Biotinylated DNA Probes Research Articles

Electrochemical Assay of Nonlabeled DNA Chip and SNOM Imaging by Using Streptavidin-Biotin Interaction

The assay of DNA biosensor-based nucleic acid recognition using microfabrication technology provides for high sensitivity, good surface coverage and reproducibility. We have achieved efficient immobilization and hybridization of nonlabeled DNA using cyclic voltammetry (CV), square wave voltammetry (SWV) and scanning near-field optical microscopy (SNOM) techniques. The increased electrochemical response observed following the immobilization of biotinlyated ssDNA probe suggests that nucleic acid is a somewhat better medium for electronic transfer. We demonstrated the high coverage of immobilized FITC-labeled biotinylated DNA probe on a streptavidin-modified surface using SNOM imaging. SNOM imaging of FITC-labeled complementary DNA also exhibited fluorescent light spots of hybridization distributed throughout. No fluorescent light was observed with the hybridization of non-complementary DNA.

Biotin/avidin system for the generation of fully renewable DNA sensor based on biotinylated polypyrrole film

A DNA sensor based on electropolymerized biotinylated polypyrrole film was prepared. Biotinylated DNA probes were immobilized on the polypyrrole-biotin film via an intercalated layer of avidin creating the sensing layer, polypyrrole-biotin/avidin/DNA probe. From this sensor, gravimetric measurements performed with a quartz crystal microbalance (QCM) showed that the amount of DNA probe immobilized is controlled by the quantity of biotin units included within the polypyrrole-biotin film. The maximum coverage of DNA probes was achieved with a copolymer electrosynthesized from a pyrrole-biotin and un-biotinylated pyrrole monomer ratio of 1/5. Furthermore, the sensing layer can be re-generated several times with a minimum loss of activity. After the hybridization between immobilized DNA probes and complementary DNA strands, two ways of regeneration are possible. The complementary oligonucleotide (ODNc) strands can be selectively removed: (i) by solubilization of the avidin layer, which can be lifted off to give the polypyrrole-biotin film on which a new DNA sensor can be built (strategy 1) or (ii) by denaturation of the DNA duplex, leaving the polypyrrole-biotin/avidin/ODN probes sensor ready for use in a subsequent recognition processor (strategy 2). For both strategies, the first regeneration step lead to a loss of activity of 15–20%. Subsequent regenerations can be achieved without further loss of activity.

Single nucleotide polymorphism typing on DNA array with hydrophobic surface fabricated by plasma-polymerization technique

A DNA array has been fabricated on glass substrates, which enables high-throughput analysis of single-base mismatches. In this work, microfabrication-compatible plasma-polymerization (PP) method was used for immobilizing probe DNAs to study the hybridization behavior by changing surface properties. The immobilization matrix consisting of 35 Å of PP layer, applied additionally on the streptavidin absorbed hexamethyldisiloxane (HMDS)–PP layer, was constructed on the substrates to anchor biotinylated DNA probes onto the surface. The hydrophobic immobilization matrix was considered to enhance hybridization accuracy and efficiency, compared with its hydrophilic acetonitrile–PP layers. The oligonucleotide arrays fabricated on HMDS–PP surface were shown to be effective in detection of single nucleotide polymorphisms (SNPs) of ApoE gene.

Piezoelectric biosensors for real-time monitoring of hybridization and detection of hepatitis C virus

The piezoelectric quartz crystal resonators modified with oligonucleotide probes were used for detection of hepatitis C virus (HCV) in serum. The gold electrodes on either rough or smooth surface crystals were modified with a self-assembled monolayer of cystamine. After activation with glutaraldehyde, either avidin or streptavidin were immobilized and used for attachment of biotinylated DNA probes (four different sequences). Piezoelectric biosensors were used in a flow-through setup for direct monitoring of DNA resulting from the reverse transcriptase-linked polymerase chain reaction (RT-PCR) amplification of the original viral RNA. The samples of patients with hepatitis C were analyzed and the results were compared with the standard RT-PCR procedure (Amplicor test kit of Roche, microwell format with spectrophotometric evaluation). The piezoelectric hybridization assay was completed in 10 min and the same sensing surface was suitable for repeated use.

Quantitative analysis of binding of transcription factor complex to biotinylated DNA probe by a streptavidin–agarose pulldown assay

Gene expression is regulated by a large complex of proteins that bind to the promoter/enhancer region of a gene. We determined whether a streptavidin–bead binding assay might be useful in detecting individual proteins in the complex comprising transactivators, coactivators, mediators, and general transcription factors. We used biotinylated cyclooxygenase-2 promoter probes as a model. Nuclear extracts obtained from human fibroblasts treated with or without an agonist were incubated with a 5 ′-biotinylated probe and streptavidin–agarose beads at room temperature for 1 h. After centrifugation, the pellet was washed and proteins in the complex were assessed by immunoblots. An array of transcription factors was detectable concurrently in the same batch of pellets at basal state. p300 and its associated factor PCAF levels but not Srb7, Med7, or TFII B were increased by phorbol ester or tumor necrosis factor α stimulation. Only trace of CREB-binding protein was detected. These results suggest that p300 and PCAF are the predominant coactivators for COX-2 promoter activation. Our findings indicate that the streptavidin–bead pulldown assay is valuable for determining the binding of a large number of transcription factors to promoter/enhancer and evaluating the relationship of protein binding with regulation of gene expression.

KMP032 Use of Ca3 DNA fingerprinting for epidemiological study of candida albicans strains isolated from multiple geographical regions

An enzyme-linked immunosorbent assay of nucleic acid sequence-based amplification (NASBA-ELISA) was developed for molecular detection of Mycobacterium tuberculosis. The primers targeting 16S rRNA were used for the amplification of bacterial RNA by the isothermal digoxigenin (DIG)-labeling NASBA process, resulting in the accumulation of DIG-labeled RNA amplicons. The amplicons were hybridized with a specific biotinylated DNA probe which was non-covalently immobilized on streptavidin-coated microtiter plate. The RNA–DNA hybrids were colorimetrically detected by the addition of an anti-DIG antibody HRP conjugate and 2,2-azino-di-(3-ethylbenzthiazolinsulfonate) substrate. Using this method, as little as 1 × 102 CFU ml−1 of M. tuberculosis was detected within less than 5 h. Results obtained from the clinical specimens showed 85.7% and 96% sensitivity and specificity, respectively. No interference was encountered in the amplification and detection of M. tuberculosis in the presence of non-target bacteria, confirming the specificity of the method.

Ureaplasma in lung: 1. localization by in situ hybridization in a mouse model

Ureaplasma urealyticum is a common inhabitant of mucosal surfaces but is also associated with a higher incidence of pneumonia and bronchopulmonary dysplasia in preterm infants. Culture and polymerase chain reaction demonstrate high isolation rates of ureaplasma in clinical specimens documenting their presence but do not associate the organism directly with the diseased tissue. In this study, lung tissue samples from newborn mice inoculated intranasally with U. urealyticum were used to develop an in situ hybridization (ISH) test for the organism. In situ hybridization allows the localization of gene expression for visualization within the context of tissue morphology. New techniques which use biotinyl-tyramide based signal amplification have been able to greatly enhance the sensitivity of ISH. Using the Dako GenPoint Catalyzed Signal Amplification system to detect a biotinylated DNA probe specific for an internal nucleotide sequence within the urease gene of U. urealyticum, the organism was detected within the infected murine lung tissues. Electron microscopy was used to verify the presence of the organisms in the positive ISH areas. The ISH procedure developed in this study can be used to analyze the presence of ureaplasma in human neonatal lung tissue with the corresponding histopathology.

Independently-addressable micron-sized biosensor elements

With the continuing development of micro-total analysis systems and sensitive biosensing technologies, it is often desirable to immobilize biomolecules onto a surface in a small well-defined area. A novel method was developed to electrochemically attach DNA probes to micron-sized regions of a gold surface using biotin-LC-hydrazide (BH). Previously, we have found that the radical produced during the oxidation of BH will attach to a wide variety of electroactive surfaces. An array of micron-sized gold band electrodes (75 μm wide) was fabricated onto glass microscope slides and BH was deposited onto each electrode through the application of an oxidizing potential. Subsequent attachment of avidin to the biotinylated surface created the ‘molecular sandwich’ architecture necessary for further immobilization of biotinylated biomolecules to the surface. In this work, we utilized biotinylated DNA probes of varying sequence to illustrate the specificity of the attachment scheme. The immobilization of avidin, DNA probe, and hybridization of DNA target is visualized with fluorescence tags and the spatially selective attachment and hybridization of unique DNA sequences is demonstrated.

In vitro selection of RNA aptamers that bind to domain II of HCV IRES.

The translation of HCV starts at the internal ribosomal entry site (IRES) within the 5' untranslated region and domain II of IRES is essential for translation activity. However, the information of function is limited. We attempted to obtain RNA that bind HCV IRES domain II. Selected aptamers will give us much information, such as RNA-RNA interaction between IRES and other cellular RNAs as well as target sites for anti-HCV drugs. We developed a novel selection method using biotinylated DNA probe. This method can be applied to many RNAs and would identify the most suitable target position experimentally and simply. The binding kinetics of aptamers were analyzed by using Biacore. Furthermore, inhibition of translation by aptamers was analyzed.

Semi-quantitative RT-PCR-based assay, improved by Southern hybridization technique, for polarity-specific influenza virus RNAs in cultured cells

Complementary (c) DNAs against viral (v) RNA of negative polarity and complementary and/or messenger (c/m) RNA of positive polarity for influenza virus hemagglutinin (HA) were synthesized from total cellular RNA extracted from influenza virus- and mock-infected cells using polarity-specific primers, respectively. HA vRNA and c/mRNA were amplified readily by polymerase chain reaction (PCR) from influenza virus-infected cells during a virus productive period; however, non-specific PCR product was prone to amplification from mock-infected cells and cells at once after virus infection. Southern blots of the PCR products were hybridized with biotinylated DNA probe, which enabled the generation of specific signals to HA vRNA and c/mRNA. Mock-infected cells produced no signals. Furthermore, titration analyses revealed linear relationships between amount of target RNAs and generated signals. Accordingly, Southern hybridization made possible the quantitation of specific PCR products for HA vRNA and c/mRNA in cell culture and proved the lack of HA RNAs in mock-infected cells in the absence of virus. The RT-PCR based assay combined with Southern hybridization methodology was useful with respect for investigating the processes of replication and transcription of viral genes in cell culture before and during the virus productive period.

Microbial identification by immunohybridization assay of artificial RNA labels

Ribosomal RNA (rRNA) and engineered stable artificial RNAs (aRNAs) are frequently used to monitor bacteria in complex ecosystems. In this work, we describe a solid-phase immunocapture hybridization assay that can be used with low molecular weight RNA targets. A biotinylated DNA probe is efficiently hybridized in solution with the target RNA, and the DNA–RNA hybrids are captured on streptavidin-coated plates and quantified using a DNA–RNA heteroduplex-specific antibody conjugated to alkaline phosphatase. The assay was shown to be specific for both 5S rRNA and low molecular weight (LMW) artificial RNAs and highly sensitive, allowing detection of as little as 5.2 ng (0.15 pmol) in the case of 5S rRNA. Target RNAs were readily detected even in the presence of excess nontarget RNA. Detection using DNA probes as small as 17 bases targeting a repetitive artificial RNA sequence in an engineered RNA was more efficient than the detection of a unique sequence.

Immobilization and detection of DNA on microfluidic chips

With the rapid development of micro-Total Analysis Systems (μTAS) and sensitive DNA recognition technologies, it is possible to immobilize DNA probes to small areas of surfaces other than silicon. To this end, photolithographic techniques were used to derivatize micron-sized, spatially segregated DNA recognition elements in Polydimethylsiloxane (PDMS) microfluidic structures. UV light was used to initiate attachment of a photoactive biotin molecule to the substrate surface. Once biotin was attached to a substrate, biotin/avidin/biotin chemistry was used to attach fluorescently labeled or non-labeled avidin and biotinylated DNA probes. These techniques were applied to create a prototype microfluidic sensor device that was used to separate and identify synthetic DNA targets that were fluorescently-labeled.

Factor V R506Q mutation-Leiden: an independent risk factor for venous thrombosis but not coronary artery disease.

A specific point G-A transition at nucleotide position 1691 in the factor V (FV) gene, FV-Leiden, was associated with increased risk of venous thromboembolism (VTE). Insofar as the association of FV-Leiden with coronary artery disease (CAD) remains poorly defined, the aim of this study was to determine the prevalence of FV-Leiden in a sample of 68 VTE patients, 69 CAD patients, and 192 randomly selected healthy subjects. Total genomic DNA was extracted from the peripheral blood of study subjects and was used for PCR analysis. The presence (or absence) of FV-Leiden was assessed by PCR using primers flanking the mutant site (nt 1691), followed by hybridization with wild-type ('G') and mutant ('A') biotinylated DNA probes; detection was by DNA enzyme immunoassay (DEIA). While the prevalence of FV-Leiden in CAD patients was not statistically different from that of healthy subjects (14.5 % vs. 15.1 %; P=0.890, odds ratio 0.95; 95 % confidence interval 0.43--2.06), a significant increase in FV-Leiden prevalence was seen in VTE patients (70.6 % in VTE patients; P<0.001, odds ratio 13.4, 95 % confidence interval 6.9--25.8). Of the 48 VTE patients who tested positive for FV-Leiden, 42 were heterozygotes (G/A), while 6 were homozygotes (A/A) (allele frequency 0.397). All 10 CAD patients positive for FV-Leiden were heterozygote carriers (allele frequency 0.072). While gender was not a factor in FV-Leiden expression, higher prevalence in FV-Leiden was seen in younger (< or =45 years) VTE patients (38/51 vs. 10/17). FV-Leiden is a major inherited risk factor for VTE, with a peak incidence in younger patients, but does not appear to play any role in CAD pathogenesis in the population studied.

Microgravimetric DNA sensor based on quartz crystal microbalance: comparison of oligonucleotide immobilization methods and the application in genetic diagnosis

We report on the study of immobilization DNA probes onto quartz crystal oscillators by self-assembly technique to form variety types of mono- and multi-layered sensing films towards the realization of DNA diagnostic devices. A 18-mer DNA probe complementary to the site of genetic β-thalassaemia mutations was immobilized on the electrodes of QCM by covalent bonding or electrostatic adsorption on polyelectrolyte films to form mono- or multi-layered sensing films by self-assembled process. Hybridization was induced by exposure of the QCMs immobilized with DNA probe to a test solution containing the target nucleic acid sequences. The kinetics of DNA probe immobilization and hybridization with the fabricated DNA sensors were studied via in-situ frequency changes. The characteristics of QCM sensors containing mono- or multi-layered DNA probe constructed by direct chemical bonding, avidin–biotin interaction or electrostatic adsorption on polyelectrolyte films were compared. Results indicated that the DNA sensing films fabricated by immobilization of biotinylated DNA probe to avidin provide fast sensor response and high hybridization efficiencies. The effects of ionic strength of the buffer solution and the concentration of target nucleic acid used in hybridization were also studied. The fabricated DNA biosensor was used to detect a set of real samples. We conclude that the microgravimetric DNA sensor with its direct detection of amplified products provide a rapid, low cost and convenient diagnostic method for genetic disease.

Escherichia coli proline tRNA: structure and recognition sites for prolyl-tRNA synthetase.

A major proline tRNA was purified from bulk Escherichia coli A19 tRNA by affinity chromatography with a biotinylated DNA probe. Its nucleotide sequence including modified nucleotides was determined by the post-labelling technique. In order to study the recognition sites of this proline tRNA for prolyl-tRNA synthetase, various mutant transcripts were prepared using an in vitro transcription system with T7 RNA polymerase. Based on the results of in vitro kinetic analyses of mutant transcripts, it was concluded that the second and third letters, G35 and G36, of the anticodon, G37 of the anticodon loop, the discriminator base A73, G72 of the acceptor stem, G49 and U17A that existed in the corner of an L-shaped structure are the recognition sites of proline tRNA for prolyl-tRNA synthetase.

Histidine Decarboxylase Expression in Human Melanoma

Histamine has been implicated as one of the mediators involved in regulation of proliferation in both normal and neoplastic tissues. Histidine decarboxylase, the only enzyme that catalyzes the formation of histamine from L-histidine, is an essential regulator of histamine levels. In this study, we investigated the gene and protein expression of histidine decarboxylase in melanoma. Reverse transcriptase polymerase chain reaction and in situ hybridization studies of WM-35, WM-983/B, HT-168, and M1 human melanoma cell lines both resulted in positive signals for histidine decarboxylase messenger RNA. A polyclonal chicken antibody was developed against human histidine decarboxylase and protein expression was confirmed by western blot analysis of the cell lysates, revealing a predominant immunoreactive band at approximately 54 kDa corresponding to monomeric histidine decarboxylase. Protein expression of histidine decarboxylase was also shown by flow cytometric analysis and strong punctate cytoplasmic staining of melanoma cell lines. Moreover, both primary and metastatic human melanoma tissues were brightly stained for histidine decarboxylase. When compared with the very weak or no reactions on cultivated human melanocytes both western blot and immunohistochemical studies showed much stronger histidine decarboxylase expression in melanoma cells. These findings suggest that expression of histidine decarboxylase is elevated in human melanoma.

Hybridization Assay at a Disposable Electrochemical Biosensor for the Attomole Detection of Amplified Human Cytomegalovirus DNA

A disposable electrochemical biosensor for the detection of DNA sequences related to the human cytomegalovirus (HCMV) is described. The sensor relies on the adsorption of an amplified human cytomegalovirus DNA strand onto the sensing surface of a screen-printed carbon electrode, and to its hybridization to a complementary single-stranded biotinylated DNA probe. The extent of hybrids formed was determined with streptavidin conjugated to horseradish peroxidase. The peroxidase label was indirectly quantified by measuring the amount of the chromophore and electroactive product 2,2′-diaminoazobenzene generated from the o-phenylenediamine substrate. The intensity of differential pulse voltammetric peak currents resulting from the reduction of the enzyme-generated product was related to the number of target HCMV-amplified DNA molecules present in the sample, and the results were compared to those obtained with standard methods, i.e., agarose gel electrophoresis quantification and colorimetric hybridization assay in a microtiter plate. A detection limit of 0.6 amol/ml of HCMV-amplified DNA fragment was obtained with the electrochemical DNA biosensor. The electrochemical method was 23,000-fold more sensitive than the gel electrophoresis technique and 83-fold more sensitive than the colorimetric hybridization assay in a microtiter plate.

RecA-mediated affinity capture: a method for full-length cDNA cloning.

We describe an improved method for rapid cloning of full-length cDNA from cDNA libraries. This approach is based on the ability of Escherichia coli RecA protein to form a stable nucleoprotein complex with a linear single-stranded DNA probe and homologous sequences in circular double-stranded DNA. Hybridization of RecA-coated biotinylated DNA probes to homologous plasmid DNA creates triple-stranded complexes, which are then captured on streptavidin-coated magnetic beads. Following magnetic separation of the hybrid molecules, the enriched plasmid population is recovered by alkaline treatment, precipitated, resuspended and used to transform bacteria. Typically, many clones can then be recovered by colony hybridization screening of a single plate of the enriched library. We have used this technology to clone full-length and alternatively spliced forms of the human bcl-xL cDNA from a human liver cDNA library.

Time-Resolved Fluorometric Detection of DNA Using a Tetradentate β-Diketonate Europium Chelate as a Label

A tetradentate β-diketonate europium chelate, 4,4′-bis(1″,1″,1″,2″,2″,3″,3″-heptafluoro-4″,6″-hexanedion-6″-yl)-chlorosulfo-o-terphenyl(BHHCT)-Eu3+, was labeled to a streptavidin-bovine serum albumin conjugate for time-resolved fluorometric detection of DNA by hybridization with a biotinylated probe DNA in a microtiter well. The method gave a detection limit of 23 pg/well (solid-phase measurement) and 4 pg/well (solution-phase measurement) for specific DNA.

Ultrastructural Localization of Human Papillomavirus Sequences by In Situ Hybridization and Image Analysis

Genital infection caused by human papillomavirus (HPV) is of great sanitary importance because of the role it can play in the development of squamous intraepithelial lesions and carcinomas. Because not all lesions have a malignant transformation, it is important that clinical and biological parameters contributing to the evolution of a specific lesion be determined.We used CaSki cells with 400–600 integrated copies of HPV-16 genome for in situ hybridization (ISH) with electron microscopy (EM) to locate HPV sequences. Three biotinylated DNA probes for HPV types 6/11, 16/18 and 31/ 33/51 were used, and hybrid detection was done with an anti-biotin antibody conjugated with 15 nm gold colloids. Electron microphotographs with positive labeling were studied by image analysis to skeletonize them to highlight the heterochromatin fibers and to determinate the spatial localization of colloidal-gold particles. Electron microscopy and ISH technique revealed 2 different distribution patterns: colloidal-gold particles spread over the whole nuclear surface, individually or in small clusters, and large accumulations with many gold particles in the nuclear periphery, next to the membrane. Image analysis revealed there was a spatial association between the gold particles and heterochromatin fibers in all cases.We believe this association is the consequence of viral genome integration. With these results and knowledge that integration of viral DNA is linked to a worse prognosis, we propose the application of this methodology for the future study of HPV infected biopsies to obtain prognostic information in lesions caused by this virus. (The J Histotechnol 21: 295–298, 1998)