides, which permitted identification of conditions for optimal base mismatch discrimination. The oligonucleotide probes were tethered to the glass using a simple, improved attachment chemistry in which a 3'-aminopropanol function introduced into the probe during chemical synthesis binds covalently to silanol groups on clean, underivatized glass. "Operating parameters" examined in the stacking hybridization system included length of capture probe, position, type and number of mismatches between the probe and the target, temperature of hybridization and length of washing, and the presence of terminal phosphate group in the probe, at its junction with the stacking oligomer. The results suggest that in the stacking hybridization configuration: 1. Optimal mismatch discrimination with 9-mer probes occurs at 45 degrees C, after which little or no improvement in mispair rejection occurred on lengthy continued washing at 45 degrees C. 2. At 25 degrees C optimal mismatch discrimination occurred with 7- or 8-mer probes, or with 9-mer probes containing an additional internal mismatch. 3. The presence of a phosphate group on the 5'-end of the glass-tethered probe had no general effect on mismatch discrimination, but influenced the relative stability of different mismatches in the sequence context studied. These results provide a motivation for continued development of the stacking hybridization technique for nucleic acid sequence analysis. This approach offers several advantages over the traditional allele-specific oligonucleotide hybridization technique, and is distinct from the contiguous stacking hybridization sitrategy that the Mirzabekov laboratory has introduced (Yershov et al. (1996) Proc. Natl. Acad. Sci. USA 93, 4913-4918; Parinov et al. (1996) Nucleic Acids Res. 24, 2998-3004).     

A new and versatile reagent for incorporating multiple primary aliphatic amines into synthetic oligonucleotides.

A novel and versatile phosphoramidite, N-Fmoc-O1-DMT-O2-cyanoethoxydiisopropylamino-phosphinyl-3-am ino-1,2-propanediol (1, Fig. 1), has been synthesized and used to incorporate primary aliphatic amines into synthetic oligonucleotides. Its convenient preparation and use in solid phase oligonucleotide synthesis is described. Using phosphoramidite 1, an amino-modified oligonucleotide probe complementary to M13mp18 DNA was constructed with five primary amines attached to the 5'-terminus. The amino-modified oligonucleotide was subsequently labeled with biotin and employed in a dot-blot hybridization assay. As little as 0.5 ng of M13mp18 target DNA was colorimetrically detected.     

Universal DNA array detection of small insertions and deletions in BRCA1 and BRCA2

Array-based mutation detection methodology typically relies on direct hybridization of the fluorescently labeled query sequence to surface-bound oligonucleotide probes. These probes contain either small sequence variations or perfect-match sequence. The intensity of fluorescence bound to each oligonucleotide probe is intended to reveal which sequence is perfectly complementary to the query sequence. However, these approaches have not always been successful, especially for detection of small frameshift mutations. Here we describe a multiplex assay to detect small insertions and deletions by using a modified PCR to evenly amplify each amplicon (PCR/PCR), followed by ligase detection reaction (LDR). Mutations were identified by screening reaction products with a universal DNA microarray, which uncouples mutation detection from array hybridization and provides for high sensitivity. Using the three BRCA1 and BRCA2 founder mutations in the Ashkenazi Jewish population (BRCA1 185delAG; BRCA1 5382insC; BRCA2 6174delT) as a model system, the assay readily detected these mutations in multiplexed reactions. Our results demonstrate that universal microarray analysis of PCR/PCR/LDR products permits rapid identification of small insertion and deletion mutations in the context of both clinical diagnosis and population studies.     

A two-step hybridization method for chemiluminescent detection of single copy genes.

We have developed a technique for the chemiluminescent detection of single copy genes that eliminates the high backgrounds and problems with probe labeling associated with existing methods. The procedure employs a primary hybridization of single-stranded probe DNA to immobilized target DNA, a secondary hybridization with a covalently cross-linked oligonucleotide-alkaline phosphatase conjugate, followed by incubation in the chemiluminescent substrate AMPPD and detection on x-ray film. The key to the success of this method is that the primary probe contains a region complementary to the target DNA as well as to the oligonucleotide sequence of the secondary probe-alkaline phosphatase conjugate. Here we report our results using the two-step hybridization procedure to detect single copy genes from genomic Southern blots.