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Sample records for rrna-targeted oligonucleotide probe

  1. Differential sensitivity of 16S rRNA targeted oligonucleotide probes used for fluorescence in situ hybridization is a result of ribosomal higher order structure.

    PubMed

    Frischer, M E; Floriani, P J; Nierzwicki-Bauer, S A

    1996-10-01

    The use of 16S rRNA targeted gene probes for the direct analysis of microbial communities has revolutionized the field of microbial ecology, yet a comprehensive approach for the design of such probes does not exist. The development of 16S rRNA targeted oligonucleotide probes for use with fluorescence in situ hybridization (FISH) procedures has been especially difficult as a result of the complex nature of the rRNA target molecule. In this study a systematic comparison of 16S rRNA targeted oligonucleotide gene probes was conducted to determine if target location influences the hybridization efficiency of oligonucleotide probes when used with in situ hybridization protocols for the detection of whole microbial cells. Five unique universal 12-mer oligonucleotide sequences, located at different regions of the 16S rRNA molecule, were identified by a computer-aided sequence analysis of over 1000 partial and complete 16S rRNA sequences. The complements of these oligomeric sequences were chemically synthesized for use as probes and end labeled with either [gamma-32P]ATP or the fluorescent molecule tetramethylrhodamine-5/-6. Hybridization sensitivity for each of the probes was determined by hybridization to heat-denatured RNA immobilized on blots or to formaldehyde fixed whole cells. All of the probes hybridized with equal efficiency to denatured RNA. However, the probes exhibited a wide range of sensitivity (from none to very strong) when hybridized with whole cells using a previously developed FISH procedure. Differential hybridization efficiencies against whole cells could not be attributed to cell wall type, since the relative probe efficiency was preserved when either Gram-negative or -positive cells were used. These studies represent one of the first attempts to systematically define criteria for 16S rRNA targeted probe design for use against whole cells and establish target site location as a critical parameter in probe design.

  2. Gene cloning based on long oligonucleotide probes

    SciTech Connect

    Wood, W.I.

    1987-01-01

    The most commonly used technique for gene cloning has been to utilize oligonucleotide probe based on protein sequence data. Of course this approach requires characterized and purified protein so that at least a portion of amino acid sequence can be determined and used to infer the corresponding DNA sequence. Based on the amino acid sequence information, either short or long oligonucleotide probes can be synthesized chemically. Long probes are typically 30-100 nucleotides long and are a single sequence based on a best guess for each codon. The long probe approach was first used to screen for three different genes: bovine trypsin inhibitor, human insulin-like growth factor I, and human factor IX. There are three advantages of long probes. (1) Any stretch of amino acid sequence 10 or longer can be used. (2) The amino acid sequence need not be absolutely correct. (3) These probes can be used to screen high-complexity libraries with fewer false positives. In spite of the uncertainties over codon selection, the long probe approach is currently the method of choice in screening for genes based on protein sequence data.

  3. Design and analysis of mismatch probes for long oligonucleotide microarrays

    SciTech Connect

    Deng, Ye; He, Zhili; Van Nostrand, Joy D.; Zhou, Jizhong

    2008-08-15

    Nonspecific hybridization is currently a major concern with microarray technology. One of most effective approaches to estimating nonspecific hybridizations in oligonucleotide microarrays is the utilization of mismatch probes; however, this approach has not been used for longer oligonucleotide probes. Here, an oligonucleotide microarray was constructed to evaluate and optimize parameters for 50-mer mismatch probe design. A perfect match (PM) and 28 mismatch (MM) probes were designed for each of ten target genes selected from three microorganisms. The microarrays were hybridized with synthesized complementary oligonucleotide targets at different temperatures (e.g., 42, 45 and 50 C). In general, the probes with evenly distributed mismatches were more distinguishable than those with randomly distributed mismatches. MM probes with 3, 4 and 5 mismatched nucleotides were differentiated for 50-mer oligonucleotide probes hybridized at 50, 45 and 42 C, respectively. Based on the experimental data generated from this study, a modified positional dependent nearest neighbor (MPDNN) model was constructed to adjust the thermodynamic parameters of matched and mismatched dimer nucleotides in the microarray environment. The MM probes with four flexible positional mismatches were designed using the newly established MPDNN model and the experimental results demonstrated that the redesigned MM probes could yield more consistent hybridizations. Conclusions: This study provides guidance on the design of MM probes for long oligonucleotides (e.g., 50 mers). The novel MPDNN model has improved the consistency for long MM probes, and this modeling method can potentially be used for the prediction of oligonucleotide microarray hybridizations.

  4. Oligonucleotide probe for detection and identification of Campylobacter pylori.

    PubMed Central

    Morotomi, M; Hoshina, S; Green, P; Neu, H C; LoGerfo, P; Watanabe, I; Mutai, M; Weinstein, I B

    1989-01-01

    We have developed a novel and practical DNA-RNA hybridization assay for the detection and identification of Campylobacter pylori in the gastric mucosa. This technique utilizes a [32P]ddATP-labeled synthetic oligonucleotide probe complementary to a nucleotide sequence present in C. pylori 16S rRNA. This probe is very sensitive and reacted with all 23 strains of C. pylori tested. It is also highly specific, since there was no cross-reactivity with the heterologous organisms Campylobacter coli, C. fetus subsp. fetus, C. jejuni, and C. laridis or with Escherichia coli. Hybridization of the oligonucleotide probe with C. pylori RNA was completely inhibited by treatment of the membrane filters with RNase but not DNase. Although a gastric mucosa tissue homogenate slightly inhibited the hybridization, as few as 10(4) C. pylori cells could be detected even in the presence of 5 mg of gastric mucosa. Gastric biopsy specimens obtained from patients referred for upper gastrointestinal tract endoscopy were tested for C. pylori infection by direct oligonucleotide hybridization, and the results were compared with those of bacteriological cultures, the urease test, and histological observations. A comparison of the urease test and the oligonucleotide hybridization results showed an excellent correlation between the two methods. The clinical usefulness of this oligonucleotide-RNA hybridization method is discussed. Images PMID:2480360

  5. Empirical evaluation of oligonucleotide probe selection for DNA microarrays.

    PubMed

    Mulle, Jennifer G; Patel, Viren C; Warren, Stephen T; Hegde, Madhuri R; Cutler, David J; Zwick, Michael E

    2010-03-29

    DNA-based microarrays are increasingly central to biomedical research. Selecting oligonucleotide sequences that will behave consistently across experiments is essential to the design, production and performance of DNA microarrays. Here our aim was to improve on probe design parameters by empirically and systematically evaluating probe performance in a multivariate context. We used experimental data from 19 array CGH hybridizations to assess the probe performance of 385,474 probes tiled in the Duchenne muscular dystrophy (DMD) region of the X chromosome. Our results demonstrate that probe melting temperature, single nucleotide polymorphisms (SNPs), and homocytosine motifs all have a strong effect on probe behavior. These findings, when incorporated into future microarray probe selection algorithms, may improve microarray performance for a wide variety of applications.

  6. Oligonucleotide probes functionalization of nanogap electrodes.

    PubMed

    Zaffino, Rosa Letizia; Mir, Mònica; Samitier, Josep

    2017-05-15

    Nanogap electrodes have attracted a lot of consideration as promising platform for molecular electronic and biomolecules detection. This is mainly for their higher aspect ratio, and because their electrical properties are easily accessed by current-voltage measurements. Nevertheless, application of standard current-voltages measurements used to characterize nanogap response, and/or to modify specific nanogap electrodes properties, represents an issue. Since the strength of electrical fields in nanoscaled devices can reach high values, even at low voltages. Here, we analyzed the effects induced by different methods of surface modification of nanogap electrodes, in test-voltage application, employed for the electrical detection of a desoxyribonucleic acid (DNA) target. Nanogap electrodes were functionalized with two antisymmetric oligo-probes designed to have 20 terminal bases complementary to the edges of the target, which after hybridization bridges the nanogap, closing the electrical circuit. Two methods of functionalization were studied for this purpose; a random self-assembling of a mixture of the two oligo-probes (OPs) used in the platform, and a selective method that controls the position of each OP at selected side of nanogap electrodes. We used for this aim, the electrophoretic effect induced on negatively charged probes by the application of an external direct current voltage. The results obtained with both functionalization methods where characterized and compared in terms of electrode surface covering, calculated by using voltammetry analysis. Moreover, we contrasted the electrical detection of a DNA target in the nanogap platform either in site-selective and in randomly assembled nanogap. According to our results, a denser, although not selective surface functionalization, is advantageous for such kind of applications. © 2017 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

  7. Microarray oligonucleotide probe designer (MOPeD): A web service.

    PubMed

    Patel, Viren C; Mondal, Kajari; Shetty, Amol Carl; Horner, Vanessa L; Bedoyan, Jirair K; Martin, Donna; Caspary, Tamara; Cutler, David J; Zwick, Michael E

    2010-11-01

    Methods of genomic selection that combine high-density oligonucleotide microarrays with next-generation DNA sequencing allow investigators to characterize genomic variation in selected portions of complex eukaryotic genomes. Yet choosing which specific oligonucleotides to be use can pose a major technical challenge. To address this issue, we have developed a software package called MOPeD (Microarray Oligonucleotide Probe Designer), which automates the process of designing genomic selection microarrays. This web-based software allows individual investigators to design custom genomic selection microarrays optimized for synthesis with Roche NimbleGen's maskless photolithography. Design parameters include uniqueness of the probe sequences, melting temperature, hairpin formation, and the presence of single nucleotide polymorphisms. We generated probe databases for the human, mouse, and rhesus macaque genomes and conducted experimental validation of MOPeD-designed microarrays in human samples by sequencing the human X chromosome exome, where relevant sequence metrics indicated superior performance relative to a microarray designed by the Roche NimbleGen proprietary algorithm. We also performed validation in the mouse to identify known mutations contained within a 487-kb region from mouse chromosome 16, the mouse chromosome 16 exome (1.7 Mb), and the mouse chromosome 12 exome (3.3 Mb). Our results suggest that the open source MOPeD software package and website (http://moped.genetics.emory.edu/) will make a valuable resource for investigators in their sequence-based studies of complex eukaryotic genomes.

  8. EvoOligo: oligonucleotide probe design with multiobjective evolutionary algorithms.

    PubMed

    Shin, Soo-Yong; Lee, In-Hee; Cho, Young-Min; Yang, Kyung-Ae; Zhang, Byoung-Tak

    2009-12-01

    Probe design is one of the most important tasks in successful deoxyribonucleic acid microarray experiments. We propose a multiobjective evolutionary optimization method for oligonucleotide probe design based on the multiobjective nature of the probe design problem. The proposed multiobjective evolutionary approach has several distinguished features, compared with previous methods. First, the evolutionary approach can find better probe sets than existing simple filtering methods with fixed threshold values. Second, the multiobjective approach can easily incorporate the user's custom criteria or change the existing criteria. Third, our approach tries to optimize the combination of probes for the given set of genes, in contrast to other tools that independently search each gene for qualifying probes. Lastly, the multiobjective optimization method provides various sets of probe combinations, among which the user can choose, depending on the target application. The proposed method is implemented as a platform called EvoOligo and is available for service on the web. We test the performance of EvoOligo by designing probe sets for 19 types of Human Papillomavirus and 52 genes in the Arabidopsis Calmodulin multigene family. The design results from EvoOligo are proven to be superior to those from well-known existing probe design tools, such as OligoArray and OligoWiz.

  9. A simple and rapid method for the preparation of homologous DNA oligonucleotide hybridization probes from heterologous gene sequences and probes.

    PubMed

    Maxwell, E S; Sarge, K D

    1988-11-30

    We describe a simple and rapid method for the preparation of homologous DNA oligonucleotide probes for hybridization analysis and/or cDNA/genomic library screening. With this method, a synthetic DNA oligonucleotide derived from a known heterologous DNA/RNA/protein sequence is annealed to an RNA preparation containing the gene transcript of interest. Any unpaired 3'-terminal oligonucleotides of the heterologous DNA primer are then removed using the 3' exonuclease activity of the DNA Polymerase I Klenow fragment before primer extension/dideoxynucleotide sequencing of the annealed RNA species with AMV reverse transcriptase. From the determined RNA sequence, a completely homologous DNA oligonucleotide probe is then prepared. This approach has been used to prepare a homologous DNA oligonucleotide probe for the successful library screening of the yeast hybRNA gene starting with a heterologous mouse hybRNA DNA oligonucleotide probe.

  10. Identifying members of the domain Archaea with rRNA-targeted oligonucleotide probes.

    PubMed

    Burggraf, S; Mayer, T; Amann, R; Schadhauser, S; Woese, C R; Stetter, K O

    1994-09-01

    Two 16S rRNA-targeted oligonucleotide probes were designed for the archaeal kingdoms Euryachaeota and Crenarchaeota. Probe specificities were evaluated by nonradioactive dot blot hybridization against selected reference organisms. The successful application of fluorescent-probe derivatives for whole-cell hybridization required organism-specific optimizations of fixation and hybridization conditions to assure probe penetration and morphological integrity of the cells. The probes allowed preliminary grouping of three new hyperthermophilic isolates. Together with other group-specific rRNA-targeted oligonucleotide probes, these probes will facilitate rapid in situ monitoring of the populations present in hydrothermal systems and support cultivation attempts.

  11. Combined in vitro transcription and reverse transcription to amplify and label complex synthetic oligonucleotide probe libraries.

    PubMed

    Murgha, Yusuf; Beliveau, Brian; Semrau, Kassandra; Schwartz, Donald; Wu, Chao-Ting; Gulari, Erdogan; Rouillard, Jean-Marie

    2015-06-01

    Oligonucleotide microarrays allow the production of complex custom oligonucleotide libraries for nucleic acid detection-based applications such as fluorescence in situ hybridization (FISH). We have developed a PCR-free method to make single-stranded DNA (ssDNA) fluorescent probes through an intermediate RNA library. A double-stranded oligonucleotide library is amplified by transcription to create an RNA library. Next, dye- or hapten-conjugate primers are used to reverse transcribe the RNA to produce a dye-labeled cDNA library. Finally the RNA is hydrolyzed under alkaline conditions to obtain the single-stranded fluorescent probes library. Starting from unique oligonucleotide library constructs, we present two methods to produce single-stranded probe libraries. The two methods differ in the type of reverse transcription (RT) primer, the incorporation of fluorescent dye, and the purification of fluorescent probes. The first method employs dye-labeled reverse transcription primers to produce multiple differentially single-labeled probe subsets from one microarray library. The fluorescent probes are purified from excess primers by oligonucleotide-bead capture. The second method uses an RNA:DNA chimeric primer and amino-modified nucleotides to produce amino-allyl probes. The excess primers and RNA are hydrolyzed under alkaline conditions, followed by probe purification and labeling with amino-reactive dyes. The fluorescent probes created by the combination of transcription and reverse transcription can be used for FISH and to detect any RNA and DNA targets via hybridization.

  12. Evaluation of an rRNA-derived oligonucleotide probe for culture confirmation of Neisseria gonorrhoeae.

    PubMed Central

    Rossau, R; Duhamel, M; Van Dyck, E; Piot, P; Van Heuverswyn, H

    1990-01-01

    The reliability of an rRNA-derived oligonucleotide probe for Neisseria gonorrhoeae was tested with 187 N. gonorrhoeae isolates, 81 Neisseria meningitidis isolates, and several strains of other bacterial species. The probe proved to be 100% specific and 100% sensitive. N. gonorrhoeae cells could also be reliably identified in contaminated cultures with the oligonucleotide probe. The 2.6-megadalton cryptic plasmid used as a probe for N. gonorrhoeae was shown to be less sensitive, detecting 179 of 181 N. gonorrhoeae isolates. Images PMID:1693630

  13. [Oligonucleotide derivatives in the nucleic acid hybridization analysis. I. Covalent immobilization of oligonucleotide probes onto the nylon].

    PubMed

    Dmitrienko, E V; Pyshnaia, I A; Pyshnyĭ, D V

    2010-01-01

    The features of UV-induced immobilization of oligonucleotides on a nylon membranes and the effectiveness of enzymatic labeling of immobilized probes at heterophase detection of nucleic acids are studied. Short terminal oligothymidilate (up to 10 nt) sequences are suggested to attach to the probe via a flexible ethylene glycol based linker. The presence of such fragment enhances the intensity of immobilization and reduces UV-dependent degradation of the targeted (sequence-specific) part of the probe by reducing the dose needed for the immobilization of DNA. The optimum dose of UV-irradiation is determined to be ~0.4 J/cm(2) at the wavelength 254 nm. This dose provides high level of hybridization signal for immobilized probes with various nucleotide composition of the sequence specific moiety. The amide groups of the polyamide are shown to play the key role in the photoinduced immobilization of nucleic acids, whereas the primary amino groups in the structure of PA is not the center responsible for the covalent binding of DNA by UV-irradiation, as previously believed. Various additives in the soaking solution during the membrane of UV-dependent immobilization of probes are shown to influence its effectiveness. The use of alternative to UV-irradiation system of radical generation are shown to provide the immobilization of oligonucleotides onto the nylon membrane.

  14. Identification of enterotoxigenic Escherichia coli with synthetic alkaline phosphatase-conjugated oligonucleotide DNA probes.

    PubMed Central

    Seriwatana, J; Echeverria, P; Taylor, D N; Sakuldaipeara, T; Changchawalit, S; Chivoratanond, O

    1987-01-01

    Alkaline phosphatase-conjugated (AP) 26-base oligonucleotide DNA probes were compared with the same probes labeled with gamma-32P for the identification of heat-labile (LT) and heat-stable (ST) enterotoxigenic Escherichia coli (ETEC). The AP oligonucleotide probes were as sensitive as the radiolabeled (RL) probes in detecting LT and STA-2 target cell DNA, but the AP ST probe, which differed from STA-1 by two bases, was less sensitive than the RL probe in detecting STA-1 DNA (6.25 versus 0.78 ng). Of 94 ETEC that were identified with the RL probe, the AP probes detected 93% (28 of 30) of ST, 73% (25 of 34) of LT, and 67% (20 of 30) of LTST ETEC. When colony lysates of these ETEC were examined, the AP probes identified all 94 ETEC. In examinations of stool blots, the RL and AP probes were shown to have sensitivities of 71 and 59%, specificities of 91 and 86%, positive predictive values of 87 and 73%, and negative predictive values of 86 and 74%, respectively. AP oligonucleotide probes to detect ETEC were less sensitive in detecting ETEC by colony or stool blot hybridization than the RL probes but could be used by laboratories without access to radioisotopes to examine colony lysates. Images PMID:3305559

  15. Kinetic effects on signal normalization in oligonucleotide microchips with labeled immobilized probes.

    PubMed

    Pan'kov, S V; Chechetkin, V R; Somova, O G; Antonova, O V; Moiseeva, O V; Prokopenko, D V; Yurasov, R A; Gryadunov, D A; Chudinov, A V

    2009-10-01

    Among various factors affecting operation of oligonucleotide microchips, the variations in concentration and in homogeneous distribution of immobilized probes over the cells are one of the most important. The labeling of immobilized probes ensures the complete current monitoring on the probe distribution and is reliable and convenient. Using hydrogel-based oligonucleotide microchips, the applicability of Cy3-labeled immobilized probes for quality control and signal normalization after hybridization with Cy5-labeled target DNA was investigated. This study showed that proper signal normalization should be different in thermodynamic conditions and in transient regime with hybridization far from saturation. This kinetic effect holds for both hydrogel-based and surface oligonucleotide microchips. Besides proving basic features, the technique was assessed on a sampling batch of 50 microchips developed for identifying mutations responsible for rifampicin and isoniazid resistance of Mycobacterium tuberculosis.

  16. Oligonucleotide primers, probes and molecular methods for the environmental monitoring of methanogenic archaea

    PubMed Central

    Narihiro, Takashi; Sekiguchi, Yuji

    2011-01-01

    Summary For the identification and quantification of methanogenic archaea (methanogens) in environmental samples, various oligonucleotide probes/primers targeting phylogenetic markers of methanogens, such as 16S rRNA, 16S rRNA gene and the gene for the α‐subunit of methyl coenzyme M reductase (mcrA), have been extensively developed and characterized experimentally. These oligonucleotides were designed to resolve different groups of methanogens at different taxonomic levels, and have been widely used as hybridization probes or polymerase chain reaction primers for membrane hybridization, fluorescence in situ hybridization, rRNA cleavage method, gene cloning, DNA microarray and quantitative polymerase chain reaction for studies in environmental and determinative microbiology. In this review, we present a comprehensive list of such oligonucleotide probes/primers, which enable us to determine methanogen populations in an environment quantitatively and hierarchically, with examples of the practical applications of the probes and primers. PMID:21375721

  17. Identification of enterotoxigenic Escherichia coli isolates with enzyme-labeled synthetic oligonucleotide probes.

    PubMed Central

    Medon, P P; Lanser, J A; Monckton, P R; Li, P; Symons, R H

    1988-01-01

    Commercially available kits containing alkaline phosphatase-labeled oligonucleotide probes for Escherichia coli heat-stable enterotoxins (STI-H, STI-P, and STII) and the heat-labile enterotoxin were compared with bioassays and radiolabeled recombinant DNA probes to identify enterotoxigenic E. coli from 100 clinical isolates. There was very good agreement between the three methods. PMID:3053766

  18. Computer selection of oligonucleotide probes from amino acid sequences for use in gene library screening.

    PubMed

    Yang, J H; Ye, J H; Wallace, D C

    1984-01-11

    We present a computer program, FINPROBE, which utilizes known amino acid sequence data to deduce minimum redundancy oligonucleotide probes for use in screening cDNA or genomic libraries or in primer extension. The user enters the amino acid sequence of interest, the desired probe length, the number of probes sought, and the constraints on oligonucleotide synthesis. The computer generates a table of possible probes listed in increasing order of redundancy and provides the location of each probe in the protein and mRNA coding sequence. Activation of a next function provides the amino acid and mRNA sequences of each probe of interest as well as the complementary sequence and the minimum dissociation temperature of the probe. A final routine prints out the amino acid sequence of the protein in parallel with the mRNA sequence listing all possible codons for each amino acid.

  19. Fluorescent-labeled oligonucleotide probes: detection of hybrid formation in solution by fluorescence polarization spectroscopy.

    PubMed Central

    Murakami, A; Nakaura, M; Nakatsuji, Y; Nagahara, S; Tran-Cong, Q; Makino, K

    1991-01-01

    Fluorescein-labeled oligonucleotides as DNA-probes were synthesized and used to monitor hybrid formation, namely to detect DNA or oligonucleotide sequence in solution. The introduction of fluorescein to oligonucleotides was carried out by oxidation of a hydrogen phosphonate linkage with ethylenediamine or hexamethylenediamine as a tether and by a subsequent labeling of the primary amine moiety by FITC. Fluorescence anisotropy, r, was adopted as an index to monitor the behavior of F-probe in solution. An increase in the anisotropy was observed upon an increase in the chain-length of F-probe. When F-Probe formed a hybrid with its complementary oligonucleotide in solution, the r value increased compared to that of F-Probe itself. These observations clearly indicate that measurements of r in solution will readily lead to the monitoring of the presence of a hybrid in solution. Consequently, it is promising to detect a certain nucleic acid sequence in solution using fluorescent-labeled oligonucleotides. PMID:1870966

  20. Ultramild protein-mediated click chemistry creates efficient oligonucleotide probes for targeting and detecting nucleic acids.

    PubMed

    Nåbo, Lina J; Madsen, Charlotte S; Jensen, Knud J; Kongsted, Jacob; Astakhova, Kira

    2015-05-26

    Functionalized synthetic oligonucleotides are finding growing applications in research, clinical studies, and therapy. However, it is not easy to prepare them in a biocompatible and highly efficient manner. We report a new strategy to synthesize oligonucleotides with promising nucleic acid targeting and detection properties. We focus in particular on the pH sensitivity of these new probes and their high target specificity. For the first time, human copper(I)-binding chaperon Cox17 was applied to effectively catalyze click labeling of oligonucleotides. This was performed under ultramild conditions with fluorophore, peptide, and carbohydrate azide derivatives. In thermal denaturation studies, the modified probes showed specific binding to complementary DNA and RNA targets. Finally, we demonstrated the pH sensitivity of the new rhodamine-based fluorescent probes in vitro and rationalize our results by electronic structure calculations. © 2015 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

  1. Serotyping of Human Group A Rotavirus with Oligonucleotide Probes

    DTIC Science & Technology

    1990-01-01

    Cold Spring Harbor , other oligonucleotides, HuG8Ac and HuG9Ac...HuG8Ac (5’ NY: Cold Spring Harbor Laboratory, 1988;5l1-159 CGA ACT ATC TUC TAT CTC TGT CTC T 3’) was based 9. Bastardo JW, McKimm-Bresckin JL, Sonza...Coulson BS, Unicomb LE, Pitson GA, Bishop RE Simple and specific manual. Cold Spring Harbor , NY Cold Spring Harbor Laboratory. enzyme

  2. probeBase—an online resource for rRNA-targeted oligonucleotide probes and primers: new features 2016

    PubMed Central

    Greuter, Daniel; Loy, Alexander; Horn, Matthias; Rattei, Thomas

    2016-01-01

    probeBase http://www.probebase.net is a manually maintained and curated database of rRNA-targeted oligonucleotide probes and primers. Contextual information and multiple options for evaluating in silico hybridization performance against the most recent rRNA sequence databases are provided for each oligonucleotide entry, which makes probeBase an important and frequently used resource for microbiology research and diagnostics. Here we present a major update of probeBase, which was last featured in the NAR Database Issue 2007. This update describes a complete remodeling of the database architecture and environment to accommodate computationally efficient access. Improved search functions, sequence match tools and data output now extend the opportunities for finding suitable hierarchical probe sets that target an organism or taxon at different taxonomic levels. To facilitate the identification of complementary probe sets for organisms represented by short rRNA sequence reads generated by amplicon sequencing or metagenomic analysis with next generation sequencing technologies such as Illumina and IonTorrent, we introduce a novel tool that recovers surrogate near full-length rRNA sequences for short query sequences and finds matching oligonucleotides in probeBase. PMID:26586809

  3. Reproducible and inexpensive probe preparation for oligonucleotide arrays.

    PubMed

    Zhang, Y; Price, B D; Tetradis, S; Chakrabarti, S; Maulik, G; Makrigiorgos, G M

    2001-07-01

    We present a new protocol for the preparation of nucleic acids for microarray hybridization. DNA is fragmented quantitatively and reproducibly by using a hydroxyl radical-based reaction, which is initiated by hydrogen peroxide, iron(II)-EDTA and ascorbic acid. Following fragmentation, the nucleic acid fragments are densely biotinylated using a biotinylated psoralen analog plus UVA light and hybridized on microarrays. This non-enzymatic protocol circumvents several practical difficulties associated with DNA preparation for microarrays: the lack of reproducible fragmentation patterns associated with enzymatic methods; the large amount of labeled nucleic acids required by some array designs, which is often combined with a limited amount of starting material; and the high cost associated with currently used biotinylation methods. The method is applicable to any form of nucleic acid, but is particularly useful when applying double-stranded DNA on oligonucleotide arrays. Validation of this protocol is demonstrated by hybridizing PCR products with oligonucleotide-coated microspheres and PCR amplified cDNA with Affymetrix Cancer GeneChip microarrays.

  4. Specificity tests of an oligonucleotide probe against food-outbreak salmonella for biosensor detection

    NASA Astrophysics Data System (ADS)

    Chen, I.-H.; Horikawa, S.; Xi, J.; Wikle, H. C.; Barbaree, J. M.; Chin, B. A.

    2017-05-01

    Phage based magneto-elastic (ME) biosensors have been shown to be able to rapidly detect Salmonella in various food systems to serve food pathogen monitoring purposes. In this ME biosensor platform, the free-standing strip-shaped magneto-elastic sensor is the transducer and the phage probe that recognizes Salmonella in food serves as the bio-recognition element. According to Sorokulova et al. at 2005, a developed oligonucleotide probe E2 was reported to have high specificity to Salmonella enterica Typhimurium. In the report, the specificity tests were focused in most of Enterobacterace groups outside of Salmonella family. Here, to understand the specificity of phage E2 to different Salmonella enterica serotypes within Salmonella Family, we further tested the specificity of the phage probe to thirty-two Salmonella serotypes that were present in the major foodborne outbreaks during the past ten years (according to Centers for Disease Control and Prevention). The tests were conducted through an Enzyme linked Immunosorbent Assay (ELISA) format. This assay can mimic probe immobilized conditions on the magnetoelastic biosensor platform and also enable to study the binding specificity of oligonucleotide probes toward different Salmonella while avoiding phage/ sensor lot variations. Test results confirmed that this oligonucleotide probe E2 was high specific to Salmonella Typhimurium cells but showed cross reactivity to Salmonella Tennessee and four other serotypes among the thirty-two tested Salmonella serotypes.

  5. chipD: a web tool to design oligonucleotide probes for high-density tiling arrays

    PubMed Central

    Dufour, Yann S.; Wesenberg, Gary E.; Tritt, Andrew J.; Glasner, Jeremy D.; Perna, Nicole T.; Mitchell, Julie C.; Donohue, Timothy J.

    2010-01-01

    chipD is a web server that facilitates design of DNA oligonucleotide probes for high-density tiling arrays, which can be used in a number of genomic applications such as ChIP-chip or gene-expression profiling. The server implements a probe selection algorithm that takes as an input, in addition to the target sequences, a set of parameters that allow probe design to be tailored to specific applications, protocols or the array manufacturer’s requirements. The algorithm optimizes probes to meet three objectives: (i) probes should be specific; (ii) probes should have similar thermodynamic properties; and (iii) the target sequence coverage should be homogeneous and avoid significant gaps. The output provides in a text format, the list of probe sequences with their genomic locations, targeted strands and hybridization characteristics. chipD has been used successfully to design tiling arrays for bacteria and yeast. chipD is available at http://chipd.uwbacter.org/. PMID:20529880

  6. PTPan--overcoming memory limitations in oligonucleotide string matching for primer/probe design.

    PubMed

    Eissler, Tilo; Hodges, Christopher P; Meier, Harald

    2011-10-15

    Nucleic acid diagnostics has high demands for non-heuristic exact and approximate oligonucleotide string matching concerning in silico primer/probe design in huge nucleic acid sequence collections. Unfortunately, public sequence repositories grow much faster than computer hardware performance and main memory capacity do. This growth imposes severe problems on existing oligonucleotide primer/probe design applications necessitating new approaches based on space-efficient indexing structures. We developed PTPan (spoken Peter Pan, 'PT' is for Position Tree, the earlier name of suffix trees), a space-efficient indexing structure for approximate oligonucleotide string matching in nucleic acid sequence data. Based on suffix trees, it combines partitioning, truncation and a new suffix tree stream compression to deal with large amounts of aligned and unaligned data. PTPan operates efficiently in main memory and on secondary storage, balancing between memory consumption and runtime during construction and application. Based on PTPan, applications supporting similarity search and primer/probe design have been implemented, namely FindFamily, ProbeMatch and ProbeDesign. All three use a weighted Levenshtein distance metric for approximative queries to find and rate matches with indels as well as substitutions. We integrated PTPan in the worldwide used software package ARB to demonstrate usability and performance. Comparing PTPan and the original ARB index for the very large ssu-rRNA database SILVA, we recognized a shorter construction time, extended functionality and dramatically reduced memory requirements at the price of expanded, but very reasonable query times. PTPan enables indexing of huge nucleic acid sequence collections at reasonable application response times. Not being limited by main memory, PTPan constitutes a major advancement regarding rapid oligonucleotide string matching in primer/probe design now and in the future facing the enormous growth of molecular

  7. Oligonucleotide microarrays with stem-loop probes: enhancing the hybridization of nucleic acids for sensitive analysis.

    PubMed

    Mahajan, Shweta; Swami, Archana; Sethi, Dalip; Kumar, P; Gupta, K C

    2008-06-15

    We have demonstrated that the dynamics of nucleic acid hybridization in microarrays depend on the physical structure of immobilized probes. We have immobilized oligonucleotide-3'-phosphates with and without stem-loop structure on epoxylated glass surface, followed by hybridization under different conditions, viz., hybridization buffer, pH condition, temperature and ionic strength. In a comparative study, we have established that array constructed using probes with stem-loop structure displayed approximately 2.2 times higher hybridization signals than the probes without it. The stem-loop DNA array format is simple and flexible in design and thus potentially useful in various DNA diagnostic tests.

  8. Enzyme-linked synthetic oligonucleotide probes: non-radioactive detection of enterotoxigenic Escherichia coli in faecal specimens.

    PubMed Central

    Li, P; Medon, P P; Skingle, D C; Lanser, J A; Symons, R H

    1987-01-01

    Synthetic oligonucleotides, complementary to unique sequences in the heat stable enterotoxin gene of Escherichia coli specific for humans, were prepared with a 30-atom spacer arm and a 3' terminal sulfhydryl group which was coupled to bromoacetyl-derivatized alkaline phosphatase. The resulting direct enzyme-linked oligonucleotide probes, containing one enzyme molecule per oligonucleotide, successfully diagnosed enterotoxigenic Escherichia coli in clinical specimens by using a modified colony hybridization method and a colorimetric assay. The procedure is rapid, simple and reliable with a sensitivity equivalent to that using 5'-terminally labelled [32p]-oligonucleotide probes. The results indicate that the enzyme-labelled oligonucleotide probes should be applicable to the routine diagnosis of enterotoxigenic Escherichia coli and possess the potential for the detection of other microbial pathogens. Images PMID:3299267

  9. Detection of cyclin D1 mRNA by hybridization sensitive NIC-oligonucleotide probe.

    PubMed

    Kovaliov, Marina; Segal, Meirav; Kafri, Pinhas; Yavin, Eylon; Shav-Tal, Yaron; Fischer, Bilha

    2014-05-01

    A large group of fluorescent hybridization probes, includes intercalating dyes for example thiazole orange (TO). Usually TO is coupled to nucleic acids post-synthetically which severely limits its use. Here, we have developed a phosphoramidite monomer, 10, and prepared a 2'-OMe-RNA probe, labeled with 5-(trans-N-hexen-1-yl-)-TO-2'-deoxy-uridine nucleoside, dU(TO), (Nucleoside bearing an Inter-Calating moiety, NIC), for selective mRNA detection. We investigated a series of 15-mer 2'-OMe-RNA probes, targeting the cyclin D1 mRNA, containing one or several dU(TO) at various positions. dU(TO)-2'-OMe-RNA exhibited up to 7-fold enhancement of TO emission intensity upon hybridization with the complementary RNA versus that of the oligomer alone. This NIC-probe was applied for the specific detection of a very small amount of a breast cancer marker, cyclin D1 mRNA, in total RNA extract from cancerous cells (250 ng/μl). Furthermore, this NIC-probe was found to be superior to our related NIF (Nucleoside with Intrinsic Fluorescence)-probe which could detect cyclin D1 mRNA target only at high concentrations (1840 ng/μl). Additionally, dU(T) can be used as a monomer in solid-phase oligonucleotide synthesis, thus avoiding the need for post-synthetic modification of oligonucleotide probes. Hence, we propose dU(TO) oligonucleotides, as hybridization probes for the detection of specific RNA in homogeneous solutions and for the diagnosis of breast cancer. Copyright © 2014 Elsevier Ltd. All rights reserved.

  10. Intrinsically Labeled Fluorescent Oligonucleotide Probes on Quantum Dots for Transduction of Nucleic Acid Hybridization.

    PubMed

    Shahmuradyan, Anna; Krull, Ulrich J

    2016-03-15

    Quantum dots (QDs) have been widely used in chemical and biosensing due to their unique photoelectrical properties and are well suited as donors in fluorescence resonance energy transfer (FRET). Selective hybridization interactions of oligonucleotides on QDs have been determined by FRET. Typically, the QD-FRET constructs have made use of labeled targets or have implemented labeled sandwich format assays to introduce dyes in proximity to the QDs for the FRET process. The intention of this new work is to explore a method to incorporate the acceptor dye into the probe molecule. Thiazole orange (TO) derivatives are fluorescent intercalating dyes that have been used for detection of double-stranded nucleic acids. One such dye system has been reported in which single-stranded oligonucleotide probes were doubly labeled with adjacent thiazole orange derivatives. In the absence of the fully complementary (FC) oligonucleotide target, the dyes form an H-aggregate, which results in quenching of fluorescence emission due to excitonic interactions between the dyes. The hybridization of the FC target to the probe provides for dissociation of the aggregate as the dyes intercalate into the double stranded duplex, resulting in increased fluorescence. This work reports investigation of the dependence of the ratiometric signal on the type of linkage used to conjugate the dyes to the probe, the location of the dye along the length of the probe, and the distance between adjacent dye molecules. The limit of detection for 34mer and 90mer targets was found to be identical and was 10 nM (2 pmol), similar to analogous QD-FRET using labeled oligonucleotide target. The detection system could discriminate a one base pair mismatch (1BPM) target and was functional without substantial compromise of the signal in 75% serum. The 1BPM was found to reduce background signal, indicating that the structure of the mismatch affected the environment of the intercalating dyes.

  11. Evaluating bacterial activity from cell-specific ribosomal RNA content measured with oligonucleotide probes

    SciTech Connect

    Kemp, P.F.; Lee, S.; LaRoche, J.

    1992-10-01

    We describe a procedure for measuring the cell-specific quantity of ribosomal RNA (rRNA) and DNA in order to evaluate the frequency distribution of activity among cells. The procedure is inherently quantitative, does not require sample incubation and potentially can be taxon-specific. Fluorescently-labelled oligonucleotide probes are hybridized to the complementary 16S rRNA sequences in preserved, intact cells. The resulting cell fluorescence is proportional to cellular rRNA content and can be measured with a microscope-mounted photometer system, by image analysis, or by flow cytometry. Similarly, DNA content is measured as fluorescence of cells stained with the DNA specific fluorochrome DAPI. These are either prepared as separate samples for purposes of enumeration and DNA measurements, or are dual-labelled cells which are also hybridized with oligonucleotide probes.

  12. Evaluating bacterial activity from cell-specific ribosomal RNA content measured with oligonucleotide probes

    SciTech Connect

    Kemp, P.F.; Lee, S.; LaRoche, J.

    1992-01-01

    We describe a procedure for measuring the cell-specific quantity of ribosomal RNA (rRNA) and DNA in order to evaluate the frequency distribution of activity among cells. The procedure is inherently quantitative, does not require sample incubation and potentially can be taxon-specific. Fluorescently-labelled oligonucleotide probes are hybridized to the complementary 16S rRNA sequences in preserved, intact cells. The resulting cell fluorescence is proportional to cellular rRNA content and can be measured with a microscope-mounted photometer system, by image analysis, or by flow cytometry. Similarly, DNA content is measured as fluorescence of cells stained with the DNA specific fluorochrome DAPI. These are either prepared as separate samples for purposes of enumeration and DNA measurements, or are dual-labelled cells which are also hybridized with oligonucleotide probes.

  13. Specific 16S ribosomal RNA targeted oligonucleotide probe against Clavibacter michiganensis subsp. sepedonicus.

    PubMed

    Mirza, M S; Rademaker, J L; Janse, J D; Akkermans, A D

    1993-11-01

    In this article we report on the polymerase chain reaction amplification of a partial 16S rRNA gene from the plant pathogenic bacterium Clavibacter michiganensis subsp. sepedonicus. A partial sequence (about 400 base pairs) of the gene was determined that covered two variable regions important for oligonucleotide probe development. A specific 24mer oligonucleotide probe targeted against the V6 region of 16S rRNA was designed. Specificity of the probe was determined using dot blot hybridization. Under stringent conditions (60 degrees C), the probe hybridized with all 16 Cl. michiganensis subsp. sepedonicus strains tested. Hybridization did not occur with 32 plant pathogenic and saprophytic bacteria used as controls under the same conditions. Under less stringent conditions (55 degrees C) the related Clavibacter michiganensis subsp. insidiosus, Clavibacter michiganensis subsp. nebraskensis, and Clavibacter michiganensis subsp. tesselarius also showed hybridization. At even lower stringency (40 degrees C), all Cl. michiganensis subspecies tested including Clavibacter michiganensis subsp. michiganensis showed hybridization signal, suggesting that under these conditions the probe may be used as a species-specific probe for Cl. michiganensis.

  14. Selection of optimal oligonucleotide probes for microarrays usingmultiple criteria, global alignment and parameter estimation.

    SciTech Connect

    Li, Xingyuan; He, Zhili; Zhou, Jizhong

    2005-10-30

    The oligonucleotide specificity for microarray hybridizationcan be predicted by its sequence identity to non-targets, continuousstretch to non-targets, and/or binding free energy to non-targets. Mostcurrently available programs only use one or two of these criteria, whichmay choose 'false' specific oligonucleotides or miss 'true' optimalprobes in a considerable proportion. We have developed a software tool,called CommOligo using new algorithms and all three criteria forselection of optimal oligonucleotide probes. A series of filters,including sequence identity, free energy, continuous stretch, GC content,self-annealing, distance to the 3'-untranslated region (3'-UTR) andmelting temperature (Tm), are used to check each possibleoligonucleotide. A sequence identity is calculated based on gapped globalalignments. A traversal algorithm is used to generate alignments for freeenergy calculation. The optimal Tm interval is determined based on probecandidates that have passed all other filters. Final probes are pickedusing a combination of user-configurable piece-wise linear functions andan iterative process. The thresholds for identity, stretch and freeenergy filters are automatically determined from experimental data by anaccessory software tool, CommOligo_PE (CommOligo Parameter Estimator).The program was used to design probes for both whole-genome and highlyhomologous sequence data. CommOligo and CommOligo_PE are freely availableto academic users upon request.

  15. Oligonucleotides as probes for studying polymerization reactions in dilute aqueous solution. 2: Polycondensations

    NASA Technical Reports Server (NTRS)

    Kolb, Vera; Orgel, Leslie E.

    1995-01-01

    We have prepared a (P-32)-labeled oligonucleotide probe carrying a ureido (-NH-CO-NH2) function at its 3'-terminus. This labeled oligomer was used to study polycondensations of urea and formaldehyde and of various phenols and formaldehyde in aqueous solution. The formation of formaldehyde copolymers attached to the amido-function of the probe was monitored by gel electrophoresis. Our results are generally in agreement with those obtained using conventional techniques. Our method is suitable for monitoring potentially prebiotic polycondensation reactions involving formaldehyde.

  16. Oligonucleotides as probes for studying polymerization reactions in dilute aqueous solution: II. Polycondensations

    NASA Technical Reports Server (NTRS)

    Kolb, V.; Orgel, L. E.

    1995-01-01

    We have prepared a [32P]-labeled oligonucleotide probe carrying a ureido (-NH-CO-NH2) function at its 3'-terminus. This labeled oligomer was used to study polycondensations of urea and formaldehyde and of various phenols and formaldehyde in aqueous solution. The formation of formaldehyde copolymers attached to the amido-function of the probe was monitored by gel electrophoresis. Our results are generally in agreement with those obtained using conventional techniques. Our method is suitable for monitoring potentially prebiotic polycondensation reactions involving formaldehyde.

  17. Oligonucleotides as probes for studying polymerization reactions in dilute aqueous solution: II. Polycondensations

    NASA Technical Reports Server (NTRS)

    Kolb, V.; Orgel, L. E.

    1995-01-01

    We have prepared a [32P]-labeled oligonucleotide probe carrying a ureido (-NH-CO-NH2) function at its 3'-terminus. This labeled oligomer was used to study polycondensations of urea and formaldehyde and of various phenols and formaldehyde in aqueous solution. The formation of formaldehyde copolymers attached to the amido-function of the probe was monitored by gel electrophoresis. Our results are generally in agreement with those obtained using conventional techniques. Our method is suitable for monitoring potentially prebiotic polycondensation reactions involving formaldehyde.

  18. Wash-free magnetic oligonucleotide probes-based NMR sensor for detecting the Hg ion.

    PubMed

    Ma, Wenwei; Hao, Changlong; Ma, Wei; Xing, Changrui; Yan, Wenjing; Kuang, Hua; Wang, Libing; Xu, Chuanlai

    2011-12-14

    An easily applied and sensitive sensor for the detection of heavy metal ion residues based entirely on magnetic nanoparticle and oligonucleotide was developed. The tool is established on the relaxation of magnetic nanoparticles with different dispersion states. The target analyte, Hg ions, induce the aggregation of the MNP oligonucleotide probes. Accordingly, the light produced by the magnetic relaxation image and the transverse relaxation time (T(2)) all change due to the effect of the aggregation. The limit of qualitative detection of the sensor is 0.15 ppt. The recoveries from test samples range between 97.1-101.8%. Using the nuclear resonance instrument, the method is a high throughput and sensitive sensor.

  19. Design, Validation and Annotation of Transcriptome-Wide Oligonucleotide Probes for the Oligochaete Annelid Eisenia fetida

    PubMed Central

    Gong, Ping; Pirooznia, Mehdi; Guan, Xin; Perkins, Edward J.

    2010-01-01

    High density oligonucleotide probe arrays have increasingly become an important tool in genomics studies. In organisms with incomplete genome sequence, one strategy for oligo probe design is to reduce the number of unique probes that target every non-redundant transcript through bioinformatic analysis and experimental testing. Here we adopted this strategy in making oligo probes for the earthworm Eisenia fetida, a species for which we have sequenced transcriptome-scale expressed sequence tags (ESTs). Our objectives were to identify unique transcripts as targets, to select an optimal and non-redundant oligo probe for each of these target ESTs, and to annotate the selected target sequences. We developed a streamlined and easy-to-follow approach to the design, validation and annotation of species-specific array probes. Four 244K-formatted oligo arrays were designed using eArray and were hybridized to a pooled E. fetida cRNA sample. We identified 63,541 probes with unsaturated signal intensities consistently above the background level. Target transcripts of these probes were annotated using several sequence alignment algorithms. Significant hits were obtained for 37,439 (59%) probed targets. We validated and made publicly available 63.5K oligo probes so the earthworm research community can use them to pursue ecological, toxicological, and other functional genomics questions. Our approach is efficient, cost-effective and robust because it (1) does not require a major genomics core facility; (2) allows new probes to be easily added and old probes modified or eliminated when new sequence information becomes available, (3) is not bioinformatics-intensive upfront but does provide opportunities for more in-depth annotation of biological functions for target genes; and (4) if desired, EST orthologs to the UniGene clusters of a reference genome can be identified and selected in order to improve the target gene specificity of designed probes. This approach is particularly

  20. Whole-cell hybridization of Methanosarcina cells with two new oligonucleotide probes.

    PubMed Central

    Sørensen, A H; Torsvik, V L; Torsvik, T; Poulsen, L K; Ahring, B K

    1997-01-01

    Two new oligonucleotide probes targeting the 16S rRNA of the methanogenic genus Methanosarcina were developed. The probes have the following sequences (Escherichia coli numbering): probe SARCI551, 5'-GAC CCAATAATCACGATCAC-3', and probe SARCI645, 5'-TCCCGGTTCCAAGTCTGGC-3'. In situ hybridization with the fluorescently labelled probes required several modifications of standard procedures. Cells of Methanosarcina mazeii S-6 were found to lyse during the hybridization step if fixed in 3% formaldehyde and stored in 50% ethanol. Lysis was, however, not observed with cells fixed and stored in 1.6% formaldehyde-0.85% NaCl. Extensive autofluorescence of the cells was found upon hybridization in the presence of 5 mM EDTA, but successful hybridization could be obtained without addition of this compound. The mounting agent Citifluor AF1, often used in conjugation with the fluorochrome fluorescein, was found to wash the labelled probes out of the cells. Stable labelling could be obtained with rhodamine-labelled probes when the specimen was mounted in immersion oil, and high hybridization intensities of the Methanosarcina cells were found even in the presence of biomass from an anaerobic reactor. The inherent high autofluorescence of the biomass could be lowered by use of a highly specific narrow-band filter. The probes were found to be specific for Methanosarcina and useful for detection of this genus in samples from anaerobic reactors. PMID:9251192

  1. Thiol- and Biotin-Labeled Probes for Oligonucleotide Quartz Crystal Microbalance Biosensors of Microalga Alexandrium Minutum

    PubMed Central

    Lazerges, Mathieu; Perrot, Hubert; Rabehagasoa, Niriniony; Compère, Chantal

    2012-01-01

    Two quartz crystal microbalance oligonucleotide biosensors of a toxic microalga gene sequence (Alexandrium Minutum) have been designed. Grafting on a gold surface of 20-base thiol- or biotin-labeled probe, and selective hybridization with the complementary 20-base target, have been monitored in situ with a 27 MHz quartz crystal microbalance under controlled hydrodynamic conditions. The frequency of the set up is stable to within a few hertz, corresponding to the nanogram scale, for three hour experiments. DNA recognition by the two biosensors is efficient and selective. Hybridization kinetic curves indicate that the biosensor designed with the thiol-labeled probe is more sensitive, and that the biosensor designed with the biotin-labeled probe has a shorter time response and a higher hybridization efficiency. PMID:25585927

  2. Development of specific fluorescent oligonucleotide probes for in situ identification of wine lactic acid bacteria.

    PubMed

    Blasco, Lucía; Ferrer, Sergi; Pardo, Isabel

    2003-08-08

    A rapid method for the identification of lactic acid bacteria (LAB) from wine has been developed. This method is based on fluorescence in situ hybridisation (FISH), using fluorescent oligonucleotide probes, homologous to 16S rDNA of those species of LAB commonly found in wines. The protocol for the specific detection of these bacteria was established through the hybridisation of 36 reference strains. The specificity of the probes was evaluated by using pure cultures. Probes were used to identify species in different wines, making it evident that direct identification and quantification from natural samples without culturing is also possible. The results show that FISH is a promising technique for the rapid identification of LAB, allowing positive identification in a few hours (4-16 h).

  3. Thiol- and biotin-labeled probes for oligonucleotide quartz crystal microbalance biosensors of microalga alexandrium minutum.

    PubMed

    Lazerges, Mathieu; Perrot, Hubert; Rabehagasoa, Niriniony; Compère, Chantal

    2012-07-04

    Two quartz crystal microbalance oligonucleotide biosensors of a toxic microalga gene sequence (Alexandrium Minutum) have been designed. Grafting on a gold surface of 20-base thiol- or biotin-labeled probe, and selective hybridization with the complementary 20-base target, have been monitored in situ with a 27 MHz quartz crystal microbalance under controlled hydrodynamic conditions. The frequency of the set up is stable to within a few hertz, corresponding to the nanogram scale, for three hour experiments. DNA recognition by the two biosensors is efficient and selective. Hybridization kinetic curves indicate that the biosensor designed with the thiol-labeled probe is more sensitive, and that the biosensor designed with the biotin-labeled probe has a shorter time response and a higher hybridization efficiency.

  4. Permeabilization of mycolic-acid-containing actinomycetes for in situ hybridization with fluorescently labelled oligonucleotide probes.

    PubMed

    Macnaughton, S J; O'Donnell, A G; Embley, T M

    1994-10-01

    The application of whole-cell hybridization using labelled oligonucleotide probes in microbial systematics and ecology is limited by difficulties in permeabilizing many Gram-positive organisms. In this investigation paraformaldehyde treatment, acid methanolysis and acid hydrolysis were evaluated as a means of permeabilizing mycolic-acid-containing actinomycetes prior to hybridization with a fluorescently labelled oligonucleotide probe designed to bind to a conserved sequence of bacterial 16S rRNA. Methods were evaluated on stationary-phase cultures of Gordona bronchialis, Mycobacterium fortuitum, Nocardia asteroides, N. brasiliensis, Rhodococcus equi, R. erythropolis, R. fascians, R. rhodochrous and Tsukamurella paurometabola, none of which could be probed following 4% (w/v) paraformaldehyde fixation. For comparison and to test the general applicability of mild acid pretreatments, Bacillus subtilis, Lactobacillus plantarum, Escherichia coli and Pseudomonas putida were also studied. The data showed that most of the mycolic-acid-containing organisms were successfully permeabilized by mild acid hydrolysis in 1 M HCl at 37 degrees C. Cells were treated for different lengths of time. In general, the mycolic-acid-containing organisms required between 30 and 50 min hydrolysis, whereas B. subtilis, E. coli and P. putida were rendered permeable in only 10 min. Interestingly, L. plantarum could not be permeabilized using acid hydrolysis even after 60 min exposure to 1 M HCl.

  5. Oligonucleotides as probes for studying polymerization reactions in dilute aqueous solution

    NASA Technical Reports Server (NTRS)

    Kolb, V.; Orgel, L. E.; Miller, S. L. (Principal Investigator)

    1994-01-01

    We have prepared a [32P]-labled oligonucleotide probe carrying a free primary amine at its 3'-terminus. This probe is used to initiate polymerization of aziridine (ethyleneimine) in aqueous solution. The nature of the oligomeric products and the kinetics of their formation are then monitored by gel electrophoresis. Our results are generally consistent with those obtained using conventional techniques. We have also investigated the effect of polyanionic templates on the rate of oligomerization of aziridine. We find that water-soluble polyanions generally accelerate the polymerization. The sodium salt of polymethacrylic acid is the most effective of the templates that we studied. The methods introduced in this paper should be applicable to a variety of polymerization reactions in aqueous solution. They should greatly simplify the screening of potentially prebiotic polymerization reactions.

  6. Localization of miRNAs by In Situ Hybridization in Plants Using Conventional Oligonucleotide Probes.

    PubMed

    Hernández-Castellano, Sara; Nic-Can, Geovanny I; De-la-Peña, Clelia

    2017-01-01

    Among the epigenetic mechanisms studied with a greater interest in the last decade are the microRNAs (miRNAs). These small noncoding RNA sequences that are approximately 17-22 nucleotides in length play an essential role in many biological processes of various organisms, including plants. The analysis of spatiotemporal expression of miRNAs provides a better understanding of the role of these small molecules in plant development, cell differentiation, and other processes; but such analysis is also an important method for the validation of biological functions. In this work, we describe the optimization of an efficient protocol for the spatiotemporal analysis of miRNA by in situ hybridization using different plant tissues embedded in paraffin. Instead of LNA-modified probes that are typically used for this work, we use conventional oligonucleotide probes that yield a high specificity and clean distribution of miRNAs.

  7. Non-radioactive detection of oligonucleotide probes by photochemical amplification of dioxetanes.

    PubMed Central

    Schubert, F; Knaf, A; Möller, U; Cech, D

    1995-01-01

    We describe a new method of non-radioactive labelling and detection of oligonucleotide probes. The approach is based on a simple chemical principle. Oligonucleotides labelled with methylene blue (a photosensitizer) are hybridized on a membrane to immobilized DNA target sequences. After hybridization and stringency washing 2(-)[3-(hydroxyphenyl)methoxymethylene] adamantane is added to the membrane and the membrane is irradiated with a tungsten lamp light source through a cut-off filter. Thermally stable dioxetanes are amplified during irradiation at the positions of the labelled probe. These amplified dioxetanes are detected using chemically triggered chemiluminescent decay. Signals are recorded on commercial X-ray film. Detection is possible immediately after the last washing step and a hard copy of the blot is obtained within 1 h. Dependent on the level of the target sequences, the sensitivity of the method allows detection of 0.3 pg single-stranded M13mp18(+) plasmid DNA in dot blots and 75 pg in Southern blots. Additional immunological reaction steps and washing steps with blocking reagents and buffers are avoided. Furthermore, expensive reagents and equipment for physical detection are not necessary. The method might be particularly useful for fast routine analysis in forensic and medical applications. The synthesis of the olefin, conditions of hybridization and the protocol of detection are described in detail. Images PMID:8524657

  8. Selectivity of Enzymatic Conversion of Oligonucleotide Probes during Nucleotide Polymorphism Analysis of DNA

    PubMed Central

    Vinogradova, O.A.

    2010-01-01

    The analysis of DNA nucleotide polymorphisms is one of the main goals of DNA diagnostics. DNA–dependent enzymes (DNA polymerases and DNA ligases) are widely used to enhance the sensitivity and reliability of systems intended for the detection of point mutations in genetic material. In this article, we have summarized the data on the selectiveness of DNA–dependent enzymes and on the structural factors in enzymes and DNA which influence the effectiveness of mismatch discrimination during enzymatic conversion of oligonucleotide probes on a DNA template. The data presented characterize the sensitivity of a series of DNA–dependent enzymes that are widely used in the detection of noncomplementary base pairs in nucleic acid substrate complexes. We have analyzed the spatial properties of the enzyme–substrate complexes. These properties are vital for the enzymatic reaction and the recognition of perfect DNA–substrates. We also discuss relevant approaches to increasing the selectivity of enzyme–dependent reactions. These approaches involve the use of modified oligonucleotide probes which “disturb” the native structure of the DNA–substrate complexes. PMID:22649627

  9. An in vitro selection scheme for oligonucleotide probes to discriminate between closely related DNA sequences

    PubMed Central

    Brukner, Ivan; El-Ramahi, Razan; Gorska-Flipot, Izabella; Krajinovic, Maja; Labuda, Damian

    2007-01-01

    Using an in vitro selection, we have obtained oligonucleotide probes with high discriminatory power against multiple, similar nucleic acid sequences, which is often required in diagnostic applications for simultaneous testing of such sequences. We have tested this approach, referred to as iterative hybridizations, by selecting probes against six 22-nt-long sequence variants representing human papillomavirus, (HPV). We have obtained probes that efficiently discriminate between HPV types that differ by 3–7 nt. The probes were found effective to recognize HPV sequences of the type 6, 11, 16, 18 and a pair of type 31 and 33, either when immobilized on a solid support or in a reverse configuration, as well to discriminate HPV types from the clinical samples. This methodology can be extended to generate diagnostic kits that rely on nucleic acid hybridization between closely related sequences. In this approach, instead of adjusting hybridization conditions to the intended set of probe–target pairs, we ‘adjust’, through in vitro selection, the probes to the conditions we have chosen. Importantly, these conditions have to be ‘relaxed’, allowing the formation of a variety of not fully complementary complexes from which those that efficiently recognize and discriminate intended from non-intended targets can be readily selected. PMID:17426126

  10. Interfacing click chemistry with automated oligonucleotide synthesis for the preparation of fluorescent DNA probes containing internal xanthene and cyanine dyes.

    PubMed

    Astakhova, I Kira; Wengel, Jesper

    2013-01-14

    Double-labeled oligonucleotide probes containing fluorophores interacting by energy-transfer mechanisms are essential for modern bioanalysis, molecular diagnostics, and in vivo imaging techniques. Although bright xanthene and cyanine dyes are gaining increased prominence within these fields, little attention has thus far been paid to probes containing these dyes internally attached, a fact which is mainly due to the quite challenging synthesis of such oligonucleotide probes. Herein, by using 2'-O-propargyl uridine phosphoramidite and a series of xanthenes and cyanine azide derivatives, we have for the first time performed solid-phase copper(I)-catalyzed azide-alkyne cycloaddition (CuAAC) click labeling during the automated phosphoramidite oligonucleotide synthesis followed by postsynthetic click reactions in solution. We demonstrate that our novel strategy is rapid and efficient for the preparation of novel oligonucleotide probes containing internally positioned xanthene and cyanine dye pairs and thus represents a significant step forward for the preparation of advanced fluorescent oligonucleotide probes. Furthermore, we demonstrate that the novel xanthene and cyanine labeled probes display unusual and very promising photophysical properties resulting from energy-transfer interactions between the fluorophores controlled by nucleic acid assembly. Potential benefits of using these novel fluorescent probes within, for example, molecular diagnostics and fluorescence microscopy include: Considerable Stokes shifts (40-110 nm), quenched fluorescence of single-stranded probes accompanied by up to 7.7-fold light-up effect of emission upon target DNA/RNA binding, remarkable sensitivity to single-nucleotide mismatches, generally high fluorescence brightness values (FB up to 26), and hence low limit of target detection values (LOD down to <5 nM). Copyright © 2013 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

  11. Aptamer-based electrochemical sensors with aptamer-complementary DNA oligonucleotides as probe.

    PubMed

    Lu, Ying; Li, Xianchan; Zhang, Limin; Yu, Ping; Su, Lei; Mao, Lanqun

    2008-03-15

    This study describes a facile and general strategy for the development of aptamer-based electrochemical sensors with a high specificity toward the targets and a ready regeneration feature. Very different from the existing strategies for the development of electrochemical aptasensors with the aptamers as the probes, the strategy proposed here is essentially based on the utilization of the aptamer-complementary DNA (cDNA) oligonucleotides as the probes for electrochemical sensing. In this context, the sequences at both ends of the cDNA are tailor-made to be complementary and both the redox moiety (i.e., ferrocene in this study) and thiol group are labeled onto the cDNA. The labeled cDNA are hybridized with their respective aptamers (i.e., ATP- and thrombin-binding aptamers in this study) to form double-stranded DNA (ds-DNA) and the electrochemical aptasensors are prepared by self-assembling the labeled ds-DNA onto Au electrodes. Upon target binding, the aptamers confined onto electrode surface dissociate from their respective cDNA oligonucleotides into the solution and the single-stranded cDNA could thus tend to form a hairpin structure through the hybridization of the complementary sequences at both its ends. Such a conformational change of the cDNA resulting from the target binding-induced dissociation of the aptamers essentially leads to the change in the voltammetric signal of the redox moiety labeled onto the cDNA and thus constitutes the mechanism for the electrochemical aptasensors for specific target sensing. The aptasensors demonstrated here with the cDNA as the probe are readily regenerated and show good responses toward the targets. This study may offer a new and relatively general approach to electrochemical aptasensors with good analytical properties and potential applications.

  12. Analysis of microRNA expression by in situ hybridization with RNA oligonucleotide probes

    PubMed Central

    Thompson, Robert C.; Deo, Monika; Turner, David L.

    2007-01-01

    In situ hybridization is an important tool for analyzing gene expression and developing hypotheses about gene functions. The discovery of hundreds of microRNA (miRNA) genes in animals has provided new challenges for analyzing gene expression and functions. The small size of the mature miRNAs (∼20-24 nucleotides in length) presents difficulties for conventional in situ hybridization methods. However, we have developed a modified in situ hybridization method for detection of mammalian miRNAs in tissue sections, based upon the use of RNA oligonucleotide probes in combination with highly specific wash conditions. Here we present detailed procedures for detection of miRNAs in tissue sections or cultured cells. The methods described can utilize either nonradioactive hapten-conjugated probes that are detected by enzyme-coupled antibodies, or radioactively labeled probes that are detected by autoradiography. The ability to visualize miRNA expression patterns in tissue sections provides an additional tool for the analyses of miRNA expression and function. In addition, the use of radioactively labeled probes should facilitate quantitative analyses of changes in miRNA gene expression. PMID:17889803

  13. Using triplex-forming oligonucleotide probes for the reagentless, electrochemical detection of double-stranded DNA.

    PubMed

    Patterson, Adriana; Caprio, Felice; Vallée-Bélisle, Alexis; Moscone, Danila; Plaxco, Kevin W; Palleschi, Giuseppe; Ricci, Francesco

    2010-11-01

    We report a reagentless, electrochemical sensor for the detection of double-stranded DNA targets that employs triplex-forming oligonucleotides (TFOs) as its recognition element. These sensors are based on redox-tagged TFO probes strongly chemisorbed onto an interrogating gold electrode. Upon the addition of the relevant double-stranded DNA target, the probe forms a rigid triplex structure via reverse Hoogsteen base pairing in the major groove. The formation of the triplex impedes contact between the probe's redox moiety and the interrogating electrode, thus signaling the presence of the target. We first demonstrated the proof of principle of this approach by using a well-characterized 22-base polypurine TFO sequence that readily detects a synthetic, double-stranded DNA target. We then confirmed the generalizability of our platform with a second probe, a 19-base polypyrimidine TFO sequence that targets a polypurine tract (PPT) sequence conserved in all HIV-1 strains. Both sensors rapidly and specifically detect their double-stranded DNA targets at concentrations as low as ~10 nM and are selective enough to be employed directly in complex sample matrices such as blood serum. Moreover, to demonstrate real-world applicability of this new sensor platform, we have successfully detected unpurified, double-stranded PCR amplicons containing the relevant conserved HIV-1 sequence.

  14. Rapid and multiplexed transcript analysis of microbial cultures using capillary electophoresis-detectable oligonucleotide probe pools.

    PubMed

    Rautio, Jari J; Kataja, Kari; Satokari, Reetta; Penttilä, Merja; Söderlund, Hans; Saloheimo, Markku

    2006-06-01

    A rapid assay for multiplex transcript analysis based on solution hybridization with pools of oligonucleotide probes was developed. In this assay called TRAC (transcript analysis with aid of affinity capture) the mRNAs to be studied are hybridized with gene-specific detection probe pools and biotinylated oligo(dT) and captured on streptavidin-coated magnetic particles. Unbound sample material and nonspecifically bound detection probes are removed and the target-specific probes are eluted and detected by capillary electrophoresis. Simultaneous treatment of 96 samples was automated using a magnetic bead particle processor. The assay enabled detection of in vitro transcribed RNA at the level of 30 amol (20 pg) and over a 300-fold linear range. Besides extracted RNA, crude cell lysates were directly used as samples. The assay was used for transcriptional analysis of selected mRNAs in the filamentous fungus Trichoderma reesei in two experimental conditions. TRAC analysis was highly reproducible, providing expression results that were consistent with conventional Northern blot analysis. The whole procedure starting from sample collecting can be carried out in 2 h, making this assay suitable for high-throughput analysis of a limited set of mRNAs e.g. in gene expression monitoring of production organism in microbial bioprocesses.

  15. Development of mercury (II) ion biosensors based on mercury-specific oligonucleotide probes.

    PubMed

    Li, Lanying; Wen, Yanli; Xu, Li; Xu, Qin; Song, Shiping; Zuo, Xiaolei; Yan, Juan; Zhang, Weijia; Liu, Gang

    2016-01-15

    Mercury (II) ion (Hg(2+)) contamination can be accumulated along the food chain and cause serious threat to the public health. Plenty of research effort thus has been devoted to the development of fast, sensitive and selective biosensors for monitoring Hg(2+). Thymine was demonstrated to specifically combine with Hg(2+) and form a thymine-Hg(2+)-thymine (T-Hg(2+)-T) structure, with binding constant even higher than T-A Watson-Crick pair in DNA duplex. Recently, various novel Hg(2+) biosensors have been developed based on T-rich Mercury-Specific Oligonucleotide (MSO) probes, and exhibited advanced selectivity and excellent sensitivity for Hg(2+) detection. In this review, we explained recent development of MSO-based Hg(2+) biosensors mainly in 3 groups: fluorescent biosensors, colorimetric biosensors and electrochemical biosensors.

  16. Fluorescent oligonucleotide probe based on G-quadruplex scaffold for signal-on ultrasensitive protein assay.

    PubMed

    Wu, Zai-Sheng; Hu, Peng; Zhou, Hui; Shen, Guoli; Yu, Ruqin

    2010-03-01

    This work reported the G-quadruplex structure of pyrene-labeled G-rich DNA probe and its application in the immunoglobulin E (IgE) detection, providing plausibly an insight into the biological function of human telomere. Based on the intermolecular G-quadruplex, a terminal-single-pyrene-labeled oligonucleotide signaling probe was developed and a novel protein assay strategy was proposed via combining specific DNA cleavage by S1 nuclease with target-recognizing aptamer. This assay platform not only circumvented the optimization of specific sites for reporter attachment and the pyrene monomer fluorescence quenching by flanking nucleotide bases but also presented a signal-on mechanism. Thus, ultrasensitive homogeneous detection of IgE was successfully conducted. A linear dynamic range of 4.72 x 10(-12) to 7.56 x 10(-9)m, a regression coefficient of 0.9941 and a detection limit of 9.45 x 10(-14)m were given. Additionally, a preliminary concept of the single-pyrene-labeled excimer fluorescence probes associated with G-quadruplex for screening biological markers was described. Importantly, the unexpected structural property of G-quadruplex discovered seems to provide valuable information to allow understanding of the structure and function of human telomere and exploring of useful structure-based anticancer drug.

  17. Rapid identification of Vibrio vulnificus on nonselective media with an alkaline phosphatase-labeled oligonucleotide probe.

    PubMed Central

    Wright, A C; Miceli, G A; Landry, W L; Christy, J B; Watkins, W D; Morris, J G

    1993-01-01

    An oligonucleotide DNA probe (VVAP) was constructed from a portion of the Vibrio vulnificus cytolysin gene (hylA) sequence and labeled with alkaline phosphatase covalently linked to the DNA. Control and environmental isolates probed with VVAP showed an exact correlation with results obtained with a plasmid DNA probe (derived from pCVD702) previously described as having 100% specificity and sensitivity for this organism. Identification of V. vulnificus strains was confirmed independently by analysis of the cellular fatty acid composition and by API 20E. Naturally occurring V. vulnificus bacteria were detected without enrichment or selective media by VVAP in unseeded oyster homogenates and seawater collected from a single site in Chesapeake Bay during June at concentrations of 6 x 10(2) and 2 x 10(1) bacteria per ml, respectively. V. vulnificus bacteria were also enumerated by VVAP in oysters seeded with known concentrations of bacteria and plated on nonselective medium. The VVAP method provides a rapid, accurate means of identifying and enumerating V. vulnificus in seawater and oysters without the use of selective media or additional biochemical tests. Images PMID:8434919

  18. Using Triplex-Forming Oligonucleotide Probes for the Reagentless, Electrochemical Detection of Double-Stranded DNA

    PubMed Central

    Patterson, Adriana; Caprio, Felice; Vallée-Bélisle, Alexis; Moscone, Danila; Plaxco, Kevin W.; Palleschi, Giuseppe; Ricci, Francesco

    2011-01-01

    We report a reagentless, electrochemical sensor for the detection of double-stranded DNA targets that employs triplex-forming oligonucleotides (TFOs) as its recognition element. These sensors are based on redox-tagged TFO probes strongly chemisorbed onto an interrogating gold electrode. Upon the addition of the relevant double-stranded DNA target, the probe forms a rigid triplex structure via reverse Hoogsteen base pairing in the major groove. The formation of the triplex impedes contact between the probe’s redox moiety and the interrogating electrode, thus signaling the presence of the target. We first demonstrated the proof of principle of this approach by using a well-characterized 22-base polypurine TFO sequence that readily detects a synthetic, double-stranded DNA target. We then confirmed the generalizability of our platform with a second probe, a 19-base polypyrimidine TFO sequence that targets a polypurine tract (PPT) sequence conserved in all HIV-1 strains. Both sensors rapidly and specifically detect their double-stranded DNA targets at concentrations as low as ~10 nM and are selective enough to be employed directly in complex sample matrices such as blood serum. Moreover, to demonstrate real-world applicability of this new sensor platform, we have successfully detected unpurified, double-stranded PCR amplicons containing the relevant conserved HIV-1 sequence. PMID:20936782

  19. A new strategy for site-specific alkylation of DNA using oligonucleotides containing an abasic site and alkylating probes.

    PubMed

    Sato, Norihiro; Tsuji, Genichiro; Sasaki, Yoshihiro; Usami, Akira; Moki, Takuma; Onizuka, Kazumitsu; Yamada, Ken; Nagatsugi, Fumi

    2015-10-14

    Selective chemical reactions with DNA, such as its labelling, are very useful in many applications. In this paper, we discuss a new strategy for the selective alkylation of DNA using an oligonucleotide containing an abasic site and alkylating probes. We designed three probes consisting of 2-AVP as a reactive moiety and three kinds of binding moiety with high affinity to duplex DNA. Among these probes, Hoechst-AVP probe exhibited high selectivity and efficient reactivity to thymine bases at the site opposite an abasic site in DNA. Our method is potentially useful for inducing site-directed reactions aimed at inhibiting polymerase reactions.

  20. A dual-colored ratiometric-fluorescent oligonucleotide probe for the detection of human telomerase RNA in cell extracts.

    PubMed

    Ning, Dianhua; He, Changtian; Liu, Zhengjie; Liu, Cui; Wu, Qilong; Zhao, TingTing; Liu, Renyong

    2017-04-10

    Human telomerase RNA (hTR), which is one component of telomerase, was deemed to be a biomarker to monitor tumor cells due to its different expression levels in tumor cells and normal somatic cells. Thus far, plentiful fluorescent probes have been designed to investigate nucleic acids. However, most of them are limited since they are time-consuming, require professional operators and even result in false positive signals in the cellular environment. Herein, we report a dual-colored ratiometric-fluorescent oligonucleotide probe to achieve the reliable detection of human telomerase RNA in cell extracts. The probe is constructed using a dual-labeled fluorescent oligonucleotide hybridized with target-complemented Dabcyl-labeled oligonucleotide. In the presence of the target, the dual-labeled fluorescent oligonucleotide translates into a hairpin structure, which leads to the generation of the fluorescence resonance energy transfer (FRET) phenomenon under UV excitation. Compared to conventional methods, this strategy could effectively avoid false positive signals, and it not only possesses the advantages of simplicity and high specificity but also has the merits of signal stability and distinguishable color variation. Moreover, the quantitative assay of hTR would have a far-reaching impact on the telomerase mechanism and even tumor diagnosis research.

  1. Mouse centromere mapping using oligonucleotide probes that detect variants of the minor satellite.

    PubMed

    Kipling, D; Wilson, H E; Mitchell, A R; Taylor, B A; Cooke, H J

    1994-03-01

    Cytologically, the centromere is found at the very end of most Mus musculus chromosomes, co-localizing with an array of minor satellite sequences. It is separated from the euchromatin of the long arm by a large domain of heterochromatin, composed in part of arrays of major satellite sequences. We used oligonucleotide probes that specifically detect regions of sequence variation found in certain cloned minor satellite sequences. They detect a limited subset of the minor satellite arrays in the mouse genome, based on both pulsed-field gel electrophoresis and in situ hybridization data, and provide direct molecular genetic markers for individual centromeres in some inbred mouse strains. Array size polymorphisms detected by these probes map to positions consistent with the centromeres of chromosomes 1 and 14 in the BXD recombinant inbred (RI) strains. The genetic distances between these minor satellite arrays and loci on the long arms of chromosomes 1 and 14 are consistent with repression of meiotic recombination in the heterochromatic domains separating them. The existence of chromosome-specific minor satellite sequences implies that the rate of sequence exchange between non-homologous chromosomes relative to the rate between homologous chromosomes is much lower than has previously been postulated. We suggest that the high degree of sequence homogeneity of mouse satellite sequences may instead reflect recent common ancestry.

  2. Genotyping male-specific RNA coliphages by hybridization with oligonucleotide probes.

    PubMed Central

    Hsu, F C; Shieh, Y S; van Duin, J; Beekwilder, M J; Sobsey, M D

    1995-01-01

    F-specific (F+) RNA coliphages are prevalent in sewage and other fecal wastes of humans and animals. There are four antigenically distinct serogroups of F+ RNA coliphages, and those predominating in humans (groups II and III) differ from those predominating in animals (groups I and IV). Hence, it may be possible to distinguish between human and animal wastes by serotyping F+ RNA coliphage isolates. Because serotyping is laborious and requires scarce antiserum reagents, we investigated genotyping using synthetic oligonucleotide probes as an alternative approach to distinguishing the four groups of F+ RNA coliphages. Oligoprobes I, II, III, IV, A, and B were selected to detect group I, II, III, IV, I plus II, and III plus IV phages, respectively. Methods for phage transfer from zones of lysis on a host cell lawn to candidate membrane filters and fixation of genomic nucleic acid on the membranes were optimized. The oligoprobes, which were end labeled with digoxigenin, were applied in DNA-RNA hybridization, and hybrids were observed by colorimetric, immunoenzymatic detection. Of 203 isolates of F+ RNA coliphages from environmental samples of water, wastes, and shellfish, 99.5 and 96.6% could be classified into each group by serotyping and genotyping, respectively. Probes A and B correctly identified 100% of the isolates. On the basis of these results, this method for genotyping F+ RNA coliphages appears to be practical and reliable for typing isolates in field samples. PMID:8526509

  3. Cloned polynucleotide and synthetic oligonucleotide probes used in colony hybridization are equally efficient in the identification of enterotoxigenic Escherichia coli

    SciTech Connect

    Sommerfelt, H.; Kalland, K.H.; Raj, P.; Moseley, S.L.; Bhan, M.K.; Bjorvatn, B.

    1988-11-01

    Restriction endonuclease-generated polynucleotide and synthetically produced oligonucleotide gene probes used in colony hybridization assays proved to be efficient for the detection and differentiation of enterotoxigenic Escherichia coli. To compare their relative efficiencies, these two sets of probes were radiolabeled with /sup 32/P and were applied to 74 strains of E. coli with known enterotoxin profiles and to 156 previously unexamined E. coli isolates. The enterotoxigenic bacteria Vibrio cholerae O1, Vibrio cholerae non-O1 (NAG), Yersinia enterocolitica, and E. coli harboring the plasmid vectors of the polynucleotide gene probes were examined for further evaluation of probe specificity. The two classes of probes showed a perfect concordance in their specific detection and differentiation of enterotoxigenic E. coli. In the analysis of six strains, the signal strength on autoradiography after hybridization with oligonucleotides was weaker than that obtained after hybridization with polynucleotide probes. The probes did not hybridize with DNA from V. cholerae O1, V. cholerae non-O1 (NAG), or Y. enterocolitica. The strains of E. coli harboring the plasmid vectors of the polynucleotide gene probes were, likewise, negative in the hybridization assays.

  4. Enzyme-Free Detection of Mutations in Cancer DNA Using Synthetic Oligonucleotide Probes and Fluorescence Microscopy.

    PubMed

    Miotke, Laura; Maity, Arindam; Ji, Hanlee; Brewer, Jonathan; Astakhova, Kira

    2015-01-01

    Rapid reliable diagnostics of DNA mutations are highly desirable in research and clinical assays. Current development in this field goes simultaneously in two directions: 1) high-throughput methods, and 2) portable assays. Non-enzymatic approaches are attractive for both types of methods since they would allow rapid and relatively inexpensive detection of nucleic acids. Modern fluorescence microscopy is having a huge impact on detection of biomolecules at previously unachievable resolution. However, no straightforward methods to detect DNA in a non-enzymatic way using fluorescence microscopy and nucleic acid analogues have been proposed so far. Here we report a novel enzyme-free approach to efficiently detect cancer mutations. This assay includes gene-specific target enrichment followed by annealing to oligonucleotides containing locked nucleic acids (LNAs) and finally, detection by fluorescence microscopy. The LNA containing probes display high binding affinity and specificity to DNA containing mutations, which allows for the detection of mutation abundance with an intercalating EvaGreen dye. We used a second probe, which increases the overall number of base pairs in order to produce a higher fluorescence signal by incorporating more dye molecules. Indeed we show here that using EvaGreen dye and LNA probes, genomic DNA containing BRAF V600E mutation could be detected by fluorescence microscopy at low femtomolar concentrations. Notably, this was at least 1000-fold above the potential detection limit. Overall, the novel assay we describe could become a new approach to rapid, reliable and enzyme-free diagnostics of cancer or other associated DNA targets. Importantly, stoichiometry of wild type and mutant targets is conserved in our assay, which allows for an accurate estimation of mutant abundance when the detection limit requirement is met. Using fluorescence microscopy, this approach presents the opportunity to detect DNA at single-molecule resolution and directly

  5. Kinetics of Oligonucleotide Hybridization to DNA Probe Arrays on High-Capacity Porous Silica Substrates

    PubMed Central

    Glazer, Marc I.; Fidanza, Jacqueline A.; McGall, Glenn H.; Trulson, Mark O.; Forman, Jonathan E.; Frank, Curtis W.

    2007-01-01

    We have investigated the kinetics of DNA hybridization to oligonucleotide arrays on high-capacity porous silica films that were deposited by two techniques. Films created by spin coating pure colloidal silica suspensions onto a substrate had pores of ∼23 nm, relatively low porosity (35%), and a surface area of 17 times flat glass (for a 0.3-μm film). In the second method, latex particles were codeposited with the silica by spin coating and then pyrolyzed, which resulted in larger pores (36 nm), higher porosity (65%), and higher surface area (26 times flat glass for a 0.3-μm film). As a result of these favorable properties, the templated silica hybridized more quickly and reached a higher adsorbed target density (11 vs. 8 times flat glass at 22°C) than the pure silica. Adsorption of DNA onto the high-capacity films is controlled by traditional adsorption and desorption coefficients, as well as by morphology factors and transient binding interactions between the target and the probes. To describe these effects, we have developed a model based on the analogy to diffusion of a reactant in a porous catalyst. Adsorption values (ka, kd, and K) measured on planar arrays for the same probe/target system provide the parameters for the model and also provide an internally consistent comparison for the stability of the transient complexes. The interpretation of the model takes into account factors not previously considered for hybridization in three-dimensional films, including the potential effects of heterogeneous probe populations, partial probe/target complexes during diffusion, and non-1:1 binding structures. The transient complexes are much less stable than full duplexes (binding constants for full duplexes higher by three orders of magnitude or more), which may be a result of the unique probe density and distribution that is characteristic of the photolithographically patterned arrays. The behavior at 22°C is described well by the predictive equations for

  6. Transcription profiling of the early gravitropic response in Arabidopsis using high-density oligonucleotide probe microarrays

    NASA Technical Reports Server (NTRS)

    Moseyko, Nick; Zhu, Tong; Chang, Hur-Song; Wang, Xun; Feldman, Lewis J.

    2002-01-01

    Studies of plant tropisms, the directed growth toward or away from external stimuli such as light and gravity, began more than a century ago. Yet biochemical, physiological, and especially molecular mechanisms of plant tropic responses remain for the most part unclear. We examined expression of 8,300 genes during early stages of the gravitropic response using high-density oligonucleotide probe microarrays. Approximately 1.7% of the genes represented on the array exhibited significant expression changes within the first 30 min of gravity stimulation. Among gravity-induced genes were a number of genes previously implicated to be involved in gravitropism. However, a much larger number of the identified genes have not been previously associated with gravitropism. Because reorientation of plants may also expose plants to mechanical perturbations, we also compared the effects of a gentle mechanical perturbation on mRNA levels during the gravity response. It was found that approximately 39% of apparently gravity-regulated genes were also regulated by the mechanical perturbation caused by plant reorientation. Our study revealed the induction of complex gene expression patterns as a consequence of gravitropic reorientation and points to an interplay between the gravitropic and mechanical responses and to the extreme sensitivity of plants to even very gentle mechanical perturbations.

  7. Transcription profiling of the early gravitropic response in Arabidopsis using high-density oligonucleotide probe microarrays

    NASA Technical Reports Server (NTRS)

    Moseyko, Nick; Zhu, Tong; Chang, Hur-Song; Wang, Xun; Feldman, Lewis J.

    2002-01-01

    Studies of plant tropisms, the directed growth toward or away from external stimuli such as light and gravity, began more than a century ago. Yet biochemical, physiological, and especially molecular mechanisms of plant tropic responses remain for the most part unclear. We examined expression of 8,300 genes during early stages of the gravitropic response using high-density oligonucleotide probe microarrays. Approximately 1.7% of the genes represented on the array exhibited significant expression changes within the first 30 min of gravity stimulation. Among gravity-induced genes were a number of genes previously implicated to be involved in gravitropism. However, a much larger number of the identified genes have not been previously associated with gravitropism. Because reorientation of plants may also expose plants to mechanical perturbations, we also compared the effects of a gentle mechanical perturbation on mRNA levels during the gravity response. It was found that approximately 39% of apparently gravity-regulated genes were also regulated by the mechanical perturbation caused by plant reorientation. Our study revealed the induction of complex gene expression patterns as a consequence of gravitropic reorientation and points to an interplay between the gravitropic and mechanical responses and to the extreme sensitivity of plants to even very gentle mechanical perturbations.

  8. Hairpin oligonucleotides anchored terbium ion: a fluorescent probe to specifically detect lead(II) at sub-nM levels.

    PubMed

    Wei, Yueteng; Liu, Ru; Wang, Yaling; Zhao, Yuliang; Cai, Zhifang; Gao, Xueyun

    2013-04-21

    A terbium based fluorescent probe was synthesized by coordinating terbium ions with a designed oligonucleotides (5'-ATATGGGGGATAT-3', termed GH5). GH5 improved the fluorescence of terbium ions by four orders of magnitude. The fluorescence enhancement of terbium ions by different oligonucleotides sequences indicated that the polyguanine loop of the hairpin GH5 is key to enhance terbium ion emission. The quantum yield of Tb-GH5 probe was 10.5% and the probe was photo-stable. The result of conductivity titration indicated that the stoichiometry of the probe is 3.5 Tb: 1 GH5, which is confirmed by fluorescence titration. This probe had high sensitivity and specificity for the detection of lead ions. The fluorescence intensity of this probe was linear with respect to lead concentration over a range 0.3-2.1 nM (R(2) = 0.99). The limit of detection for lead ions was 0.1 nM at a signal-to-noise ratio of 3.

  9. Microarray-based long oligonucleotides probe designed for Brucella Spp. detection and identification of antibiotic susceptibility pattern.

    PubMed

    Khazaei, Zahra; Najafi, Ali; Piranfar, Vahhab; Mirnejad, Reza

    2016-04-01

    Brucella spp. is a common zoonotic infection referred to as Brucellosis, and it is a serious public health problem around the world. There are currently six classical species (pathogenic species in both animals and humans) within the genus Brucella. The ability and practicality facilitated by a microarray experiment help us to recognize Brucella spp. and its antibiotic resistant gene. Rapid phenotypic determination of antibiotic resistance is not possible by disk diffusion methods. Thus, evaluating antibiotics pattern and Brucella detection appear necessary technique by molecular methods in brucellosis. So, the aim of this study was to design a microarray long oligonucleotides probe and primer for the complete diagnosis of Brucella spp. and obtaining genetic profiles for antibiotic resistance in bacteria at the same time. In this study, we designed 16 antibiotic-resistant gene solid-phase primers with similar melting temperatures of 60 °C and 16 long oligonucleotide probes. These primers and probes can identify tetracycline-, chloramphenicol-, and aminoglycoside-resistant genes, respectively. The design of microarray probes is a versatile process that be done in a wide range of selections. Since the long oligo microarray probes are the best choices for specific diagnosis and definite treatment, this group of probes was designed in the present survey.

  10. Microarray-based long oligonucleotides probe designed for Brucella Spp. detection and identification of antibiotic susceptibility pattern

    PubMed Central

    Khazaei, Zahra; Najafi, Ali; Piranfar, Vahhab; Mirnejad, Reza

    2016-01-01

    Brucella spp. is a common zoonotic infection referred to as Brucellosis, and it is a serious public health problem around the world. There are currently six classical species (pathogenic species in both animals and humans) within the genus Brucella. The ability and practicality facilitated by a microarray experiment help us to recognize Brucella spp. and its antibiotic resistant gene. Rapid phenotypic determination of antibiotic resistance is not possible by disk diffusion methods. Thus, evaluating antibiotics pattern and Brucella detection appear necessary technique by molecular methods in brucellosis. So, the aim of this study was to design a microarray long oligonucleotides probe and primer for the complete diagnosis of Brucella spp. and obtaining genetic profiles for antibiotic resistance in bacteria at the same time. In this study, we designed 16 antibiotic-resistant gene solid-phase primers with similar melting temperatures of 60 °C and 16 long oligonucleotide probes. These primers and probes can identify tetracycline-, chloramphenicol-, and aminoglycoside-resistant genes, respectively. The design of microarray probes is a versatile process that be done in a wide range of selections. Since the long oligo microarray probes are the best choices for specific diagnosis and definite treatment, this group of probes was designed in the present survey. PMID:27280008

  11. Design and synthesis of triazolyl-donor/acceptor unnatural nucleosides and oligonucleotide probes containing triazolyl-phenanthrene nucleoside.

    PubMed

    Bag, Subhendu Sekhar; Talukdar, Sangita; Das, Suman Kalyan

    2014-09-08

    In the context of abasic DNA or DNA duplex stabilization, several unnatural nucleosidic/non-nucleosidic base surrogates have been reported. Toward this end, we have designed and synthesized triazolyl-aromatic donor chomophores as unnatural nucleoside analogs. These modifications display markedly higher thermal stabilization of abasic DNA duplex in comparison to the stabilization offered by other nucleoside/non-nucleoside base surrogates reported in the literature. The same oligonucleotide probe containing triazolylphenanthrene nucleotide also offers very good stability of the self-pair duplex via π-π stacking interaction and hetero-pair duplex via charge transfer interaction when paired against triazolyl acceptor aromatic nucleoside. Moreover, the probe in the reverse sequence containing triazolylphenanthrene nucleotide has shown FRET efficiency in a chimeric DNA duplex. The triazolyl nucleotides would expectedly show stability toward exonuclease activity. This unit describes protocols for chemical synthesis of unnatural triazolyl nucleosides and one oligonucleotide probe. The unit also provides a summary of various thermal and photophysical applications of triazolylphenantherene-containing oligonucleotides. Copyright © 2014 John Wiley & Sons, Inc.

  12. Detection of human papilloma viruses in paraffin wax sections with biotinylated synthetic oligonucleotide probes and immunogold staining.

    PubMed

    Cubie, H A; Norval, M

    1989-09-01

    Human papilloma virus was detected by in situ hybridisation in routinely processed paraffin wax sections using a synthetically produced oligonucleotide probe, end-labelled with biotin, and amplified with anti-biotin-immunogold silver staining (anti-biotin-IGSS). This system proved more sensitive than amplification with streptavidin-biotinylated alkaline phosphatase for detecting human papilloma virus type 16 in cervical tissues. The method was successfully combined with antigen staining for papilloma virus common antigens in skin and genital warts. This simple and quick method, using non-radioactively labelled synthetic probes, may be useful for the detection of other viruses in stored material and may be suitable for other double staining procedures.

  13. Environment-responsive fluorescent nucleoside analogue probe for studying oligonucleotide dynamics in a model cell-like compartment.

    PubMed

    Pawar, Maroti G; Srivatsan, Seergazhi G

    2013-11-21

    The majority of fluorescent nucleoside analogue probes that have been used in the in vitro study of nucleic acids are not suitable for cell-based biophysical assays because they exhibit excitation maxima in the UV region and low quantum yields within oligonucleotides. Therefore, we propose that the photophysical characterization of oligonucleotides labeled with a fluorescent nucleoside analogue in reverse micelles (RM), which are good biological membrane models and UV-transparent, could provide an alternative approach to studying the properties of nucleic acids in a cell-like confined environment. In this context, we describe the photophysical properties of an environment-sensitive fluorescent uridine analogue (1), based on the 5-(benzo[b]thiophen-2-yl)pyrimidine core, in micelles and RM. The emissive nucleoside, which is polarity- and viscosity-sensitive, reports the environment of the surfactant assemblies via changes in its fluorescence properties. The nucleoside analogue, incorporated into an RNA oligonucleotide and hybridized to its complementary DNA and RNA oligonucleotides, exhibits a significantly higher fluorescence intensity, lifetime, and anisotropy in RM than in aqueous buffer, which is consistent with the environment of RM. Collectively, our results demonstrate that nucleoside 1 could be utilized as a fluorescent label to study the function of nucleic acids in a model cellular milieu.

  14. Optimal Probe Length and Target Location for Electrochemical Detection of Selected Uropathogens at Ambient Temperature▿

    PubMed Central

    Mastali, Mitra; Babbitt, Jane T.; Li, Yang; Landaw, Elliot M.; Gau, Vincent; Churchill, Bernard M.; Haake, David A.

    2008-01-01

    We have previously demonstrated the clinical validity of the rapid detection of uropathogens by use of a DNA biosensor. This assay involves the hybridization of capture and detector probe pairs with bacterial 16S rRNA target molecules to form a DNA-RNA sandwich on the sensor surface. Horseradish peroxidase-conjugated antibody binds to the detector probe to enzymatically amplify the hybridization signal. These previous studies involved the hybridization of bacterial 16S rRNA target sequences with 35-mer oligonucleotide probe pairs at 65°C. Achievement of point-of-care technology will be greatly facilitated by ambient-temperature detection. The purpose of this study was to examine the effects of probe length and target location on signal intensity using hybridization temperatures of 20 to 25°C. Signal intensity was found to vary dramatically with hybridization location in the species-specific bulge region of 16S rRNA helix 18. Probe pairs of as short as 10 nucleotides in length were able to produce a significant electrochemical signal, and signal intensity was correlated with probe length for probes of 10 to 20 nucleotides in length. The sensitivity of the Escherichia coli-specific 15-mer probe pairs was approximately 330 cells. These shorter probes allowed differentiation of Klebsiella pneumoniae from Proteus mirabilis 16S rRNA target sequences differing by a single nucleotide. A panel of oligonucleotide probe pairs ranging from 11 to 23 nucleotides in length was able to distinguish among seven groups of urinary tract pathogens. In conclusion, we have developed short oligonucleotide probe pairs for the species-specific identification of uropathogens at ambient temperature by use of an electrochemical sensor. PMID:18562584

  15. Multiplexed quantification of bacterial 16S rRNA by solution hybridization with oligonucleotide probes and affinity capture.

    PubMed

    Satokari, Reetta M; Kataja, Kari; Söderlund, Hans

    2005-07-01

    Multiplexed and quantitative analysis of nucleic acid sequences in complex mixtures is essential in various applications of microbiological research. We have developed a method based on solution hybridization between biotinylated nucleic acid targets and multiple fluorophore-labeled oligonucleotide probes of distinct sizes. The biotin-nucleic acid-probe complexes are captured on magnetic streptavidin-coated microparticles and washed. The hybridized probes are eluted and their identity and quantity are determined by capillary electrophoresis. The signal intensities of the recorded probes correspond to the amount of target nucleic acid in the mixture, and the size indicates the target. Based on this principle and 16S rRNA-specific oligonucleotide probes, we set up an application for the relative quantification of different groups of clostridia and related organisms in a mixed bacterial population. The lower detection limit is 0.05 ng of total RNA and the linear range of measurement is 10(2). The method allowed accurate and highly repeatable quantification of the proportion of clostridia in human feces. Further, we discuss other applications of the method such as quantitative transcriptional analysis of eukaryotic microorganisms, which can be performed without conversion of mRNA to cDNA.

  16. New Oligonucleotide Probes for ND-FISH Analysis to Identify Barley Chromosomes and to Investigate Polymorphisms of Wheat Chromosomes

    PubMed Central

    Tang, Shuyao; Qiu, Ling; Xiao, Zhiqiang; Fu, Shulan; Tang, Zongxiang

    2016-01-01

    Oligonucleotide probes that can be used for non-denaturing fluorescence in situ hybridization (ND-FISH) analysis are convenient tools for identifying chromosomes of wheat (Triticum aestivum L.) and its relatives. New oligonucleotide probes, Oligo-HvT01, Oligo-pTa71-1, Oligo-s120.1, Oligo-s120.2, Oligo-s120.3, Oligo-275.1, Oligo-275.2, Oligo-k566 and Oligo-713, were designed based on the repetitive sequences HVT01, pTa71, pTa-s120, pTa-275, pTa-k566 and pTa-713. All these probes can be used for ND-FISH analysis and some of them can be used to detect polymorphisms of wheat chromosomes. Probes Oligo-HvT01, Oligo-pTa71-1, Oligo-s120.3, Oligo-275.1, Oligo-k566 and Oligo-713 can, respectively, replace the roles of their original sequences to identify chromosomes of some barley (Hordeum vulgare ssp. vulgare) and the common wheat variety Chinese Spring. Oligo-s120.1, Oligo-s120.2 and Oligo-275.2 produced different hybridization patterns from the ones generated by their original sequences. In addition, Oligo-s120.1, Oligo-s120.2 and Oligo-s120.3, which were derived from pTa-s120, revealed different signal patterns. Likewise, Oligo-275.1 and Oligo-275.2, which were derived from pTa-275, also displayed different hybridization patterns. These results imply that differently arranged or altered structural statuses of tandem repeats might exist on different chromosome regions. These new oligonucleotide probes provide extra convenience for identifying some wheat and barley chromosomes, and they can display polymorphisms of wheat chromosomes. PMID:27929398

  17. New Concepts of Fluorescent Probes for Specific Detection of DNA Sequences: Bis-Modified Oligonucleotides in Excimer and Exciplex Detection

    PubMed Central

    Gbaj, A; Bichenkova, EV; Walsh, L; Savage, HE; Sardarian, AR; Etchells, LL; Gulati, A; Hawisa, S; Douglas, KT

    2009-01-01

    The detection of single base mismatches in DNA is important for diagnostics, treatment of genetic diseases, and identification of single nucleotide polymorphisms. Highly sensitive, specific assays are needed to investigate genetic samples from patients. The use of a simple fluorescent nucleoside analogue in detection of DNA sequence and point mutations by hybridisation in solution is described in this study. The 5′-bispyrene and 3′-naphthalene oligonucleotide probes form an exciplex on hybridisation to target in water and the 5′-bispyrene oligonucleotide alone is an adequate probe to determine concentration of target present. It was also indicated that this system has a potential to identify mismatches and insertions. The aim of this work was to investigate experimental structures and conditions that permit strong exciplex emission for nucleic acid detectors, and show how such exciplexes can register the presence of mismatches as required in SNP analysis. This study revealed that the hybridisation of 5′-bispyrenyl fluorophore to a DNA target results in formation of a fluorescent probe with high signal intensity change and specificity for detecting a complementary target in a homogeneous system. Detection of SNP mutations using this split-probe system is a highly specific, simple, and accessible method to meet the rigorous requirements of pharmacogenomic studies. Thus, it is possible for the system to act as SNP detectors and it shows promise for future applications in genetic testing. PMID:21483539

  18. Pigeons: A Novel GUI Software for Analysing and Parsing High Density Heterologous Oligonucleotide Microarray Probe Level Data

    PubMed Central

    Lai, Hung-Ming; May, Sean T.; Mayes, Sean

    2014-01-01

    Genomic DNA-based probe selection by using high density oligonucleotide arrays has recently been applied to heterologous species (Xspecies). With the advent of this new approach, researchers are able to study the genome and transcriptome of a non-model or an underutilised crop species through current state-of-the-art microarray platforms. However, a software package with a graphical user interface (GUI) to analyse and parse the oligonucleotide probe pair level data is still lacking when an experiment is designed on the basis of this cross species approach. A novel computer program called Pigeons has been developed for customised array data analysis to allow the user to import and analyse Affymetrix GeneChip® probe level data through XSpecies. One can determine empirical boundaries for removing poor probes based on genomic hybridisation of the test species to the Xspecies array, followed by making a species-specific Chip Description File (CDF) file for transcriptomics in the heterologous species, or Pigeons can be used to examine an experimental design to identify potential Single-Feature Polymorphisms (SFPs) at the DNA or RNA level. Pigeons is also focused around visualization and interactive analysis of the datasets. The software with its manual (the current release number version 1.2.1) is freely available at the website of the Nottingham Arabidopsis Stock Centre (NASC). PMID:27605027

  19. Design and application of two oligonucleotide probes for the identification of Geodermatophilaceae strains using fluorescence in situ hybridization (FISH).

    PubMed

    Urzì, Clara; La Cono, Violetta; Stackebrandt, Erko

    2004-07-01

    Bacteria of the family of Geodermatophilaceae are actively involved in the decay processes [Urzì, C. and Realini, M. (1998) Int Biodeterior Biodegrad 42: 45-54; Urzì, C., Salamone, P., Schumann, P., and Stackebrandt, E. (2000) Int J Syst Evol Microbiol 50: 529-536] of stone monuments. Characterization of isolates includes phenotypic, chemotaxonomic and genetic analysis often requiring long-term procedures. The use of specific probes for members of Geodermatophilaceae family could be useful for the easy detection of those strains colonizing rock surfaces and involved in the biodeterioration. Two 16S rRNA-targeted oligonucleotide probes were designed for the specific detection of members of the family Geodermatophilaceae using fluorescence in situ hybridization (FISH); one probe specific for members of the two genera Geodermatophilus/Blastococcus and the second for members of the genus Modestobacter.

  20. Fluorescent in situ hybridization with rRNA-targeted oligonucleotide probes to identify small phytoplankton by flow cytometry.

    PubMed Central

    Simon, N; LeBot, N; Marie, D; Partensky, F; Vaulot, D

    1995-01-01

    Because of their tiny size (0.2 to 2 microns), oceanic picophytoplanktonic cells (either cultured strains or natural communities) are difficult to identify, and some basic questions concerning their taxonomy, physiology, and ecology are still largely unanswered. The present study was designed to test the suitability of in situ hybridization with rRNA fluorescent probes detected by flow cytometry for the identification of small photosynthetic eukaryotes. Oligonucleotide probes targeted against regions of the 18S rRNAs of Chlorophyta lineage (CHLO probe) and of non-Chlorophyta (NCHLO probe) algal species were designed. The CHLO and NCHLO probes, which differed by a single nucleotide, allowed discrimination of chlorophyte from nonchlorophyte cultured strains. The sensitivity of each probe was dependent upon the size of the cells and upon their growth stage. The mean fluorescence was 8 to 80 times higher for specifically labeled than for nonspecifically labeled cells in exponential growth phase, but it decreased sharply in stationary phase. Such taxon-specific probes should increase the applicability of flow cytometry for the rapid identification of cultured pico- and nanoplanktonic strains, especially those that lack taxonomically useful morphological features. PMID:7618862

  1. The Human OligoGenome Resource: a database of oligonucleotide capture probes for resequencing target regions across the human genome.

    PubMed

    Newburger, Daniel E; Natsoulis, Georges; Grimes, Sue; Bell, John M; Davis, Ronald W; Batzoglou, Serafim; Ji, Hanlee P

    2012-01-01

    Recent exponential growth in the throughput of next-generation DNA sequencing platforms has dramatically spurred the use of accessible and scalable targeted resequencing approaches. This includes candidate region diagnostic resequencing and novel variant validation from whole genome or exome sequencing analysis. We have previously demonstrated that selective genomic circularization is a robust in-solution approach for capturing and resequencing thousands of target human genome loci such as exons and regulatory sequences. To facilitate the design and production of customized capture assays for any given region in the human genome, we developed the Human OligoGenome Resource (http://oligogenome.stanford.edu/). This online database contains over 21 million capture oligonucleotide sequences. It enables one to create customized and highly multiplexed resequencing assays of target regions across the human genome and is not restricted to coding regions. In total, this resource provides 92.1% in silico coverage of the human genome. The online server allows researchers to download a complete repository of oligonucleotide probes and design customized capture assays to target multiple regions throughout the human genome. The website has query tools for selecting and evaluating capture oligonucleotides from specified genomic regions.

  2. Development of oligonucleotides and multiplex probes for quick and accurate identification of wheat and Thinopyrum bessarabicum chromosomes.

    PubMed

    Du, Pei; Zhuang, Lifang; Wang, Yanzhi; Yuan, Li; Wang, Qing; Wang, Danrui; Dawadondup; Tan, Lijun; Shen, Jian; Xu, Haibin; Zhao, Han; Chu, Chenggen; Qi, Zengjun

    2017-02-01

    In comparison with general FISH for preparing probes in terms of time and cost, synthesized oligonucleotide (oligo hereafter) probes for FISH have many advantages such as ease of design, synthesis, and labeling. Low cost and high sensitivity and resolution of oligo probes greatly simplify the FISH procedure as a simple, fast, and efficient method of chromosome identification. In this study, we developed new oligo and oligo multiplex probes to accurately and efficiently distinguish wheat (Triticum aestivum, 2n = 6x, AABBDD) and Thinopyrum bessarabicum (2n = 2x = 14, JJ) chromosomes. The oligo probes contained more nucleotides or more repeat units that produced stronger signals for more efficient chromosome painting. Four Th. bessarabicum-specific oligo probes were developed based on genomic DNA sequences of Th. bessarabicum chromosome arm 4JL, and one of them (oligo DP4J27982) was pooled with the oligo multiplex #1 to simultaneously detect wheat and Th. bessarabicum chromosomes for quick and accurate identification of Chinese Spring (CS) - Th. bessarabicum alien chromosome introgression lines. Oligo multiplex #4 revealed chromosome variations among CS and eight wheat cultivars by a single round of FISH analysis. This research demonstrated the high efficiency of using oligos and oligo multiplexes in chromosome identification and manipulation.

  3. Quantification of Gordona amarae Strains in Foaming Activated Sludge and Anaerobic Digester Systems with Oligonucleotide Hybridization Probes

    PubMed Central

    de los Reyes, M. Fiorella; de los Reyes, Francis L.; Hernandez, Mark; Raskin, Lutgarde

    1998-01-01

    Previous studies have shown the predominance of mycolic acid-containing filamentous actinomycetes (mycolata) in foam layers in activated sludge systems. Gordona (formerly Nocardia) amarae often is considered the major representative of this group in activated sludge foam. In this study, small-subunit rRNA genes of four G. amarae strains were sequenced, and the resulting sequences were compared to the sequence of G. amarae type strain SE-6. Comparative sequence analysis showed that the five strains used represent two lines of evolutionary descent; group 1 consists of strains NM23 and ASAC1, and group 2 contains strains SE-6, SE-102, and ASF3. The following three oligonucleotide probes were designed: a species-specific probe for G. amarae, a probe specific for group 1, and a probe targeting group 2. The probes were characterized by dissociation temperature and specificity studies, and the species-specific probe was evaluated for use in fluorescent in situ hybridizations. By using the group-specific probes, it was possible to place additional G. amarae isolates in their respective groups. The probes were used along with previously designed probes in membrane hybridizations to determine the abundance of G. amarae, group 1, group 2, bacterial, mycolata, and Gordona rRNAs in samples obtained from foaming activated sludge systems in California, Illinois, and Wisconsin. The target groups were present in significantly greater concentrations in activated sludge foam than in mixed liquor and persisted in anaerobic digesters. Hybridization results indicated that the presence of certain G. amarae strains may be regional or treatment plant specific and that previously uncharacterized G. amarae strains may be present in some systems. PMID:9647822

  4. Detection of human papilloma viruses in paraffin wax sections with biotinylated synthetic oligonucleotide probes and immunogold staining.

    PubMed Central

    Cubie, H A; Norval, M

    1989-01-01

    Human papilloma virus was detected by in situ hybridisation in routinely processed paraffin wax sections using a synthetically produced oligonucleotide probe, end-labelled with biotin, and amplified with anti-biotin-immunogold silver staining (anti-biotin-IGSS). This system proved more sensitive than amplification with streptavidin-biotinylated alkaline phosphatase for detecting human papilloma virus type 16 in cervical tissues. The method was successfully combined with antigen staining for papilloma virus common antigens in skin and genital warts. This simple and quick method, using non-radioactively labelled synthetic probes, may be useful for the detection of other viruses in stored material and may be suitable for other double staining procedures. Images PMID:2551931

  5. Novel oligonucleotide probes for in situ detection of pederin-producing endosymbionts of Paederus riparius rove beetles (Coleoptera: Staphylinidae).

    PubMed

    Kador, Matthias; Horn, Marcus A; Dettner, Konrad

    2011-06-01

    Bacterial endosymbionts from female Paederus rove beetles are hitherto uncultured, phylogenetically related to Pseudomonas sp., and produce the polyketide pederin, which exhibits strong cytotoxic effects and antitumoral activities. The location of such endosymbionts inside beetles and on beetles' eggs is hypothesized based on indirect evidence rather than elucidated. Thus, an endosymbiont-specific and a competitor oligonucleotide probe (Cy3-labelled PAE444 and unlabelled cPAE444, respectively) were designed and utilized for FISH with semi-thin sections of Paederus riparius eggs. Cy3-PAE444-positive cells were densely packed and covered the whole eggshell. Hundred percent of EUB338-Mix-positive total bacterial cells were PAE444 positive, indicating a biofilm dominated by Paederus endosymbionts. Analysis of different egg deposition stadiums by electron microscopy and pks (polyketide synthase gene, a structural gene associated with pederin biosynthesis)-PCR supported results obtained by FISH and revealed that the endosymbiont-containing layer is applied to the eggshell inside the efferent duct. These findings suggest that P. riparius endosymbionts are located inside unknown structures of the female genitalia, which allow for a well-regulated release of endosymbionts during oviposition. The novel oligonucleotide probes developed in this study will facilitate (1) the identification of symbiont-containing structures within genitalia of their beetle hosts and (2) directed cultivation approaches in the future.

  6. A regenerated electrochemical biosensor for label-free detection of glucose and urea based on conformational switch of i-motif oligonucleotide probe.

    PubMed

    Gao, Zhong Feng; Chen, Dong Mei; Lei, Jing Lei; Luo, Hong Qun; Li, Nian Bing

    2015-10-15

    Improving the reproducibility of electrochemical signal remains a great challenge over the past decades. In this work, i-motif oligonucleotide probe-based electrochemical DNA (E-DNA) sensor is introduced for the first time as a regenerated sensing platform, which enhances the reproducibility of electrochemical signal, for label-free detection of glucose and urea. The addition of glucose or urea is able to activate glucose oxidase-catalyzed or urease-catalyzed reaction, inducing or destroying the formation of i-motif oligonucleotide probe. The conformational switch of oligonucleotide probe can be recorded by electrochemical impedance spectroscopy. Thus, the difference of electron transfer resistance is utilized for the quantitative determination of glucose and urea. We further demonstrate that the E-DNA sensor exhibits high selectivity, excellent stability, and remarkable regenerated ability. The human serum analysis indicates that this simple and regenerated strategy holds promising potential in future biosensing applications.

  7. Highly specific identification of single nucleic polymorphism in M. tuberculosis using smart probes and single-molecule fluorescence spectroscopy in combination with blocking oligonucleotides

    NASA Astrophysics Data System (ADS)

    Friedrich, Achim; Müller, Matthias; Nolte, Oliver; Wolfrum, Jürgen; Sauer, Markus; Hoheisel, Jörg D.; Knemeyer, Jens-Peter; Marme, Nicole

    2008-02-01

    In this article we present a method for the highly specific identification of single nucleotide polymorphism (SNP) responsible for rifampicin resistance of Mycobacterium tuberculosis. This approach applies fluorescently labeled hairpin-structured oligonucleotides (smart probes) and confocal single-molecule fluorescence spectroscopy. Smart probes are fluorescently labeled at the 5'-end. The dye's fluorescence is quenched in the closed hairpin conformation due to close proximity of the guanosine residues located at the 3'-end. As a result of the hybridization to the complementary target sequence the hairpin structure and thus fluorescence quenching gets lost and a strong fluorescence increase appears. To enhance the specificity of the SNP detection unlabeled "blocking oligonucleotides" were added to the sample. These oligonucleotides hybridizes to the DNA sequence containing the mismatch thus masking this sequence and hereby preventing the smart probe from hybridizing to the mismatched sequence.

  8. Design and validation of an oligonucleotide probe for detection of protozoa from the order Trichomonadida using chromogenic in situ hybridization

    PubMed Central

    Mostegl, Meike Marissa; Richter, Barbara; Nedorost, Nora; Maderner, Anton; Dinhopl, Nora; Kulda, Jaroslav; Liebhart, Dieter; Hess, Michael; Weissenböck, Herbert

    2010-01-01

    Infections with protozoal parasites of the order Trichomonadida are often observed in veterinary medicine. Based on the trichomonad species involved these infections are either asymptomatic or can lead to sometimes serious disease. To further study protozoal agents of the order Trichomonadida the establishment of a method to detect trichomonads directly in the tissue, allowing parasite-lesion correlation, is necessary. Here we describe the design and evaluation of an oligonucleotide probe for chromogenic in situ hybridization, theoretically allowing detection of all hitherto known members of the order Trichomonadida. The probe was designed on a region of the 18S ribosomal RNA gene homologue for all representatives of the order Trichomonadida available in the GenBank. Functionality of the probe was proven using protozoal cultures containing different trichomonads (Monocercomonas colubrorum, Hypotrichomonas acosta, Pentatrichomonas hominis, Trichomitus batrachorum, Trichomonas gallinae, Tetratrichomonas gallinarum, Tritrichomonas foetus, and Tritrichomonas augusta). Furthermore, three different tissue sections containing either Trichomonas gallinae, Tritrichomonas foetus or Histomonas meleagridis were tested positive. Additionally, to rule out cross reactivity of the probe a large number of different pathogenic protozoal agents, fungi, bacteria and viruses were tested and gave negative results. The probe presented here can be considered an important tool for diagnosis of all to date described relevant protozoal parasites of the order Trichomonadida in tissue samples. PMID:20395049

  9. UPS 2.0: unique probe selector for probe design and oligonucleotide microarrays at the pangenomic/genomic level.

    PubMed

    Chen, Shu-Hwa; Lo, Chen-Zen; Su, Sheng-Yao; Kuo, Bao-Han; Hsiung, Chao A; Lin, Chung-Yen

    2010-12-02

    Nucleic acid hybridization is an extensively adopted principle in biomedical research, in which the performance of any hybridization-based method depends on the specificity of probes to their targets. To determine the optimal probe(s) for detecting target(s) from a sample cocktail, we developed a novel algorithm, which has been implemented into a web platform for probe designing. This probe design workflow is now upgraded to satisfy experiments that require a probe designing tool to take the increasing volume of sequence datasets. Algorithms and probe parameters applied in UPS 2.0 include GC content, the secondary structure, melting temperature (Tm), the stability of the probe-target duplex estimated by the thermodynamic model, sequence complexity, similarity of probes to non-target sequences, and other empirical parameters used in the laboratory. Several probe background options,Unique probe within a group,Unique probe in a specific Unigene set,Unique probe based on the pangenomic level, and Unique Probe in the user-defined genome/transcriptome, are available to meet the scenarios that the experiments will be conducted. Parameters, such as salt concentration and the lower-bound Tm of probes, are available for users to optimize their probe design query. Output files are available for download on the result page. Probes designed by the UPS algorithm are suitable for generating microarrays, and the performance of UPS-designed probes has been validated by experiments. The UPS 2.0 evaluates probe-to-target hybridization under a user-defined condition to ensure high-performance hybridization with minimal chance of non-specific binding at the pangenomic and genomic levels. The UPS algorithm mimics the target/non-target mixture in an experiment and is very useful in developing diagnostic kits and microarrays. The UPS 2.0 website has had more than 1,300 visits and 360,000 sequences performed the probe designing task in the last 30 months. It is freely accessible at http

  10. Characterization of the Cricket Hindgut Microbiota with Fluorescently Labeled rRNA-Targeted Oligonucleotide Probes

    PubMed Central

    Santo Domingo, Jorge W.; Kaufman, Michael G.; Klug, Michael J.; Tiedje, James M.

    1998-01-01

    Most cricket hindgut microorganisms (60 to 80%) were detected with a universal fluorescent rRNA-targeted probe and found to be eubacteria. Group-specific probes showed that the hindguts of five different cricket species harbor similar bacterial groups, although in different proportions, and that different diets shifted the structure of the hindgut microbial community. The Bacteroides-Prevotella probe, of the eight eubacterial probes tested, stained the largest percentage of cells in all crickets. PMID:16349506

  11. Use of a synthetic DNA oligonucleotide to probe the precision of RNA splicing in a yeast mitochondrial petite mutant.

    PubMed Central

    Tabak, H F; van der Laan, J; Osinga, K A; Schouten, J P; van Boom, J H; Veeneman, G H

    1981-01-01

    In some strains of Saccharomyces cerevisiae the mitochondrial gene coding for 21S rRNA is interrupted by an intron of 1143 bp. This intron contains a reading frame for 235 amino acids: Unassigned Reading Frame (URF). In order to check whether expression of this URF is required for proper splicing of precursors to 21S rRNA, the precision of RNA splicing was analysed in a petite mutant, where no mitochondrial protein synthesis is possible anymore. We have devised a new assay to monitor the precision of the splicing event. The method is of general application, provided that the sequence of the splice boundaries is known. In the case of the 21S rRNA it involves the synthesis of the DNA oligonucleotide d(CGATCCCTATTGTC( complementary to the 5' d(CGATCCCTAT) and 3' d(TGTC) borders flanking the intron in the 21S rRNA gene. The oligonucleotide is labelled with 32p at the 5'-end, hybridised to RNA and subsequently subjected to digestion with S1 nuclease. Resistance to digestion will only be observed if the correct splice-junction is made. The petite mutant we have studied contains a 21S rRNA with the same migration behaviour as wildtype 21S rRNA. In RNA blotting experiments, using an intron specific hybridisation probe, the same intermediates in splicing are found both in wild type and petite mutant. Finally the synthetic oligonucleotide hybridises to petite 21S rRNA and its thermal dissociation behaviour is indistinguishable from a hybrid formed with wildtype 21S rRNA. We conclude that expression of the URF, present in the intron of the 21S rRNA gene, is not required for processing and correct splicing of 21S ribosomal precursor RNA. Images PMID:7029466

  12. Detection of oligonucleotide hybridization on a single microparticle by time-resolved fluorometry: hybridization assays on polymer particles obtained by direct solid phase assembly of the oligonucleotide probes.

    PubMed

    Hakala, H; Heinonen, P; Iitiä, A; Lönnberg, H

    1997-01-01

    Oligodeoxyribonucleotides were assembled by conventional phosphoramidite chemistry on uniformly sized (50 microns) porous glycidyl methacrylate/ethylene dimethacrylate (SINTEF) and compact polystyrene (Dynosphere) particles, the aminoalkyl side chains of which were further derivatized with DMTrO-acetyl groups. The linker was completely resistant toward ammonolytic deprotection of the base moieties. The quality of oligonucleotides was assessed by repeating the synthesis on the same particles derivatized with a cleavable ester linker. The ability of the oligonucleotide-coated particles to bind complementary sequences via hybridization was examined by following the attachment of oligonucleotides bearing a photoluminescent europium(III) chelate to the particles. The fluorescence emission was measured directly on a single particle. The effects of the following factors on the kinetics and efficiency of hybridization were studied: number of particles in a given volume of the assay solution, loading of oligonucleotide on the particle, concentration of the target oligonucleotide in solution, length of the hybridizing sequence, presence of noncomplementary sequences, and ionic strength. The fluorescence signal measured on a single particle after hybridization was observed to be proportional to the concentration of the target oligonucleotide in solution over a concentration range of 5 orders of magnitude.

  13. Synthetic oligonucleotide probes deduced from amino acid sequence data. Theoretical and practical considerations.

    PubMed

    Lathe, R

    1985-05-05

    Synthetic probes deduced from amino acid sequence data are widely used to detect cognate coding sequences in libraries of cloned DNA segments. The redundancy of the genetic code dictates that a choice must be made between (1) a mixture of probes reflecting all codon combinations, and (2) a single longer "optimal" probe. The second strategy is examined in detail. The frequency of sequences matching a given probe by chance alone can be determined and also the frequency of sequences closely resembling the probe and contributing to the hybridization background. Gene banks cannot be treated as random associations of the four nucleotides, and probe sequences deduced from amino acid sequence data occur more often than predicted by chance alone. Probe lengths must be increased to confer the necessary specificity. Examination of hybrids formed between unique homologous probes and their cognate targets reveals that short stretches of perfect homology occurring by chance make a significant contribution to the hybridization background. Statistical methods for improving homology are examined, taking human coding sequences as an example, and considerations of codon utilization and dinucleotide frequencies yield an overall homology of greater than 82%. Recommendations for probe design and hybridization are presented, and the choice between using multiple probes reflecting all codon possibilities and a unique optimal probe is discussed.

  14. Empirical Establishment of Oligonucleotide Probe Design Criteria; Use of Microarrays with Different Probe Sizes for Monitoring Gene Expression; Temporal Transcriptomic Analysis as Desulfovibrio vulgaris Hildenborough Transitions into Stationary Phase during Electron Donor Depletion

    SciTech Connect

    He, Q.; He, Z.; Huang, K. H.; Alm, E. J.; Wan, X. F.; Hazen, T. C.; Arkin, A. P.; Wall, J. D.; Zhou, J. Z.; Fields, M. W.

    2005-07-15

    In order to experimentally establish the criteria for designing gene-specific and group-specific oligonucleotide probes, an oligonucleotide array was constructed that contained perfect match (PM) and mismatch (MM) probes (50mers and 70mers) based upon 4 genes. The effects of probe-target identity, continuous stretch, mismatch position, and hybridization free energy on specificity were examined. Little hybridization was observed at a probe-target identity of <85% for both 50mer and 70mer probes........Based on the experimental results, a set of criteria are suggested for the design of gene-specific and group-specific oligonucleotide probes, and these criteria should provide valuable information for the development of new software and algorithms for microarray-based studies.; Microarrays with oligonucleotides of different lengths were used to monitor gene expression at a wholegenome level. To determine what length of oligonucleotide is a better alternative to PCR-generated probes, the performance of oligonucleotide probes was systematically compared to that of their PCR-generated counterparts for 96 genes from Shewanella oneidensis MR-1 in terms of overall signal intensity, numbers of genes detected, specificity, sensitivity, and differential gene expression under experimental conditions. .......To evaluate differential gene expression under experimental conditions, S. oneidensis MR-1 cells were exposed to low- or high-pH conditions for 30 and 60 min, and the transcriptional profiles detected by oligonucleotide probes (50-mer, 60-mer, and 70-mer) were closely correlated with those detected by the PCR probes. The results demonstrated that 70-mer oligonucleotides can provide the performance most comparable to the performance obtained with PCR-generated probes.; Desulfovibrio vulgaris was cultivated in a defined medium, and biomass was sampled for approximately 70 h to characterize the shifts in gene expression as cells transitioned from the exponential to the

  15. Application of DNA fingerprinting with digoxigenated oligonucleotide probe (CAC)5 to analysis of the genetic variation within Taenia taeniaeformis.

    PubMed

    Okamoto, M; Ueda, H; Hayashi, M; Oku, Y; Kurosawa, T; Kamiya, M

    1995-04-01

    DNA from T. taeniaeformis digested with the restriction endonuclease was hybridized with digoxigenated oligonucleotide probe (CAC)5. Metacestode and adult showed same clear multibanding patterns, which were characteristic of multilocus DNA fingerprinting. The fingerprinting patterns were quite different from those of the rodent hosts. Genetic variations in 4 laboratory-reared isolates of T. taeniaeformis, including 3 isolates which have been reported to be indistinguishable by infectivity, morphology and protein composition of metacestode, were investigated using this technique. Each of the 4 isolates exhibited isolate-specific fingerprinting patterns and were easily distinguished from one another, thus it was considered that (CAC)5 was a highly resolvable and informative probe for cestodes. However, it was also indicated that (CAC)5 was so sensitive that applying fingerprinting with (CAC)5 to taxonomical or phylogenetic analysis was limited where habitat of the host was restricted to the small area. In comparison to fingerprinting with 32P-labeled (CAC)5, fingerprinting with digoxigenated (CAC)5 represented more and sharper bands. It was considered that a digoxigenated probe was more useful for genetic analysis of cestodes.

  16. Designation of Streptomycete 16S and 23S rRNA-based target regions for oligonucleotide probes.

    PubMed

    Stackebrandt, E; Witt, D; Kemmerling, C; Kroppenstedt, R; Liesack, W

    1991-05-01

    The 16S and 23S rRNA of various Streptomyces species were partially sequenced and screened for the presence of stretches that could define all members of the genus, groups of species, or individual species. Nucleotide 929 (Streptomyces ambofaciens nomenclature [J.L. Pernodet, M.T. Alegre, F. Boccard, and M. Guerineau, Gene 79:33-46, 1989]) is a nucleotide highly unique to Streptomyces species which, in combination with flanking regions, allowed the designation of a genus-specific probe. Regions 158 through 203 of the 16S rRNA and 1518 through 1645 of the 23S rRNA (helix 54 [Pernodet et al., Gene 79:33-46, 1989]) have a high potential to define species, whereas the degree of variation in regions 982 through 998 and 1102 through 1122 of the 16S rRNA is less pronounced but characteristic for at least certain species. Alone or in combination with each other, these regions may serve as target sites for synthetic oligonucleotide probes and primers to be used in the determination of pure cultures and in the characterization of community structures. The specificity of several probes is demonstrated by dot blot hybridization.

  17. A G-quadruplex based fluorescent oligonucleotide turn-on probe towards iodides detection in real samples.

    PubMed

    Li, Qian; Li, Shuaihua; Chen, Xiu; Bian, Liujiao

    2017-09-01

    A basket-type G-quadruplex (GQ) fluorescent oligonucleotide (OND) probe is designed to detect iodides dependent on thymine-Hg(II)-thymine (T-Hg(II)-T) base pairs and the intrinsic fluorescence quenching capacity of GQ. In the presence of Hg(II) ions (Hg(2+)), the two hexachloro-fluorescein-labeled ONDs form a hairpin structure and the fluorophores are dragged close to the GQ, leading to fluorescence quenching of the probe due to photoinduced electron transfer. Upon addition of iodide anions, Hg(2+) are extracted from T-Hg(II)-T complexes which attributes to the stronger binding with iodide anions, resulting in the fluorescence recovery. Through performing the fluorescence quenching and recovery processes, this probe developed a fluorescence turn-on sensor for iodide anions determination over a linear range of 20-200nmol/L with a limit of detection of 5nmol/L. The practical use of the turn-on technology was demonstrated by its application in determination of iodides in water, food, pharmaceutical products and biological samples. Copyright © 2017 Elsevier Ltd. All rights reserved.

  18. A novel single-labeled fluorescent oligonucleotide probe for silver(I) ion detection in water, drugs, and food.

    PubMed

    Bian, Liujiao; Ji, Xu; Hu, Wei

    2014-05-28

    Due to the high toxicity of silver(I) ions, a method for the rapid, sensitive, and selective detection for silver(I) ions in water, pharmaceutical products, and food is of great importance. Herein, a novel single-labeled fluorescent oligonucleotide (OND) probe based on cytosine-Ag(I)-cytosine coordination and the inherent fluorescence quenching ability of the G-quadruplex is designed to detect silver(I) ions. The formation of a hairpin structure in the OND-Ag(I) complex brings the hexachloro fluorescein (HEX) labeled at the 5'-end of the OND probe close to the G-quadruplex located at the 3'-end of the OND probe, leading to a fluorescence quenching due to photoinduced electron transfer between HEX and the G-quadruplex. Through this method, silver(I) ions can be detected quantitatively, the linear response range is from 1 to 100 nmol/L with a detection limit of 50 pmol/L, and no obvious interference occurs with other metal ions with a 10-fold concentration. This assay is simple, sensitive, and selective, and it can be used to detect silver(I) ions in actual water, drug, and food samples.

  19. Use of a multiplexed CMOS microarray to optimize and compare oligonucleotide binding to DNA probes synthesized or immobilized on individual electrodes.

    PubMed

    Maurer, Karl; Yazvenko, Nina; Wilmoth, Jodi; Cooper, John; Lyon, Wanda; Danley, David

    2010-01-01

    The CombiMatrix microarray with 12,544 electrodes supports in situ electrochemical synthesis of user-defined DNA probes. As an alternative, we immobilized commercially synthesized DNA probes on individual electrodes coated with electropolymerized polypyrrole (Ppy). Hybridization was measured using a biotinylated target oligonucleotide and either Cy5-streptavidin and fluorescence detection or horseradish peroxidase-streptavidin and enzyme-enhanced electrochemical detection. Detection efficiencies were optimized by varying the deposition of the Ppy, the terminal groups on the DNA probes, and other factors that impacted fluorescence quenching and electrical conductivity. Optimized results were compared against those obtained using a microarray with the same DNA sequences synthesized in situ. Immobilized probes produced higher fluorescence signals, possibly by providing a greater stand off between the Cy5 on the target oligonucleotide and the quenching effects of the Ppy and the platinum electrode.

  20. Use of a Multiplexed CMOS Microarray to Optimize and Compare Oligonucleotide Binding to DNA Probes Synthesized or Immobilized on Individual Electrodes

    PubMed Central

    Maurer, Karl; Yazvenko, Nina; Wilmoth, Jodi; Cooper, John; Lyon, Wanda; Danley, David

    2010-01-01

    The CombiMatrix microarray with 12,544 electrodes supports in situ electrochemical synthesis of user-defined DNA probes. As an alternative, we immobilized commercially synthesized DNA probes on individual electrodes coated with electropolymerized polypyrrole (Ppy). Hybridization was measured using a biotinylated target oligonucleotide and either Cy5-streptavidin and fluorescence detection or horseradish peroxidase-streptavidin and enzyme-enhanced electrochemical detection. Detection efficiencies were optimized by varying the deposition of the Ppy, the terminal groups on the DNA probes, and other factors that impacted fluorescence quenching and electrical conductivity. Optimized results were compared against those obtained using a microarray with the same DNA sequences synthesized in situ. Immobilized probes produced higher fluorescence signals, possibly by providing a greater stand off between the Cy5 on the target oligonucleotide and the quenching effects of the Ppy and the platinum electrode. PMID:22163607

  1. Use of fluorescent oligonucleotide probes for differentiation between Paracoccidioides brasiliensis and Paracoccidioides lutzii in yeast and mycelial phase

    PubMed Central

    Arantes, Thales Domingos; Theodoro, Raquel Cordeiro; Teixeira, Marcus de Melo; Bagagli, Eduardo

    2017-01-01

    BACKGROUND Fluorescence in situ hybridisation (FISH) associated with Tyramide Signal Amplification (TSA) using oligonucleotides labeled with non-radioactive fluorophores is a promising technique for detection and differentiation of fungal species in environmental or clinical samples, being suitable for microorganisms which are difficult or even impossible to culture. OBJECTIVE In this study, we aimed to standardise an in situ hybridisation technique for the differentiation between the pathogenic species Paracoccidioides brasiliensis and Paracoccidioides lutzii, by using species-specific DNA probes targeting the internal transcribed spacer-1 (ITS-1) of the rRNA gene. METHODS Yeast and mycelial phase of each Paracoccidioides species, were tested by two different detection/differentiation techniques: TSA-FISH for P. brasiliensis with HRP (Horseradish Peroxidase) linked to the probe 5’ end; and FISH for P. lutzii with the fluorophore TEXAS RED-X® also linked to the probe 5’ end. After testing different protocols, the optimised procedure for both techniques was accomplished without cross-positivity with other pathogenic fungi. FINDINGS The in silico and in vitro tests show no reaction with controls, like Candida and Cryptococcus (in silico) and Histoplasma capsulatum and Aspergillus spp. (in vitro). For both phases (mycelial and yeast) the in situ hybridisation showed dots of hybridisation, with no cross-reaction between them, with a lower signal for Texas Red probe than HRP-TSA probe. The dots of hybridisation was confirmed with genetic material marked with 4’,6-diamidino-2-phenylindole (DAPI), visualised in a different filter (WU) on fluorescent microscopic. MAIN CONCLUSION Our results indicated that TSA-FISH and/or FISH are suitable for in situ detection and differentiation of Paracoccidioides species. This approach has the potential for future application in clinical samples for the improvement of paracoccidioidomycosis patients prognosis. PMID:28177048

  2. Structurally responsive oligonucleotide-based single-probe lateral-flow test for detection of miRNA-21 mimics.

    PubMed

    Kor, Kamalodin; Turner, Anthony P F; Zarei, Kobra; Atabati, Morteza; Beni, Valerio; Mak, Wing Cheung

    2016-02-01

    A single-probe strip test for the rapid and sensitive detection of miRNA-21 mimics is reported herein. Highly specific structurally responsive bi-functional, thiol and biotin, DNA/LNA oligonucleotide probes (molecular beacons-MB) were designed and conjugated with gold nanoparticles (AuNPs) (i.e. biotin-MB-AuNPs). The proposed design had the ability to modulate the accessibility of the biotin group as a function of the presence of a miRNA target allowing the interaction of the boilable with the streptavidin test zone only in the presence of the miRNA-21 mimics. For quantitative evaluation, images of the strip tests were recorded using a flatbed scanner (Epson Perfection V370 Photo). The colour intensities of the test zones of the strip tests were analysed with the ImageJ software (Scion Corp., USA) and quantified as a function of pixel intensity. The response of the strip test was linear over the range 0.5 to 20 nM miRNA-21 (limit of detection of 115 pM) and showed good reproducibility (intra and inter CVs below 8%); furthermore, the assay was shown to be highly selective, discriminating other interference miRNAs mimics (e.g. miRNA-221 and miRNA-205). Finally, the proposed strip test was used for detection of miRNA-21 mimics in spiked serum samples, demonstrating its potential for point-of-care clinical applications. Main advantages of the single-probe strip test design are its versatility, simplicity and robustness, which can be easily extended to other miRNA targets by tuning the sequence of the single probe. Furthermore, the use of the structurally responsive single probe is particularly relevant in the case of short-length targets, such as miRNA, whereas a conventional sandwich approach might require a careful control of assay conditions such as hybridization temperature and salt concentration.

  3. mathFISH, a Web Tool That Uses Thermodynamics-Based Mathematical Models for In Silico Evaluation of Oligonucleotide Probes for Fluorescence In Situ Hybridization▿ †

    PubMed Central

    Yilmaz, L. Safak; Parnerkar, Shreyas; Noguera, Daniel R.

    2011-01-01

    Mathematical models of RNA-targeted fluorescence in situ hybridization (FISH) for perfectly matched and mismatched probe/target pairs are organized and automated in web-based mathFISH (http://mathfish.cee.wisc.edu). Offering the users up-to-date knowledge of hybridization thermodynamics within a theoretical framework, mathFISH is expected to maximize the probability of success during oligonucleotide probe design. PMID:21148691

  4. Dominant microbial composition and its vertical distribution in saline meromictic Lake Kaiike (Japan) as revealed by quantitative oligonucleotide probe membrane hybridization.

    PubMed

    Koizumi, Yoshikazu; Kojima, Hisaya; Fukui, Manabu

    2004-08-01

    Vertical distributions of dominant bacterial populations in saline meromictic Lake Kaiike were investigated throughout the water column and sediment by quantitative oligonucleotide probe membrane hybridization. Three oligonucleotide probes specific for the small-subunit (SSU) rRNA of three groups of Chlorobiaceae were newly designed. In addition, three general domain (Bacteria, Archaea, and Eukarya)-specific probes, two delta-Proteobacteria-specific probes, a Chlorobiaceae-specific probe, and a Chloroflexi-specific probe were used after optimization of their washing conditions. The abundance of the sum of SSU rRNAs hybridizing with probes specific for three groups of Chlorobiaceae relative to total SSU rRNA peaked in the chemocline, accounting for up to 68%. The abundance of the delta-proteobacterial SSU rRNA relative to total SSU rRNA rapidly increased just below the chemocline up to 29% in anoxic water and peaked at the 2- to 3-cm sediment depth at ca. 34%. The abundance of SSU rRNAs hybridizing with the probe specific for the phylum Chloroflexi relative to total SSU rRNA was highest (31 to 54%) in the top of the sediment but then steeply declined with depth and became stable at 11 to 19%, indicating the robust coexistence of sulfate-reducing bacteria and Chloroflexi in the top of the sediment. Any SSU rRNA of Chloroflexi in the water column was under the detection limit. The summation of the signals of group-specific probes used in this study accounted for up to 89% of total SSU rRNA, suggesting that the DGGE-oligonucleotide probe hybridization approach, in contrast to conventional culture-dependent approaches, was very effective in covering dominant populations.

  5. Enumeration of bacteria from the Clostridium leptum subgroup in human faecal microbiota using Clep1156 16S rRNA probe in combination with helper and competitor oligonucleotides.

    PubMed

    Saunier, Katiana; Rougé, Carole; Lay, Christophe; Rigottier-Gois, Lionel; Doré, Joël

    2005-07-01

    Target site inaccessibility represents a significant problem for fluorescent in situ hybridisation (FISH) of 16S rRNA oligonucleotide probes. For this reason, the Clep1156 probe targeting 16S rRNA of the Clostridium leptum phylogenetic subgroup used for dot blot experiments could not be used until now for FISH. Considering that bacteria from the C. leptum subgroup are very abundant in the human faecal microbiota and may play a significant role in host health, we have used unlabelled helper and competitor oligonucleotides to improve the 16S rRNA in situ accessibility and specificity of the Clep1156 probe and applied this approach to enumerate C. leptum bacteria in this ecosystem. Nine C. leptum target strains and five non-target strains were selected to develop and validate the helper-competitor strategy. Depending on the target strains, the use of helpers enhanced the fluorescence intensity signal of Clep1156 from 0.4-fold to 8.4-fold with a mean value of 3.6-fold, switching this probe from the brightness class V-VI (masked sites) to III-IV (accessible sites). The simultaneous use of helper and competitor oligonucleotides with Clep1156 probe allowed the expected specificity without disturbing in situ accessibility. Quantified by FISH combined with flow cytometry, C. leptum bacteria in human faecal samples (n=22) represented 19 +/- 7% of bacteria on average [4.9-37.5]. We conclude that helper oligonucleotides are very useful to circumvent the problem of target site in situ accessibility, especially when probe design is limited to only one 16S rRNA area and that helpers and competitors may be efficiently combined.

  6. Probing the microenvironments in the grooves of Z-DNA using dan-modified oligonucleotides.

    PubMed

    Kimura, Takumi; Kawai, Kiyohiko; Majima, Tetsuro

    2006-04-14

    The environment-sensitive fluorophore dan (6-dimethylamino-2-acyl-naphthalene)- modified dC or dG bases were introduced into the Z-DNA forming sequence. It was demonstrated that both grooves of Z-DNA are more hydrated than those of B-DNA. Dan will be useful for probing the microenvironments in the grooves among the DNA polymorphs.

  7. Electrochemical impedance probing of DNA hybridisation on oligonucleotide-functionalised polypyrrole.

    PubMed

    Tlili, Chaker; Korri-Youssoufi, Hafsa; Ponsonnet, Laurence; Martelet, Claude; Jaffrezic-Renault, Nicole J

    2005-11-15

    We report a new approach for detecting DNA hybridisation using non faradaic electrochemical impedance spectroscopy. The technique was applied to a system of DNA probes bearing amine groups that are immobilized by covalent grafting on a supporting polypyrrole matrix functionalised with activated ester groups. The kinetics of the attachment of the ss-DNA probe was monitored using the temporal evolution of the open circuit potential (OCP). This measurement allows the determination of the time necessary for the chemical reaction of ss-DNA probe into the polypyrrole backbone. The hybridisation reactions with the DNA complementary target and non complementary target were investigated by non faradaic electrochemical impedance spectroscopy. Results show a significant modification in the Nyquist plot upon addition of the complementary target whereas, in presence of the non complementary target, the Nyquist plot is not modified. The spectra, in the form of Nyquist plot, were analysed with the Randles circuit. The transfer charge resistance R(2) shows a linear variation versus the complementary target concentration. Sensitivity and detection limit (0.2nM) were determined and detection limit was lower of one order of magnitude than that obtained with the same system and measuring variation of the oxidation current at constant potential.

  8. A 27-mer oligonucleotide probe for the detection and measurement of the mRNA for uncoupling protein in brown adipose tissue of different species.

    PubMed

    Brander, F; Keith, J S; Trayhurn, P

    1993-01-01

    1. Data from rats, cattle, mice, rabbits and humans indicate considerable species heterogeneity in the sequence of the gene coding for the mitochondrial uncoupling protein (UCP) in brown adipose tissue. A 27-base sequence of an exon region of the gene is, however, identical in rats and cattle; in mice, rabbits, and humans this same region shows only a single base difference from the sequence in rats and cattle. 2. A 27-mer oligonucleotide (3'-TGGAAGGGCGACCTGTGGCGGTTTCAG-5') complementary to the conserved region of the rat and cattle UCP genes has been synthesized as a potential probe for UCP mRNA in widely differing species. 3. Northern blots of RNA from rat brown fat showed that the oligonucleotide hybridized with a 1.5 kbase mRNA, indicative of UCP mRNA. No hybridization was observed with RNA from white fat (subcutaneous, internal), liver, kidney, skeletal muscle, heart and brain. Acute cold-exposure of rats and mice led to an increase in UCP mRNA level, while streptozotocin-induced diabetes resulted in a decrease. 4. The oligonucleotide hybridized with a 1.5 (or 1.9) kbase mRNA from brown fat of rats, mice, golden hamsters, Djungarian hamsters, and newborn rabbits, pipistrelle bats, lambs, goats and red deer. 5. The 27-mer oligonucleotide provides a simple probe for UCP mRNA across a wide range of mammals, obviating any need to obtain species-specific cDNAs.

  9. Programmable oligonucleotide probes design and applications for in situ and in vivo RNA imaging in cells

    NASA Astrophysics Data System (ADS)

    Cheglakov, Zoya

    Unequal spreading of mRNA is a frequent experience observed in varied cell lines. The study of cellular processes dynamics and precise localization of mRNAs offers a vital toolbox to target specific proteins in precise cytoplasmic areas and provides a convenient instrument to uncover their mechanisms and functions. Latest methodological innovations have allowed imaging of a single mRNA molecule in situ and in vivo. Today, Fluorescent In Situ Hybridization (FISH) methods allow the studying of mRNA expression and offer a vital toolbox for accurate biological models. Studies enable analysis of the dynamics of an individual mRNA, have uncovered the multiplex RNA transport systems. With all current approaches, a single mRNA tracking in the mammalian cells is still challenging. This thesis describes mRNA detection methods based on programmable fluorophore-labeled DNA structures complimentary to native targets providing an accurate mRNA imaging in mammalian cells. First method represents beta-actin (ACTB) transcripts in situ detection in human cells, the technique strategy is based on programmable DNA probes, amplified by rolling circle amplification (RCA). The method reports precise localization of molecule of interest with an accuracy of a single-cell. Visualization and localization of specific endogenous mRNA molecules in real-time in vivo has the promising to innovate cellular biology studies, medical analysis and to provide a vital toolbox in drugs invention area. Second method described in this thesis represents miR-21 miRNA detection within a single live-cell resolution. The method using fluorophore-labeled short synthetic DNAs probes forming a stem-loop shape and generating Fluorescent Resonance Energy Transfer (FRET) as a result of target-probes hybridization. Catalytic nucleic acid (DNAzymes) probes are cooperative tool for precise detection of different mRNA targets. With assistance of a complementary fluorophore-quencher labeled substrate, the DNAzymes provide

  10. Platinated DNA oligonucleotides: new probes forming ultrastable conjugates with graphene oxide

    NASA Astrophysics Data System (ADS)

    Wang, Feng; Liu, Juewen

    2014-05-01

    Metal containing polymers have expanded the property of polymers by involving covalently associated metal complexes. DNA is a special block copolymer. While metal ions are known to influence DNA, little is explored on its polymer property when strong metal complexes are associated. In this work, we study cisplatin modified DNA as a new polymer and probe. Out of the complexes formed between cisplatin-A15, HAuCl4-A15, Hg2+-T15 and Ag+-C15, only the cisplatin adduct is stable under the denaturing gel electrophoresis condition. Each Pt-nucleobase bond gives a positive charge and thus makes DNA a zwitterionic polymer. This allows ultrafast adsorption of DNA by graphene oxide (GO) and the adsorbed complex is highly stable. Non-specific DNA, protein, surfactants and thiolated compounds cannot displace platinated DNA from GO, while non-modified DNA is easily displaced in most cases. The stable GO/DNA conjugate is further tested for surface hybridization. This is the first demonstration of using metallated DNA as a polymeric material for interfacing with nanoscale materials.Metal containing polymers have expanded the property of polymers by involving covalently associated metal complexes. DNA is a special block copolymer. While metal ions are known to influence DNA, little is explored on its polymer property when strong metal complexes are associated. In this work, we study cisplatin modified DNA as a new polymer and probe. Out of the complexes formed between cisplatin-A15, HAuCl4-A15, Hg2+-T15 and Ag+-C15, only the cisplatin adduct is stable under the denaturing gel electrophoresis condition. Each Pt-nucleobase bond gives a positive charge and thus makes DNA a zwitterionic polymer. This allows ultrafast adsorption of DNA by graphene oxide (GO) and the adsorbed complex is highly stable. Non-specific DNA, protein, surfactants and thiolated compounds cannot displace platinated DNA from GO, while non-modified DNA is easily displaced in most cases. The stable GO/DNA conjugate

  11. B- to Z-DNA transition probed by oligonucleotides containing methylphosphonates.

    PubMed Central

    Callahan, L; Han, F S; Watt, W; Duchamp, D; Kézdy, F J; Agarwal, K

    1986-01-01

    The simulation of the B--Z-DNA transition by using space-filling models of the dimer d(C-G) shows the possibility of hydrogen-bond formation between the N-2 amino group of the partially rotated guanine and one of the 5'-phosphate oxygens of deoxyguanylic acid. To probe the importance of this postulated interaction, analogs of the hexamer d(C-G)3 were synthesized. These analogs contained a methylphosphonate linkage, of distinct stereochemistry, which replaced the first 5'-phosphate linkage of deoxyguanosine. The CD spectra in high salt concentration showed that the hexamer containing a methylphosphonate linkage with the RP stereochemistry formed Z-DNA to the same extent as d(C-G)3, whereas the hexamer containing a methylphosphonate linkage with the SP stereochemistry did not form Z-DNA. These results are consistent with a mechanism in which an interaction between the N-2 amino group of guanine and the prochiral SP oxygen of deoxyguanosine 5'-phosphate kinetically controls the formation of Z-DNA. A water bridge between the N-2 amino group of guanine and the 3'-phosphate oxygen of deoxyguanylic acid has been implicated in the stabilization of Z-DNA. To probe the importance of this water bridge, two additional analogs of the hexamer d(C-G)3 were synthesized. These analogs contained a methylphosphonate linkage, of distinct stereochemistry, that replaced the first deoxyguanosine 3'-phosphate. The CD spectra showed that the hexamer containing a methylphosphonate linkage of the RP stereochemistry underwent the transition to Z-DNA to the same extent as d(C-G)3, whereas the hexamer containing a methylphosphonate linkage of the SP stereochemistry underwent the transition to Z-DNA to a 35% lesser extent. Thus the water bridge involving the prochiral SP oxygen provides modest stabilization energy for Z-DNA. These studies, therefore, suggest that the B--Z-DNA transition is regulated both thermodynamically and kinetically through hydrogen-bond interactions involving phosphate

  12. 18S rRNA Gene Variation among Common Airborne Fungi, and Development of Specific Oligonucleotide Probes for the Detection of Fungal Isolates

    PubMed Central

    Wu, Zhihong; Tsumura, Yoshihiko; Blomquist, Göran; Wang, Xiao-Ru

    2003-01-01

    In this study, we sequenced 18S rRNA genes (rDNA) from 49 fungal strains representing 31 species from 15 genera. Most of these species are common airborne fungi and pathogens that may cause various public health concerns. Sequence analysis revealed distinct divergence between Zygomycota and Ascomycota. Within Ascomycota, several strongly supported clades were identified that facilitate the taxonomic placement of several little-studied fungi. Wallemia appeared as the group most diverged from all the other Ascomycota species. Based on the 18S rDNA sequence variation, 108 oligonucleotide probes were designed for each genus and species included in this study. After homology searches and DNA hybridization evaluations, 33 probes were verified as genus or species specific. The optimal hybridization temperatures to achieve the best specificity for these 33 probes were determined. These new probes can contribute to the molecular diagnostic research for environmental monitoring. PMID:12957927

  13. Oligonucleotide probes for detection and differentiation of Staphylococcus aureus strains containing genes for enterotoxins A, B, and C and toxic shock syndrome toxin 1.

    PubMed Central

    Neill, R J; Fanning, G R; Delahoz, F; Wolff, R; Gemski, P

    1990-01-01

    Different synthetic DNA nucleotide sequences were evaluated as gene probes for the specific detection and differentiation of Staphylococcus aureus strains encoding enterotoxins A (SEA), B (SEB), and C (SEC) and toxic shock syndrome toxin 1 (TSST-1). Identification of sequences unique to each toxin, based on knowledge of their nucleotide sequences, led to preparation of the specific 18-base oligonucleotide probes EA1 (encoding amino acids 177 to 182 of SEA), EB2 (encoding amino acids 105 to 110 of SEB), EC5 (encoding amino acids 125 to 131 of SEC1), and TS1 (encoding amino acids 160 to 166 of TSST-1). In colony blot hybridization analyses, these probes hybridized specifically with DNA from strains that produced the respective toxin serotypes. An excellent (greater than or equal to 93%) correlation between hybridization results (genotype) and toxin protein detection by an enzyme-linked immunosorbent assay (phenotype) was observed in the characterization of both reference and clinical strains of S. aureus for SEA, SEB, and TSST-1. A lower correlation (64%) for SEC reflected a lack of sensitivity in detecting toxin production. Our findings demonstrate that molecular DNA hybridization with synthetic oligonucleotide probes provides another approach for establishing the toxigenicity of S. aureus. Images PMID:2380378

  14. Variations of Bacterial Populations in Human Feces Measured by Fluorescent In Situ Hybridization with Group-Specific 16S rRNA-Targeted Oligonucleotide Probes

    PubMed Central

    Franks, Alison H.; Harmsen, Hermie J. M.; Raangs, Gerwin C.; Jansen, Gijsbert J.; Schut, Frits; Welling, Gjalt W.

    1998-01-01

    Six 16S rRNA-targeted oligonucleotide probes were designed, validated, and used to quantify predominant groups of anaerobic bacteria in human fecal samples. A set of two probes was specific for species of the Bacteroides fragilis group and the species Bacteroides distasonis. Two others were designed to detect species of the Clostridium histolyticum and the Clostridium lituseburense groups. Another probe was designed for the genera Streptococcus and Lactococcus, and the final probe was designed for the species of the Clostridium coccoides-Eubacterium rectale group. The temperature of dissociation of each of the probes was determined. The specificities of the probes for a collection of target and reference organisms were tested by dot blot hybridization and fluorescent in situ hybridization (FISH). The new probes were used in initial FISH experiments to enumerate human fecal bacteria. The combination of the two Bacteroides-specific probes detected a mean of 5.4 × 1010 cells per g (dry weight) of feces; the Clostridium coccoides-Eubacterium rectale group-specific probe detected a mean of 7.2 × 1010 cells per g (dry weight) of feces. The Clostridium histolyticum, Clostridium lituseburense, and Streptococcus-Lactococcus group-specific probes detected only numbers of cells ranging from 1 × 107 to 7 × 108 per g (dry weight) of feces. Three of the newly designed probes and three additional probes were used in further FISH experiments to study the fecal flora composition of nine volunteers over a period of 8 months. The combination of probes was able to detect at least two-thirds of the fecal flora. The normal biological variations within the fecal populations of the volunteers were determined and indicated that these variations should be considered when evaluating the effects of agents modulating the flora. PMID:9726880

  15. Distribution of sulfate-reducing bacteria, O2, and H2S in photosynthetic biofilms determined by oligonucleotide probes and microelectrodes.

    PubMed Central

    Ramsing, N B; Kühl, M; Jørgensen, B B

    1993-01-01

    The vertical distribution of sulfate-reducing bacteria (SRB) in photosynthetic biofilms from the trickling filter of a sewage treatment plant was investigated with oligonucleotide probes binding to 16S rRNA. To demonstrate the effect of daylight and photosynthesis and thereby of increased oxygen penetration, we incubated two 4-mm-thick biofilm samples in darkness or exposed to light at natural intensity. Gradients of O2, H2S, and pH were examined with microelectrodes during incubation. The samples were subsequently frozen with liquid nitrogen and sliced on a cryomicrotome in 20-microns vertical slices. Fluorescent-dye-conjugated oligonucleotides were used as "phylogenetic" probes to identify single cells in the slices. Oligonucleotide sequences were selected which were complementary to short sequence elements (16 to 20 nucleotides) within the 16S rRNA of sulfate-reducing bacteria. The probes were labeled with fluorescein or rhodamine derivatives for subsequent visualization by epifluorescence microscopy. Five probes were synthesized for eukaryotes, eubacteria, SRB (including most species of the delta group of purple bacteria), Desulfobacter spp., and a nonhybridizing control. The SRB were unevenly distributed in the biofilm, being present in all states from single scattered cells to dense clusters of several thousand cells. To quantify the vertical distribution of SRB, we counted cells along vertical transects through the biofilm. This was done in a blind experiment to ascertain the reliability of the staining. A negative correlation between the vertical distribution of positively stained SRB cells and the measured O2 profiles was found. The distribution differed in light- and dark-incubated samples presumably because of the different extensions of the oxic surface layer. In both cases the SRB were largely restricted to anoxic layers. Images PMID:7506896

  16. An integrated approach for the design and synthesis of oligonucleotide probes and their interfacing to a QCM-based RNA biosensor.

    PubMed

    Tedeschi, Lorena; Citti, Lorenzo; Domenici, Claudio

    2005-05-15

    The quantitative determination of specific cellular messenger-RNA is extremely important both in basic and applied research, especially in diagnostic and pharmacological fields. In order to perform a direct and easy quantification of transcripts on cell extracts, the feasibility of an analytical device able to selectively detect a defined target RNA in a complex mixture while avoiding labelling, retrotranscription and amplification steps, has been explored. In particular, several aspects necessary to obtain good selectivity in target recognition, stability, reusability and sensitivity of a gene specific biosensor were considered. For the development of suitable probe-receptors, analysis of the nucleotide sequence of the target mRNA was carried out to localise the preferred binding regions. As criteria for optimisation, we selected accessibility and uniqueness. Oligonucleotide probes, designed to specifically bind these sequences, were synthesised by using particular monomers producing nuclease-resistant RNA strands with high affinity towards the target. Quartz crystal microbalance (QCM) technology was selected to realise a microgravimetric sensor able to bind the RNA under investigation through a complementary oligonucleotide probe. Covalent immobilisation of bioreceptor molecules to the transducer sensitive surface ensured a stable integration between the two. The binding ability of immobilised probes was tested evaluating their annealing behaviour with both complementary oligonucleotides and full-length target mRNA. The conditions necessary for the regeneration of biosensor were also assessed. Measurements of shift in QCM resonant frequency, performed by hybridisation experiments in liquido, demonstrate that a label-free RNA-biosensor with high specificity, reusability and the ability to give quantitative information, can be realised.

  17. Quantification of different Eubacterium spp. in human fecal samples with species-specific 16S rRNA-targeted oligonucleotide probes.

    PubMed

    Schwiertz, A; Le Blay, G; Blaut, M

    2000-01-01

    Species-specific 16S rRNA-targeted, Cy3 (indocarbocyanine)-labeled oligonucleotide probes were designed and validated to quantify different Eubacterium species in human fecal samples. Probes were directed at Eubacterium barkeri, E. biforme, E. contortum, E. cylindroides (two probes), E. dolichum, E. hadrum, E. lentum, E. limosum, E. moniliforme, and E. ventriosum. The specificity of the probes was tested with the type strains and a range of common intestinal bacteria. With one exception, none of the probes showed cross-hybridization under stringent conditions. The species-specific probes were applied to fecal samples obtained from 12 healthy volunteers. E. biforme, E. cylindroides, E. hadrum, E. lentum, and E. ventriosum could be determined. All other Eubacterium species for which probes had been designed were under the detection limit of 10(7) cells g (dry weight) of feces(-1). The cell counts obtained are essentially in accordance with the literature data, which are based on colony counts. This shows that whole-cell in situ hybridization with species-specific probes is a valuable tool for the enumeration of Eubacterium species in feces.

  18. Oligonucleotide probes containing pyrimidine analogs reveal diminished hydrogen bonding capacity of the DNA adduct O⁶-methyl-G in DNA duplexes.

    PubMed

    Angelov, Todor; Dahlmann, Heidi A; Sturla, Shana J

    2013-10-15

    Oligonucleotide hybridization probes containing nucleoside analogs offer a potential strategy for binding specific DNA sequences that bear pro-mutagenic O(6)-G alkylation adducts. To optimize O(6)-Me-G-targeting probes, an understanding of how base pairs with O(6)-Me-G are stabilized is needed. In this study, we compared the ability of O(6)-Me-G and G to hydrogen bond with three pyrimidine-like nucleobases (Z, 4-thio-U, and 3-deaza-C) bearing varied hydrogen bond donor and acceptor groups. We found that duplexes containing the pyrimidine analog nucleoside:G pairs were more thermodynamically stable than those containing pyrimidine analog nucleoside:O(6)-alkyl-G pairs. Thus, hydrogen bonding alone was not sufficient to impart selectivity to probes that target O(6)-G alkylation adducts in DNA.

  19. In Situ Localization of Azospirillum brasilense in the Rhizosphere of Wheat with Fluorescently Labeled, rRNA-Targeted Oligonucleotide Probes and Scanning Confocal Laser Microscopy

    PubMed Central

    Assmus, B.; Hutzler, P.; Kirchhof, G.; Amann, R.; Lawrence, J. R.; Hartmann, A.

    1995-01-01

    The colonization of wheat roots by Azospirillum brasilense was used as a model system to evaluate the utility of whole-cell hybridization with fluorescently labeled, rRNA-targeted oligonucleotide probes for the in situ monitoring of rhizosphere microbial communities. Root samples of agar- or soil-grown 10- and 30-day-old wheat seedlings inoculated with different strains of A. brasilense were hybridized with a species-specific probe for A. brasilense, a probe hybridizing to alpha subclass proteobacteria, and a probe specific for the domain Bacteria to identify and localize the target bacteria. After hybridization, about 10 to 25% of the rhizosphere bacteria as visualized with 4(prm1),6-diamidino-2-phenylindole (DAPI) gave sufficient fluorescence signals to be detected with rRNA-targeted probes. Scanning confocal laser microscopy was used to overcome disturbing effects arising from autofluorescence of the object or narrow depth of focus in thick specimens. This technique also allowed high-resolution analysis of the spatial distribution of bacteria in the rhizosphere. Occurrence of cells of A. brasilense Sp7 and Wa3 was restricted to the rhizosphere soil, mainly to the root hair zone. C-forms of A. brasilense were demonstrated to be physiologically active forms in the rhizosphere. Strain Sp245 also was found repeatedly at high density in the interior of root hair cells. In general, the combination of fluorescently labeled oligonucleotide probes and scanning confocal laser microscopy provided a very suitable strategy for detailed studies of rhizosphere microbial ecology. PMID:16534951

  20. Electrochemical detection of DNA binding by tumor suppressor p53 protein using osmium-labeled oligonucleotide probes and catalytic hydrogen evolution at the mercury electrode.

    PubMed

    Němcová, Kateřina; Sebest, Peter; Havran, Luděk; Orság, Petr; Fojta, Miroslav; Pivoňková, Hana

    2014-09-01

    In this paper, we present an electrochemical DNA-protein interaction assay based on a combination of protein-specific immunoprecipitation at magnetic beads (MBIP) with application of oligonucleotide (ON) probes labeled with an electroactive oxoosmium complex (Os,bipy). We show that double-stranded ONs bearing a dT20 tail labeled with Os,bipy are specifically recognized by the tumor suppressor p53 protein according to the presence or absence of a specific binding site (p53CON) in the double-stranded segment. We demonstrate the applicability of the Os,bipy-labeled probes in titration as well as competition MBIP assays to evaluate p53 relative affinity to various sequence-specific or structurally distinct unlabeled DNA substrates upon modulation of the p53-DNA binding by monoclonal antibodies used for the immunoprecipitation. To detect the p53-bound osmium-labeled probes, we took advantage of a catalytic peak yielded by Os,bipy-modified DNA at the mercury-based electrodes, allowing facile determination of subnanogram quantities of the labeled oligonucleotides. Versatility of the electrochemical MBIP technique and its general applicability in studies of any DNA-binding protein is discussed.

  1. Flow Cytometric Analysis of Characteristics of Hybridization of Species-Specific Fluorescent Oligonucleotide Probes to rRNA of Marine Nanoflagellates

    PubMed Central

    Rice, J.; Sleigh, M. A.; Burkill, P. H.; Tarran, G. A.; O'Connor, C. D.; Zubkov, M. V.

    1997-01-01

    Identification problems restrict quantitative ecological research on specific nanoflagellates. Identification by specific oligonucleotide probes permits use of flow cytometry for enumeration and measurement of size of nanoflagellates in statistically meaningful samples. Flow cytometry also permits measurement of intensity of probe binding by cells. Five fluorescent probes targeted to different regions of the small subunit rRNA of the common marine flagellate Paraphysomonas vestita all hybridized with cells of this flagellate. Cells fixed with trichloroacetic acid gave detectable signals at a probe concentration of 15 aM and specific fluorescence increased almost linearly to 1.5 fM, but at higher concentrations nonspecific binding increased sharply. Three flagellates, P. vestita, Paraphysomonas imperforata, and Pteridomonas danica, all bound a general eukaryotic probe approximately in proportion to their cell size, but the specific P. vestita probe gave 14 times more fluorescence with P. vestita than with either of the other flagellates. Cell fluorescence increased during the early growth of a batch culture and decreased toward the stationary phase; cell size changed in a comparable manner. Cell fluorescence intensity may allow inferences about growth rate, but whether fluorescence (assumed to reflect ribosome number) merely correlates with cell biomass or changes in a more complex manner remains unresolved. PMID:16535558

  2. Discrimination between Gyrodactylus salaris, G. derjavini and G. truttae (Platyhelminthes: Monogenea) using restriction fragment length polymorphisms and an oligonucleotide probe within the small subunit ribosomal RNA gene.

    PubMed

    Cunningham, C O; McGillivray, D M; MacKenzie, K; Melvin, W T

    1995-07-01

    The small subunit ribosomal RNA (srRNA) gene was amplified from Gyrodactylus salaris using the polymerase chain reaction (PCR), cloned, and the complete gene sequence of 1966 bp determined. The V4 region of the srRNA gene was identified and amplified from single specimens of G. salaris, G. derjavini and G. truttae. Comparison of the V4 sequences from these three species revealed sequence differences from which restriction fragment length polymorphisms (RFLPs) were predicted and an oligonucleotide probe (GsV4) specific to G. salaris designed. Digestion of the amplified V4 region of the srRNA gene with Hae III and either Alw I, BstY I, Dde I or Mbo I provided a means of discriminating between G. salaris, G. derjavini and G. truttae. The GsV4 probe was used to detect the srRNA gene from G. salaris in Southern and dot blots of the amplified V4 region.

  3. Large scale explorative oligonucleotide probe selection for thousands of genetic groups on a computing grid: application to phylogenetic probe design using a curated small subunit ribosomal RNA gene database.

    PubMed

    Jaziri, Faouzi; Peyretaillade, Eric; Missaoui, Mohieddine; Parisot, Nicolas; Cipière, Sébastien; Denonfoux, Jérémie; Mahul, Antoine; Peyret, Pierre; Hill, David R C

    2014-01-01

    Phylogenetic Oligonucleotide Arrays (POAs) were recently adapted for studying the huge microbial communities in a flexible and easy-to-use way. POA coupled with the use of explorative probes to detect the unknown part is now one of the most powerful approaches for a better understanding of microbial community functioning. However, the selection of probes remains a very difficult task. The rapid growth of environmental databases has led to an exponential increase of data to be managed for an efficient design. Consequently, the use of high performance computing facilities is mandatory. In this paper, we present an efficient parallelization method to select known and explorative oligonucleotide probes at large scale using computing grids. We implemented a software that generates and monitors thousands of jobs over the European Computing Grid Infrastructure (EGI). We also developed a new algorithm for the construction of a high-quality curated phylogenetic database to avoid erroneous design due to bad sequence affiliation. We present here the performance and statistics of our method on real biological datasets based on a phylogenetic prokaryotic database at the genus level and a complete design of about 20,000 probes for 2,069 genera of prokaryotes.

  4. Design and validation of an oligonucleotide probe for the detection of protozoa from the order Trichomonadida using chromogenic in situ hybridization.

    PubMed

    Mostegl, Meike Marissa; Richter, Barbara; Nedorost, Nora; Maderner, Anton; Dinhopl, Nora; Kulda, Jaroslav; Liebhart, Dieter; Hess, Michael; Weissenböck, Herbert

    2010-07-15

    Infections with protozoal parasites of the order Trichomonadida are often observed in veterinary medicine. Based on the trichomonad species involved these infections are either asymptomatic or can lead to sometimes serious disease. To further study protozoal agents of the order Trichomonadida the establishment of a method to detect trichomonads directly in the tissue, allowing parasite-lesion correlation, is necessary. Here we describe the design and evaluation of an oligonucleotide probe for chromogenic in situ hybridization, theoretically allowing detection of all hitherto known members of the order Trichomonadida. The probe was designed on a region of the 18S ribosomal RNA gene homologue for all representatives of the order Trichomonadida available in the GenBank. Functionality of the probe was proven using protozoal cultures containing different trichomonads (Monocercomonas colubrorum, Hypotrichomonas acosta, Pentatrichomonas hominis, Trichomitus batrachorum, Trichomonas gallinae, Tetratrichomonas gallinarum, Tritrichomonas foetus, and Tritrichomonas augusta). Furthermore, three different tissue sections containing either T. gallinae, T. foetus or Histomonas meleagridis were tested positive. Additionally, to rule out cross-reactivity of the probe a large number of different pathogenic protozoal agents, fungi, bacteria and viruses were tested and gave negative results. The probe presented here can be considered an important tool for diagnosis of all to date described relevant protozoal parasites of the order Trichomonadida in tissue samples.

  5. Superiorities of time-correlated single-photon counting against standard fluorimetry in exploiting the potential of fluorochromized oligonucleotide probes for biomedical investigation

    NASA Astrophysics Data System (ADS)

    Lamperti, Marco; Nardo, Luca; Bondani, Maria

    2015-05-01

    Site-specific fluorescence-resonance-energy-transfer donor-acceptor dual-labelled oligonucleotide probes are widely used in state-of-art biotechnological applications. Such applications include their usage as primers in polymerase chain reaction. However, the steady-state fluorescence intensity signal emitted by these molecular tools strongly depends from the specificities of the probe conformation. For this reason, the information which can be reliably inferred by steady-state fluorimetry performed on such samples is forcedly confined to a semi-qualitative level. Namely, fluorescent emission is frequently used as ON/OFF indicator of the probe hybridization state, i.e. detection of fluorescence signals indicates either hybridization to or detachment from the template DNA of the probe. Nonetheless, a fully quantitative analysis of their fluorescence emission properties would disclose other exciting applications of dual-labelled probes in biosensing. Here we show how time-correlated single-photon counting can be applied to get rid of the technical limitations and interpretational ambiguities plaguing the intensity analysis, and to derive information on the template DNA reaching single-base.

  6. Highly sensitive electrochemical sensor for mercury(II) ions by using a mercury-specific oligonucleotide probe and gold nanoparticle-based amplification.

    PubMed

    Zhu, Zhiqiang; Su, Yuanyuan; Li, Jiang; Li, Di; Zhang, Jiong; Song, Shiping; Zhao, Yun; Li, Genxi; Fan, Chunhai

    2009-09-15

    We report a highly sensitive electrochemical sensor for the detection of Hg(2+) ions in aqueous solution by using a thymine (T)-rich, mercury-specific oligonucleotide (MSO) probe and gold nanoparticles (Au NPs)-based signal amplification. The MSO probe contains seven thymine bases at both ends and a "mute" spacer in the middle, which, in the presence of Hg(2+), forms a hairpin structure via the Hg(2+)-mediated coordination of T-Hg(2+)-T base pairs. The thiolated MSO probe is immobilized on Au electrodes to capture free Hg(2+) in aqueous media, and the MSO-bound Hg(2+) can be electrochemically reduced to Hg(+), which provides a readout signal for quantitative detection of Hg(2+). This direct immobilization strategy leads to a detection limit of 1 microM. In order to improve the sensitivity, MSO probe-modified Au NPs are employed to amplify the electrochemical signals. Au NPs are comodified with the MSO probe and a linking probe that is complementary to a capture DNA probe immobilized on gold electrodes. We demonstrated that this Au NPs-based sensing strategy brings about an amplification factor of more than 3 orders of magnitude, leading to a limit of detection of 0.5 nM (100 ppt), which satisfactorily meets the sensitivity requirement of U.S. Environmental Protection Agency (EPA). This Au NPs-based Hg(2+) sensor also exhibits excellent selectivity over a spectrum of interference metal ions. Considering the high sensitivity and selectivity of this sensor, as well as the cost-effective and portable features of electrochemical techniques, we expect this Au NPs amplified electrochemical sensor will be a promising candidate for field detection of environmentally toxic mercury.

  7. Detection of harmful algal bloom causing microalgae using covalently immobilised capture oligonucleotide probes on glass and poly(dimethylsiloxane) surfaces

    NASA Astrophysics Data System (ADS)

    Bruce, Karen L.; Ellis, Amanda V.; Leterme, Sophie C.; Khodakov, Dmitriy A.; Lenehan, Claire E.

    2013-12-01

    Harmful algal bloom (HAB) events have been on the rise in the last few decades with some of the causative microalgae exhibiting toxic properties. Therefore, detection is essential in order to prevent mortality of aquatic life and poisoning events from consumption of these biotoxins. Here, oligonucleotide modified glass and poly(dimethylsiloxane) (PDMS) surfaces have been developed for the detection of the HAB causing microalgae, Alexandrium catenella, in a model system. Our preliminary studies show that the glass surface offers superior stability and analytical response when compared to those prepared from PDMS.

  8. Development and evaluation of two novel oligonucleotide probes based on 16S rRNA sequence for the identification of Salmonella in foods.

    PubMed

    Lin, C K; Tsen, H Y

    1995-05-01

    DNA sequence in the V3 to V6 region of the 16S rRNA gene of Salmonella enteritidis was determined. By comparison of this sequence with those of Escherichia coli and Proteus vulgaris obtained from GenBank/EMBL database, three oligonucleotides termed as 16S I, 16S II and 16S III were synthesized. Hybridization of these oligonucleotides with 325 Salmonella isolates and some non-Salmonella isolates including the Salmonella closely related species of the family of Enterobacteriaceae showed that 16S II could not be used as a Salmonella specific-probe. 16S I and 16S III hybridized with all the Salmonella isolates tested, the former also hybridizing with Citrobacter spp. and the latter hybridizing with Klebsiella pneumoniae as well as Serratia marcescens. Since enrichment of the target cells in food samples was usually required prior to the DNA hybridization assay, the interference from those non-Salmonella isolates could be prevented by enrichment by culturing in lactose-combined tetrathionate (CTET) broth followed by Gram-negative (GN) broth at 37 degrees C and/or 43 degrees C. Such a culture step could inhibit the growth of Klebsiella spp., Ser. marcescens and/or Citrobacter spp. and allowed the specific detection of Salmonella.

  9. Development of a Flow Cytometry-Based Method for Rapid Detection of Escherichia coli and Shigella Spp. Using an Oligonucleotide Probe

    PubMed Central

    Xue, Yong; Wilkes, Jon G.; Moskal, Ted J.; Williams, Anna J.; Cooper, Willie M.; Nayak, Rajesh; Rafii, Fatemeh; Buzatu, Dan A.

    2016-01-01

    Standard methods to detect Escherichia coli contamination in food use the polymerase chain reaction (PCR) and agar culture plates. These methods require multiple incubation steps and take a long time to results. An improved rapid flow-cytometry based detection method was developed, using a fluorescence-labeled oligonucleotide probe specifically binding a16S rRNA sequence. The method positively detected 51 E. coli isolates as well as 4 Shigella species. All 27 non-E. coli strains tested gave negative results. Comparison of the new genetic assay with a total plate count (TPC) assay and agar plate counting indicated similar sensitivity, agreement between cytometry cell and colony counts. This method can detect a small number of E.coli cells in the presence of large numbers of other bacteria. This method can be used for rapid, economical, and stable detection of E. coli and Shigella contamination in the food industry and other contexts. PMID:26913737

  10. Fluorescent Whole-Cell Hybridization with 16S rRNA-Targeted Oligonucleotide Probes To Identify Brucella spp. by Flow Cytometry

    PubMed Central

    Fernández-Lago, Luis; Vallejo, F. Javier; Trujillano, Ignacio; Vizcaíno, Nieves

    2000-01-01

    A whole-cell hybridization assay with fluorescent oligonucleotide probes derived from the 16S rRNA sequence of Brucella abortus in combination with flow cytometry has been developed. With the three fluorescent probes selected, a positive signal was observed with all the representative strains of the species and biovars of Brucella and with a total of nine different Brucella clinical isolates. Using the B9 probe in the hybridization assay, it was possible to discriminate between Brucella suis biovars 2, 3, 4, and 5 and almost all the other Brucella spp. On the basis of differences in fluorescence intensities, no discrimination was established between Brucella spp. and other phylogenetically related microorganisms. No positive fluorescence signals were detected with any of the bacteria showing serological cross-reactions with Brucella spp. and with a total of 17 clinical isolates not belonging to the genus Brucella. These results suggest that the 16S rRNA whole-cell hybridization technique could be a valuable diagnostic tool for the detection and identification of Brucella spp. PMID:10878084

  11. Phylogenetic group- and species-specific oligonucleotide probes for single-cell detection of lactic acid bacteria in oral biofilms

    PubMed Central

    2011-01-01

    Background The purpose of this study was to design and evaluate fluorescent in situ hybridization (FISH) probes for the single-cell detection and enumeration of lactic acid bacteria, in particular organisms belonging to the major phylogenetic groups and species of oral lactobacilli and to Abiotrophia/Granulicatella. Results As lactobacilli are known for notorious resistance to probe penetration, probe-specific assay protocols were experimentally developed to provide maximum cell wall permeability, probe accessibility, hybridization stringency, and fluorescence intensity. The new assays were then applied in a pilot study to three biofilm samples harvested from variably demineralized bovine enamel discs that had been carried in situ for 10 days by different volunteers. Best probe penetration and fluorescent labeling of reference strains were obtained after combined lysozyme and achromopeptidase treatment followed by exposure to lipase. Hybridization stringency had to be established strictly for each probe. Thereafter all probes showed the expected specificity with reference strains and labeled the anticipated morphotypes in dental plaques. Applied to in situ grown biofilms the set of probes detected only Lactobacillus fermentum and bacteria of the Lactobacillus casei group. The most cariogenic biofilm contained two orders of magnitude higher L. fermentum cell numbers than the other biofilms. Abiotrophia/Granulicatella and streptococci from the mitis group were found in all samples at high levels, whereas Streptococcus mutans was detected in only one sample in very low numbers. Conclusions Application of these new group- and species-specific FISH probes to oral biofilm-forming lactic acid bacteria will allow a clearer understanding of the supragingival biome, its spatial architecture and of structure-function relationships implicated during plaque homeostasis and caries development. The probes should prove of value far beyond the field of oral microbiology, as many of

  12. Detection and haplotype differentiation of Southeast Asian alpha-thalassemia using polymerase chain reaction and a piezoelectric biosensor immobilized with a single oligonucleotide probe.

    PubMed

    Vattanaviboon, Phantip; Sangseekhiow, Kulphassorn; Winichagoon, Pranee; Promptmas, Chamras

    2008-05-01

    DNA-based diagnosis of alpha-thalassemias routinely relies on polymerase chain reaction (PCR) and gel electrophoresis. Here, we developed a new procedure for the detection and haplotype differentiation of Southeast Asian (SEA) alpha-thalassemia using a 3-primer system for PCR coupling with a DNA-based piezoelectric biosensor. PCR products amplified from genomic DNA were differentiated directly by using a quartz crystal microbalance immobilized with a single oligonucleotide probe. The frequency changes after hybridization of the PCR products amplified from a representative sample of normal alpha-globin, SEA alpha-thalassemia heterozygote, and homozygote were 206+/-11, 256+/-5, and 307+/-3 Hz, respectively. The fabricated biosensor was evaluated through an examination of 18 blind specimens. It could accurately discriminate between normal and SEA alpha-thalassemic samples, which suggests that this biosensor system is a promising alternative technique to detect SEA alpha-thalassemia because of its specificity and less hazardous exposure as compared with conventional methods.

  13. Quantifying filamentous microorganisms in activated sludge before, during, and after an incident of foaming by oligonucleotide probe hybridizations and antibody staining.

    PubMed

    Oerther, D B; de los Reyes, F L; de los Reyes, M F; Raskin, L

    2001-10-01

    Quantitative oligonucleotide probe hybridizations, immunostaining, and a simple foaming potential test were used to follow an incident of seasonal filamentous foaming at the Urbana-Champaign Sanitary District, Northeast Wastewater Treatment Plant. A positive correlation was observed between an increase in foaming potential and the appearance of foam on the surfaces of aeration basins and secondary clarifiers. In addition, during the occurrence of foaming, the mass and activity of Gordonia spp. increased as measured by fluorescence in situ hybridization, antibody staining, and quantitative membrane hybridization of RNA extracts. An increase in Gordonia spp. rRNA levels from 0.25 to 1.4% of total rRNA was observed using quantitative membrane hybridizations, whereas during the same period, the fraction of mixed liquor volatile suspended solids attributed to Gordonia spp. increased from 4% to more than 32% of the total mixed liquor volatile suspended solids. These results indicate that both the activity and biomass level of Gordonia spp. in activated sludge increased relative to the activity aid the biomass level of the complete microbial community during a seasonal occurrence of filamentous foaming. Thus, Gordonia spp. may represent a numerically dominant but metabolically limited fraction of the total biomass, and the role of Gordonia spp. in filamentous foaming may be linked more tightly to the physical presence of filamentous microorganisms than to the metabolic activity of the cells.

  14. Synthesis and characterization of 8-methoxy-2'- deoxyadenosine-containing oligonucleotides to probe the syn glycosidic conformation of 2'-deoxyadenosine within DNA.

    PubMed Central

    Eason, R G; Burkhardt, D M; Phillips, S J; Smith, D P; David, S S

    1996-01-01

    The synthesis of 8-methoxy-2'-deoxyadenosine (moA) protected at N6 as an N,N-dimethylformamidine derivative and incorporation of the modified nucleoside into oligodeoxynucleotides via the phosphoramidite method are described. UV thermal denaturation studies were conducted on duplexes containing moA:G, moA:C and moA:T base pairs to determine the thermodynamic stability of duplexes containing moA relative to their adenosine (A)-containing counterparts. In the case of moA:G base pairs the effect of moA substitution is sequence dependent. In A:G mismatch-containing sequences, which have been shown by structural characterization to have a syn conformational preference at the glycosidic bond of A, moA substitution results in stabilization of the duplex. In contrast, in sequences where the A in the A:G mismatch has been shown to prefer the anti conformation moA substitution is destabilizing to the duplex. Thus moA may be a useful probe for investigating the conformational preferences of the N-glycosidic bond of adenosine within DNA. In addition, moA nucleoside is more resistant to acid-catalyzed depurination than previously described 8-bromo-2'-deoxyadenosine, allowing for facile incorporation into oligonucleotides via automated solid phase DNA synthesis. PMID:8600457

  15. High levels of Gardnerella vaginalis detected with an oligonucleotide probe combined with elevated pH as a diagnostic indicator of bacterial vaginosis.

    PubMed Central

    Sheiness, D; Dix, K; Watanabe, S; Hillier, S L

    1992-01-01

    We have demonstrated a new approach to diagnosing bacterial vaginosis (BV) that is based on measuring the concentration of Gardnerella vaginalis in vaginal fluid with DNA probes. G. vaginalis is virtually always present at high concentrations in women who have BV but is also detected frequently in normal women, usually at concentrations of less than 10(7) CFU/ml of vaginal fluid. Elevated vaginal pH is another sensitive indicator of BV, although it can occur in conjunction with other conditions. We have proposed that quantitative measurements of G. vaginalis using specific DNA probes can serve as a useful aid in diagnosing BV, provided the vaginal pH is above 4.5. To test this hypothesis, a group of 113 women were first evaluated for BV by the standard set of clinical signs. Vaginal washes were collected, and aliquots were analyzed by quantitative culture for the concentration of G. vaginalis. Portions of these same samples were immobilized on nylon filters, along with standards for quantitation. The filters were incubated with a radiolabelled oligonucleotide specific for G. vaginalis 16S rRNA, and the subsequent autoradiographs were examined to determine levels of G. vaginalis in each sample. G. vaginalis at concentrations of greater than or equal to 2 x 10(7) CFU/ml and vaginal pH of greater than 4.5 were then analyzed for concurrence with the diagnoses based on clinical criteria. Results of this slot blot analysis gave a sensitivity of 95%, correctly categorizing 41 of 43 BV-positive specimens, and a specificity of 99%, correctly identifying 69 of 70 BV-negative specimens, compared with diagnosis based on clinical criteria. Images PMID:1372621

  16. One-year monitoring of an oligonucleotide fluorescence in situ hybridization probe panel laboratory-developed test for bladder cancer detection

    PubMed Central

    Tinawi-Aljundi, Rima; King, Lauren; Knuth, Shannon T; Gildea, Michael; Ng, Carrie; Kahl, Josh; Dion, Jacqueline; Young, Chris; Schervish, Edward W; Frontera, J Rene; Hafron, Jason; Kernen, Kenneth M; Di Loreto, Robert; Aurich-Costa, Joan

    2015-01-01

    Background Previously, we had developed and manufactured an oligonucleotide fluorescence in situ hybridization (OligoFISH) probe panel based on the most clinically sensitive chromosomes found in a reference set of bladder carcinoma cases. The panel was clinically validated for use as a diagnostic and monitoring assay for bladder cancer, reaching 100% correlation with the results of the UroVysion test. After 1 year of using this probe panel, we present here the comparison of cytology, cystoscopy, and pathology findings to the OligoFISH probe panel results to calculate its clinical performance. Materials and methods In order to calculate clinical performance, we compared the OligoFISH results to the cytology and cystoscopy/pathology findings for 147 initial diagnoses and 399 recurrence monitorings. Finally, we compared clinical performance to published values for the UroVysion test, including both low- and high-grade tumors. Results Chromosomes 3, 6, 7, and 20 were highly involved in bladder carcinoma aneuploidy. At the initial diagnosis, we obtained 90.5% (95% confidence interval [CI]: 84.5%–94.7%) accuracy, 96.8% sensitivity (95% CI: 91.0%–99.3%), 79.2% specificity (95% CI: 65.9%–87.8%), 89.2% positive predictive value (PPV; 95% CI: 81.5%–94.5%), and 93.3% negative predictive value (NPV; 95% CI: 81.7%–97.3%). When monitoring for recurrence, we obtained 85.2% accuracy (95% CI: 81.3%–88.5%), 82.0% sensitivity (95% CI: 76.0%–87.1%), 88.4% specificity (95% CI: 83.2%–92.5%), 87.7% PPV (95% CI: 82.1%–92.0%), and 83.0% NPV (95% CI: 77.3%–87.8%). When looking at low- and high-grade tumors, the test showed 100% sensitivity for high-grade tumors (95% CI: 92.5%–100%) and 87.5% sensitivity (95% CI: 68.8%–95.5%) for low-grade tumors. All the clinical parameters for the OligoFISH panel were higher than the UroVysion test’s published performance. We found significantly higher clinical sensitivity and NPV at initial diagnosis and significantly higher

  17. Human leukocyte antigen typing using a knowledge base coupled with a high-throughput oligonucleotide probe array analysis.

    PubMed

    Zhang, Guang Lan; Keskin, Derin B; Lin, Hsin-Nan; Lin, Hong Huang; DeLuca, David S; Leppanen, Scott; Milford, Edgar L; Reinherz, Ellis L; Brusic, Vladimir

    2014-01-01

    Human leukocyte antigens (HLA) are important biomarkers because multiple diseases, drug toxicity, and vaccine responses reveal strong HLA associations. Current clinical HLA typing is an elimination process requiring serial testing. We present an alternative in situ synthesized DNA-based microarray method that contains hundreds of thousands of probes representing a complete overlapping set covering 1,610 clinically relevant HLA class I alleles accompanied by computational tools for assigning HLA type to 4-digit resolution. Our proof-of-concept experiment included 21 blood samples, 18 cell lines, and multiple controls. The method is accurate, robust, and amenable to automation. Typing errors were restricted to homozygous samples or those with very closely related alleles from the same locus, but readily resolved by targeted DNA sequencing validation of flagged samples. High-throughput HLA typing technologies that are effective, yet inexpensive, can be used to analyze the world's populations, benefiting both global public health and personalized health care.

  18. Radiolabeled oligonucleotides for antisense imaging

    PubMed Central

    Iyer, Arun K; He, Jiang

    2011-01-01

    Oligonucleotides radiolabeled with isotopes emitting γ-rays (for SPECT imaging) or positrons (for PET imaging) can be useful for targeting messenger RNA (mRNA) thereby serving as non-invasive imaging tools for detection of gene expression in vivo (antisense imaging). Radiolabeled oligonucleotides may also be used for monitoring their in vivo fate, thereby helping us better understand the barriers to its delivery for antisense targeting. These developments have led to a new area of molecular imaging and targeting, utilizing radiolabeled antisense oligonucleotides. However, the success of antisense imaging relies heavily on overcoming the barriers for its targeted delivery in vivo. Furthermore, the low ability of the radiolabeled antisense oligonucleotide to subsequently internalize into the cell and hybridize with its target mRNA poses additional challenges in realizing its potentials. This review covers the advances in the antisense imaging probe development for PET and SPECT, with an emphasis on radiolabeling strategies, stability, delivery and in vivo targeting. PMID:21822406

  19. PCR/oligonucleotide probe typing of HLA class II alleles in a Filipino population reveals an unusual distribution of HLA haplotypes.

    PubMed Central

    Bugawan, T. L.; Chang, J. D.; Klitz, W.; Erlich, H. A.

    1994-01-01

    We have analyzed the distribution of HLA class II alleles and haplotypes in a Filipino population by PCR amplification of the DRB1, DQB1, and DPB1 second-exon sequences from buccal swabs obtained from 124 family members and 53 unrelated individuals. The amplified DNA was typed by using nonradioactive sequence-specific oligonucleotide probes. Twenty-two different DRB1 alleles, including the novel Filipino *1105, and 46 different DRB1/DQB1 haplotypes, including the unusual DRB1*0405-DQB1*0503, were identified. An unusually high frequency (f = .383) of DPB1*0101, a rare allele in other Asian populations, was also observed. In addition, an unusual distribution of DRB1 alleles and haplotypes was seen in this population, with DR2 (f = .415) and DRB1*1502-DQB1*0502 (f = .233) present at high frequencies. This distribution of DRB1 alleles differs from the typical HLA population distribution, in which the allele frequencies are more evenly balanced. The distribution of HLA class II alleles and haplotypes in this Filipino population is different from that of other Asian and Pacific groups: of those populations studied to date; the Indonesian population is the most similar. DRB1*1502-DQB1*0502 was in strong linkage disequilibrium (D' = .41) with DPB1*0101 (f = .126, for the extended haplotype), which is consistent with selection for this DR, DQ, DP haplotype being responsible for the high frequency of these three class II alleles in this population. PMID:8304349

  20. Visualized detection of single-base difference in multiplexed loop-mediated isothermal amplification amplicons by invasive reaction coupled with oligonucleotide probe-modified gold nanoparticles.

    PubMed

    Lu, Yan; Ma, Xueping; Wang, Jianping; Sheng, Nan; Dong, Tianhui; Song, Qinxin; Rui, Jianzhong; Zou, Bingjie; Zhou, Guohua

    2017-04-15

    Loop-mediated isothermal amplification (LAMP) is a well-developed DNA amplification method with an ultra-high sensitivity, but it is difficult to recognize a single-base difference (like genotyping) in target-specific amplicons by conventional detection ways, such as the intercalation of dyes into dsDNA amplicons or the increase of solution turbidity along with the polymerization process. To allow genotyping based on LAMP suitable for POCT (point-of-care testing) or on-site testing, here we proposed a highly specific and cost-effective method for detecting a single-base difference in LAMP amplicons. The method includes three key steps, sequence amplifier to amplify multiple fragments containing the single nucleotide polymorphisms (SNPs) of interest, allele identifier to recognize a targeted base in the amplicons by invasive reaction, and signal generator to yield signals by hybridization-induced assembly of oligonucleotide probe-modified gold nanoparticles. Because the allele identifier is sensitive to one base difference, it is possible to use multiplexed LAMP (mLAMP) to generate amplicon mixtures for multiple SNP typing. Genotyping of 3 different SNPs (CYP2C19*2, CYP2C19*3 and MDR1-C3435T) for guiding the dosage of clopidogrel is successfully carried out in a 3-plex LAMP on real clinical samples. As our method relies on the naked-eye detection and constant-temperature reaction, no expensive instrument is required for both target amplification and sequence identification, thus much suitable for inexpensive gene-guided personalized medicine in source-limited regions.

  1. Design of species-specific oligonucleotide probes for the detection of Bacteroides and Parabacteroides by fluorescence in situ hybridization and their application to the analysis of mouse caecal Bacteroides-Parabacteroides microbiota.

    PubMed

    Momose, Y; Park, S H; Miyamoto, Y; Itoh, K

    2011-07-01

    To develop species-specific monitoring techniques for rapid detection of Bacteroides and Parabacteroides inhabiting the mouse intestine by fluorescence in situ hybridization. The specificity of oligonucleotide probes was evaluated by fluorescence whole-cell hybridization. Oligonucleotide probes specific for each species hybridized only with the target bacteria. Using these probes, caecal Bacteroides-Parabacteroides microbiota of conventional mice and specific pathogen-free (SPF) mice from three different breeders were analysed. It was shown that Bacteroides acidifaciens Group-1, Group-2 and Group-3 were dominant in conventional mice and SPF mice from two out of three breeders. Bacteroides vulgatus and Parabacteroides distasonis were detected in one of these two SPF breeding colonies in addition to Bact. acidifaciens. SPF mice of the remaining breeder harboured characteristic Bacteroides-Parabacteroides microbiota, consisting of Bacteroides sp. ASF519 and Bacteroides caccae. Bacteroides acidifaciens is the dominant and most typical species in the mouse Bacteroides-Parabacteroides microbiota. The Group-3 was identified as a novel group and revealed to occupy a major niche together with Bact. acidifaciens Group-1 and Group-2. The species-specific probe set developed in this study was the efficient tool for rapid detection of target bacterial groups inhabiting the mouse intestine. The results of this study provide important new information on the mouse Bacteroides-Parabacteroides community. © 2011 The Authors. Journal of Applied Microbiology © 2011 The Society for Applied Microbiology.

  2. A signal-on electrochemical DNA biosensor based on potential-assisted Cu(I)-catalyzed azide-alkyne cycloaddition mediated labeling of hairpin-like oligonucleotide with electroactive probe.

    PubMed

    Hu, Qiong; Kong, Jinming; Li, Yajie; Zhang, Xueji

    2016-01-15

    A novel electrochemical biosensor was developed for the signal-on detection of sequence-specific DNA by exploiting potential-assisted Cu(I)-catalyzed azide-alkyne cycloaddition (φCuAAC) as an efficient approach for the labeling of hairpin-like oligonucleotide (hairpin) with electroactive probe. The hairpins, dually labeled with thiol and azide at either terminal, were firstly self-assembled on gold electrode and served as the capture probes for the specific recognition of target DNA. Upon hybridization with target DNA, the surface-confined hairpins were unfolded, liberating the azide-containing terminals away from electrode surface. Subsequently, the unfolded hairpins were conveniently and efficiently labeled with ethynylferrocene (EFC) via the φCuAAC. The quantitatively labeled EFC was finally measured via differential pulse voltammetry (DPV) for the signal-on electrochemical detection of sequence-specific DNA. The biosensor presented a good linear response over the range from 1pM to 1nM with a detection limit of 0.62pM. Results also revealed that it was highly specific and held a good detection capability in serum samples. Furthermore, the ability to chemoselectively label hairpin-like oligonucleotide with signal reporter by electrical addressing, together with the simplicity and efficiency of the φCuAAC, makes it compatible with microfluidic devices and microelectrode arrays to achieve the miniaturized and multiplexed detections. Copyright © 2015 Elsevier B.V. All rights reserved.

  3. Pentopyranosyl Oligonucleotide Systems

    NASA Technical Reports Server (NTRS)

    Wagner, Thomas; Huyuh, Hoan K.; Krishnamurthy, Ramanarayanan; Eschenmoser, Albert

    2002-01-01

    Beta-D-Xylopyranosyl-(4 - 2 )-oligonucleotides containing adenine and thymine as nucleohases were synthesized as a part of a systematic study of the pairing properties of pentopyranosyl oligonucleotides. Contrary to earlier expectations based on qualitative conformational criteria, Beta-D-xylopyranosyl(4 - 2 )- oligonucleotides show Watson-Crick pairing comparable in strength to that shown by pyranosyl-RNA.

  4. Pentopyranosyl Oligonucleotide Systems

    NASA Technical Reports Server (NTRS)

    Wagner, Thomas; Huyuh, Hoan K.; Krishnamurthy, Ramanarayanan; Eschenmoser, Albert

    2002-01-01

    Beta-D-Xylopyranosyl-(4 - 2 )-oligonucleotides containing adenine and thymine as nucleohases were synthesized as a part of a systematic study of the pairing properties of pentopyranosyl oligonucleotides. Contrary to earlier expectations based on qualitative conformational criteria, Beta-D-xylopyranosyl(4 - 2 )- oligonucleotides show Watson-Crick pairing comparable in strength to that shown by pyranosyl-RNA.

  5. Drug targeting: synthesis and endocytosis of oligonucleotide-neoglycoprotein conjugates.

    PubMed Central

    Bonfils, E; Depierreux, C; Midoux, P; Thuong, N T; Monsigny, M; Roche, A C

    1992-01-01

    Inhibition of gene expression by antisense oligonucleotides is limited by their low ability to enter cells. Knowing that sugar binding receptors, also called membrane lectins, efficiently internalize neoglycoproteins bearing the relevant sugar, 6-phosphomannose, for instance, oligonucleotides--substituted on their 5'-end with either a fluorescent probe or a radioactive label on the one hand, and bearing a thiol function on their 3'-end, on the other hand,--were coupled onto 6-phosphomannosylated proteins via a disulfide bridge. The oligonucleotide bound to 6-phosphomannosylated serum albumin is much more efficiently internalized roughly 20 times than the free oligonucleotide. Although most of the oligonucleotides are associated with vesicular compartments, oligonucleotides after releasing from the carrier by reduction of the disulfide bridge may find their way to reach the cytosol and then lead to an increase in the efficiency of the oligonucleotides. Images PMID:1408764

  6. Evolution of the MIDTAL microarray: the adaption and testing of oligonucleotide 18S and 28S rDNA probes and evaluation of subsequent microarray generations with Prymnesium spp. cultures and field samples.

    PubMed

    McCoy, Gary R; Touzet, Nicolas; Fleming, Gerard T A; Raine, Robin

    2015-07-01

    The toxic microalgal species Prymnesium parvum and Prymnesium polylepis are responsible for numerous fish kills causing economic stress on the aquaculture industry and, through the consumption of contaminated shellfish, can potentially impact on human health. Monitoring of toxic phytoplankton is traditionally carried out by light microscopy. However, molecular methods of identification and quantification are becoming more common place. This study documents the optimisation of the novel Microarrays for the Detection of Toxic Algae (MIDTAL) microarray from its initial stages to the final commercial version now available from Microbia Environnement (France). Existing oligonucleotide probes used in whole-cell fluorescent in situ hybridisation (FISH) for Prymnesium species from higher group probes to species-level probes were adapted and tested on the first-generation microarray. The combination and interaction of numerous other probes specific for a whole range of phytoplankton taxa also spotted on the chip surface caused high cross reactivity, resulting in false-positive results on the microarray. The probe sequences were extended for the subsequent second-generation microarray, and further adaptations of the hybridisation protocol and incubation temperatures significantly reduced false-positive readings from the first to the second-generation chip, thereby increasing the specificity of the MIDTAL microarray. Additional refinement of the subsequent third-generation microarray protocols with the addition of a poly-T amino linker to the 5' end of each probe further enhanced the microarray performance but also highlighted the importance of optimising RNA labelling efficiency when testing with natural seawater samples from Killary Harbour, Ireland.

  7. Enhanced oligonucleotide-nanoparticle conjugate stability using thioctic acid modified oligonucleotides.

    PubMed

    Dougan, Jennifer A; Karlsson, Camilla; Smith, W Ewen; Graham, Duncan

    2007-01-01

    Metallic nanoparticles of gold functionalized with oligonucleotides conventionally use a terminal thiol modification and have been used in a wide range of applications. Although readily available, the oligonucleotide-nanoparticle conjugates prepared in this way suffer from a lack of stability when exposed to a variety of small molecules or elevated temperatures. If silver is used in place of gold then this lack of stability is even more pronounced. In this study we report the synthesis of highly stabilized oligonucleotide-nanoparticle conjugates using a simple oligonucleotide modification. A modified solid support was used to generate 3'-thioctic acid modified oligonucleotides by treatment with an N-hydroxysuccimidyl ester of thioctic acid. Unusually, both gold and silver nanoparticles have been investigated in this study and show that these disulphide-modified oligonucleotide probes offer significant improvements in nanoparticle stability when treated with dithiothreitol (DTT) compared with monothiol analogues. This is a significant advance in oligonucleotide-nanoparticle conjugate stability and for the first time allows silver nanoparticles to be prepared that are more stable than standard gold-thiol functionalized nanoparticles. This opens up the possibility of using silver nanoparticles functionalized with oligonucleotides as an alternative to gold.

  8. The use of 16S rRNA-targeted oligonucleotide probes to study competition between ruminal fibrolytic bacteria: pure-culture studies with cellulose and alkaline peroxide-treated wheat straw.

    PubMed

    Odenyo, A A; Mackie, R I; Stahl, D A; White, B A

    1994-10-01

    Specific oligonucleotide probes targeted to sites on the 16S rRNA of Ruminococcus albus 8, Ruminococcus flavefaciens FD-1, and Fibrobacter succinogenes S85 and a domain Bacteria probe were used to study bacterial interactions during the fermentation of cellulose and alkaline hydrogen peroxide-treated wheat straw in monocultures, dicultures, and tricultures. Results showed that R. albus 8 inhibited the growth of R. flavefaciens FD-1 when grown as a diculture with cellulose or alkaline hydrogen peroxide-treated wheat straw as the carbon source. In dicultures containing R. albus 8 and F. succinogenes S85 grown on cellulose or alkaline hydrogen peroxide-treated wheat straw, competition was not detected. R. flavefaciens FD-1 outcompeted F. succinogenes S85 when cellulose was used as the carbon source. In tricultures with cellulose as the carbon source, R. flavefaciens FD-1 was inhibited, R. albus 8 appeared to dominate during the early phase of degradation (12 to 48 h), while F. succinogenes S85 became predominant during the later phase of degradation (60 to 70 h). When alkaline hydrogen peroxide-treated wheat straw was used as a growth substrate, F. succinogenes S85 showed better growth than either R. albus 8 or R. flavefaciens FD-1. However, R. flavefaciens FD-1 was present in small numbers throughout the incubation period, unlike the growth patterns when cellulose was the carbon source.

  9. Linker immobilization of protein and oligonucleotide on indium-tin-oxide for detection of probe-target interactions by Kelvin physics.

    PubMed

    Saoud, Marwan; Blaszykowski, Christophe; Ballantyne, Scott M; Thompson, Michael

    2009-05-01

    This work describes a label-free microarray analysis technique capable of detecting biomolecule target interactions with probes being anchored with a new linker-diluent system on indium-tin-oxide. The method is based on the differential work function characteristics of the substrate measured by a scanning Kelvin nanoprobe in terms of contact potential difference signals.

  10. A nucleic acid probe labeled with desmethyl thiazole orange: a new type of hybridization-sensitive fluorescent oligonucleotide for live-cell RNA imaging.

    PubMed

    Okamoto, Akimitsu; Sugizaki, Kaori; Yuki, Mizue; Yanagisawa, Hiroyuki; Ikeda, Shuji; Sueoka, Takuma; Hayashi, Gosuke; Wang, Dan Ohtan

    2013-01-14

    A new fluorescent nucleotide with desmethyl thiazole orange dyes, D'(505), has been developed for expansion of the function of fluorescent probes for live-cell RNA imaging. The nucleoside unit of D'(505) for DNA autosynthesis was soluble in organic solvents, which made the preparation of nucleoside units and the reactions in the cycles of DNA synthesis more efficient. The dyes of D'(505)-containing oligodeoxynucleotide were protonated below pH 7 and the oligodeoxynucleotide exhibited hybridization-sensitive fluorescence emission through the control of excitonic interactions of the dyes of D'(505). The simplified procedure and effective hybridization-sensitive fluorescence emission produced multicolored hybridization-sensitive fluorescent probes, which were useful for live-cell RNA imaging. The acceptor-bleaching method gave us information on RNA in a specific cell among many living cells.

  11. Oligonucleotide Immobilization and Hybridization on Aldehyde-Functionalized Poly(2-hydroxyethyl methacrylate) Brushes.

    PubMed

    Bilgic, Tugba; Klok, Harm-Anton

    2015-11-09

    DNA biosensing requires high oligonucleotide binding capacity interface chemistries that can be tuned to maximize probe presentation as well as hybridization efficiency. This contribution investigates the feasibility of aldehyde-functionalized poly(2-hydroxyethyl methacrylate) (PHEMA) brush-based interfaces for oligonucleotide binding and hybridization. These polymer brushes, which allow covalent immobilization of oligonucleotides, are prepared by surface-initiated atom transfer radical polymerization (SI-ATRP) of HEMA followed by a postpolymerization oxidation step to generate side chain aldehyde groups. A series of polymer brushes covering a range of film thicknesses and grafting densities was investigated with regard to their oligonucleotide binding capacity as well as their ability to support oligonucleotide hybridization. Densely grafted brushes were found to have probe oligonucleotide binding capacities of up to ∼30 pmol/cm(2). Increasing the thickness of these densely grafted brush films, however, resulted in a decrease in the oligonucleotide binding capacity. Less densely grafted brushes possess binding capacities of ∼10 pmol/cm(2), which did not significantly depend on film thickness. The oligonucleotide hybridization efficiencies, however, were highest (93%) on those brushes that present the lowest surface concentration of the probe oligonucleotide. These results highlight the importance of optimizing the probe oligonucleotide surface concentration and binding interface chemistry. The versatility and tunability of the PHEMA-based brushes presented herein makes these films a very attractive platform for the immobilization and hybridization of oligonucleotides.

  12. Submolecular Structure and Orientation of Oligonucleotide Duplexes Tethered to Gold Electrodes Probed by Infrared Reflection Absorption Spectroscopy: Effect of the Electrode Potentials.

    PubMed

    Kékedy-Nagy, László; Ferapontova, Elena E; Brand, Izabella

    2017-02-23

    at a submolecular level. These pioneer results on the potential-driven changes in the submolecular structure of double stranded DNA adsorbed on conductive supports contribute to further understanding of the potential-driven sequence-specific electronic properties of surface-tethered oligonucleotides.

  13. In situ hybridisation for the detection of Leishmania species in paraffin wax-embedded canine tissues using a digoxigenin-labelled oligonucleotide probe.

    PubMed

    Dinhopl, N; Mostegl, M M; Richter, B; Nedorost, N; Maderner, A; Fragner, K; Weissenböck, H

    2011-11-12

    The diagnosis of canine leishmaniosis (CanL) is currently predominantly achieved by cytological or histological identification of amastigotes in biopsy samples, demonstration of specific anti-Leishmania antibodies and PCR-based approaches. All these methods have the advantage of being sensitive and more or less specific; nevertheless, most of them also have disadvantages. A chromogenic in situ hybridisation (ISH) procedure with a digoxigenin-labelled probe, targeting a fragment of the 5.8S rRNA was developed for the detection of all species of Leishmania parasites in routinely paraffin wax-embedded canine tissues. This method was validated in comparison with traditional techniques (histology, PCR), on various tissues from three dogs with histological changes consistent with a florid leishmaniosis. Amastigote forms of Leishmania gave clear signals and were easily identified using ISH. Various tissues from 10 additional dogs with clinical suspicion or/and a positive serological test but without histological presence of amastigotes did not show any ISH signals. Potential cross-reactivity of the probe was ruled out by negative outcome of the ISH against selected protozoa (including the related Trypanosoma cruzi) and fungi. Thus, ISH proved to be a powerful tool for unambiguous detection of Leishmania parasites in paraffin wax-embedded tissues.

  14. In situ hybridisation for the detection of Leishmania species in paraffin wax-embedded canine tissues using a digoxigenin-labelled oligonucleotide probe

    PubMed Central

    Dinhopl, N.; Mostegl, M. M.; Richter, B.; Nedorost, N.; Maderner, A.; Fragner, K.; Weissenböck, H.

    2011-01-01

    The diagnosis of canine leishmaniosis (CanL) is currently predominantly achieved by cytological or histological identification of amastigotes in biopsy samples, demonstration of specific anti-Leishmania antibodies and PCR-based approaches. All these methods have the advantage of being sensitive and more or less specific; nevertheless, most of them also have disadvantages. A chromogenic in situ hybridisation (ISH) procedure with a digoxigenin-labelled probe, targeting a fragment of the 5.8S rRNA was developed for the detection of all species of Leishmania parasites in routinely paraffin wax-embedded canine tissues. This method was validated in comparison with traditional techniques (histology, PCR), on various tissues from three dogs with histological changes consistent with a florid leishmaniosis. Amastigote forms of Leishmania gave clear signals and were easily identified using ISH. Various tissues from 10 additional dogs with clinical suspicion or/and a positive serological test but without histological presence of amastigotes did not show any ISH signals. Potential cross-reactivity of the probe was ruled out by negative outcome of the ISH against selected protozoa (including the related Trypanosoma cruzi) and fungi. Thus, ISH proved to be a powerful tool for unambiguous detection of Leishmania parasites in paraffin wax-embedded tissues. PMID:21921059

  15. BOND: Basic OligoNucleotide Design

    PubMed Central

    2013-01-01

    Background DNA microarrays have become ubiquitous in biological and medical research. The most difficult problem that needs to be solved is the design of DNA oligonucleotides that (i) are highly specific, that is, bind only to the intended target, (ii) cover the highest possible number of genes, that is, all genes that allow such unique regions, and (iii) are computed fast. None of the existing programs meet all these criteria. Results We introduce a new approach with our software program BOND (Basic OligoNucleotide Design). According to Kane’s criteria for oligo design, BOND computes highly specific DNA oligonucleotides, for all the genes that admit unique probes, while running orders of magnitude faster than the existing programs. The same approach enables us to introduce also an evaluation procedure that correctly measures the quality of the oligonucleotides. Extensive comparison is performed to prove our claims. BOND is flexible, easy to use, requires no additional software, and is freely available for non-commercial use from http://www.csd.uwo.ca/∼ilie/BOND/. Conclusions We provide an improved solution to the important problem of oligonucleotide design, including a thorough evaluation of oligo design programs. We hope BOND will become a useful tool for researchers in biological and medical sciences by making the microarray procedures faster and more accurate. PMID:23444904

  16. Array of Synthetic Oligonucleotides to Generate Unique Multi-Target Artificial Positive Controls and Molecular Probe-Based Discrimination of Liposcelis Species

    PubMed Central

    Arif, Mohammad; Opit, George; Mendoza-Yerbafría, Abigail; Dobhal, Shefali; Li, Zhihong; Kučerová, Zuzana; Ochoa-Corona, Francisco M.

    2015-01-01

    Several species of the genus Liposcelis are common insect pests that cause serious qualitative and quantitative losses to various stored grains and processed grain products. They also can contaminate foods, transmit pathogenic microorganisms and cause allergies in humans. The common occurrence of multi-species infestations and the fact that it is difficult to identify and discriminate Liposcelis spp. make accurate, rapid detection and discriminatory tools absolutely necessary for confirmation of their identity. In this study, PCR primers and probes specific to different Liposcelis spp. were designed based on nucleotide sequences of the cytochrome oxidase 1 (CO1) gene. Primer sets ObsCo13F/13R, PeaCo15F/14R, BosCO7F/7R, BruCo5F/5R, and DecCo11F/11R were used to specifically detect Liposcelis obscura Broadhead, Liposcelis pearmani Lienhard, Liposcelis bostrychophila Badonnel, Liposcelis brunnea Motschulsky and Liposcelis decolor (Pearman) in multiplex endpoint PCRs, which amplified products of 438-, 351-, 191-, 140-, and 87-bp, respectively. In multiplex TaqMan qPCR assays, orange, yellow, red, crimson and green channels corresponding to reporter dyes 6-ROXN, HEX, Cy5, Quasar705 and 6-FAM specifically detected L. obscura, L. brunnea, L. bostrychophila, L. pearmani and L. decolor, respectively. All developed primer and probe sets allowed specific amplification of corresponding targeted Liposcelis species. The development of multiplex endpoint PCR and multiplex TaqMan qPCR will greatly facilitate psocid identification and their management. The use of APCs will streamline and standardize PCR assays. APC will also provide the opportunity to have all positive controls in a single tube, which reduces maintenance cost and labor, but increases the accuracy and reliability of the assays. These novel methods from our study will have applications in pest management, biosecurity, quarantine, food safety, and routine diagnostics. PMID:26086728

  17. Pentopyranosyl Oligonucleotide Systems

    NASA Technical Reports Server (NTRS)

    Reck, Folkert; Kudick, Rene; Krishnamurthy, Ramanarayanan; Eschenmoser, Albert; Wippo, Harald

    2001-01-01

    To determine whether the remarkable chemical properties of the pyranosyl isomer of RNA as an informational Watson-Crick base-pairing system are unique to the pentopyranosyl-(4 + 2)-oligonucleotide isomer derived from the RNA-building block D-ribose, studies on the entire family of diastereoisomeric pyranosyL(4 - Z)-oligonucleotide systems deriving from D-ribose. L-lyxose. D-xylose, and L-arabinose were carried out. The result of these extended studies is unambiguous: not only pyranosyl-RNA, but all members of the pentopyranosyl(4 + 2)-oligonucleotide family are highly efficient Watson-Crick base-pairing systems. Their synthesis and pairing properties will be described in a series of publications in this journal.

  18. The delivery of therapeutic oligonucleotides

    PubMed Central

    Juliano, Rudolph L.

    2016-01-01

    The oligonucleotide therapeutics field has seen remarkable progress over the last few years with the approval of the first antisense drug and with promising developments in late stage clinical trials using siRNA or splice switching oligonucleotides. However, effective delivery of oligonucleotides to their intracellular sites of action remains a major issue. This review will describe the biological basis of oligonucleotide delivery including the nature of various tissue barriers and the mechanisms of cellular uptake and intracellular trafficking of oligonucleotides. It will then examine a variety of current approaches for enhancing the delivery of oligonucleotides. This includes molecular scale targeted ligand-oligonucleotide conjugates, lipid- and polymer-based nanoparticles, antibody conjugates and small molecules that improve oligonucleotide delivery. The merits and liabilities of these approaches will be discussed in the context of the underlying basic biology. PMID:27084936

  19. The delivery of therapeutic oligonucleotides.

    PubMed

    Juliano, Rudolph L

    2016-08-19

    The oligonucleotide therapeutics field has seen remarkable progress over the last few years with the approval of the first antisense drug and with promising developments in late stage clinical trials using siRNA or splice switching oligonucleotides. However, effective delivery of oligonucleotides to their intracellular sites of action remains a major issue. This review will describe the biological basis of oligonucleotide delivery including the nature of various tissue barriers and the mechanisms of cellular uptake and intracellular trafficking of oligonucleotides. It will then examine a variety of current approaches for enhancing the delivery of oligonucleotides. This includes molecular scale targeted ligand-oligonucleotide conjugates, lipid- and polymer-based nanoparticles, antibody conjugates and small molecules that improve oligonucleotide delivery. The merits and liabilities of these approaches will be discussed in the context of the underlying basic biology. © The Author 2016. Published by Oxford University Press on behalf of Nucleic Acids Research.

  20. Application of rRNA probes and fluorescence in situ hybridization for rapid detection of the toxic dinoflagellate Alexandrium minutum

    NASA Astrophysics Data System (ADS)

    Tang, Xianghai; Yu, Rencheng; Zhou, Mingjiang; Yu, Zhigang

    2012-03-01

    The dinoflagellate Alexandrium minutum is often associated with harmful algal blooms (HABs). This species consists of many strains that differ in their ability to produce toxins but have similar morphology, making identification difficult. In this study, species-specific rRNA probes were designed for whole-cell fluorescence in situ hybridization (FISH) to distinguish A. minutum from two phylogenetic clades. We acquired the complete SSU to LSU rDNA sequences (GenBank accession numbers JF906989-JF906999) of 11 Alexandrium strains and used these to design rRNA targeted oligonucleotide probes. Three ribotype-specific probes, M-GC-1, M-PC-2, and M-PC-3, were designed. The former is specific for the GC clade ("Global clade") of A. minutum, the majority of which have been found non-toxic, and the latter two are specific for the PSP (paralytic shellfish poisoning)-producing PC clade ("Pacific clade"). The specificity of these three probes was confirmed by FISH. All cells in observed fields of view were fluorescently labeled when probes and target species were incubated under optimized FISH conditions. However, the accessibility of rRNA molecules in ribosomes varied among the probe binding positions. Thus, there was variation in the distribution of positive signals in labeled cells within nucleolus and cytosol (M-GC-1, M-PC-3), or just nucleolus (M-PC-2). Our results provide a methodological basis for studying the biogeography and population dynamics of A. minutum, and providing an early warning of toxic HABs.

  1. A facile method for the construction of oligonucleotide microarrays.

    PubMed

    Sethi, Dalip; Kumar, A; Gupta, K C; Kumar, P

    2008-11-19

    In recent years, the oligonucleotide-based microarray technique has emerged as a powerful and promising tool for various molecular biological studies. Here, a facile protocol for the construction of an oligonucleotide microarray is demonstrated that involves immobilization of oligonucleotide-trimethoxysilyl conjugates onto virgin glass microslides. The projected immobilization strategy reflects high immobilization efficiency ( approximately 36-40%) and signal-to-noise ratio ( approximately 98), and hybridization efficiency ( approximately 32-35%). Using the proposed protocol, aminoalkyl, mercaptoalkyl, and phosphorylated oligonucleotides were immobilized onto virgin glass microslides. Briefly, modified oligonucleotides were reacted first with 3-glycidyloxypropyltriethoxysilane (GOPTS), and subsequently, the resultant conjugates were directly immobilized onto the virgin glass surface by making use of silanization chemistry. The constructed microarrays were then used for discrimination of base mismatches. On subjecting to different pH and thermal conditions, the microarray showed sufficient stability. Application of this chemistry to manufacture oligonucleotide probe-based microarrays for detection of bacterial meningitis is demonstrated. Single-step reaction for the formation of conjugates with the commercially available reagent (GOPTS), omission of capping step and surface modification, and efficient immobilization of oligonucleotides onto the virgin glass surface are the key features of the proposed strategy.

  2. Electrophoresis for genotyping: temporal thermal gradient gel electrophoresis for profiling of oligonucleotide dissociation.

    PubMed Central

    Day, I N; O'Dell, S D; Cash, I D; Humphries, S E; Weavind, G P

    1995-01-01

    Traditional use of an oligonucleotide probe to determine genotype depends on perfect base pairing to a single-stranded target which is stable to a higher temperature than when imperfect binding occurs due to a mismatch in the target sequence. Bound oligonucleotide is detected at a predetermined single temperature 'snapshot' of the melting profile, allowing the distinction of perfect from imperfect base pairing. In heterozygotes, the presence of the alternative sequence must be verified with a second oligonucleotide complementary to the variant. Here we describe a system of real-time variable temperature electrophoresis during which the oligonucleotide dissociates from its target. In 20% polyacrylamide the target strand has minimal mobility and released oligonucleotide migrates extremely quickly so that the 'freed' rather than the 'bound' is displayed. The full profile of oligonucleotide dissociation during gel electrophoresis is represented along the gel track, and a single oligonucleotide is sufficient to confirm heterozygosity, since the profile displays two separate peaks. Resolution is great, with use of short track lengths enabling analysis of dense arrays of samples. Each gel track can contain a different target or oligonucleotide and the temperature gradient can accommodate oligonucleotides of different melting temperatures. This provides a convenient system to examine the interaction of many different oligonucleotides and target sequences simultaneously and requires no prior knowledge of the mutant sequence(s) nor of oligonucleotide melting temperatures. The application of the technique is described for screening of a hotspot for mutations in the LDL receptor gene in patients with familial hypercholesterolaemia. Images PMID:7630718

  3. Scalable amplification of strand subsets from chip-synthesized oligonucleotide libraries

    NASA Astrophysics Data System (ADS)

    Schmidt, Thorsten L.; Beliveau, Brian J.; Uca, Yavuz O.; Theilmann, Mark; da Cruz, Felipe; Wu, Chao-Ting; Shih, William M.

    2015-11-01

    Synthetic oligonucleotides are the main cost factor for studies in DNA nanotechnology, genetics and synthetic biology, which all require thousands of these at high quality. Inexpensive chip-synthesized oligonucleotide libraries can contain hundreds of thousands of distinct sequences, however only at sub-femtomole quantities per strand. Here we present a selective oligonucleotide amplification method, based on three rounds of rolling-circle amplification, that produces nanomole amounts of single-stranded oligonucleotides per millilitre reaction. In a multistep one-pot procedure, subsets of hundreds or thousands of single-stranded DNAs with different lengths can selectively be amplified and purified together. These oligonucleotides are used to fold several DNA nanostructures and as primary fluorescence in situ hybridization probes. The amplification cost is lower than other reported methods (typically around US$ 20 per nanomole total oligonucleotides produced) and is dominated by the use of commercial enzymes.

  4. Scalable amplification of strand subsets from chip-synthesized oligonucleotide libraries

    PubMed Central

    Schmidt, Thorsten L.; Beliveau, Brian J.; Uca, Yavuz O.; Theilmann, Mark; Da Cruz, Felipe; Wu, Chao-Ting; Shih, William M.

    2015-01-01

    Synthetic oligonucleotides are the main cost factor for studies in DNA nanotechnology, genetics and synthetic biology, which all require thousands of these at high quality. Inexpensive chip-synthesized oligonucleotide libraries can contain hundreds of thousands of distinct sequences, however only at sub-femtomole quantities per strand. Here we present a selective oligonucleotide amplification method, based on three rounds of rolling-circle amplification, that produces nanomole amounts of single-stranded oligonucleotides per millilitre reaction. In a multistep one-pot procedure, subsets of hundreds or thousands of single-stranded DNAs with different lengths can selectively be amplified and purified together. These oligonucleotides are used to fold several DNA nanostructures and as primary fluorescence in situ hybridization probes. The amplification cost is lower than other reported methods (typically around US$ 20 per nanomole total oligonucleotides produced) and is dominated by the use of commercial enzymes. PMID:26567534

  5. Thermodynamic criteria for high hit rate antisense oligonucleotide design

    PubMed Central

    Matveeva, O. V.; Mathews, D. H.; Tsodikov, A. D.; Shabalina, S. A.; Gesteland, R. F.; Atkins, J. F.; Freier, S. M.

    2003-01-01

    Antisense oligonucleotides are used for therapeutic applications and in functional genomic studies. In practice, however, many of the oligonucleotides complementary to an mRNA have little or no antisense activity. Theoretical strategies to improve the ‘hit rate’ in antisense screens will reduce the cost of discovery and may lead to identification of antisense oligonucleotides with increased potency. Statistical analysis performed on data collected from more than 1000 experiments with phosphorothioate-modified oligonucleotides revealed that the oligo-probes, which form stable duplexes with RNA (ΔGo37 ≤ –30 kcal/mol) and have small self-interaction potential, are more frequently efficient than molecules that form less stable oligonucleotide–RNA hybrids or more stable self-structures. To achieve optimal statistical preference, the values for self-interaction should be (ΔGo37) ≥ –8 kcal/mol for inter-oligonucleotide pairing and (ΔGo37) ≥ –1.1 kcal/mol for intra-molecular pairing. Selection of oligonucleotides with these thermodynamic values in the analyzed experiments would have increased the ‘hit rate’ by as much as 6-fold. PMID:12930948

  6. Improving signal intensities for genes with low-expression on oligonucleotide microarrays

    PubMed Central

    Ramdas, Latha; Cogdell, David E; Jia, Jack Y; Taylor, Ellen E; Dunmire, Valerie R; Hu, Limei; Hamilton, Stanley R; Zhang, Wei

    2004-01-01

    Background DNA microarrays using long oligonucleotide probes are widely used to evaluate gene expression in biological samples. These oligonucleotides are pre-synthesized and sequence-optimized to represent specific genes with minimal cross-hybridization to homologous genes. Probe length and concentration are critical factors for signal sensitivity, particularly when genes with various expression levels are being tested. We evaluated the effects of oligonucleotide probe length and concentration on signal intensity measurements of the expression levels of genes in a target sample. Results Selected genes of various expression levels in a single cell line were hybridized to oligonucleotide arrays of four lengths and four concentrations of probes to determine how these critical parameters affected the intensity of the signal representing their expression. We found that oligonucleotides of longer length significantly increased the signals of genes with low-expression in the target. High-expressing gene signals were also boosted but to a lesser degree. Increasing the probe concentration, however, did not linearly increase the signal intensity for either low- or high-expressing genes. Conclusions We conclude that the longer the oligonuclotide probe the better the signal intensities of low expressing genes on oligonucleotide arrays. PMID:15196312

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

    PubMed Central

    Guschin, D Y; Mobarry, B K; Proudnikov, D; Stahl, D A; Rittmann, B E; Mirzabekov, A D

    1997-01-01

    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. PMID:9172361

  8. Finding a most likely clone ordering from oligonucleotide hybridization data

    SciTech Connect

    Newberg, L.A.

    1994-06-01

    Using an extension of a statistical model given by E. Lander and M. Waterman, the authors define the a posteriori probability of a clone ordering based upon oligonucleotide hybridization data. They give algorithms for computing the likelihood of a clone ordering and for finding a clone ordering of maximum likelihood. The dynamic programming algorithm for computing likelihoods runs in time O(mnc), where m is the number of oligonucleotide probes, n is the number of clones, and c is the coverage of the clone library. They use the Expectation-Maximization technique to maximize likelihoods. 21 refs., 3 figs.

  9. 'Specific' oligonucleotides often recognize more than one gene: the limits of in situ hybridization applied to GABA receptors.

    PubMed

    Mladinic, M; Didelon, F; Cherubini, E; Bradbury, A

    2000-05-15

    As exquisite probes for gene sequences, oligonucleotides are one of the most powerful tools of recombinant molecular biology. In studying the GABA receptor subunits in the neonatal hippocampus we have used oligonucleotide probes in in situ hybridization and cloning techniques. The oligonucleotides used and assumed to be specific for the target gene, actually recognized more than one gene, leading to surprising and contradictory results. In particular, we found that a GABA(A)-rho specific oligonucleotide recognized an abundant, previously unknown, transcription factor in both in situ and library screening, while oligos 'specific' for GABA(A) subunits were able to recognize 30 additional unrelated genes in library screening. This suggests that positive results obtained with oligonucleotides should be interpreted with caution unless confirmed by identical results with oligonucleotides from different parts of the same gene, or cDNA library screening excludes the presence of other hybridizing species.

  10. EvOligo: A Novel Software to Design and Group Libraries of Oligonucleotides Applicable for Nucleic Acid-Based Experiments.

    PubMed

    Milewski, Marek C; Kamel, Karol; Kurzynska-Kokorniak, Anna; Chmielewski, Marcin K; Figlerowicz, Marek

    2017-03-15

    Experimental methods based on DNA and RNA hybridization, such as multiplex polymerase chain reaction, multiplex ligation-dependent probe amplification, or microarray analysis, require the use of mixtures of multiple oligonucleotides (primers or probes) in a single test tube. To provide an optimal reaction environment, minimal self- and cross-hybridization must be achieved among these oligonucleotides. To address this problem, we developed EvOligo, which is a software package that provides the means to design and group DNA and RNA molecules with defined lengths. EvOligo combines two modules. The first module performs oligonucleotide design, and the second module performs oligonucleotide grouping. The software applies a nearest-neighbor model of nucleic acid interactions coupled with a parallel evolutionary algorithm to construct individual oligonucleotides, and to group the molecules that are characterized by the weakest possible cross-interactions. To provide optimal solutions, the evolutionary algorithm sorts oligonucleotides into sets, preserves preselected parts of the oligonucleotides, and shapes their remaining parts. In addition, the oligonucleotide sets can be designed and grouped based on their melting temperatures. For the user's convenience, EvOligo is provided with a user-friendly graphical interface. EvOligo was used to design individual oligonucleotides, oligonucleotide pairs, and groups of oligonucleotide pairs that are characterized by the following parameters: (1) weaker cross-interactions between the non-complementary oligonucleotides and (2) more uniform ranges of the oligonucleotide pair melting temperatures than other available software products. In addition, in contrast to other grouping algorithms, EvOligo offers time-efficient sorting of paired and unpaired oligonucleotides based on various parameters defined by the user.

  11. Amplification-Free Detection of Circulating microRNA Biomarkers from Body Fluids Based on Fluorogenic Oligonucleotide-Templated Reaction between Engineered Peptide Nucleic Acid Probes: Application to Prostate Cancer Diagnosis.

    PubMed

    Metcalf, Gavin A D; Shibakawa, Akifumi; Patel, Hinesh; Sita-Lumsden, Ailsa; Zivi, Andrea; Rama, Nona; Bevan, Charlotte L; Ladame, Sylvain

    2016-08-16

    Highly abundant in cells, microRNAs (or miRs) play a key role as regulators of gene expression. A proportion of them are also detectable in biofluids making them ideal noninvasive biomarkers for pathologies in which miR levels are aberrantly expressed, such as cancer. Peptide nucleic acids (PNAs) are engineered uncharged oligonucleotide analogues capable of hybridizing to complementary nucleic acids with high affinity and high specificity. Herein, novel PNA-based fluorogenic biosensors have been designed and synthesized that target miR biomarkers for prostate cancer (PCa). The sensing strategy is based on oligonucleotide-templated reactions where the only miR of interest serves as a matrix to catalyze an otherwise highly unfavorable fluorogenic reaction. Validated in vitro using synthetic RNAs, these newly developed biosensors were then shown to detect endogenous concentrations of miR in human blood samples without the need for any amplification step and with minimal sample processing. This low-cost, quantitative, and versatile sensing technology has been technically validated using gold-standard RT-qPCR. Compared to RT-qPCR however, this enzyme-free, isothermal blood test is amenable to incorporation into low-cost portable devices and could therefore be suitable for widespread public screening.

  12. Base Composition-Independent Hybridization in Tetramethylammonium Chloride: A Method for Oligonucleotide Screening of Highly Complex Gene Libraries

    NASA Astrophysics Data System (ADS)

    Wood, William I.; Gitschier, Jane; Lasky, Laurence A.; Lawn, Richard M.

    1985-03-01

    An oligonucleotide hybridization procedure has been developed that eliminates the preferential melting of A\\cdot T versus G\\cdot C base pairs, allowing the stringency of the hybridization to be controlled as a function of probe length only. This technique, which uses tetramethylammonium chloride, is especially helpful whenever a highly complex library is screened with a pool of oligonucleotide probes, which usually vary widely in base composition. The procedure can also be applied advantageously whenever an exact match to an oligonucleotide probe is desired, such as in screening for clones having as little as a single-base alteration generated by in vitro mutagenesis.

  13. SERS beacons for multiplexed oligonucleotide detection

    NASA Astrophysics Data System (ADS)

    Sun, Jian; Cullum, Brian M.

    2007-09-01

    Gold-based surface-enhanced Raman scattering (SERS) beacons have been developed, which represent a simple, biocompatible and rapid means of performing multiplexed DNA sequence detection in a non-arrayed format. These SERS beacons consist of a simple stem-loop oligonucleotide probe in its native form with one end attached to a SERS active dye molecule and the other to a gold nanoparticle, approximately 50 nm in diameter. The probe sequence is designed to achieve a stem-loop structure, with the loop portion complementary to the target sequence, similar to fluorescent molecular beacons. In the absence of the target DNA sequence, the SERS signal of the associated dye molecule is detected, representing the "ON" state of the probe. When the target sequence is hybridized to the probe, which results in an open conformation, its respective reporter dye is separated from the gold nanoparticle, producing diminished SERS signal. In this paper, the fabrication and characterization of these SERS beacons is described. We also demonstrate selective hybridization of a target sequence to one beacon in a mixture, revealing their potential for use in a multiplexed fashion.

  14. Optical detection and discrimination of cystic fibrosis-related genetic mutations using oligonucleotide-nanoparticle conjugates.

    PubMed

    Murphy, Deirdre; Redmond, Gareth

    2005-03-01

    Novel methods for application of oligonucleotide-gold nanoparticle conjugates to selective colorimetric detection and discrimination of cystic fibrosis (CF) related genetic mutations in model oligonucleotide systems are presented. Three-strand oligonucleotide complexes are employed, wherein two probe oligonucleotide-gold nanoparticle conjugates are linked together by a third target oligonucleotide strand bearing the CF-related mutation(s). By monitoring the temperature dependence of the optical properties of the complexes, either in solution or on silica gel plates, melting behaviors may be accurately and reproducibly compared. Using this approach, fully complementary sequences are successfully distinguished from mismatched sequences, with single base mismatch resolution, for Delta F 508, M470V, R74W and R75Q mutations.

  15. Probing the Influence of Stereoelectronic Effects on the Biophysical Properties of Oligonucleotides: Comprehensive Analysis of the RNA Affinity, Nuclease Resistance, and Crystal Structure of Ten 2'-O-Ribonucleic Acid Modifications

    SciTech Connect

    Egli, Martin; Minasov, George; Tereshko, Valentina; Pallan, Pradeep S.; Teplova, Marianna; Inamati, Gopal B.; Lesnik, Elena A.; Owens, Steve R.; Ross, Bruce S.; Prakash, Thazha P.; Manoharan, Muthiah

    2010-03-05

    The syntheses of 10 new RNA 2'-O-modifications, their incorporation into oligonucleotides, and an evaluation of their properties such as RNA affinity and nuclease resistance relevant to antisense activity are presented. All modifications combined with the natural phosphate backbone lead to significant gains in terms of the stability of hybridization to RNA relative to the first-generation DNA phosphorothioates (PS-DNA). The nuclease resistance afforded in particular by the 2'-O-modifications carrying a positive charge surpasses that of PS-DNA. However, small electronegative 2'-O-substituents, while enhancing the RNA affinity, do not sufficiently protect against degradation by nucleases. Similarly, oligonucleotides containing 3'-terminal residues modified with the relatively large 2'-O-[2-(benzyloxy)ethyl] substituent are rapidly degraded by exonucleases, proving wrong the assumption that steric bulk will generally improve protection against nuclease digestion. To analyze the factors that contribute to the enhanced RNA affinity and nuclease resistance we determined crystal structures of self-complementary A-form DNA decamer duplexes containing single 2'-O-modified thymidines per strand. Conformational preorganization of substituents, favorable electrostatic interactions between substituent and sugar-phosphate backbone, and a stable water structure in the vicinity of the 2'-O-modification all appear to contribute to the improved RNA affinity. Close association of positively charged substituents and phosphate groups was observed in the structures with modifications that protect most effectively against nucleases. The promising properties exhibited by some of the analyzed 2'-O-modifications may warrant a more detailed evaluation of their potential for in vivo antisense applications. Chemical modification of RNA can also be expected to significantly improve the efficacy of small interfering RNAs (siRNA). Therefore, the 2'-O-modifications introduced here may benefit the

  16. Time-resolved optical sensing of oligonucleotide hybridization via Au colloidal nanoparticles.

    PubMed

    Liu, Gang L; Rodriguez, Victoria B; Lee, Luke P

    2005-11-01

    Au nanoparticles have distinctive absorption spectra whose peak position or particle plasmon resonance wavelength is highly sensitive to molecule adsorption on their surfaces. Spherical Au nanoparticles are surface-modified by amino-functionalized self-assembly-monolayer and used as optical probes in the fluorescence-label-free spectroscopic detection of sub-nanomole oligonucleotides. Time-resolved studies of the immobilization and hybridization of oligonucleotides on the surface of Au nanoparticles were carried out. By measuring peak shift of absorption spectra of the Au colloidal nanoparticles over time, the samples of 15 nM 20 mer target and mismatched oligonucleotides are distinguished by their different influences on the particle plasmon resonance wavelength. The approach presented in this paper extends the application of Au nanoparticles as the optical probe in oligonucleotide recognitions without prior sample labeling.

  17. Electrochemical uranyl cation biosensor with DNA oligonucleotides as receptor layer.

    PubMed

    Jarczewska, Marta; Ziółkowski, Robert; Górski, Łukasz; Malinowska, Elżbieta

    2014-04-01

    The present study aims at the further development of the uranyl oligonucleotide-based voltammetric biosensor, which takes advantage of strong interaction between UO2(2+) and phosphate DNA backbone. Herein we report the optimization of working parameters of previously elaborated electrochemical DNA biosensor. It is shown that the sensor sensitivity is highly dependent on the oligonucleotide probe length and the incubation time of sensor in a sample solution. Consequently, the highest sensitivity was obtained for 10-nucleotide sequence and 60 min incubation time. The lower detection limit towards uranyl cation for developed biosensor was 30 nM. The influence of mixed monolayers and the possibility of developing a non-calibration device were also investigated. The selectivity of the proposed biosensor was significantly improved via elimination of adenine nucleobases from the DNA probe. Moreover, the regeneration procedure was elaborated and tested to prolong the use of the same biosensor for 4 subsequent determinations of UO2(2+).

  18. Oligonucleotide conjugates for therapeutic applications.

    PubMed

    Winkler, Johannes

    2013-07-01

    Insufficient pharmacokinetic properties and poor cellular uptake are the main hurdles for successful therapeutic development of oligonucleotide agents. The covalent attachment of various ligands designed to influence the biodistribution and cellular uptake or for targeting specific tissues is an attractive possibility to advance therapeutic applications and to expand development options. In contrast to advanced formulations, which often consist of multiple reagents and are sensitive to a variety of preparation conditions, oligonucleotide conjugates are defined molecules, enabling structure-based analytics and quality control techniques. This review gives an overview of current developments of oligonucleotide conjugates for therapeutic applications. Attached ligands comprise peptides, proteins, carbohydrates, aptamers and small molecules, including cholesterol, tocopherol and folic acid. Important linkage types and conjugation methods are summarized. The distinct ligands directly influence biochemical parameters, uptake mechanisms and pharmacokinetic properties.

  19. Oligonucleotide conjugates for therapeutic applications

    PubMed Central

    Winkler, Johannes

    2013-01-01

    Insufficient pharmacokinetic properties and poor cellular uptake are the main hurdles for successful therapeutic development of oligonucleotide agents. The covalent attachment of various ligands designed to influence the biodistribution and cellular uptake or for targeting specific tissues is an attractive possibility to advance therapeutic applications and to expand development options. In contrast to advanced formulations, which often consist of multiple reagents and are sensitive to a variety of preparation conditions, oligonucleotide conjugates are defined molecules, enabling structure-based analytics and quality control techniques. This review gives an overview of current developments of oligonucleotide conjugates for therapeutic applications. Attached ligands comprise peptides, proteins, carbohydrates, aptamers and small molecules, including cholesterol, tocopherol and folic acid. Important linkage types and conjugation methods are summarized. The distinct ligands directly influence biochemical parameters, uptake machanisms and pharmacokinetic properties. PMID:23883124

  20. Advanced surface-enhanced Raman gene probe systems and methods thereof

    DOEpatents

    Vo-Dinh, Tuan

    2001-01-01

    The subject invention is a series of methods and systems for using the Surface-Enhanced Raman (SER)-labeled Gene Probe for hybridization, detection and identification of SER-labeled hybridized target oligonucleotide material comprising the steps of immobilizing SER-labeled hybridized target oligonucleotide material on a support means, wherein the SER-labeled hybridized target oligonucleotide material comprise a SER label attached either to a target oligonucleotide of unknown sequence or to a gene probe of known sequence complementary to the target oligonucleotide sequence, the SER label is unique for the target oligonucleotide strands of a particular sequence wherein the SER-labeled oligonucleotide is hybridized to its complementary oligonucleotide strand, then the support means having the SER-labeled hybridized target oligonucleotide material adsorbed thereon is SERS activated with a SERS activating means, then the support means is analyzed.

  1. Thermodynamics of Oligonucleotide Duplex Melting

    ERIC Educational Resources Information Center

    Schreiber-Gosche, Sherrie; Edwards, Robert A.

    2009-01-01

    Melting temperatures of oligonucleotides are useful for a number of molecular biology applications, such as the polymerase chain reaction (PCR). Although melting temperatures are often calculated with simplistic empirical equations, application of thermodynamics provides more accurate melting temperatures and an opportunity for students to apply…

  2. Thermodynamics of Oligonucleotide Duplex Melting

    NASA Astrophysics Data System (ADS)

    Schreiber-Gosche, Sherrie; Edwards, Robert A.

    2009-05-01

    Melting temperatures of oligonucleotides are useful for a number of molecular biology applications, such as the polymerase chain reaction (PCR). Although melting temperatures are often calculated with simplistic empirical equations, application of thermodynamics provides more accurate melting temperatures and an opportunity for students to apply rigorous thermodynamic analysis to an important biochemical problem. Because the stacking of base pairs on top of one another is a significant factor in the energetics of oligonucleotide melting, several investigators have applied van't Hoff analysis to melting temperature data using a nearest-neighbor model and have obtained entropies and enthalpies for the stacking of bases. The present article explains how the equilibrium constant for the dissociation of strands from double-stranded oligonucleotides can be expressed in terms of the total strand concentration and thus how the total strand concentration influences the melting temperature. It also presents a simplified analysis based on the entropies and enthalpies of stacking that is manually tractable so that students can work examples to help them understand the thermodynamics of oligonucleotide melting.

  3. Molecular Mechanisms of Antisense Oligonucleotides.

    PubMed

    Crooke, Stanley T

    2017-04-01

    In 1987, when I became interested in the notion of antisense technology, I returned to my roots in RNA biochemistry and began work to understand how oligonucleotides behave in biological systems. Since 1989, my research has focused primarily on this topic, although I have been involved in most areas of research in antisense technology. I believe that the art of excellent science is to frame large important questions that are perhaps not immediately answerable with existing knowledge and methods, and then conceive a long-term (multiyear) research strategy that begins by answering the most pressing answerable questions on the path to the long-term goals. Then, a step-by-step research pathway that will address the strategic questions posed must be implemented, adjusting the plan as new things are learned. This is the approach we have taken at Ionis. Obviously, to create antisense technology, we have had to address a wide array of strategic questions, for example, the medicinal chemistry of oligonucleotides, manufacturing and analytical methods, pharmacokinetics and toxicology, as well as questions about the molecular pharmacology of antisense oligonucleotides (ASOs). Each of these endeavors has consumed nearly three decades of scientific effort, is still very much a work-in-progress, and has resulted in hundreds of publications. As a recipient of the Lifetime Achievement Award 2016 granted by the Oligonucleotide Therapeutic Society, in this note, my goal is to summarize the contributions of my group to the efforts to understand the molecular mechanisms of ASOs.

  4. Molecular Mechanisms of Antisense Oligonucleotides

    PubMed Central

    2017-01-01

    In 1987, when I became interested in the notion of antisense technology, I returned to my roots in RNA biochemistry and began work to understand how oligonucleotides behave in biological systems. Since 1989, my research has focused primarily on this topic, although I have been involved in most areas of research in antisense technology. I believe that the art of excellent science is to frame large important questions that are perhaps not immediately answerable with existing knowledge and methods, and then conceive a long-term (multiyear) research strategy that begins by answering the most pressing answerable questions on the path to the long-term goals. Then, a step-by-step research pathway that will address the strategic questions posed must be implemented, adjusting the plan as new things are learned. This is the approach we have taken at Ionis. Obviously, to create antisense technology, we have had to address a wide array of strategic questions, for example, the medicinal chemistry of oligonucleotides, manufacturing and analytical methods, pharmacokinetics and toxicology, as well as questions about the molecular pharmacology of antisense oligonucleotides (ASOs). Each of these endeavors has consumed nearly three decades of scientific effort, is still very much a work-in-progress, and has resulted in hundreds of publications. As a recipient of the Lifetime Achievement Award 2016 granted by the Oligonucleotide Therapeutic Society, in this note, my goal is to summarize the contributions of my group to the efforts to understand the molecular mechanisms of ASOs. PMID:28080221

  5. Thermodynamics of Oligonucleotide Duplex Melting

    ERIC Educational Resources Information Center

    Schreiber-Gosche, Sherrie; Edwards, Robert A.

    2009-01-01

    Melting temperatures of oligonucleotides are useful for a number of molecular biology applications, such as the polymerase chain reaction (PCR). Although melting temperatures are often calculated with simplistic empirical equations, application of thermodynamics provides more accurate melting temperatures and an opportunity for students to apply…

  6. Application of oligonucleotide array technology for the rapid detection of pathogenic bacteria of foodborne infections.

    PubMed

    Hong, Bang-Xing; Jiang, Li-Fang; Hu, Yu-Shan; Fang, Dan-Yun; Guo, Hui-Yu

    2004-09-01

    A rapid and accurate method for detection for common pathogenic bacteria in foodborne infections was established by using oligonucleotide array technology. Nylon membrane was used as the array support. A mutation region of the 23S rRNA gene was selected as the discrimination target from 14 species (genera) of bacteria causing foodborne infections and two unrelated bacterial species. A pair of universal primers was designed for PCR amplification of the 23S rRNA gene. Twenty-one species (genera)-specific oligonucleotide detection probes were synthesized and spotted onto the nylon membranes. The 23S rRNA gene amplification products of 14 species of pathogenic bacteria were hybridized to the oligonucleotide array. Hybridization results were analyzed with digoxigenin-linked enzyme reaction. Results indicated that nine species of pathogenic bacteria (Escherichia coli, Campylobacter jejuni, Shigella dysenteriae, Vibrio cholerae, Vibrio parahaemolyticus, Proteus vulgaris, Bacillus cereus, Listeria monocytogenes and Clostridium botulinum) showed high sensitivity and specificity for the oligonucleotide array. Two other species (Salmonella enterica and Yersinia enterocolitica) gave weak cross-reaction with E. coli, but the reaction did not affect their detection. After redesigning the probes, positive hybridization results were obtained with Staphylococcus aureus, but not with Clostridium perfringens and Streptococcus pyogenes. The oligonucleotide array can also be applied to samples collected in clinical settings of foodborne infections. The superiority of oligonucleotide array over other tests lies on its rapidity, accuracy and efficiency in the diagnosis, treatment and control of foodborne infections.

  7. In situ synthesis of oligonucleotide arrays by using soft lithography

    NASA Astrophysics Data System (ADS)

    Xiao, P. F.; He, N. Y.; Liu, Z. C.; He, Q. G.; Sun, X.; Lu, Z. H.

    2002-12-01

    This paper describes the in situ synthesis of oligonucleotide arrays on glass surfaces by using soft lithography. In this method, based on the standard phosphoramidite chemistry protocol, the coupling was achieved by the glass slide being printed with a set of polydimethylsiloxane (PDMS) microstamps, on which was spread nucleoside monomer and tetrazole mixed solution. The elastic characteristic of PDMS allowed it to make conformal contact with the glass slide in the printing coupling. With regard to the efficiency of the printing coupling, the hybridization microscope images of 20-mer oligonucleotide synthesized via the directly drip-dropped coupling and the contact coupling were compared; the fluorescence intensities of the two methods showed no significant differences. The coupling efficiency was also investigated via an end-labelled fluorescence nucleotide method and a stepwise yield of 97% was obtained. A high-quality, high spatial resolution and large-scale PDMS stamp, which was developed by integrating 168 different microstamps on one glass substrate for synthesizing oligonucleotide arrays. The stamp was modified to improve the surface wettability by plasma discharge treatments, so that microstamps could be used to fabricate oligonucleotide arrays. A motional printing head was developed to improve the contact effect of the glass slide with different microstamps. A higher boiling point solvent was used in the printing coupling to inhibit solvent volatilization and to maintain the consistency of reagents on different features of the microstamp. An effective method was used to eliminate residual reactive nucleosides on chips with small molecules containing a hydroxyl group. A specific oligonucleotide array of four probes both matched and mismatched with the target sequence was fabricated to identify the perfect match and mismatch sequences.

  8. Quantitative surface-enhanced resonance Raman scattering of phthalocyanine-labelled oligonucleotides

    PubMed Central

    Macaskill, A.; Chernonosov, A. A.; Koval, V. V.; Lukyanets, E. A.; Fedorova, O. S.; Smith, W. E.; Faulds, K.; Graham, D.

    2007-01-01

    The evaluation of phthalocyanine labels for the surface-enhanced resonance Raman scattering (SERRS) detection of oligonucleotides is reported. Three phthalocyanine-labelled oligonucleotides were assessed, each containing a different metal centre. Detection limits for each labelled oligonucleotide were determined using two excitation frequencies where possible. Limits of detection as low as 2.8 × 10−11 mol. dm−3 were obtained which are comparable to standard fluorescently labelled probes used in previous SERRS studies. The identification of two phthalocyanine-labelled oligonucleotides without separation was also demonstrated indicating their suitability for multiplexing. This study extends the range of labels suitable for quantitative surface-enhanced resonance Raman scattering with silver nanoparticles and offers more flexibility and choice when considering SERRS for quantitative DNA detection. PMID:17289751

  9. Orthogonal ion pairing reversed phase liquid chromatography purification of oligonucleotides with bulky fluorophores.

    PubMed

    Anacleto, Concordio; Ouye, Randall; Schoenbrunner, Nancy

    2014-02-14

    A dual labeled oligonucleotide used as TaqMan® or 5' nuclease probe for in vitro diagnostic has been purified through orthogonal ion-pairing reversed phase chromatography, using polymeric semi-preparative and preparative PRP-1 column. We studied the mechanism of separation of oligonucleotides using ion-pairing reversed phase chromatography. We found that elution profiles of dye labeled oligonucleotides can be controlled by use of specific ion-pairing reagents. Here, we report a method for purification of an oligonucleotide containing an internally positioned rhodamine dye using two orthogonal chromatographic steps, in which the primary step resolves mostly by differences in hydrophobicity by using a weak ion-pairing reagent, and a secondary step uses a strong ion-pairing reagent for separation of length variants. Purification is demonstrated for both 1 and 15μmol scale syntheses, and amenable to further scale up for commercial lot production.

  10. Functionalization of PVC membrane with ss oligonucleotides for a potentiometric biosensor.

    PubMed

    Shishkanova, T V; Volf, R; Krondak, M; Král, V

    2007-05-15

    A novel application of a single stranded (ss) oligonucleotide as an active component of polymeric membrane in an ion-selective electrode (ISE) is described. The original oligonucleotides, oligo(dA)(15), modified by cholesterol, triphenylmethyl and hexadecyl derivatives, were immobilized into poly(vinyl chloride) (PVC) membrane using extraction protocol. In parallel, the adsorption protocol was used to immobilize unmodified oligo(dA)(15) on the PVC membrane based on tridodecylmethyammonium chloride (TDDMA(+)Cl(-)). Immobilization of ss oligonucleotide probe through spacer was more effective for the potentiometric detection of the hybridization between complementary oligonucleotides. It was found that cholesterol-oligo(dA)(15) modified membranes were sensitive toward complementary oligo(dT)(15) in the concentration range 2-80 nM at pH 7. An explanation for the detection mechanism is proposed.

  11. H5 avian influenza virus pathotyping using oligonucleotide microarray.

    PubMed

    Wang, Lih-Chiann; Huang, Dean; Cheng, Ming-Chu; Lee, Shu-Hwae; Wang, Ching-Ho

    2015-08-01

    The H5 avian influenza virus subtype has huge impact on the poultry industry. Rapid diagnosis and accurate identification of the highly pathogenic avian influenza virus and low-pathogenicity avian influenza virus is essential, especially during H5 outbreaks and surveillance. To this end, a novel and rapid strategy for H5 virus molecular pathotyping is presented. The specific hemagglutinin gene of the H5 virus and the basic amino acid number of the motif at the hemagglutinin precursor protein cleavage site were detected using oligonucleotide microarray. Highly pathogenic and low-pathogenicity avian influenza viruses in Taiwan were differentiated using 13 microarray probes with the naked eye. The detection limit reached 3.4 viral RNA copies, 1000 times more sensitive than reverse transcription polymerase chain reaction. Thus, the oligonucleotide microarray would provide an alternative H5 pathogenicity determination using the naked eye for laboratories lacking facilities. Copyright © 2015 Elsevier B.V. All rights reserved.

  12. Palladium-catalyzed modification of unprotected nucleosides, nucleotides, and oligonucleotides.

    PubMed

    Shaughnessy, Kevin H

    2015-05-22

    Synthetic modification of nucleoside structures provides access to molecules of interest as pharmaceuticals, biochemical probes, and models to study diseases. Covalent modification of the purine and pyrimidine bases is an important strategy for the synthesis of these adducts. Palladium-catalyzed cross-coupling is a powerful method to attach groups to the base heterocycles through the formation of new carbon-carbon and carbon-heteroatom bonds. In this review, approaches to palladium-catalyzed modification of unprotected nucleosides, nucleotides, and oligonucleotides are reviewed. Polar reaction media, such as water or polar aprotic solvents, allow reactions to be performed directly on the hydrophilic nucleosides and nucleotides without the need to use protecting groups. Homogeneous aqueous-phase coupling reactions catalyzed by palladium complexes of water-soluble ligands provide a general approach to the synthesis of modified nucleosides, nucleotides, and oligonucleotides.

  13. Oligonucleotide microarray for the identification of potential mycotoxigenic fungi

    PubMed Central

    2010-01-01

    Background Mycotoxins are secondary metabolites which are produced by numerous fungi and pose a continuous challenge to the safety and quality of food commodities in South Africa. These toxins have toxicologically relevant effects on humans and animals that eat contaminated foods. In this study, a diagnostic DNA microarray was developed for the identification of the most common food-borne fungi, as well as the genes leading to toxin production. Results A total of 40 potentially mycotoxigenic fungi isolated from different food commodities, as well as the genes that are involved in the mycotoxin synthetic pathways, were analyzed. For fungal identification, oligonucleotide probes were designed by exploiting the sequence variations of the elongation factor 1-alpha (EF-1 α) coding regions and the internal transcribed spacer (ITS) regions of the rRNA gene cassette. For the detection of fungi able to produce mycotoxins, oligonucleotide probes directed towards genes leading to toxin production from different fungal strains were identified in data available in the public domain. The probes selected for fungal identification and the probes specific for toxin producing genes were spotted onto microarray slides. Conclusions The diagnostic microarray developed can be used to identify single pure strains or cultures of potentially mycotoxigenic fungi as well as genes leading to toxin production in both laboratory samples and maize-derived foods offering an interesting potential for microbiological laboratories. PMID:20307326

  14. Sex determination of bovine preimplantation embryos by oligonucleotide microarray.

    PubMed

    Yang, Hua; Zhong, Fagang; Yang, Yonglin; Wang, Xinhua; Liu, Shouren; Zhu, Bin

    2013-06-01

    The aim has been to set up a rapid and accurate microarray assay using sandwich mode for sex determination of bovine preimplantation embryos. Twelve sequence-specific oligonucleotide capture probes used to discriminate 12 samples were spotted onto the aldehyde-modified glass slides by Arrayer. The 2 recognition probes used to identify coding regions of the sex-determining region of the Y chromosome gene (SRY) and β-casein (CSN2) reference gene were coupled with biotin. The assay was optimized by using genomic DNA extracted from blood samples of known sex individuals. Polymerase chain reaction (PCR) was used to amplify the fragments in the HMG box region of SRY gene and CSN2 gene with sequence-specific primers. The sex of samples was identified by detecting both the SRY and CSN2 genes simultaneously in 2 reaction cells of microarrays, with the male having SRY and CSN2 signals and the female only CSN2. The sex of 20 bovine preimplantation embryos was determined by oligonucleotide microarray. The protocol was run with a blind test that showed a 100% (82/82) specificity and accuracy in sexing of leukocytes. The bovine embryos were transferred into 20 bovine recipients, with a pregnant rate of 40% (8/20). Three calves were born at term, and 5 fetuses were miscarried. Their sexes were fully in accordance with the embryonic sex predetermination predicted by oligonucleotide microarray. This suggests that the oligonucleotide microarray method of SRY gene analysis can be used in early sex prediction of bovine embryos in breeding programs.

  15. Oligonucleotide-based antiviral strategies.

    PubMed

    Schubert, S; Kurreck, J

    2006-01-01

    In the age of extensive global traffic systems, the close neighborhood of man and livestock in some regions of the world, as well as inadequate prevention measures and medical care in poorer countries, greatly facilitates the emergence and dissemination of new virus strains. The appearance of avian influenza viruses that can infect humans, the spread of the severe acute respiratory syndrome (SARS) virus, and the unprecedented raging of human immunodeficiency virus (HIV) illustrate the threat of a global virus pandemic. In addition, viruses like hepatitis B and C claim more than one million lives every year for want of efficient therapy. Thus, new approaches to prevent virus propagation are urgently needed. Antisense strategies are considered a very attractive means of inhibiting viral replication, as oligonucleotides can be designed to interact with any viral RNA, provided its sequence is known. The ensuing targeted destruction of viral RNA should interfere with viral replication without entailing negative effects on ongoing cellular processes. In this review, we will give some examples of the employment of antisense oligonucleotides, ribozymes, and RNA interference strategies for antiviral purposes. Currently, in spite of encouraging results in preclinical studies, only a few antisense oligonucleotides and ribozymes have turned out to be efficient antiviral compounds in clinical trials. The advent of RNA interference now seems to be refueling hopes for decisive progress in the field of therapeutic employment of antisense strategies.

  16. Oligonucleotide microarrays for the detection and identification of viable beer spoilage bacteria.

    PubMed

    Weber, D G; Sahm, K; Polen, T; Wendisch, V F; Antranikian, G

    2008-10-01

    The design and evaluation of an oligonucleotide microarray in order to detect and identify viable bacterial species that play a significant role in beer spoilage. These belong to the species of the genera Lactobacillus, Megasphaera, Pediococcus and Pectinatus. Oligonucleotide probes specific to beer spoilage bacteria were designed. In order to detect viable bacteria, the probes were designed to target the intergenic spacer regions (ISR) between 16S and 23S rRNA. Prior to hybridization the ISR were amplified by combining reverse transcriptase and polymerase chain reactions using a designed consenus primer. The developed oligonucleotide microarrays allows the detection of viable beer spoilage bacteria. This method allows the detection and discrimination of single bacterial species in a sample containing complex microbial community. Furthermore, microarrays using oligonucleotide probes targeting the ISR allow the distinction between viable bacteria with the potential to grow and non growing bacteria. The results demonstrate the feasibility of oligonucleotide microarrays as a contamination control in food industry for the detection and identification of spoilage micro-organisms within a mixed population.

  17. Automated Solid-Phase Click Synthesis of Oligonucleotide Conjugates: From Small Molecules to Diverse N-Acetylgalactosamine Clusters.

    PubMed

    Farzan, Valentina M; Ulashchik, Egor A; Martynenko-Makaev, Yury V; Kvach, Maksim V; Aparin, Ilya O; Brylev, Vladimir A; Prikazchikova, Tatiana A; Maklakova, Svetlana Yu; Majouga, Alexander G; Ustinov, Alexey V; Shipulin, German A; Shmanai, Vadim V; Korshun, Vladimir A; Zatsepin, Timofei S

    2017-10-04

    We developed a novel technique for the efficient conjugation of oligonucleotides with various alkyl azides such as fluorescent dyes, biotin, cholesterol, N-acetylgalactosamine (GalNAc), etc. using copper-catalysed alkyne-azide cycloaddition on the solid phase and CuI·P(OEt)3 as a catalyst. Conjugation is carried out in an oligonucleotide synthesizer in fully automated mode and is coupled to oligonucleotide synthesis and on-column deprotection. We also suggest a set of reagents for the construction of diverse conjugates. The sequential double-click procedure using a pentaerythritol-derived tetraazide followed by the addition of a GalNAc or Tris-GalNAc alkyne gives oligonucleotide-GalNAc dendrimer conjugates in good yields with minimal excess of sophisticated alkyne reagents. The approach is suitable for high-throughput synthesis of oligonucleotide conjugates ranging from fluorescent DNA probes to various multi-GalNAc derivatives of 2'-modified siRNA.

  18. Novel photocleavable universal support for oligonucleotide synthesis.

    PubMed

    Anderson, Emma; Brown, Tom; Picken, Douglas

    2003-01-01

    A novel photocleavable universal support for the automated solid phase synthesis of oligonucleotides is described. The linker between the growing oligonucleotide chain and CPG support contains a nucleophilic amine protected with a photocleavable group. On exposure to UV light, this group is detached and the free amine affords cleavage of the oligonucleotide from the support. The use of long wavelength UV light avoids damage to the DNA.

  19. Light-generated oligonucleotide arrays for rapid DNA sequence analysis.

    PubMed Central

    Pease, A C; Solas, D; Sullivan, E J; Cronin, M T; Holmes, C P; Fodor, S P

    1994-01-01

    In many areas of molecular biology there is a need to rapidly extract and analyze genetic information; however, current technologies for DNA sequence analysis are slow and labor intensive. We report here how modern photolithographic techniques can be used to facilitate sequence analysis by generating miniaturized arrays of densely packed oligonucleotide probes. These probe arrays, or DNA chips, can then be applied to parallel DNA hybridization analysis, directly yielding sequence information. In a preliminary experiment, a 1.28 x 1.28 cm array of 256 different octanucleotides was produced in 16 chemical reaction cycles, requiring 4 hr to complete. The hybridization pattern of fluorescently labeled oligonucleotide targets was then detected by epifluorescence microscopy. The fluorescence signals from complementary probes were 5-35 times stronger than those with single or double base-pair hybridization mismatches, demonstrating specificity in the identification of complementary sequences. This method should prove to be a powerful tool for rapid investigations in human genetics and diagnostics, pathogen detection, and DNA molecular recognition. Images PMID:8197176

  20. The prebiotic synthesis of oligonucleotides

    NASA Technical Reports Server (NTRS)

    Oro, J.; Stephen-Sherwood, E.

    1974-01-01

    This paper is primarily a review of recent developments in the abiotic synthesis of nucleotides, short chain oligonucleotides, and their mode of replication in solution. It also presents preliminary results from this laboratory on the prebiotic synthesis of thymidine oligodeoxynucleotides. A discussion, based on the physicochemical properties of RNA and DNA oligomers, relevant to the molecular evolution of these compounds leads to the tentative hypothesis that oligodeoxyribonucleotides of about 12 units may have been of sufficient length to initiate a self replicating coding system. Two models are suggested to account for the synthesis of high molecular weight oligomers using short chain templates and primers.

  1. SOAP: short oligonucleotide alignment program.

    PubMed

    Li, Ruiqiang; Li, Yingrui; Kristiansen, Karsten; Wang, Jun

    2008-03-01

    We have developed a program SOAP for efficient gapped and ungapped alignment of short oligonucleotides onto reference sequences. The program is designed to handle the huge amounts of short reads generated by parallel sequencing using the new generation Illumina-Solexa sequencing technology. SOAP is compatible with numerous applications, including single-read or pair-end resequencing, small RNA discovery and mRNA tag sequence mapping. SOAP is a command-driven program, which supports multi-threaded parallel computing, and has a batch module for multiple query sets. http://soap.genomics.org.cn.

  2. Detection and Typing of "Candidatus Phytoplasma " spp. in Host DNA Extracts Using Oligonucleotide-Coupled Fluorescent Microspheres.

    PubMed

    Pérez-López, Edel; Hammond, Christine; Olivier, Chrystel; Dumonceaux, Tim J

    2017-01-01

    The use of oligonucleotide-coupled fluorescent microspheres is a rapid, sequencing-independent, and reliable way to diagnose bacterial diseases. Previously described applications of oligonucleotide-coupled fluorescent microspheres for the detection and identification of bacteria in human clinical samples have been successfully adapted to detect and differentiate "Ca. Phytoplasma" species using as a target the chaperonin 60-encoding gene. In this chapter, we describe in detail the design and validation of oligonucleotide capture probes, and their application in the assay aiming to differentiate phytoplasma strains infecting Brassica napus and Camelina sativa plants grown in the same geographic location at the same time.

  3. Ultrathin oligonucleotide layers for fluorescence-based DNA sensors

    NASA Astrophysics Data System (ADS)

    Furch, M.; Ueberfeld, J.; Hartmann, Andreas; Bock, Daniel; Seeger, Stefan

    1996-11-01

    Preliminary investigations into the design of an affinity sensor using evanescent wave technology concentrate upon the means of immobilization of the receptor molecules. In this work DNA served as the selective recognition element. The molecular principle of a sequence-selective biosensor for DNA is based on a sandwich-hybridization assay wherein the analyte, a single-stranded (ss)DNA, bound specifically to both an immobilized capture probe and a dye-labeled oligonucleotide in free solution. The efficiency of the capture array depends on the density of highly organized oligonucleotides on the waveguide surface and correlates therefore directly with the specificity and the sensitivity of the sensor. In the present approach using the Langmuir- Blodgett technique cinnamoylbutylether-cellulose monolayers were transferred onto optical fibers or planar waveguides. These films served as matrices for the immobilization of biotinylated oligonucleotides via streptavidin. For the first time streptavidin was immobilized by that manner. The specificity of the streptavidin layer or the following bounded nucleic acid molecules were controlled by an enzyme- linked immunosorbent assay (ELISA). Finally, this application has also shown to be suitable for the detection of Salmonella, which is an important pathogen associated with acute gastroenteritidis and food borne diseases.

  4. Diagnostic Oligonucleotide Microarray Fingerprinting of Bacillus Isolates

    SciTech Connect

    Chandler, Darrell P.; Alferov, Oleg; Chernov, Boris; Daly, Don S.; Golova, Julia; Perov, Alexander N.; Protic, Miroslava; Robison, Richard; Shipma, Matthew; White, Amanda M.; Willse, Alan R.

    2006-01-01

    A diagnostic, genome-independent microbial fingerprinting method using DNA oligonucleotide microarrays was used for high-resolution differentiation between closely related Bacillus strains, including two strains of Bacillus anthracis that are monomorphic (indistinguishable) via amplified fragment length polymorphism fingerprinting techniques. Replicated hybridizations on 391-probe nonamer arrays were used to construct a prototype fingerprint library for quantitative comparisons. Descriptive analysis of the fingerprints, including phylogenetic reconstruction, is consistent with previous taxonomic organization of the genus. Newly developed statistical analysis methods were used to quantitatively compare and objectively confirm apparent differences in microarray fingerprints with the statistical rigor required for microbial forensics and clinical diagnostics. These data suggest that a relatively simple fingerprinting microarray and statistical analysis method can differentiate between species in the Bacillus cereus complex, and between strains of B. anthracis. A synthetic DNA standard was used to understand underlying microarray and process-level variability, leading to specific recommendations for the development of a standard operating procedure and/or continued technology enhancements for microbial forensics and diagnostics.

  5. A novel, one-step amplification and oligonucleotide ligation procedure for multiplex genetic typing

    SciTech Connect

    Eggerding, F.A.

    1994-09-01

    A new technique, coupled amplification and oligonucleotide ligation (CAL), has been developed for simultaneous multiplex amplification and genotyping of DNA. CAL is a biphasic method which combines in one assay DNA amplification by the polymerase chain reaction (PCR) with DNA genotyping by the oligonucleotide ligation assay (OLA). By virtue of a difference in the melting temperatures of PCR primer-target DNA and OLA probe-target DNA hybrids, the method allows preferential amplification of DNA during stage I and oligonucleotide ligation during stage II of the reaction. In stage I target DNA is amplified using high-melting primers in a two-step PCR cycle that employs a 72{degrees}C anneal-elongation step. In stage II genotyping of PCR products by competitive oligonucleotide ligation with oligonucleotide probes located between PCR primers is accomplished by several cycles of denaturation at 94{degrees}C followed by anneal-ligation at 55{degrees}C. Ligation products are fluorochrome-labeled at their 3{prime}-ends and analyzed electrophoretically on a fluorescent DNA sequencer. The CAL procedure has been used for multiplex detection of 30 cystic fibrosis mutations and for analysis of ras gene point mutations. Because mutation detection occurs concurrently with target amplification, the technique is rapid, highly sensitive and specific, easily automatable, and requires minimal sample processing.

  6. Oligonucleotide therapeutics for human leukaemia.

    PubMed

    Gewirtz, A M

    1997-01-01

    The concept of antisense oligonucleotide 'therapeutics' has generated a great deal of controversy. Questions abound regarding the mechanism of action of these compounds, their reliability and their ultimate utility. These problems are compounded by the 'hype', which has attended their development, and the inability of workers in this area to meet the expectations raised by its most zealous proponents. Nevertheless, it is worth pointing out that there have been some notable gene disruption successes with this technique that have stood up to rigorous scrutiny. Our own work with c-myb as a target is perhaps a reasonable example. Though much remains to be accomplished before antisense drugs are commonly, and usefully, employed in the clinic, it is important to remember what motivates their development. Gene-targeted drugs have the promise of exquisite specificity and the potential to do much good with little toxicity. Accordingly, antisense oligonucleotides can serve as a paradigm of rational drug development. For all these reasons then, we believe that efforts should be increased to decipher the mechanism of action of antisense oligodeoxynucleotides, and to learn how they may be successfully employed in the clinic.

  7. Mutational analysis using oligonucleotide microarrays

    PubMed Central

    Hacia, J.; Collins, F.

    1999-01-01

    The development of inexpensive high throughput methods to identify individual DNA sequence differences is important to the future growth of medical genetics. This has become increasingly apparent as epidemiologists, pathologists, and clinical geneticists focus more attention on the molecular basis of complex multifactorial diseases. Such undertakings will rely upon genetic maps based upon newly discovered, common, single nucleotide polymorphisms. Furthermore, candidate gene approaches used in identifying disease associated genes necessitate screening large sequence blocks for changes tracking with the disease state. Even after such genes are isolated, large scale mutational analyses will often be needed for risk assessment studies to define the likely medical consequences of carrying a mutated gene.
This review concentrates on the use of oligonucleotide arrays for hybridisation based comparative sequence analysis. Technological advances within the past decade have made it possible to apply this technology to many different aspects of medical genetics. These applications range from the detection and scoring of single nucleotide polymorphisms to mutational analysis of large genes. Although we discuss published scientific reports, unpublished work from the private sector12 could also significantly affect the future of this technology.


Keywords: mutational analysis; oligonucleotide microarrays; DNA chips PMID:10528850

  8. Surface plasmon resonance detection of oligonucleotide sequences of the rpoB genes of Mycobacterium tuberculosis.

    PubMed

    Rachkov, A; Patskovsky, S; Soldatkin, A; Meunier, M

    2011-09-30

    Oligonucleotide sequences related to the normal and mutated rpoB genes of Mycobacterium tuberculosis are detected using a surface plasmon resonance (SPR) biosensor system. A bioselective element was prepared by immobilizing the thiol-modified oligonucleotides of the selected sequence (the capture probe P2) that contains the mutated TCG→TTG codon 531 (evoking drug resistance) of the rpoB gene of M. tuberculosis on a gold sensor surface. Specific hybridization between immobilized probe P2 and complementary target T2 gave the highest sensor response, single-base mismatched oligonucleotide TN (corresponding to the normal gene sequence) produced somewhat smaller response and no response was observed at injection of noncomplementary oligonucleotide TC. The P2-T2 hybridization efficiency is calculated ca. 30% (5 × 10(12) molecules cm(-2)), and the lowest detection limit of T2 was 10nM. An extended T2E oligonucleotide sequence consisting of T2 sequence and additional 24 nucleotides was shown to cause more pronounced sensor response (at least 5 nM T2E was easily detected). Injection into the sensor cell of the oligonucleotides complementary to the free additional part of T2E after P2-T2E hybridization gave a significant additional SPR response, thus showing that the sandwich hybridization format further improves the sensor sensitivity and decreases the lowest detection limit. The experimental results on surface hybridization between the studied oligonucleotides were in good agreement with thermodynamic parameters of the hybridization calculated for solution conditions. The described approach could be proposed as a basis for creating a biosensor for real-time and label-free diagnostics of drug resistant tuberculosis. Copyright © 2011 Elsevier B.V. All rights reserved.

  9. P-chiral oligonucleotides in biological recognition processes.

    PubMed

    Guga, Piotr

    2007-01-01

    Internucleotide phosphodiester linkages in non-modified oligonucleotides are quickly degraded by nucleolytic enzymes present in the cells and this feature practically eliminates natural DNA and RNA molecules from medical applications and from many structural and mechanistic studies. P-chiral oligonucleotide analogs, in which one of the non-bridging phosphate oxygen atoms is substituted with another heteroatom (e.g. S, Se) or a chemical group (e.g. CH3, BH3(-)), have significantly greater nuclease resistance and also offer important possibilities for detailed studies of interactions with other biomolecules at the molecular level. Notably, these substitutions do not disrupt hydrogen bonding between nucleobases and affect the overall geometry of the oligomers to only low or moderate extent, although important changes of hydration patterns and changes of interactions with metal ions are observed. Such the probes, including isotopomeric species labeled with a heavy oxygen isotope, possessing phosphorus atoms of selected absolute configurations, have been used for elucidation of the mode of action of many enzymes (nucleases, transferases, kinases), ribozymes and DNA-zymes, as well as for investigations on thermodynamic stability of nucleic acids complexes (duplexes, triplexes, i-motif) and for studies on a mechanism of conformational changes of B-Z type. They are also useful tools for analysis of interactions of the phosphoryl oxygen atoms in natural precursors with functional groups of proteins. The synthetic routes to stereodefined forms of selected types of P-chiral oligonucleotides are presented, as well as recently developed methods for their configurational analysis at micromolar concentration. Selected examples of application of diastereomerically pure P-chiral oligonucleotides for structural, biochemical and biological experiments are discussed.

  10. Cellular Uptake and Intracellular Trafficking of Oligonucleotides: Implications for Oligonucleotide Pharmacology

    PubMed Central

    Ming, Xin; Carver, Kyle; Laing, Brian

    2014-01-01

    One of the major constraints on the therapeutic use of oligonucleotides is inefficient delivery to their sites of action in the cytosol or nucleus. Recently it has become evident that the pathways of cellular uptake and intracellular trafficking of oligonucleotides can strongly influence their pharmacological actions. Here we provide background information on the basic processes of endocytosis and trafficking and then review recent literature on targeted delivery and subcellular trafficking of oligonucleotides in that context. A variety of approaches including molecular scale ligand-oligonucleotide conjugates, ligand-targeted nanocarriers, and the use of small molecules to enhance oligonucleotide effects are discussed. PMID:24383421

  11. Drop drying on surfaces determines chemical reactivity - the specific case of immobilization of oligonucleotides on microarrays

    PubMed Central

    2013-01-01

    Background Drop drying is a key factor in a wide range of technical applications, including spotted microarrays. The applied nL liquid volume provides specific reaction conditions for the immobilization of probe molecules to a chemically modified surface. Results We investigated the influence of nL and μL liquid drop volumes on the process of probe immobilization and compare the results obtained to the situation in liquid solution. In our data, we observe a strong relationship between drop drying effects on immobilization and surface chemistry. In this work, we present results on the immobilization of dye labeled 20mer oligonucleotides with and without an activating 5′-aminoheptyl linker onto a 2D epoxysilane and a 3D NHS activated hydrogel surface. Conclusions Our experiments identified two basic processes determining immobilization. First, the rate of drop drying that depends on the drop volume and the ambient relative humidity. Oligonucleotides in a dried spot react unspecifically with the surface and long reaction times are needed. 3D hydrogel surfaces allow for immobilization in a liquid environment under diffusive conditions. Here, oligonucleotide immobilization is much faster and a specific reaction with the reactive linker group is observed. Second, the effect of increasing probe concentration as a result of drop drying. On a 3D hydrogel, the increasing concentration of probe molecules in nL spotting volumes accelerates immobilization dramatically. In case of μL volumes, immobilization depends on whether the drop is allowed to dry completely. At non-drying conditions, very limited immobilization is observed due to the low oligonucleotide concentration used in microarray spotting solutions. The results of our study provide a general guideline for microarray assay development. They allow for the initial definition and further optimization of reaction conditions for the immobilization of oligonucleotides and other probe molecule classes to different

  12. Bromodeoxyuridine-labeled oligonucleotides as tools for oligonucleotide uptake studies.

    PubMed

    Maszewska, Maria; Kobylańska, Anna; Gendaszewska-Darmach, Edyta; Koziołkiewicz, Maria

    2002-12-01

    The mechanisms by which various oligonucleotides (ODNs) and their analogs enter cells are not fully understood. A common technique used in studies on cellular uptake of ODNs is their conjugation with fluorochromes. However, fluorescently labeled ODNs may vary from the parent compounds in charge and hydrophilicity, and they may interact differently with some components of cellular membranes. In this report, we present an alternative method based on the immunofluorescent detection of ODNs with incorporated 5-bromo-2'-deoxyuridine (BrdUrd). Localization of BrdUrd-modified ODNs has been achieved using FITC-labeled anti-BrdUrd antibodies. This technique allowed determination of the differences in cellular uptake of phosphodiester (PO) and phosphorothioate (PS) ODNs and their derivatives conjugated with cholesterol and menthol. The immunocytochemical method also has shown that the cellular uptake of some ODNs may be influenced by specific sequences that are responsible for the formation of higher-order structures.

  13. Fluorescence detection of specific sequence of nucleic acids by oxazole yellow-linked oligonucleotides. Homogeneous quantitative monitoring of in vitro transcription.

    PubMed

    Ishiguro, T; Saitoh, J; Yawata, H; Otsuka, M; Inoue, T; Sugiura, Y

    1996-12-15

    We have developed a fluorescent DNA probe, oxazole yellow (YO)-linked oligonucleotide complementary to a target DNA/RNA, which can enhance the fluorescence on hybridizing with a target nucleotide. We demonstrated the applicability of the YO-linked oligonucleotide probe to real-time monitoring of the in vitro transcription process of a plasmid DNA constructed containing the 5'-terminus non-coded region of hepatitis C virus RNA. In the process of in vitro transcription in the presence of YO-linked complementary oligonucleotide, the fluorescence of the reaction mixture showed a time-dependent linear increase corresponding to the generated target RNA product.

  14. Fluorescence detection of specific sequence of nucleic acids by oxazole yellow-linked oligonucleotides. Homogeneous quantitative monitoring of in vitro transcription.

    PubMed Central

    Ishiguro, T; Saitoh, J; Yawata, H; Otsuka, M; Inoue, T; Sugiura, Y

    1996-01-01

    We have developed a fluorescent DNA probe, oxazole yellow (YO)-linked oligonucleotide complementary to a target DNA/RNA, which can enhance the fluorescence on hybridizing with a target nucleotide. We demonstrated the applicability of the YO-linked oligonucleotide probe to real-time monitoring of the in vitro transcription process of a plasmid DNA constructed containing the 5'-terminus non-coded region of hepatitis C virus RNA. In the process of in vitro transcription in the presence of YO-linked complementary oligonucleotide, the fluorescence of the reaction mixture showed a time-dependent linear increase corresponding to the generated target RNA product. PMID:9016671

  15. [Recent progress and prospect in oligonucleotide therapeutics].

    PubMed

    Yoshioka, Kotaro; Kuwahara, Hiroya; Nishina, Kazutaka; Nagata, Tetsuya; Yokota, Takanori

    2015-06-01

    Remarkable progress has been made in chemical modification and nonviral delivery systems that improve the properties and efficacy of therapeutics oligonucleotides therapeutics, such as antisense oligonucleotide (ASO) and small interfering RNA(siRNA). ASOs act through various mechanisms including the degradation of mRNA by RNase H (gapmer-type ASO) and the modulation alternative splicing patterns(splice switching oligonucleotide). Recent favorable outcomes in clinical trials for cancers and genetic diseases such as familial amyloid polyneuropathy and Duchenne muscular dystrophy indicate high clinical potency of oligonucleotide therapeutics. Here we reviewed recent advances in basic properties and clinical applications of ASO and siRNA, and provide future perspective on oligonucleotide therapeutics.

  16. Spatially Defined Oligonucleotide Arrays. Technical Report for Phase II

    SciTech Connect

    2000-06-15

    The goal of the Human Genome Project is to sequence all 3 billion base pairs of the human genome. Progress in this has been rapid; GenBank{reg_sign} finished 1994 with 286 million bases of sequence and grew by 2470 in the first quarter of 1995. The challenge to the scientific community is to understand the biological relevance of this genetic information. In most cases the sequence being generated for any single region of the genome represents the genotype of a single individual. A complete understanding of the function of specific genes and other regions of the genome and their role in human disease and development will only become apparent when the sequence of many more individuals is known. Access to genetic information is ultimately limited by the ability to screen DNA sequence. Although the pioneering sequencing methods of Sanger et al. (15) and Maxam and Gilbert (11) have become standard in virtually all molecular biology laboratories, the basic protocols remain largely unchanged. The throughput of this sequencing technology is now becoming the rate-limiting step in both large-scale sequencing projects such as the Human Genome Project and the subsequent efforts to understand genetic diversity. This has inspired the development of advanced DNA sequencing technologies (9), Incremental improvements to Sanger sequencing have been made in DNA labeling and detection. High-speed electrophoresis methods using ultrathin gels or capillary arrays are now being more widely employed. However, these methods are throughput-limited by their sequential nature and the speed and resolution of separations. This limitation will become more pronounced as the need to rapidly screen newly discovered genes for biologically relevant polymorphisms increases. An alternative to gel-based sequencing is to use high-density oligonucleotide probe arrays. Oligonucleotide probe arrays display specific oligonucleotide probes at precise locations in a high density, information-rich format (5

  17. Avian oncogenic virus differential diagnosis in chickens using oligonucleotide microarray.

    PubMed

    Wang, Lih-Chiann; Huang, Dean; Pu, Chang-En; Wang, Ching-Ho

    2014-12-15

    Avian oncogenic viruses include the avian leukosis virus (ALV), reticuloendotheliosis virus (REV) and Marek's disease virus (MDV). Multiple oncogenic viral infections are frequently seen, with even Marek's disease vaccines reported to be contaminated with ALV and REV. The gross lesions caused by avian oncogenic viruses often overlap, making differentiation diagnosis based on histopathology difficult. The objective of this study is to develop a rapid approach to simultaneously differentiate, subgroup and pathotype the avian oncogenic viruses. The oligonucleotide microarray was employed in this study. Particular DNA sequences were recognized using specific hybridization between the DNA target and probe on the microarray, followed with colorimetric development through enzyme reaction. With 10 designed probes, ALV-A, ALV-E, ALV-J, REV, MDV pathogenic and vaccine strains were clearly discriminated on the microarray with the naked eyes. The detection limit was 27 copy numbers, which was 10-100 times lower than multiplex PCR. Of 102 field samples screened using the oligonucleotide microarray, 32 samples were positive for ALV-E, 17 samples were positive for ALV-J, 6 samples were positive for REV, 4 samples were positive for MDV, 7 samples were positive for both ALV-A and ALV-E, 5 samples were positive for ALV-A, ALV-E and ALV-J, one sample was positive for both ALV-J and MDV, and 3 samples were positive for both REV and MDV. The oligonucleotide microarray, an easy-to-use, high-specificity, high-sensitivity and extendable assay, presents a potent technique for rapid differential diagnosis of avian oncogenic viruses and the detection of multiple avian oncogenic viral infections under field conditions. Copyright © 2014 Elsevier B.V. All rights reserved.

  18. Adaptive resolution simulation of oligonucleotides.

    PubMed

    Netz, Paulo A; Potestio, Raffaello; Kremer, Kurt

    2016-12-21

    Nucleic acids are characterized by a complex hierarchical structure and a variety of interaction mechanisms with other molecules. These features suggest the need of multiscale simulation methods in order to grasp the relevant physical properties of deoxyribonucleic acid (DNA) and RNA using in silico experiments. Here we report an implementation of a dual-resolution modeling of a DNA oligonucleotide in physiological conditions; in the presented setup only the nucleotide molecule and the solvent and ions in its proximity are described at the atomistic level; in contrast, the water molecules and ions far from the DNA are represented as computationally less expensive coarse-grained particles. Through the analysis of several structural and dynamical parameters, we show that this setup reliably reproduces the physical properties of the DNA molecule as observed in reference atomistic simulations. These results represent a first step towards a realistic multiscale modeling of nucleic acids and provide a quantitatively solid ground for their simulation using dual-resolution methods.

  19. Adaptive resolution simulation of oligonucleotides

    NASA Astrophysics Data System (ADS)

    Netz, Paulo A.; Potestio, Raffaello; Kremer, Kurt

    2016-12-01

    Nucleic acids are characterized by a complex hierarchical structure and a variety of interaction mechanisms with other molecules. These features suggest the need of multiscale simulation methods in order to grasp the relevant physical properties of deoxyribonucleic acid (DNA) and RNA using in silico experiments. Here we report an implementation of a dual-resolution modeling of a DNA oligonucleotide in physiological conditions; in the presented setup only the nucleotide molecule and the solvent and ions in its proximity are described at the atomistic level; in contrast, the water molecules and ions far from the DNA are represented as computationally less expensive coarse-grained particles. Through the analysis of several structural and dynamical parameters, we show that this setup reliably reproduces the physical properties of the DNA molecule as observed in reference atomistic simulations. These results represent a first step towards a realistic multiscale modeling of nucleic acids and provide a quantitatively solid ground for their simulation using dual-resolution methods.

  20. The illusion of specific capture: surface and solution studies of suboptimal oligonucleotide hybridization

    PubMed Central

    2013-01-01

    Background Hybridization based assays and capture systems depend on the specificity of hybridization between a probe and its intended target. A common guideline in the construction of DNA microarrays, for instance, is that avoiding complementary stretches of more than 15 nucleic acids in a 50 or 60-mer probe will eliminate sequence specific cross-hybridization reactions. Here we present a study of the behavior of partially matched oligonucleotide pairs with complementary stretches starting well below this threshold complementarity length – in silico, in solution, and at the microarray surface. The modeled behavior of pairs of oligonucleotide probes and their targets suggests that even a complementary stretch of sequence 12 nt in length would give rise to specific cross-hybridization. We designed a set of binding partners to a 50-mer oligonucleotide containing complementary stretches from 6 nt to 21 nt in length. Results Solution melting experiments demonstrate that stable partial duplexes can form when only 12 bp of complementary sequence are present; surface hybridization experiments confirm that a signal close in magnitude to full-strength signal can be obtained from hybridization of a 12 bp duplex within a 50mer oligonucleotide. Conclusions Microarray and other molecular capture strategies that rely on a 15 nt lower complementarity bound for eliminating specific cross-hybridization may not be sufficiently conservative. PMID:23445545

  1. Optimal design of oligonucleotide microarrays for measurement of DNA copy-number.

    PubMed

    Sharp, Andrew J; Itsara, Andy; Cheng, Ze; Alkan, Can; Schwartz, Stuart; Eichler, Evan E

    2007-11-15

    Copy-number variants (CNVs) occur frequently within the human genome, and may be associated with many human phenotypes. If disease association studies of CNVs are to be performed routinely, it is essential that the copy-number status be accurately genotyped. We systematically assessed the dynamic range response of an oligonucleotide microarray platform to accurately predict copy-number in a set of seven patients who had previously been shown to carry between 1 and 6 copies of an approximately 4 Mb region of 15q12.2-q13.1. We identify probe uniqueness, probe length, uniformity of probe melting temperature, overlap with SNPs and common repeats (particularly Alu elements) and guanine homopolymer content as parameters that significantly affect probe performance. Further, we prove the influence of these criteria on array performance by using these parameters to prospectively filter data from a second array design covering an independent genomic region and observing significant improvements in data quality. The informed selection of probes which have superior performance characteristics allows the prospective design of oligonucleotide arrays which show increased sensitivity and specificity compared with current designs. Although based on the analysis of data from comparative genomic hybridization experiments, we anticipate that our results are relevant to the design of improved oligonucleotide arrays for high-throughput copy-number genotyping of complex regions of the human genome.

  2. Rapid, high-resolution in situ hybridization histochemistry with radioiodinated synthetic oligonucleotides

    SciTech Connect

    Lewis, M.E.; Arentzen, R.; Baldino, F. Jr.

    1986-01-01

    In situ hybridization histochemistry is a valuable technique for localizing specific messenger RNA (mRNA) and detecting changes in gene expression. Generally, the mRNA of interest has been detected by probes obtained from cloned DNA and labelled to high specific activity by nick translation. Such probes have a number of disadvantages which can be circumvented by the use of short synthetic oligonucleotides designed to be complementary to a known mRNA sequence. We report here that synthetic oligonucleotides complementary to part of the mRNA coding for rat arginine-vasopressin (AVP) can be labelled to high specific activity with (/sup 125/I), using either the primer extension method with the Klenow fragment of DNA polymerase I or the 3'-tailing method with terminal deoxynucleotidyl transferase. Both AVP probes hybridized well to the magnocellular neurons of the hypothalamic paraventricular and supraoptic nuclei. A strong autoradiographic signal was present by 2 days, with grains largely confined to the perikaryon. These results compare favorably to those obtained with (/sup 32/P)- or (/sup 3/H)-labelled probes. Given the ease of the 3'-tailing method, (/sup 125/I)-labelled oligonucleotides appear to be especially useful probes for in situ hybridization histochemistry.

  3. Minimizing DNA microarrays to a single molecule per spot: using zero-mode waveguide technology to obtain kinetic data for a large number of short oligonucleotide hybridization reactions

    NASA Astrophysics Data System (ADS)

    Sobek, Jens; Rehrauer, Hubert; Kuhn, Gerrit; Schlapbach, Ralph

    2016-03-01

    We have shown recently that the hybridization of short oligonucleotides can be studied in a zero-mode waveguide nanostructure (ZMW) chip using a modified DNA sequencer.[1] Here we present an extension of this method enabling the parallel measurement of kinetic constants of a large number of hybridization reactions on a single chip. This can be achieved by immobilization of a mixture of oligonucleotides, which leads to a statistical and random distribution of single molecules in the 150'000 ZMWs of a SMRT™ cell. This setup is comparable to a classical microarray with ZMWs in place of spots but unknown allocation of probes. The probe surface density is reduced by a factor of ~1010 allowing the study of hybridization in the absence of interactions with neighboring probes. Hybridization with a dye labelled oligonucleotide results in trains of fluorescence pulses from which interpulse durations (IPDs) and pulse widths (PWs) can be extracted. Since the identity of a probe in a ZMW is unknown, the immobilized oligonucleotide is sequenced in a subsequent step. After mapping the fluorescence traces to the sequence, the association and dissociation rate constant for each oligonucleotide can be calculated. By selecting suitable probes, the method can be used to determine rate constants of hybridization for a large number of mismatch oligonucleotides in a single measurement and at single-molecule level.

  4. Ruthenium versus platinum: interactions of anticancer metallodrugs with duplex oligonucleotides characterised by electrospray ionisation mass spectrometry.

    PubMed

    Groessl, Michael; Tsybin, Yury O; Hartinger, Christian G; Keppler, Bernhard K; Dyson, Paul J

    2010-06-01

    The binding of the ruthenium-based anticancer drug candidates KP1019, NAMI-A and RAPTA-T towards different double-stranded oligonucleotides was probed by electrospray ionisation mass spectrometry and compared with that of the widely used platinum-based chemotherapeutics cisplatin, carboplatin and oxaliplatin. It was found that the extent of adduct formation decreased in the following order: cisplatin > oxaliplatin > NAMI-A > RAPTA-T > carboplatin > KP1019. In addition to the characterisation of the adducts formed with the DNA models, the binding sites of the metallodrugs on the oligonucleotides were elucidated employing top-down tandem mass spectrometry and were found to be similar for all the metallodrugs studied, irrespective of the sequence of the oligonucleotide. A strong preference for guanine residues was established.

  5. Streptavidin-coated gold nanoparticles: critical role of oligonucleotides on stability and fractal aggregation

    PubMed Central

    D'Agata, Roberta; Palladino, Pasquale

    2017-01-01

    Gold nanoparticles (AuNPs) exhibit unique properties that can be modulated through a tailored surface functionalization, enabling their targeted use in biochemical sensing and medical diagnostics. In particular, streptavidin-modified AuNPs are increasingly used for biosensing purposes. We report here a study of AuNPs surface-functionalized with streptavidin-biotinylated oligonucleotide, focussing on the role played by the oligonucleotide probes in the stabilization/destabilization of the functionalized nanoparticle dispersion. The behaviour of the modified AuNP dispersion as a consequence of the competitive displacement of the biotinylated oligonucleotide has been investigated and the critical role of displaced oligonucletides in triggering the quasi one-dimensional aggregation of nanoparticles is demonstrated for the first time. The thorough understanding of the fundamental properties of bioconjugated AuNPs is of great importance for the design of highly sensitive and reliable functionalized AuNP-based assays. PMID:28144559

  6. Hydrolysis of microporous polyamide-6 membranes as substrate for in situ synthesis of oligonucleotides

    NASA Astrophysics Data System (ADS)

    Tang, Jianxin; He, Nongyue; Nie, Libo; Xiao, Pengfeng; Chen, Hong

    2004-02-01

    This article provides a novel method of preparing substrate for in situ synthesis of oligonucleotide by hydrolyzing microporous polyamide-6 membranes in a 0.01 mol/l/NaOH/(H 2O-CH 3OH) mixture medium with refluxing about 36 h. Attenuated total reflectance Fourier transform infrared spectroscopy (ATR-FTIR) demonstrated the emergence of amines (NH 2) on the surface. Optimum hydrolyzing conditions were determined through the ultra-violet (UV) spectra. A pH value of 12 and a hydrolysis time of 36 h are the preferred conditions for the modification. The treated membrane can be applied to in situ synthesis of oligonucleotide and, for example, the oligonucleotide probes of 5 '-AAC CAC CAA ACA CAC-3 ' were successfully synthesized on the hydrolyzed membrane. The single step coupling efficiency determined by ultraviolet (UV) spectra is above 98%.

  7. New protocol for oligonucleotide microarray fabrication using SU-8-coated glass microslides.

    PubMed

    Sethi, D; Kumar, A; Gandhi, R P; Kumar, P; Gupta, K C

    2010-09-15

    Microarray technology has become an important tool for detection and analysis of nucleic acid targets. Immobilization of modified and unmodified oligonucleotides on epoxy-functionalized glass surfaces is often used in microarray fabrication. Here, we demonstrate a protocol that employs coating of SU-8 (glycidyl ether of bisphenol A) onto glass microslides to obtain high density of epoxy functions for efficient immobilization of aminoalkyl-, thiophosphoryl-, and phosphorylated oligonucleotides with uniform spot morphology. The resulting microarrays exhibited high immobilization (∼65%) and hybridization efficiency (30-36%) and were sufficiently stable over a range of temperature and pH conditions. The prominent feature of the protocol is that spots can be visualized distinctly at 0.05 μM probe (a 20-mer oligonucleotide) concentration. The constructed microarrays were subsequently used for detection of base mismatches and bacterial diseases (meningitis and typhoid).

  8. Complex DNA nanostructures from oligonucleotide ensembles.

    PubMed

    Mathur, Divita; Henderson, Eric R

    2013-04-19

    The first synthetic DNA nanostructures were created by self-assembly of a small number of oligonucleotides. Introduction of the DNA origami method provided a new paradigm for designing and creating two- and three-dimensional DNA nanostructures by folding a large single-stranded DNA and 'stapling' it together with a library of oligonucleotides. Despite its power and wide-ranging implementation, the DNA origami technique suffers from some limitations. Foremost among these is the limited number of useful single-stranded scaffolds of biological origin. This report describes a new approach to creating large DNA nanostructures exclusively from synthetic oligonucleotides. The essence of this approach is to replace the single-stranded scaffold in DNA origami with a library of oligonucleotides termed "scaples" (scaffold staples). Scaples eliminate the need for scaffolds of biological origin and create new opportunities for producing larger and more diverse DNA nanostructures as well as simultaneous assembly of distinct structures in a "single-pot" reaction.

  9. Enhanced oligonucleotide–nanoparticle conjugate stability using thioctic acid modified oligonucleotides

    PubMed Central

    Dougan, Jennifer A.; Karlsson, Camilla; Smith, W. Ewen; Graham, Duncan

    2007-01-01

    Metallic nanoparticles of gold functionalized with oligonucleotides conventionally use a terminal thiol modification and have been used in a wide range of applications. Although readily available, the oligonucleotide–nanoparticle conjugates prepared in this way suffer from a lack of stability when exposed to a variety of small molecules or elevated temperatures. If silver is used in place of gold then this lack of stability is even more pronounced. In this study we report the synthesis of highly stabilized oligonucleotide–nanoparticle conjugates using a simple oligonucleotide modification. A modified solid support was used to generate 3′-thioctic acid modified oligonucleotides by treatment with an N-hydroxysuccimidyl ester of thioctic acid. Unusually, both gold and silver nanoparticles have been investigated in this study and show that these disulphide-modified oligonucleotide probes offer significant improvements in nanoparticle stability when treated with dithiothreitol (DTT) compared with monothiol analogues. This is a significant advance in oligonucleotide–nanoparticle conjugate stability and for the first time allows silver nanoparticles to be prepared that are more stable than standard gold-thiol functionalized nanoparticles. This opens up the possibility of using silver nanoparticles functionalized with oligonucleotides as an alternative to gold. PMID:17488844

  10. Antisense oligonucleotides as therapeutics for malignant diseases.

    PubMed

    Ho, P T; Parkinson, D R

    1997-04-01

    The continued progress in our understanding of the biology of neoplasia and in the identification, cloning, and sequencing of genes critical to tumor cell function permits the exploitation of this information to develop specific agents that may directly modulate the function of these genes or their protein products. Antisense oligonucleotides are being investigated as a potential therapeutic modality that takes direct advantage of molecular sequencing. The antisense approach uses short oligonucleotides designed to hybridize to a target mRNA transcript through Watson-Crick base pairing. The formation of this oligonucleotide: RNA heteroduplex results in mRNA inactivation and consequent inhibition of synthesis of the protein product. A fundamental attraction of the antisense approach is that this method potentially may be applied to any gene product, in theory, for the treatment of malignant and non-malignant diseases. However, this simple and attractive model has proven to be much more complex in practice. A number of important challenges in the preclinical development of antisense oligonucleotides have been identified, including stability, sequence length, cellular uptake, target sequence selection, appropriate negative controls, oligonucleotide: protein interactions, and cost of manufacture. Although the biological activity of an oligonucleotide against its molecular target is theoretically sequence-dependent, the animal pharmacokinetics and toxicology of phosphorothioate analogues directed against vastly disparate gene products appear relatively non-sequence-specific. In oncology, a number of clinical trials have been initiated with antisense oligonucleotides directed against molecular targets including: p53; bcl-2; raf kinase; protein kinase C-alpha; c-myb. The experience gained from these early clinical trials will be applicable to the next generation of antisense agents in development. These may include molecules with novel backbones or other structural

  11. Incorporation of an aldehyde function in oligonucleotides.

    PubMed

    Tilquin, J M; Dechamps, M; Sonveaux, E

    2001-01-01

    A nucleotide-like phosphoramidite building block that has the nucleic base replaced by the tert-butyldimethylsilyl-protected styrene glycol was synthesized. After the automatic synthesis of an oligonucleotide incorporating this synthon, the benzaldehyde function was generated by fluoride deprotection and oxidation by sodium periodate. In a similar manner, an oligonucleotide where a nucleic base was replaced by the (CH2)8CH=O chain was synthesized and conjugated with biotin derivatives.

  12. Zip nucleic acids are potent hydrolysis probes for quantitative PCR

    PubMed Central

    Paris, Clément; Moreau, Valérie; Deglane, Gaëlle; Voirin, Emilie; Erbacher, Patrick; Lenne-Samuel, Nathalie

    2010-01-01

    Zip nucleic acids (ZNAs) are oligonucleotides conjugated with cationic spermine units that increase affinity for their target. ZNAs were recently shown to enable specific and sensitive reactions when used as primers for polymerase chain reaction (PCR) and reverse-transcription. Here, we report their use as quantitative PCR hydrolysis probes. Ultraviolet duplex melting data demonstrate that attachment of cationic residues to the 3′ end of an oligonucleotide does not alter its ability to discriminate nucleotides nor the destabilization pattern relative to mismatch location in the oligonucleotide sequence. The stability increase provided by the cationic charges allows the use of short dual-labeled probes that significantly improve single-nucleotide polymorphism genotyping. Longer ZNA probes were shown to display reduced background fluorescence, therefore, generating greater sensitivity and signal level as compared to standard probes. ZNA probes thus provide broad flexibility in assay design and also represent an effective alternative to minor groove binder- and locked nucleic-acid-containing probes. PMID:20071749

  13. Chromosome-Specific Painting in Cucumis Species Using Bulked Oligonucleotides

    PubMed Central

    Han, Yonghua; Zhang, Tao; Thammapichai, Paradee; Weng, Yiqun; Jiang, Jiming

    2015-01-01

    Chromosome-specific painting is a powerful technique in molecular cytogenetic and genome research. We developed an oligonucleotide (oligo)-based chromosome painting technique in cucumber (Cucumis sativus) that will be applicable in any plant species with a sequenced genome. Oligos specific to a single chromosome of cucumber were identified using a newly developed bioinformatic pipeline and then massively synthesized de novo in parallel. The synthesized oligos were amplified and labeled with biotin or digoxigenin for use in fluorescence in situ hybridization (FISH). We developed three different probes with each containing 23,000–27,000 oligos. These probes spanned 8.3–17 Mb of DNA on targeted cucumber chromosomes and had the densities of 1.5–3.2 oligos per kilobases. These probes produced FISH signals on a single cucumber chromosome and were used to paint homeologous chromosomes in other Cucumis species diverged from cucumber for up to 12 million years. The bulked oligo probes allowed us to track a single chromosome in early stages during meiosis. We were able to precisely map the pairing between cucumber chromosome 7 and chromosome 1 of Cucumis hystrix in a F1 hybrid. These two homeologous chromosomes paired in 71% of prophase I cells but only 25% of metaphase I cells, which may provide an explanation of the higher recombination rates compared to the chiasma frequencies between homeologous chromosomes reported in plant hybrids. PMID:25971668

  14. Multipathogen oligonucleotide microarray for environmental and biodefense applications.

    PubMed

    Sergeev, Nikolay; Distler, Margaret; Courtney, Shannon; Al-Khaldi, Sufian F; Volokhov, Dmitriy; Chizhikov, Vladimir; Rasooly, Avraham

    2004-11-01

    Food-borne pathogens are a major health problem. The large and diverse number of microbial pathogens and their virulence factors has fueled interest in technologies capable of detecting multiple pathogens and multiple virulence factors simultaneously. Some of these pathogens and their toxins have potential use as bioweapons. DNA microarray technology allows the simultaneous analysis of thousands of sequences of DNA in a relatively short time, making it appropriate for biodefense and for public health uses. This paper describes methods for using DNA microarrays to detect and analyze microbial pathogens. The FDA-1 microarray was developed for the simultaneous detection of several food-borne pathogens and their virulence factors including Listeria spp., Campylobacter spp., Staphylococcus aureus enterotoxin genes and Clostridium perfringens toxin genes. Three elements were incorporated to increase confidence in the microarray detection system: redundancy of genes, redundancy of oligonucleotide probes (oligoprobes) for a specific gene, and quality control oligoprobes to monitor array spotting and target DNA hybridization. These elements enhance the reliability of detection and reduce the chance of erroneous results due to the genetic variability of microbes or technical problems with the microarray. The results presented demonstrate the potential of oligonucleotide microarrays for detection of environmental and biodefense relevant microbial pathogens.

  15. Differentiation of the seven major lyssavirus species by oligonucleotide microarray.

    PubMed

    Xi, Jin; Guo, Huancheng; Feng, Ye; Xu, Yunbin; Shao, Mingfu; Su, Nan; Wan, Jiayu; Li, Jiping; Tu, Changchun

    2012-03-01

    An oligonucleotide microarray, LyssaChip, has been developed and verified as a highly specific diagnostic tool for differentiation of the 7 major lyssavirus species. As with conventional typing microarray methods, the LyssaChip relies on sequence differences in the 371-nucleotide region coding for the nucleoprotein. This region was amplified using nested reverse transcription-PCR primers that bind to the 7 major lyssaviruses. The LyssaChip includes 57 pairs of species typing and corresponding control oligonucleotide probes (oligoprobes) immobilized on glass slides, and it can analyze 12 samples on a single slide within 8 h. Analysis of 111 clinical brain specimens (65 from animals with suspected rabies submitted to the laboratory and 46 of butchered dog brain tissues collected from restaurants) showed that the chip method was 100% sensitive and highly consistent with the "gold standard," a fluorescent antibody test (FAT). The chip method could detect rabies virus in highly decayed brain tissues, whereas the FAT did not, and therefore the chip test may be more applicable to highly decayed brain tissues than the FAT. LyssaChip may provide a convenient and inexpensive alternative for diagnosis and differentiation of rabies and rabies-related diseases.

  16. Portable system for microbial sample preparation and oligonucleotide microarray analysis.

    SciTech Connect

    Bavykin, S. G.; Akowski, J. P.; Zakhariev, V. M.; Barsky, V. E.; Mirzabekov, A. D.; Perov, A. N.; Biochip Technology Center; Engelhardt Inst. of Molecular Biology

    2001-02-01

    We have developed a three-component system for microbial identification that consists of (i) a universal syringe-operated silica minicolumn for successive DNA and RNA isolation, fractionation, fragmentation, fluorescent labeling, and removal of excess free label and short oligonucleotides; (ii) microarrays of immobilized oligonucleotide probes for 16S rRNA identification; and (iii) a portable battery-powered device for imaging the hybridization of fluorescently labeled RNA fragments with the arrays. The minicolumn combines a guanidine thiocyanate method of nucleic acid isolation with a newly developed hydroxyl radical-based technique for DNA and RNA labeling and fragmentation. DNA and RNA can also be fractionated through differential binding of double- and single-stranded forms of nucleic acids to the silica. The procedure involves sequential washing of the column with different solutions. No vacuum filtration steps, phenol extraction, or centrifugation is required. After hybridization, the overall fluorescence pattern is captured as a digital image or as a Polaroid photo. This three-component system was used to discriminate Escherichia coli, Bacillus subtilis, Bacillus thuringiensis, and human HL60 cells. The procedure is rapid: beginning with whole cells, it takes approximately 25 min to obtain labeled DNA and RNA samples and an additional 25 min to hybridize and acquire the microarray image using a stationary image analysis system or the portable imager.

  17. An oligonucleotide hybridization approach to DNA sequencing.

    PubMed

    Khrapko, K R; Lysov YuP; Khorlyn, A A; Shick, V V; Florentiev, V L; Mirzabekov, A D

    1989-10-09

    We have proposed a DNA sequencing method based on hybridization of a DNA fragment to be sequenced with the complete set of fixed-length oligonucleotides (e.g., 4(8) = 65,536 possible 8-mers) immobilized individually as dots of a 2-D matrix [(1989) Dokl. Akad. Nauk SSSR 303, 1508-1511]. It was shown that the list of hybridizing octanucleotides is sufficient for the computer-assisted reconstruction of the structures for 80% of random-sequence fragments up to 200 bases long, based on the analysis of the octanucleotide overlapping. Here a refinement of the method and some experimental data are presented. We have performed hybridizations with oligonucleotides immobilized on a glass plate, and obtained their dissociation curves down to heptanucleotides. Other approaches, e.g., an additional hybridization of short oligonucleotides which continuously extend duplexes formed between the fragment and immobilized oligonucleotides, should considerably increase either the probability of unambiguous reconstruction, or the length of reconstructed sequences, or decrease the size of immobilized oligonucleotides.

  18. Rapid and specific detection of Lassa virus by reverse transcription-PCR coupled with oligonucleotide array hybridization.

    PubMed

    Olschläger, Stephan; Günther, Stephan

    2012-07-01

    To facilitate sequence-specific detection of DNA amplified in a diagnostic reverse transcription (RT)-PCR for Lassa virus, we developed an array featuring 47 oligonucleotide probes for post-PCR hybridization of the amplicons. The array procedure may be performed with low-tech equipment and does not take longer than agarose gel detection.

  19. Rapid large-scale oligonucleotide selection for microarrays.

    PubMed

    Rahmann, Sven

    2002-01-01

    We present the first algorithm that selects oligonucleotide probes (e.g. 25-mers) for microarray experiments on a large scale. For example, oligos for human genes can be found within 50 hours. This becomes possible by using the longest common substring as a specificity measure for candidate oligos. We present an algorithm based on a suffix array with additional information that is efficient both in terms of memory usage and running time to rank all candidate oligos according to their specificity. We also introduce the concept of master sequences to describe the sequences from which oligos are to be selected. Constraints such as oligo length, melting temperature, and self-complementarity are incorporated in the master sequence at a preprocessing stage and thus kept separate from the main selection problem. As a result, custom oligos can now be designed for any sequenced genome, just as the technology for on-site chip synthesis is becoming increasingly mature.

  20. Optimization of an oligonucleotide microchip for microbial identification studies: a non-equilibrium dissociation approach

    NASA Technical Reports Server (NTRS)

    Liu, W. T.; Mirzabekov, A. D.; Stahl, D. A.

    2001-01-01

    The utility of a high-density oligonucleotide microarray (microchip) for identifying strains of five closely related bacilli (Bacillus anthracis, Bacillus cereus, Bacillus mycoides, Bacillus medusa and Bacillus subtilis) was demonstrated using an approach that compares the non-equilibrium dissociation rates ('melting curves') of all probe-target duplexes simultaneously. For this study, a hierarchical set of 30 oligonucleotide probes targeting the 16S ribosomal RNA of these bacilli at multiple levels of specificity (approximate taxonomic ranks of domain, kingdom, order, genus and species) was designed and immobilized in a high-density matrix of gel pads on a glass slide. Reproducible melting curves for probes with different levels of specificity were obtained using an optimized salt concentration. Clear discrimination between perfect match (PM) and mismatch (MM) duplexes was achieved. By normalizing the signals to an internal standard (a universal probe), a more than twofold discrimination (> 2.4x) was achieved between PM and 1-MM duplexes at the dissociation temperature at which 50% of the probe-target duplexes remained intact. This provided excellent differentiation among representatives of different Bacillus species, both individually and in mixtures of two or three. The overall pattern of hybridization derived from this hierarchical probe set also provided a clear 'chip fingerprint' for each of these closely related Bacillus species.

  1. Optimization of an oligonucleotide microchip for microbial identification studies: a non-equilibrium dissociation approach

    NASA Technical Reports Server (NTRS)

    Liu, W. T.; Mirzabekov, A. D.; Stahl, D. A.

    2001-01-01

    The utility of a high-density oligonucleotide microarray (microchip) for identifying strains of five closely related bacilli (Bacillus anthracis, Bacillus cereus, Bacillus mycoides, Bacillus medusa and Bacillus subtilis) was demonstrated using an approach that compares the non-equilibrium dissociation rates ('melting curves') of all probe-target duplexes simultaneously. For this study, a hierarchical set of 30 oligonucleotide probes targeting the 16S ribosomal RNA of these bacilli at multiple levels of specificity (approximate taxonomic ranks of domain, kingdom, order, genus and species) was designed and immobilized in a high-density matrix of gel pads on a glass slide. Reproducible melting curves for probes with different levels of specificity were obtained using an optimized salt concentration. Clear discrimination between perfect match (PM) and mismatch (MM) duplexes was achieved. By normalizing the signals to an internal standard (a universal probe), a more than twofold discrimination (> 2.4x) was achieved between PM and 1-MM duplexes at the dissociation temperature at which 50% of the probe-target duplexes remained intact. This provided excellent differentiation among representatives of different Bacillus species, both individually and in mixtures of two or three. The overall pattern of hybridization derived from this hierarchical probe set also provided a clear 'chip fingerprint' for each of these closely related Bacillus species.

  2. Detection of Ligand-Induced Conformational Changes in Oligonucleotides by Second-Harmonic Generation at a Supported Lipid Bilayer Interface.

    PubMed

    Butko, Margaret T; Moree, Ben; Mortensen, Richard B; Salafsky, Joshua

    2016-11-01

    There is a high demand for characterizing oligonucleotide structural changes associated with binding interactions as well as identifying novel binders that modulate their structure and function. In this study, second-harmonic generation (SHG) was used to study RNA and DNA oligonucleotide conformational changes associated with ligand binding. For this purpose, we developed an avidin-based biotin capture surface based on a supported lipid bilayer membrane. The technique was applied to two well-characterized aptamers, both of which undergo conformational changes upon binding either a protein or a small molecule ligand. In both cases, SHG was able to resolve conformational changes in these oligonucleotides sensitively and specifically, in solution and in real time, using nanogram amounts of material. In addition, we developed a competition assay for the oligonucleotides between the specific ligands and known, nonspecific binders, and we demonstrated that intercalators and minor groove binders affect the conformation of the DNA and RNA oligonucleotides in different ways upon binding and subsequently block specific ligand binding in all cases. Our work demonstrates the broad potential of SHG for studying oligonucleotides and their conformational changes upon interaction with ligands. As SHG offers a powerful, high-throughput screening approach, our results here also open an important new avenue for identifying novel chemical probes or sequence-targeted drugs that disrupt or modulate DNA or RNA structure and function.

  3. Bolaamphiphile-based nanocomplex delivery of phosphorothioate gapmer antisense oligonucleotides as a treatment for Clostridium difficile

    PubMed Central

    Hegarty, John P; Krzeminski, Jacek; Sharma, Arun K; Guzman-Villanueva, Diana; Weissig, Volkmar; Stewart, David B

    2016-01-01

    Despite being a conceptually appealing alternative to conventional antibiotics, a major challenge toward the successful implementation of antisense treatments for bacterial infections is the development of efficient oligonucleotide delivery systems. Cationic vesicles (bolasomes) composed of dequalinium chloride (“DQAsomes”) have been used to deliver plasmid DNA across the cardiolipin-rich inner membrane of mitochondria. As cardiolipin is also a component of many bacterial membranes, we investigated the application of cationic bolasomes to bacteria as an oligonucleotide delivery system. Antisense sequences designed in silico to target the expression of essential genes of the bacterial pathogen, Clostridium difficile, were synthesized as 2′-O-methyl phosphorothioate gapmer antisense oligonucleotides (ASO). These antisense gapmers were quantitatively assessed for their ability to block mRNA translation using luciferase reporter and C. difficile protein expression plasmid constructs in a coupled transcription–translation system. Cationic bolaamphiphile compounds (dequalinium derivatives) of varying alkyl chain length were synthesized and bolasomes were prepared via probe sonication of an aqueous suspension. Bolasomes were characterized by particle size distribution, zeta potential, and binding capacities for anionic oligonucleotide. Bolasomes and antisense gapmers were combined to form antisense nanocomplexes. Anaerobic C. difficile log phase cultures were treated with serial doses of gapmer nanocomplexes or equivalent amounts of empty bolasomes for 24 hours. Antisense gapmers for four gene targets achieved nanomolar minimum inhibitory concentrations for C. difficile, with the lowest values observed for oligonucleotides targeting polymerase genes rpoB and dnaE. No inhibition of bacterial growth was observed from treatments at matched dosages of scrambled gapmer nanocomplexes or plain, oligonucleotide-free bolasomes compared to untreated control cultures. We

  4. Detection of genetically modified canola using multiplex PCR coupled with oligonucleotide microarray hybridization.

    PubMed

    Schmidt, Anna-Mary; Sahota, Robert; Pope, Derek S; Lawrence, Tracy S; Belton, Mark P; Rott, Michael E

    2008-08-27

    A rapid method was developed for concurrent screening of transgenic elements in GM canola. This method utilizes a single multiplex PCR coupled with an oligonucleotide DNA array capable of simultaneously detecting the 12 approved GM canola lines in Canada. The assay includes construct-specific elements for identification of approved lines, common elements (e.g., CaMV 35S promoter, Agrobacterium tumefaciens nos terminator, or nptII gene) for screening of approved or unapproved lines, a canola-specific endogenous gene, and endogenous genes from heterologous crops to serve as additional controls. Oligonucleotide probes were validated individually for functionality and specificity by amplification of specific transgene sequences from appropriate GM canola lines corresponding to each probe sequence, and hybridization of amplicons to the array. Each target sequence hybridized to its corresponding oligonucleotide probe and no significant cross-hybridization was observed. The limit of detection was examined for the GM lines GT73, T45, and MS8/RF3, and was determined to be 0.1%, 0.1%, and 0.5%, respectively, well within the European food and feed labeling threshold level of 0.9% for approved GM product. Practically, the method was demonstrated to be effective for the detection of GM canola in several types of animal feed, as well as in commercial canola meal.

  5. Development, Characterization and Experimental Validation of a Cultivated Sunflower (Helianthus annuus L.) Gene Expression Oligonucleotide Microarray

    PubMed Central

    Fernandez, Paula; Soria, Marcelo; Blesa, David; DiRienzo, Julio; Moschen, Sebastian; Rivarola, Maximo; Clavijo, Bernardo Jose; Gonzalez, Sergio; Peluffo, Lucila; Príncipi, Dario; Dosio, Guillermo; Aguirrezabal, Luis; García-García, Francisco; Conesa, Ana; Hopp, Esteban; Dopazo, Joaquín; Heinz, Ruth Amelia; Paniego, Norma

    2012-01-01

    Oligonucleotide-based microarrays with accurate gene coverage represent a key strategy for transcriptional studies in orphan species such as sunflower, H. annuus L., which lacks full genome sequences. The goal of this study was the development and functional annotation of a comprehensive sunflower unigene collection and the design and validation of a custom sunflower oligonucleotide-based microarray. A large scale EST (>130,000 ESTs) curation, assembly and sequence annotation was performed using Blast2GO (www.blast2go.de). The EST assembly comprises 41,013 putative transcripts (12,924 contigs and 28,089 singletons). The resulting Sunflower Unigen Resource (SUR version 1.0) was used to design an oligonucleotide-based Agilent microarray for cultivated sunflower. This microarray includes a total of 42,326 features: 1,417 Agilent controls, 74 control probes for sunflower replicated 10 times (740 controls) and 40,169 different non-control probes. Microarray performance was validated using a model experiment examining the induction of senescence by water deficit. Pre-processing and differential expression analysis of Agilent microarrays was performed using the Bioconductor limma package. The analyses based on p-values calculated by eBayes (p<0.01) allowed the detection of 558 differentially expressed genes between water stress and control conditions; from these, ten genes were further validated by qPCR. Over-represented ontologies were identified using FatiScan in the Babelomics suite. This work generated a curated and trustable sunflower unigene collection, and a custom, validated sunflower oligonucleotide-based microarray using Agilent technology. Both the curated unigene collection and the validated oligonucleotide microarray provide key resources for sunflower genome analysis, transcriptional studies, and molecular breeding for crop improvement. PMID:23110046

  6. Development, characterization and experimental validation of a cultivated sunflower (Helianthus annuus L.) gene expression oligonucleotide microarray.

    PubMed

    Fernandez, Paula; Soria, Marcelo; Blesa, David; DiRienzo, Julio; Moschen, Sebastian; Rivarola, Maximo; Clavijo, Bernardo Jose; Gonzalez, Sergio; Peluffo, Lucila; Príncipi, Dario; Dosio, Guillermo; Aguirrezabal, Luis; García-García, Francisco; Conesa, Ana; Hopp, Esteban; Dopazo, Joaquín; Heinz, Ruth Amelia; Paniego, Norma

    2012-01-01

    Oligonucleotide-based microarrays with accurate gene coverage represent a key strategy for transcriptional studies in orphan species such as sunflower, H. annuus L., which lacks full genome sequences. The goal of this study was the development and functional annotation of a comprehensive sunflower unigene collection and the design and validation of a custom sunflower oligonucleotide-based microarray. A large scale EST (>130,000 ESTs) curation, assembly and sequence annotation was performed using Blast2GO (www.blast2go.de). The EST assembly comprises 41,013 putative transcripts (12,924 contigs and 28,089 singletons). The resulting Sunflower Unigen Resource (SUR version 1.0) was used to design an oligonucleotide-based Agilent microarray for cultivated sunflower. This microarray includes a total of 42,326 features: 1,417 Agilent controls, 74 control probes for sunflower replicated 10 times (740 controls) and 40,169 different non-control probes. Microarray performance was validated using a model experiment examining the induction of senescence by water deficit. Pre-processing and differential expression analysis of Agilent microarrays was performed using the Bioconductor limma package. The analyses based on p-values calculated by eBayes (p<0.01) allowed the detection of 558 differentially expressed genes between water stress and control conditions; from these, ten genes were further validated by qPCR. Over-represented ontologies were identified using FatiScan in the Babelomics suite. This work generated a curated and trustable sunflower unigene collection, and a custom, validated sunflower oligonucleotide-based microarray using Agilent technology. Both the curated unigene collection and the validated oligonucleotide microarray provide key resources for sunflower genome analysis, transcriptional studies, and molecular breeding for crop improvement.

  7. Design and evaluation of an oligonucleotide-microarray for the detection of different species of the genus Kitasatospora.

    PubMed

    Günther, Sebastian; Groth, Ingrid; Grabley, Susanne; Munder, Thomas

    2006-05-01

    An oligonucleotide-microarray method was developed for the detection of Kitasatospora species in soil samples. The 16S-23S rDNA internal transcribed spacer (ITS) sequence of these antibiotics-producing actinomycetes was applied to design short oligonucleotide probes. Two different 26-mers were synthesized, specific to each species used. Additionally, four oligonucleotide probes were designed to evaluate the system. The oligonucleotides were spotted onto slides of the ArrayTube microarray system and examined with a new silver-labeling detection technique. Prior to hybridization analysis, the 16S-23S rDNA were amplified by polymerase chain reaction both from bacterial cells and environmental samples using two actinomycetes specific primers containing a 5' biotin labeling. The type strains of eight Kitasatospora species included in this study were K. phosalacinea DSM 43860, K. setae DSM 43861, K. cochleata DSM 41652, K. cystarginea DSM 41680, K. azatica DSM 41650, K. mediocidica DSM 43929, K. paracochleata DSM 41656, and K. griseola DSM 43859. The actinomycetes-specific primers were shown to amplify the entire 16S-23S rDNA ITS region from all tested strains. More importantly, the described technique allows the detection of Kitasatospora strains from soil samples by extracting metagenomic DNA followed by a PCR amplification step. This indicates that the oligonucleotide-microarray method developed in this study is a reliable tool for the detection of Kitasatospora species in environmental samples.

  8. A diagnostic oligonucleotide microarray for simultaneous detection of grapevine viruses.

    PubMed

    Engel, Esteban A; Escobar, Paula F; Rojas, Luis A; Rivera, Paulina A; Fiore, Nicola; Valenzuela, Pablo D T

    2010-02-01

    At least 58 viruses have been reported to infect grapevines causing economic damage globally. Conventional detection strategies based on serological assays, biological indexing and RT-PCR targeting one or few viruses in each assay are widely used. Grapevines are prone to contain mixed infections of several viruses, making the use of these techniques time-consuming. A 70-mer oligonucleotide microarray able to detect simultaneously a broad spectrum of known viruses as well as new viruses by cross-hybridization to highly conserved probes is reported in the present study. The array contains 570 unique probes designed against highly conserved and species-specific regions of 44 plant viral genomes. In addition probes designed against plant housekeeping genes are also included. By using a random primed RT-PCR amplification strategy of grapevine double stranded RNA-enriched samples, viral agents were detected in single and mixed infections. The microarray accuracy to detect 10 grapevine viruses was compared with RT-PCR yielding consistent results. For this purpose, grapevine samples containing single or mixed infections of Grapevine leafroll-associated virus-1, -2, -3, -4, -7, -9, Grapevine fanleaf virus, Grapevine rupestris stem pitting-associated virus, Grapevine virus A, and Grapevine virus B were used. Genomic libraries containing complete viral genomes were also used as part of the validation process. The specific probe hybridization pattern obtained from each virus makes this approach a powerful tool for high throughput plant certification purposes and also for virus discovery if the new viral genomic sequences have partial similarity with the microarray probes. Three Closteroviridae members (Grapevine leafroll-associated virus -4, -7 and -9) were detected for the first time in Chilean grapevines using the microarray. 2009 Elsevier B.V. All rights reserved.

  9. Surface enhanced Raman gene probe and methods thereof

    DOEpatents

    Vo-Dinh, Tuan

    1998-01-01

    The subject invention disclosed herein is a new gene probe biosensor and methods thereof based on surface enhanced Raman scattering (SERS) label detection. The SER gene probe biosensor comprises a support means, a SER gene probe having at least one oligonucleotide strand labeled with at least one SERS label, and a SERS active substrate disposed on the support means and having at least one of the SER gene probes adsorbed thereon. Biotargets such as bacterial and viral DNA, RNA and PNA are detected using a SER gene probe via hybridization to oligonucleotide strands complementary to the SER gene probe. The support means supporting the SERS active substrate includes a fiberoptic probe, an array of fiberoptic probes for performance of multiple assays and a waveguide microsensor array with charge-coupled devices or photodiode arrays.

  10. Surface enhanced Raman gene probe and methods thereof

    DOEpatents

    Vo-Dinh, T.

    1998-07-21

    The subject invention disclosed is a new gene probe biosensor and methods based on surface enhanced Raman scattering (SERS) label detection. The SER gene probe biosensor comprises a support means, a SER gene probe having at least one oligonucleotide strand labeled with at least one SERS label, and a SERS active substrate disposed on the support means and having at least one of the SER gene probes adsorbed. Biotargets such as bacterial and viral DNA, RNA and PNA are detected using a SER gene probe via hybridization to oligonucleotide strands complementary to the SER gene probe. The support means supporting the SERS active substrate includes a fiberoptic probe, an array of fiberoptic probes for performance of multiple assays and a waveguide microsensor array with charge-coupled devices or photodiode arrays. 18 figs.

  11. Surface enhanced Raman gene probe and methods thereof

    DOEpatents

    Vo-Dinh, T.

    1998-09-29

    The subject invention disclosed herein is a new gene probe biosensor and methods based on surface enhanced Raman scattering (SERS) label detection. The SER gene probe biosensor comprises a support means, a SER gene probe having at least one oligonucleotide strand labeled with at least one SERS label, and a SERS active substrate disposed on the support means and having at least one of the SER gene probes adsorbed thereon. Biotargets such as bacterial and viral DNA, RNA and PNA are detected using a SER gene probe via hybridization to oligonucleotide strands complementary to the SER gene probe. The support means supporting the SERS active substrate includes a fiberoptic probe, an array of fiberoptic probes for performance of multiple assays and a waveguide microsensor array with charge-coupled devices or photodiode arrays. 18 figs.

  12. Surface enhanced Raman gene probe and methods thereof

    DOEpatents

    Vo-Dinh, T.

    1998-02-24

    The subject invention disclosed is a new gene probe biosensor and methods based on surface enhanced Raman scattering (SERS) label detection. The SER gene probe biosensor comprises a support means, a SER gene probe having at least one oligonucleotide strand labeled with at least one SERS label, and a SERS active substrate disposed on the support means and having at least one of the SER gene probes adsorbed thereon. Biotargets such as bacterial and viral DNA, RNA and PNA are detected using a SER gene probe via hybridization to oligonucleotide strands complementary to the SER gene probe. The support means includes a fiberoptic probe, an array of fiberoptic probes for performance of multiple assays and a waveguide microsensor array with charge-coupled devices or photodiode arrays. 18 figs.

  13. Direct fluorescence in situ hybridization on human metaphase chromosomes using quantum dot-platinum labeled DNA probes

    SciTech Connect

    Hwang, Gyoyeon; Lee, Hansol; Lee, Jiyeon

    2015-11-13

    The telomere shortening in chromosomes implies the senescence, apoptosis, or oncogenic transformation of cells. Since detecting telomeres in aging and diseases like cancer, is important, the direct detection of telomeres has been a very useful biomarker. We propose a telomere detection method using a newly synthesized quantum dot (QD) based probe with oligonucleotide conjugation and direct fluorescence in situ hybridization (FISH). QD-oligonucleotides were prepared with metal coordination bonding based on platinum-guanine binding reported in our previous work. The QD-oligonucleotide conjugation method has an advantage where any sequence containing guanine at the end can be easily bound to the starting QD-Pt conjugate. A synthesized telomeric oligonucleotide was bound to the QD-Pt conjugate successfully and this probe hybridized specifically on the telomere of fabricated MV-4-11 and MOLT-4 chromosomes. Additionally, the QD-telomeric oligonucleotide probe successfully detected the telomeres on the CGH metaphase slide. Due to the excellent photostability and high quantum yield of QDs, the QD-oligonucleotide probe has high fluorescence intensity when compared to the organic dye-oligonucleotide probe. Our QD-oligonucleotide probe, conjugation method of this QD probe, and hybridization protocol with the chromosomes can be a useful tool for chromosome painting and FISH. - Highlights: • We prepared a probe linked between QD and telomeric oligonucleotide with platinum-guanine bonding. • Telomeres were detected by our new telomere probes successfully in three different human metaphase chromosomes. • QDPt-DNA probe has high fluorescence intensity in comparison with organic dye-DNA probe.

  14. Comparison of small molecules and oligonucleotides that target a toxic, non-coding RNA.

    PubMed

    Costales, Matthew G; Rzuczek, Suzanne G; Disney, Matthew D

    2016-06-01

    Potential RNA targets for chemical probes and therapeutic modalities are pervasive in the transcriptome. Oligonucleotide-based therapeutics are commonly used to target RNA sequence. Small molecules are emerging as a modality to target RNA structures selectively, but their development is still in its infancy. In this work, we compare the activity of oligonucleotides and several classes of small molecules that target the non-coding r(CCUG) repeat expansion (r(CCUG)(exp)) that causes myotonic dystrophy type 2 (DM2), an incurable disease that is the second-most common cause of adult onset muscular dystrophy. Small molecule types investigated include monomers, dimers, and multivalent compounds synthesized on-site by using RNA-templated click chemistry. Oligonucleotides investigated include phosphorothioates that cleave their target and vivo-morpholinos that modulate target RNA activity via binding. We show that compounds assembled on-site that recognize structure have the highest potencies amongst small molecules and are similar in potency to a vivo-morpholino modified oligonucleotide that targets sequence. These studies are likely to impact the design of therapeutic modalities targeting other repeats expansions that cause fragile X syndrome and amyotrophic lateral sclerosis, for example.

  15. Oligonucleotide recombination in gram negative bacteria

    USDA-ARS?s Scientific Manuscript database

    This report describes several key aspects of a novel form of RecA-independent homologous recombination. We found that synthetic single stranded DNA oligonucleotides (oligos) introduced into bacteria by transformation can site-specifically recombine with bacterial chromosomes in the absence of any a...

  16. Construction and evaluation of a Clostridium thermocellum ATCC 27405 whole-genome oligonucleotide microarray

    SciTech Connect

    Brown, Steven David; Raman, Babu; McKeown, Catherine K; Kale, Shubhangi P; He, Zhili; Mielenz, Jonathan R

    2007-04-01

    Clostridium thermocellum is an anaerobic, thermophilic bacterium that can directly convert cellulosic substrates into ethanol. Microarray technology is a powerful tool to gain insights into cellular processes by examining gene expression under various physiological states. Oligonucleotide microarray probes were designed for 96.7% of the 3163 C. thermocellum ATCC 27405 candidate protein-encoding genes and then a partial-genome microarray containing 70 C. thermocellum specific probes was constructed and evaluated. We detected a signal-to-noise ratio of three with as little as 1.0 ng of genomic DNA and only low signals from negative control probes (nonclostridial DNA), indicating the probes were sensitive and specific. In order to further test the specificity of the array we amplified and hybridized 10 C. thermocellum polymerase chain reaction products that represented different genes and found gene specific hybridization in each case. We also constructed a whole-genome microarray and prepared total cellular RNA from the same point in early-logarithmic growth phase from two technical replicates during cellobiose fermentation. The reliability of the microarray data was assessed by cohybridization of labeled complementary DNA from the cellobiose fermentation samples and the pattern of hybridization revealed a linear correlation. These results taken together suggest that our oligonucleotide probe set can be used for sensitive and specific C. thermocellum transcriptomic studies in the future.

  17. Liver as a target for oligonucleotide therapeutics.

    PubMed

    Sehgal, Alfica; Vaishnaw, Akshay; Fitzgerald, Kevin

    2013-12-01

    Oligonucleotide-based therapeutics are an emerging class of drugs that hold the promise for silencing "un-druggable" targets,thus creating unique opportunities for innovative medicines. As opposed to gene therapy, oligonucleotides are considered to be more akin to small molecule therapeutics because they are small,completely synthetic in origin, do not integrate into the host genome,and have a defined duration of therapeutic activity after which effects recover to baseline. They offer a high degree of specificity at the genetic level, thereby reducing off-target effects.At the same time, they provide a strategy for targeting any gene in the genome, including transcripts that produce mutated proteins.Oligonucleotide-based therapeutics include short interfering RNA (siRNA), that degrade target mRNA through RISC mediated RNAi; anti-miRs, that target miRNAs; miRNA mimics, that regulate target mRNA; antisense oligonucleotides, that may be working through RNAseH mediated mRNA decay; mRNA upregulation,by targeting long non-coding RNAs; and oligonucleotides induced alternative splicing [1]. All these approaches require some minimal degree of homology at the nucleic acid sequence level for them to be functional. The different mechanisms of action and their relevant activity are outlined in Fig. 1. Besides homology,RNA secondary structure has also been exploited in the case of ribozymes and aptamers, which act by binding to nucleic acids or proteins, respectively. While there have been many reports of gene knockdown and gene modulation in cell lines and mice with all these methods, very few have advanced to clinical stages.The main obstacle to date has been the safe and effective intracellular delivery of these compounds in higher species, including humans. Indeed, their action requires direct interaction with DNA/RNA within the target cell so even when one solves the issues of tissue and cellular access, intracellular/intranuclear location represents yet another barrier to

  18. Formation of N-branched oligonucleotides as by-products in solid-phase oligonucleotide synthesis.

    PubMed

    Cazenave, Christian; Bathany, Katell; Rayner, Bernard

    2006-01-01

    During the synthesis of oligonucleotides by the standard phosphoramidite method using 2'-deoxycytidine- derivatized solid support, a side reaction was observed that gave rise to the formation of high molecular weight N-branched oligomers having two identical chains linked to the 3'-terminal 2'-deoxycytidine. Postsynthesis treatment with neat triethylamine trihydrofluoride selectively cleaved the phosphoramidate linkage and converted the N-branched oligomers back to the expected oligonucleotides.

  19. Chemical ligation of oligonucleotides using an electrophilic phosphorothioester

    PubMed Central

    Oikawa, Ryota; Hayakawa, Mayu; Takamori, Shono; Kimura, Yasuaki; Abe, Naoko; Tsuji, Genichiro; Matsuda, Akira; Shuto, Satoshi; Ito, Yoshihiro

    2017-01-01

    Abstract We developed a new approach for chemical ligation of oligonucleotides using the electrophilic phosphorothioester (EPT) group. A nucleophilic phosphorothioate group on oligonucleotides was converted into the EPT group by treatment with Sanger's reagent (1-fluoro-2,4-dinitrobenzene). EPT oligonucleotides can be isolated, stored frozen, and used for the ligation reaction. The reaction of the EPT oligonucleotide and an amino-modified oligonucleotide took place without any extra reagents at pH 7.0–8.0 at room temperature, and resulted in a ligation product with a phosphoramidate bond with a 39–85% yield. This method has potential uses in biotechnology and chemical biology. PMID:28520986

  20. Detection of Glucose with Atomic Absorption Spectroscopy by Using Oligonucleotide Functionalized Gold Nanoparticle.

    PubMed

    Zhang, Hong; Yan, Honglian; Ling, Liansheng

    2016-06-01

    A novel method for the detection of glucose was established with atomic absorption spectroscopy by using the label of gold nanoparticle (AuNP). Silver-coated glass assembled with oligonucleotide 5'-SH-T12-AGA CAA GAG AGG-3' (Oligo 1) was acted as separation probe, oligonucleotide 5'-CAA CAG AGA ACG-T12-SH-3' modified gold nanoparticle (AuNP-Oligo 2) was acted as signal-reporting probe. Oligonucleotide 5'-CGT TCT CTG TTG CCT CTC TTG TCT-3' (Oligo 3) could hybridize with Oligo 1 on the surface of silver-coated glass and AuNP-Oligo 2, and free AuNP-Oligo 2 could be removed by rinsing with buffer. Hence the concentration of Oligo 3 was transformed into the concentration of gold element. In addition, Oligo 3 could be cleaved into DNA fragments by glucose, glucose oxidase and Fe(2+)-EDTA through Fenton reaction. Thereby the concentration of glucose could be transformed to the absorbance of gold element. Under the optimum conditions, the integrated absorbance decreased proportionally to the concentration of glucose over the range from 50.0 μM to 1.0 mM with a detection limit of 40.0 μM. Moreover, satisfactory result was obtained when the assay was used to determinate glucose in human serum.

  1. Molecular probe technology detects bacteria without culture

    PubMed Central

    2012-01-01

    Background Our ultimate goal is to detect the entire human microbiome, in health and in disease, in a single reaction tube, and employing only commercially available reagents. To that end, we adapted molecular inversion probes to detect bacteria using solely a massively multiplex molecular technology. This molecular probe technology does not require growth of the bacteria in culture. Rather, the molecular probe technology requires only a sequence of forty sequential bases unique to the genome of the bacterium of interest. In this communication, we report the first results of employing our molecular probes to detect bacteria in clinical samples. Results While the assay on Affymetrix GenFlex Tag16K arrays allows the multiplexing of the detection of the bacteria in each clinical sample, one Affymetrix GenFlex Tag16K array must be used for each clinical sample. To multiplex the clinical samples, we introduce a second, independent assay for the molecular probes employing Sequencing by Oligonucleotide Ligation and Detection. By adding one unique oligonucleotide barcode for each clinical sample, we combine the samples after processing, but before sequencing, and sequence them together. Conclusions Overall, we have employed 192 molecular probes representing 40 bacteria to detect the bacteria in twenty-one vaginal swabs as assessed by the Affymetrix GenFlex Tag16K assay and fourteen of those by the Sequencing by Oligonucleotide Ligation and Detection assay. The correlations among the assays were excellent. PMID:22404909

  2. Identification of characteristic oligonucleotides in the bacterial 16S ribosomal RNA sequence dataset

    NASA Technical Reports Server (NTRS)

    Zhang, Zhengdong; Willson, Richard C.; Fox, George E.

    2002-01-01

    MOTIVATION: The phylogenetic structure of the bacterial world has been intensively studied by comparing sequences of 16S ribosomal RNA (16S rRNA). This database of sequences is now widely used to design probes for the detection of specific bacteria or groups of bacteria one at a time. The success of such methods reflects the fact that there are local sequence segments that are highly characteristic of particular organisms or groups of organisms. It is not clear, however, the extent to which such signature sequences exist in the 16S rRNA dataset. A better understanding of the numbers and distribution of highly informative oligonucleotide sequences may facilitate the design of hybridization arrays that can characterize the phylogenetic position of an unknown organism or serve as the basis for the development of novel approaches for use in bacterial identification. RESULTS: A computer-based algorithm that characterizes the extent to which any individual oligonucleotide sequence in 16S rRNA is characteristic of any particular bacterial grouping was developed. A measure of signature quality, Q(s), was formulated and subsequently calculated for every individual oligonucleotide sequence in the size range of 5-11 nucleotides and for 15mers with reference to each cluster and subcluster in a 929 organism representative phylogenetic tree. Subsequently, the perfect signature sequences were compared to the full set of 7322 sequences to see how common false positives were. The work completed here establishes beyond any doubt that highly characteristic oligonucleotides exist in the bacterial 16S rRNA sequence dataset in large numbers. Over 16,000 15mers were identified that might be useful as signatures. Signature oligonucleotides are available for over 80% of the nodes in the representative tree.

  3. Identification of characteristic oligonucleotides in the bacterial 16S ribosomal RNA sequence dataset

    NASA Technical Reports Server (NTRS)

    Zhang, Zhengdong; Willson, Richard C.; Fox, George E.

    2002-01-01

    MOTIVATION: The phylogenetic structure of the bacterial world has been intensively studied by comparing sequences of 16S ribosomal RNA (16S rRNA). This database of sequences is now widely used to design probes for the detection of specific bacteria or groups of bacteria one at a time. The success of such methods reflects the fact that there are local sequence segments that are highly characteristic of particular organisms or groups of organisms. It is not clear, however, the extent to which such signature sequences exist in the 16S rRNA dataset. A better understanding of the numbers and distribution of highly informative oligonucleotide sequences may facilitate the design of hybridization arrays that can characterize the phylogenetic position of an unknown organism or serve as the basis for the development of novel approaches for use in bacterial identification. RESULTS: A computer-based algorithm that characterizes the extent to which any individual oligonucleotide sequence in 16S rRNA is characteristic of any particular bacterial grouping was developed. A measure of signature quality, Q(s), was formulated and subsequently calculated for every individual oligonucleotide sequence in the size range of 5-11 nucleotides and for 15mers with reference to each cluster and subcluster in a 929 organism representative phylogenetic tree. Subsequently, the perfect signature sequences were compared to the full set of 7322 sequences to see how common false positives were. The work completed here establishes beyond any doubt that highly characteristic oligonucleotides exist in the bacterial 16S rRNA sequence dataset in large numbers. Over 16,000 15mers were identified that might be useful as signatures. Signature oligonucleotides are available for over 80% of the nodes in the representative tree.

  4. A synthetic strategy for regio- and stereoselective site specific modification of oligonucleotides by hydrocarbon diol epoxides

    SciTech Connect

    Chaturvedi, S.

    1993-01-01

    The primary metabolites formed by the oxidative metabolism of polycyclic aromatic hydrocarbons (PAHs) are phenols, quinones, bay region diol epoxides and their corresponding trans-dihydrodiols. These electrophilic diol epoxides intercalate and bind covalently to cellular DNA. Existing evidence suggests that covalent binding of carcinogen diol epoxides to DNA causes cell transformation either due to improper lesion repair or due to base mismatch in the vicinity of the adducted nucleoside during DNA replication which lead to point mutations. The mechanism of cell transformation and the mechanism of carcinogenesis at the molecular level is not yet understood. This has therefore encouraged the author to synthesize PAH adducted deoxyadensosines followed by their rational site specific incorporation into a defined DNA sequence of biological importance. This method developed by the author provides oligonucleotides containing both the (+) and ([minus]) PAH diol epoxide adducts in significant amounts. The adducted oligonucleotides are characterized by UV, CD and negative ion FAB spectroscopy. This dissertation describes the synthesis of model adducts and their incorporation into a pentamer (TpGpA*pGpT). The synthesis of activated phosphoramidite derivatives of B[a]P followed by their incorporation into oligonucleotides comprising codons 60-62 of the human K-ras b proto-oncogene d(5[prime]-GGTCA*CGAG) (where A* is the modified base) has been performed. These oligonucleosides having both (+) or ([minus]) isomer of diol epoxides could be used for site-directed mutagenesis. The solution structure of oligonucleotides containing the (+) and ([minus]) isomers of PAH diol epoxide could also be performed by NMR. The action of repair enzymes and their activity on oligonucleotides containing (+) and ([minus]) isomer of PAH diol epoxide could also be probed.

  5. A Solution to the Common Problem of the Synthesis and Applications of Hexachlorofluorescein Labeled Oligonucleotides

    PubMed Central

    Chuvilin, Andrey N.; Smirnov, Igor P.; Mosina, Alena G.; Varizhuk, Anna M.; Pozmogova, Galina E.

    2016-01-01

    A common problem of the preparation of hexachlorofluorescein labeled oligonucleotides is the transformation of the fluorophore to an arylacridine derivative under standard ammonolysis conditions. We show here that the arylacridine byproduct with distinct optical characteristics cannot be efficiently separated from the major product by HPLC or electrophoretic methods, which hampers precise physicochemical experiments with the labeled oligonucleotides. Studies of the transformation mechanism allowed us to select optimal conditions for avoiding the side reaction. The novel method for the post-synthetic deblocking of hexachlorofluorescein-labeled oligodeoxyribonucleotides described in this paper prevents the formation of the arylacridine derivative, enhances the yield of target oligomers, and allows them to be proper real-time PCR probes. PMID:27861573

  6. In situ oligonucleotide synthesis on carbon materials: stable substrates for microarray fabrication

    PubMed Central

    Phillips, Margaret F.; Lockett, Matthew R.; Rodesch, Matthew J.; Shortreed, Michael R.; Cerrina, Franco; Smith, Lloyd M.

    2008-01-01

    Glass has become the standard substrate for the preparation of DNA arrays. Typically, glass is modified using silane chemistries to provide an appropriate functional group for nucleic acid synthesis or oligonucleotide immobilization. We have found substantial issues with the stability of these surfaces as manifested in the unwanted release of oligomers from the surface when incubated in aqueous buffers at moderate temperatures. To address this issue, we have explored the use of carbon-based substrates. Here, we demonstrate in situ synthesis of oligonucleotide probes on carbon-based substrates using light-directed photolithographic phosphoramidite chemistry and evaluate the stabilities of the resultant DNA arrays compared to those fabricated on silanized glass slides. DNA arrays on carbon-based substrates are substantially more stable than arrays prepared on glass. This superior stability enables the use of high-density DNA arrays for applications involving high temperatures, basic conditions, or where serial hybridization and dehybridization is desired. PMID:18084027

  7. Antisense oligonucleotides in therapy for neurodegenerative disorders.

    PubMed

    Evers, Melvin M; Toonen, Lodewijk J A; van Roon-Mom, Willeke M C

    2015-06-29

    Antisense oligonucleotides are synthetic single stranded strings of nucleic acids that bind to RNA and thereby alter or reduce expression of the target RNA. They can not only reduce expression of mutant proteins by breakdown of the targeted transcript, but also restore protein expression or modify proteins through interference with pre-mRNA splicing. There has been a recent revival of interest in the use of antisense oligonucleotides to treat several neurodegenerative disorders using different approaches to prevent disease onset or halt disease progression and the first clinical trials for spinal muscular atrophy and amyotrophic lateral sclerosis showing promising results. For these trials, intrathecal delivery is being used but direct infusion into the brain ventricles and several methods of passing the blood brain barrier after peripheral administration are also under investigation.

  8. Oligonucleotide-based therapy for neurodegenerative diseases.

    PubMed

    Magen, Iddo; Hornstein, Eran

    2014-10-10

    Molecular genetics insight into the pathogenesis of several neurodegenerative diseases, such as Alzheimer׳s disease, Parkinson׳s disease, Huntington׳s disease and amyotrophic lateral sclerosis, encourages direct interference with the activity of neurotoxic genes or the molecular activation of neuroprotective pathways. Oligonucleotide-based therapies are recently emerging as an efficient strategy for drug development and these can be employed as new treatments of neurodegenerative states. Here we review advances in this field in recent years which suggest an encouraging assessment that oligonucleotide technologies for targeting of RNAs will enable the development of new therapies and will contribute to preservation of brain integrity. Copyright © 2014 Elsevier B.V. All rights reserved.

  9. AMOD: a morpholino oligonucleotide selection tool

    PubMed Central

    Klee, Eric W.; Shim, Kyong Jin; Pickart, Michael A.; Ekker, Stephen C.; Ellis, Lynda B. M.

    2005-01-01

    AMOD is a web-based program that aids in the functional evaluation of nucleotide sequences through sequence characterization and antisense morpholino oligonucleotide (target site) selection. Submitted sequences are analyzed by translation initiation site prediction algorithms and sequence-to-sequence comparisons; results are used to characterize sequence features required for morpholino design. Within a defined subsequence, base composition and homodimerization values are computed for all putative morpholino oligonucleotides. Using these properties, morpholino candidates are selected and compared with genomic and transcriptome databases with the goal to identify target-specific enriched morpholinos. AMOD has been used at the University of Minnesota to design ∼200 morpholinos for a functional genomics screen in zebrafish. The AMOD web server and a tutorial are freely available to both academic and commercial users at . PMID:15980523

  10. Comparison of gene coverage of mouse oligonucleotide microarray platforms

    PubMed Central

    Verdugo, Ricardo A; Medrano, Juan F

    2006-01-01

    Background The increasing use of DNA microarrays for genetical genomics studies generates a need for platforms with complete coverage of the genome. We have compared the effective gene coverage in the mouse genome of different commercial and noncommercial oligonucleotide microarray platforms by performing an in-house gene annotation of probes. We only used information about probes that is available from vendors and followed a process that any researcher may take to find the gene targeted by a given probe. In order to make consistent comparisons between platforms, probes in each microarray were annotated with an Entrez Gene id and the chromosomal position for each gene was obtained from the UCSC Genome Browser Database. Gene coverage was estimated as the percentage of Entrez Genes with a unique position in the UCSC Genome database that is tested by a given microarray platform. Results A MySQL relational database was created to store the mapping information for 25,416 mouse genes and for the probes in five microarray platforms (gene coverage level in parenthesis): Affymetrix430 2.0 (75.6%), ABI Genome Survey (81.24%), Agilent (79.33%), Codelink (78.09%), Sentrix (90.47%); and four array-ready oligosets: Sigma (47.95%), Operon v.3 (69.89%), Operon v.4 (84.03%), and MEEBO (84.03%). The differences in coverage between platforms were highly conserved across chromosomes. Differences in the number of redundant and unspecific probes were also found among arrays. The database can be queried to compare specific genomic regions using a web interface. The software used to create, update and query the database is freely available as a toolbox named ArrayGene. Conclusion The software developed here allows researchers to create updated custom databases by using public or proprietary information on genes for any organisms. ArrayGene allows easy comparisons of gene coverage between microarray platforms for any region of the genome. The comparison presented here reveals that the

  11. The prebiotic synthesis of deoxythymidine oligonucleotides

    NASA Technical Reports Server (NTRS)

    Stephen-Sherwood, E.; Odom, D. G.; Oro, J.

    1974-01-01

    Deoxythymidine 5 prime-triphosphate in the presence of deoxythymidine 5 prime-phosphate, cyanamide and 4-amino-5-imidazole carboxamide polymerizes under drying conditions at moderate temperatures (60 to 90 C) to yield oligonucleotides of up to four units in length. Enzymatic analysis indicated that the majority of these oligomers contained natural 3 prime-5 prime phosphodiester bonds. This reaction offers a possible method for the formation of deoxyoligonucleotides under primitive earth conditions.

  12. Cationic Nanohydrogel Particles for Therapeutic Oligonucleotide Delivery.

    PubMed

    Leber, Nadine; Nuhn, Lutz; Zentel, Rudolf

    2017-06-12

    Short pharmaceutical active oligonucleotides such as small interfering RNA (siRNA) or cytidine-phosphate-guanosine (CpG) are considered as powerful therapeutic alternatives, especially to medicate hard-to-treat diseases (e.g., liver fibrosis or cancer). Unfortunately, these molecules are equipped with poor pharmacokinetic properties that prevent them from translation. Well-defined nanosized carriers can provide opportunities to optimize their delivery and guide them to their site of action. Among several concepts, this Feature Article focuses on cationic nanohydrogel particles as a universal delivery system for small anionic molecules including siRNA and CpG. Cationic nanohydrogels are derived from preaggregated precursor block copolymers, which are further cross-linked to obtain well-defined nanoparticles of tunable sizes and with (degradable) cationic cores. Novel opportunities for oligonucleotide delivery in vitro and in vivo with respect to liver fibrosis therapies will be highlighted as well as perspectives toward modulating the immune system. In general, the approach of covalently stabilized cationic carrier systems can contribute to find advanced oligonucleotide therapeutics. © 2017 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

  13. Label-Free Electrochemical Detection of the Specific Oligonucleotide Sequence of Dengue Virus Type 1 on Pencil Graphite Electrodes

    PubMed Central

    Souza, Elaine; Nascimento, Gustavo; Santana, Nataly; Ferreira, Danielly; Lima, Manoel; Natividade, Edna; Martins, Danyelly; Lima-Filho, José

    2011-01-01

    A biosensor that relies on the adsorption immobilization of the 18-mer single-stranded nucleic acid related to dengue virus gene 1 on activated pencil graphite was developed. Hybridization between the probe and its complementary oligonucleotides (the target) was investigated by monitoring guanine oxidation by differential pulse voltammetry (DPV). The pencil graphite electrode was made of ordinary pencil lead (type 4B). The polished surface of the working electrode was activated by applying a potential of 1.8 V for 5 min. Afterward, the dengue oligonucleotides probe was immobilized on the activated electrode by applying 0.5 V to the electrode in 0.5 M acetate buffer (pH 5.0) for 5 min. The hybridization process was carried out by incubating at the annealing temperature of the oligonucleotides. A time of five minutes and concentration of 1 μM were found to be the optimal conditions for probe immobilization. The electrochemical detection of annealing between the DNA probe (TS-1P) immobilized on the modified electrode, and the target (TS-1T) was achieved. The target could be quantified in a range from 1 to 40 nM with good linearity and a detection limit of 0.92 nM. The specificity of the electrochemical biosensor was tested using non-complementary sequences of dengue virus 2 and 3. PMID:22163916

  14. Kinetics of hybridization on surface oligonucleotide microchips: theory, experiment, and comparison with hybridization on gel-based microchips.

    PubMed

    Sorokin, N V; Chechetkin, V R; Pan'kov, S V; Somova, O G; Livshits, M A; Donnikov, M Y; Turygin, A Y; Barsky, V E; Zasedatelev, A S

    2006-08-01

    The optimal design of oligonucleotide microchips and efficient discrimination between perfect and mismatch duplexes strongly depend on the external transport of target DNA to the cells with immobilized probes as well as on respective association and dissociation rates at the duplex formation. In this paper we present the relevant theory for hybridization of DNA fragments with oligonucleotide probes immobilized in the cells on flat substrate. With minor modifications, our theory also is applicable to reaction-diffusion hybridization kinetics for the probes immobilized on the surface of microbeads immersed in hybridization solution. The main theoretical predictions are verified with control experiments. Besides that, we compared the characteristics of the surface and gel-based oligonucleotide microchips. The comparison was performed for the chips printed with the same pin robot, for the signals measured with the same devices and processed by the same technique, and for the same hybridization conditions. The sets of probe oligonucleotides and the concentrations of probes in respective solutions used for immobilization on each platform were identical as well. We found that, despite the slower hybridization kinetics, the fluorescence signals and mutation discrimination efficiency appeared to be higher for the gel-based microchips with respect to their surface counterparts even for the relatively short hybridization time about 0.5-1 hour. Both the divergence between signals for perfects and the difference in mutation discrimination efficiency for the counterpart platforms rapidly grow with incubation time. In particular, for hybridization during 3 h the signals for gel-based microchips surpassed their surface counterparts in 5-20 times, while the ratios of signals for perfect-mismatch pairs for gel microchips exceeded the corresponding ratios for surface microchips in 2-4 times. These effects may be attributed to the better immobilization efficiency and to the higher

  15. Template-Directed Ligation of Peptides to Oligonucleotides

    NASA Technical Reports Server (NTRS)

    Bruick, Richard K.; Dawson, Philip E.; Kent, Stephen BH; Usman, Nassim; Joyce, Gerald F.

    1996-01-01

    Synthetic oligonucleotides and peptides have enjoyed a wide range of applications in both biology and chemistry. As a consequence, oligonucleotide-peptide conjugates have received considerable attention, most notably in the development of antisense constructs with improved pharmacological properties. In addition, oligonucleotide-peptide conjugates have been used as molecular tags, in the assembly of supramolecular arrays and in the construction of encoded combinatorial libraries. To make these chimeric molecules more accessible for a broad range of investigations, we sought to develop a facile method for joining fully deprotected oligonucleotides and peptides through a stable amide bond linkage. Furthermore, we wished to make this ligation reaction addressable, enabling one to direct the ligation of specific oligonucleotide and peptide components.To confer specificity and accelerate the rate of the reaction, the ligation process was designed to be dependent on the presence of a complementary oligonucleotide template.

  16. Detection of SPO11-oligonucleotide complexes from mouse testes.

    PubMed

    Pan, Jing; Keeney, Scott

    2009-01-01

    The SPO11 protein generates programmed DNA double-strand breaks (DSBs) that initiate meiotic recombination. Endonucleolytic cleavage 3' to the DSB sites releases SPO11 from DNA, leaving SPO11 covalently associated with an oligonucleotide. This chapter describes detection of the release product, SPO11-oligonucleotide complexes, from mouse testis lysates. The method for determining the size of SPO11-associated oligonucleotides is also provided.

  17. Distance-dependent emission from dye-labeled oligonucleotides on striped Au/Ag nanowires: effect of secondary structure and hybridization efficiency.

    PubMed

    Stoermer, Rebecca L; Keating, Christine D

    2006-10-11

    When fluorescently tagged oligonucleotides are located near metal surfaces, their emission intensity is impacted by both electromagnetic effects (i.e., quenching and/or enhancement of emission) and the structure of the nucleic acids (e.g., random coil, hairpin, or duplex). We present experiments exploring the effect of label position and secondary structure in oligonucleotide probes as a function of hybridization buffer, which impacts the percentage of double-stranded probes on the surface after exposure to complementary DNA. Nanowires containing identifiable patterns of Au and Ag segments were used as the metal substrates in this work, which enabled us to directly compare different dye positions in a single multiplexed experiment and differences in emission for probes attached to the two metals. The observed metal-dye separation dependence for unstructured surface-bound oligonucleotides is highly sensitive to hybridization efficiency, due to substantial changes in DNA extension from the surface upon hybridization. In contrast, fluorophore labeled oligonucleotides designed to form hairpin secondary structures analogous to solution-phase molecular beacon probes are relatively insensitive to hybridization efficiency, since the folded form is quenched and therefore does not appreciably impact the observed distance-dependence of the response. Differences in fluorescence patterning on Au and Ag were noted as a function of not only chromophore identity but also metal-dye separation. For example, emission intensity for TAMRA-labeled oligonucleotides changed from brighter on Ag for 24-base probes to brighter on Au for 48-base probes. We also observed fluorescence enhancement at the ends of nanowires and at surface defects where heightened electromagnetic fields affect the fluorescence.

  18. Fluorescent hybridization probes for nucleic acid detection.

    PubMed

    Guo, Jia; Ju, Jingyue; Turro, Nicholas J

    2012-04-01

    Due to their high sensitivity and selectivity, minimum interference with living biological systems, and ease of design and synthesis, fluorescent hybridization probes have been widely used to detect nucleic acids both in vivo and in vitro. Molecular beacons (MBs) and binary probes (BPs) are two very important hybridization probes that are designed based on well-established photophysical principles. These probes have shown particular applicability in a variety of studies, such as mRNA tracking, single nucleotide polymorphism (SNP) detection, polymerase chain reaction (PCR) monitoring, and microorganism identification. Molecular beacons are hairpin oligonucleotide probes that present distinctive fluorescent signatures in the presence and absence of their target. Binary probes consist of two fluorescently labeled oligonucleotide strands that can hybridize to adjacent regions of their target and generate distinctive fluorescence signals. These probes have been extensively studied and modified for different applications by modulating their structures or using various combinations of fluorophores, excimer-forming molecules, and metal complexes. This review describes the applicability and advantages of various hybridization probes that utilize novel and creative design to enhance their target detection sensitivity and specificity.

  19. A factorial analysis of silanization conditions for the immobilization of oligonucleotides on glass surfaces.

    PubMed

    Halliwell, C M; Cass, A E

    2001-06-01

    The modification of glass surfaces with (3-mercaptopropyl)trimethoxysilane and the application of this to DNA chip technology are described. A range of factors influencing the silanization method, and hence the number of surface-bound, chemically active thiol groups, were investigated using a design of experiment approach based on analysis of variance. The number of thiol groups introduced on glass substrates were measured directly using a specific radiolabel, [14C]cysteamine hydrochloride. For liquid-phase silanization, the number of surface-bound thiol groups was found to be dependent on both postsilanization thermal curing and silanization time and relatively independent of silane concentration, reaction temperature, and sample pretreatment. Depending on the conditions used in liquid-phase silanization, (1.3-9.0) x 10(12) thiol groups/cm2 on the glass samples were bound. The reliability and repeatability of liquid- and vacuum-phase silanization were also investigated. Eighteen-base oligonucleotide probes were covalently attached to the modified surfaces via a 3'-amino modification on the DNA and subsequent reaction with the cross-linking reagent N-(gamma-maleimidobutyryloxy) succinimide ester (GMBS). The resulting probe levels were determined and found to be stoichiometric with that of the introduced thiol groups. These results demonstrate that silanization of glass surfaces under specific conditions, prior to probe attachment, is of great importance in the development of DNA chips that use the simple concept of the covalent attachment of presynthesized oligonucleotides to silicon oxide surfaces.

  20. Oligonucleotide gap-fill ligation for mutation detection and sequencing in situ.

    PubMed

    Mignardi, Marco; Mezger, Anja; Qian, Xiaoyan; La Fleur, Linnea; Botling, Johan; Larsson, Chatarina; Nilsson, Mats

    2015-12-15

    In clinical diagnostics a great need exists for targeted in situ multiplex nucleic acid analysis as the mutational status can offer guidance for effective treatment. One well-established method uses padlock probes for mutation detection and multiplex expression analysis directly in cells and tissues. Here, we use oligonucleotide gap-fill ligation to further increase specificity and to capture molecular substrates for in situ sequencing. Short oligonucleotides are joined at both ends of a padlock gap probe by two ligation events and are then locally amplified by target-primed rolling circle amplification (RCA) preserving spatial information. We demonstrate the specific detection of the A3243G mutation of mitochondrial DNA and we successfully characterize a single nucleotide variant in the ACTB mRNA in cells by in situ sequencing of RCA products generated by padlock gap-fill ligation. To demonstrate the clinical applicability of our assay, we show specific detection of a point mutation in the EGFR gene in fresh frozen and formalin-fixed, paraffin-embedded (FFPE) lung cancer samples and confirm the detected mutation by in situ sequencing. This approach presents several advantages over conventional padlock probes allowing simpler assay design for multiplexed mutation detection to screen for the presence of mutations in clinically relevant mutational hotspots directly in situ.

  1. An anchored framework BAC map of mouse chromosome 11 assembled using multiplex oligonucleotide hybridization.

    PubMed

    Cai, W W; Reneker, J; Chow, C W; Vaishnav, M; Bradley, A

    1998-12-15

    Despite abundant library resources for many organisms, physical mapping of these organisms has been seriously limited due to lack of efficient library screening techniques. We have developed a highly efficient strategy for large-scale screening of genomic libraries based on multiplex oligonucleotide hybridization on high-density genomic filters. We have applied this strategy to generate a bacterial artificial chromosome (BAC) anchored map of mouse chromosome 11. Using the MIT mouse SSLP data, 320 pairs of oligonucleotide probes were designed with an "overgo" computer program that selects new primer sequences that avoid the microsatellite repeat. BACs identified by these probes are automatically anchored to the chromosome. Ninety-two percent of the probes identified positive clones from a 5.9-fold coverage mouse BAC library with an average of 7 positive clones per marker. An average of 4.2 clones was confirmed for 204 markers by PCR. Our data show that a large number of clones can be efficiently isolated from a large genomic library using this strategy with minimal effort. This strategy will have wide application for large-scale mapping and sequencing of human and other large genomes.

  2. Synthesis of oligonucleotides on a soluble support

    PubMed Central

    2017-01-01

    Oligonucleotides are usually prepared in lab scale on a solid support with the aid of a fully automated synthesizer. Scaling up of the equipment has allowed industrial synthesis up to kilogram scale. In spite of this, solution-phase synthesis has received continuous interest, on one hand as a technique that could enable synthesis of even larger amounts and, on the other hand, as a gram scale laboratory synthesis without any special equipment. The synthesis on a soluble support has been regarded as an approach that could combine the advantageous features of both the solution and solid-phase syntheses. The critical step of this approach is the separation of the support-anchored oligonucleotide chain from the monomeric building block and other small molecular reagents and byproducts after each coupling, oxidation and deprotection step. The techniques applied so far include precipitation, extraction, chromatography and nanofiltration. As regards coupling, all conventional chemistries, viz. phosphoramidite, H-phosphonate and phosphotriester strategies, have been attempted. While P(III)-based phosphoramidite and H-phosphonate chemistries are almost exclusively used on a solid support, the “outdated” P(V)-based phosphotriester chemistry still offers one major advantage for the synthesis on a soluble support; the omission of the oxidation step simplifies the coupling cycle. Several of protocols developed for the soluble-supported synthesis allow the preparation of both DNA and RNA oligomers of limited length in gram scale without any special equipment, being evidently of interest for research groups that need oligonucleotides in large amounts for research purposes. However, none of them has really tested at such a scale that the feasibility of their industrial use could be critically judged. PMID:28781703

  3. Antisense delivery using protamine–oligonucleotide particles

    PubMed Central

    Junghans, Monika; Kreuter, Jörg; Zimmer, Andreas

    2000-01-01

    Protamine, a polycationic peptide (mol. wt 4000–4500), was evaluated as a potential penetration enhancer for phosphodiester antisense oligonucleotides (ODNs). Unique complexes in the form of nanoparticles were spontaneously formed, which we call ‘proticles’. The stability of the particles and the ODNs bound into the proticles was examined in foetal calf serum and cell culture medium. FITC-labelled ODNs bound to protamine showed an increased cellular uptake into human histiocytic lymphoma U 937 cells compared to free ODNs. Proticles significantly decreased cellular growth in a cell proliferation assay using ODNs against the c-myc proto-oncogene. PMID:10773093

  4. Direct profiling of environmental microbial populations by thermal dissociation analysis of native rRNAs hybridized to oligonucleotide microarrays

    NASA Technical Reports Server (NTRS)

    El Fantroussi, Said; Urakawa, Hidetoshi; Bernhard, Anne E.; Kelly, John J.; Noble, Peter A.; Smidt, H.; Yershov, G. M.; Stahl, David A.

    2003-01-01

    Oligonucleotide microarrays were used to profile directly extracted rRNA from environmental microbial populations without PCR amplification. In our initial inspection of two distinct estuarine study sites, the hybridization patterns were reproducible and varied between estuarine sediments of differing salinities. The determination of a thermal dissociation curve (i.e., melting profile) for each probe-target duplex provided information on hybridization specificity, which is essential for confirming adequate discrimination between target and nontarget sequences.

  5. Direct profiling of environmental microbial populations by thermal dissociation analysis of native rRNAs hybridized to oligonucleotide microarrays

    NASA Technical Reports Server (NTRS)

    El Fantroussi, Said; Urakawa, Hidetoshi; Bernhard, Anne E.; Kelly, John J.; Noble, Peter A.; Smidt, H.; Yershov, G. M.; Stahl, David A.

    2003-01-01

    Oligonucleotide microarrays were used to profile directly extracted rRNA from environmental microbial populations without PCR amplification. In our initial inspection of two distinct estuarine study sites, the hybridization patterns were reproducible and varied between estuarine sediments of differing salinities. The determination of a thermal dissociation curve (i.e., melting profile) for each probe-target duplex provided information on hybridization specificity, which is essential for confirming adequate discrimination between target and nontarget sequences.

  6. Rapid detection of deletional alpha-thalassemia by an oligonucleotide microarray.

    PubMed

    Zesong, Li; Ruijun, Guo; Wen, Zhang

    2005-12-01

    A simple, fast, and highly reproducible protocol for detection of the three most common deletional alpha-thalassemias (- -SEA, - alpha3.7, -alpha4.2) using an oligonucleotide microarray was developed. PCR products were directly hybridized to the microarrays with different deletion-specific probes. Genotypes were determined by quantitative analysis of the fluorescent signals detected by fluorescence scanning. Blind assays on 400 samples validated the efficiency and specificity of the protocol. This method may be suitable for routine clinical use and population screening for deletional alpha-thalassemia. (c) 2005 Wiley-Liss, Inc.

  7. Oligonucleotide-based systems: DNA, microRNAs, DNA/RNA aptamers

    PubMed Central

    Jolly, Pawan; Estrela, Pedro

    2016-01-01

    There are an increasing number of applications that have been developed for oligonucleotide-based biosensing systems in genetics and biomedicine. Oligonucleotide-based biosensors are those where the probe to capture the analyte is a strand of deoxyribonucleic acid (DNA), ribonucleic acid (RNA) or a synthetic analogue of naturally occurring nucleic acids. This review will shed light on various types of nucleic acids such as DNA and RNA (particularly microRNAs), their role and their application in biosensing. It will also cover DNA/RNA aptamers, which can be used as bioreceptors for a wide range of targets such as proteins, small molecules, bacteria and even cells. It will also highlight how the invention of synthetic oligonucleotides such as peptide nucleic acid (PNA) or locked nucleic acid (LNA) has pushed the limits of molecular biology and biosensor development to new perspectives. These technologies are very promising albeit still in need of development in order to bridge the gap between the laboratory-based status and the reality of biomedical applications. PMID:27365033

  8. Bisulfite oligonucleotide-capture sequencing for targeted base- and strand-specific absolute 5-methylcytosine quantitation.

    PubMed

    Masser, Dustin R; Stanford, David R; Hadad, Niran; Giles, Cory B; Wren, Jonathan D; Sonntag, William E; Richardson, Arlan; Freeman, Willard M

    2016-06-01

    Epigenetic regulation through DNA methylation (5mC) plays an important role in development, aging, and a variety of diseases. Genome-wide studies of base- and strand-specific 5mC are limited by the extensive sequencing required. Targeting bisulfite sequencing to specific genomic regions through sequence capture with complimentary oligonucleotide probes retains the advantages of bisulfite sequencing while focusing sequencing reads on regions of interest, enables analysis of more samples by decreasing the amount of sequence required per sample, and provides base- and strand-specific absolute quantitation of CG and non-CG methylation levels. As an example, an oligonucleotide capture set to interrogate 5mC levels in all rat RefSeq gene promoter regions (18,814) and CG islands, shores, and shelves (18,411) was generated. Validation using whole-genome methylation standards and biological samples demonstrates enrichment of the targeted regions and accurate base-specific quantitation of CG and non-CG methylation for both forward and reverse genomic strands. A total of 170 Mb of the rat genome is covered including 6.6 million CGs and over 67 million non-CG sites, while reducing the amount of sequencing required by ~85 % as compared to existing whole-genome sequencing methods. This oligonucleotide capture targeting approach and quantitative validation workflow can also be applied to any genome of interest.

  9. Image-based detection of oligonucleotides--a low cost alternative to spectrophotometric or fluorometric methods.

    PubMed

    Ahirwar, Rajesh; Tanwar, Swati; Parween, Shahila; Kumar, Ashok; Nahar, Pradip

    2014-05-07

    Herein, we report a sensitive and low cost image-based (photocolorimetric) method for the detection of oligonucleotides on an activated polypropylene microtest plate (APPμTP). The assay was developed on the APPμTP by covalently immobilising 20-mer amino-modified oligonucleotides. Biotin-tagged complementary target sequences were then hybridised with the immobilised oligonucleotides. Colour was developed by streptavidin-HRP conjugate and the image of the coloured assay solution was taken by a desktop scanner and analysed using colour saturation. The developed method was analysed for its detection limit, accuracy, sensitivity and interference. The linearity range was found to be 1.7-170 ng mL(-1) while the lower limit of detection and limit of quantification were 1.7 and 5.6 ng mL(-1) respectively. The method showed comparable sensitivity to fluorometric methods, and was found to be correlated to fluorescence (R(2) = 0.8081, p-value < 0.0001) and absorbance (R(2) = 0.9394, p-value < 0.0001)-based quantification. It discriminates mismatched base sequences from perfectly matched sequences efficiently. Validation of the method was carried out by detecting por A DNA of Neisseria meningitidis in bacterial meningitis samples. The por A-specific probe having a 6-carbon spacer at its 5'-NH2 terminus was immobilised covalently to the APPμTP and hybridised with different samples of biotinylated single-stranded por A DNA.

  10. Application of oligonucleotide microarrays for bacterial source tracking of environmental Enterococcus sp. isolates.

    PubMed

    Indest, Karl J; Betts, Kelley; Furey, John S

    2005-04-01

    In an effort towards adapting new and defensible methods for assessing and managing the risk posed by microbial pollution, we evaluated the utility of oligonucleotide microarrays for bacterial source tracking (BST) of environmental Enterococcus sp. isolates derived from various host sources. Current bacterial source tracking approaches rely on various phenotypic and genotypic methods to identify sources of bacterial contamination resulting from point or non-point pollution. For this study Enterococcus sp. isolates originating from deer, bovine, gull, and human sources were examined using microarrays. Isolates were subjected to Box PCR amplification and the resulting amplification products labeled with Cy5. Fluorescent-labeled templates were hybridized to in-house constructed nonamer oligonucleotide microarrays consisting of 198 probes. Microarray hybridization profiles were obtained using the ArrayPro image analysis software. Principal Components Analysis (PCA) and Hierarchical Cluster Analysis (HCA) were compared for their ability to visually cluster microarray hybridization profiles based on the environmental source from which the Enterococcus sp. isolates originated. The PCA was visually superior at separating origin-specific clusters, even for as few as 3 factors. A Soft Independent Modeling (SIM) classification confirmed the PCA, resulting in zero misclassifications using 5 factors for each class. The implication of these results for the application of random oligonucleotide microarrays for BST is that, given the reproducibility issues, factor-based variable selection such as in PCA and SIM greatly outperforms dendrogram-based similarity measures such as in HCA and K-Nearest Neighbor KNN.

  11. Application of Oligonucleotide Microarrays for Bacterial Source Tracking of Environmental Enterococcus sp. Isolates

    PubMed Central

    Indest, Karl J.; Betts, Kelley; Furey, John S.

    2005-01-01

    In an effort towards adapting new and defensible methods for assessing and managing the risk posed by microbial pollution, we evaluated the utility of oligonucleotide microarrays for bacterial source tracking (BST) of environmental Enterococcus sp. isolates derived from various host sources. Current bacterial source tracking approaches rely on various phenotypic and genotypic methods to identify sources of bacterial contamination resulting from point or non-point pollution. For this study Enterococcus sp. isolates originating from deer, bovine, gull, and human sources were examined using microarrays. Isolates were subjected to Box PCR amplification and the resulting amplification products labeled with Cy5. Fluorescent-labeled templates were hybridized to in-house constructed nonamer oligonucleotide microarrays consisting of 198 probes. Microarray hybridization profiles were obtained using the ArrayPro image analysis software. Principal Components Analysis (PCA) and Hierarchical Cluster Analysis (HCA) were compared for their ability to visually cluster microarray hybridization profiles based on the environmental source from which the Enterococcus sp. isolates originated. The PCA was visually superior at separating origin-specific clusters, even for as few as 3 factors. A Soft Independent Modeling (SIM) classification confirmed the PCA, resulting in zero misclassifications using 5 factors for each class. The implication of these results for the application of random oligonucleotide microarrays for BST is that, given the reproducibility issues, factor-based variable selection such as in PCA and SIM greatly outperforms dendrogram-based similarity measures such as in HCA and K-Nearest Neighbor KNN. PMID:16705816

  12. Attomolar Zika virus oligonucleotide detection based on loop-mediated isothermal amplification and AC susceptometry.

    PubMed

    Tian, Bo; Qiu, Zhen; Ma, Jing; Zardán Gómez de la Torre, Teresa; Johansson, Christer; Svedlindh, Peter; Strömberg, Mattias

    2016-12-15

    Because of the serological cross-reactivity among the flaviviruses, molecular detection methods, such as reverse-transcription polymerase chain reaction (RT-PCR), play an important role in the recent Zika outbreak. However, due to the limited sensitivity, the detection window of RT-PCR for Zika viremia is only about one week after symptom onset. By combining loop-mediated isothermal amplification (LAMP) and AC susceptometry, we demonstrate a rapid and homogeneous detection system for the Zika virus oligonucleotide. Streptavidin-magnetic nanoparticles (streptavidin-MNPs) are premixed with LAMP reagents including the analyte and biotinylated primers, and their hydrodynamic volumes are dramatically increased after a successful LAMP reaction. Analyzed by a portable AC susceptometer, the changes of the hydrodynamic volume are probed as Brownian relaxation frequency shifts, which can be used to quantify the Zika virus oligonucleotide. The proposed detection system can recognize 1 aM synthetic Zika virus oligonucleotide in 20% serum with a total assay time of 27min, which can hopefully widen the detection window for Zika viremia and is therefore promising in worldwide Zika fever control.

  13. Sequencing by hybridization with the generic 6-mer oligonucleotide microarray : an advanced scheme for data processing.

    SciTech Connect

    Chechetkin, V. R.; Turygin, A. Y.; Proudnikov, D. Y.; Prokopenko, D. V.; Kirillov, E. V.; Mirzabekov, A. D.; Biochip Technology Center; Russian Academy of Sciences

    2000-08-01

    DNA sequencing by hybridization was carried out with a microarray of all 4{sup 6} = 4,096 hexadeoxyribonucleotides (the generic microchip). The oligonucleotides immobilized in 100 x 100 x 20-{mu}m polyacrylamide gel pads of the generic microchip were hybridized with fluorescently labeled ssDNA, providing perfect and mismatched duplexes. Melting curves were measured in parallel for all microchip duplexes with a fluorescence microscope equipped with CCD camera. This allowed us to discriminate the perfect duplexes formed by the oligonucleotides, which are complementary to the target DNA. The DNA sequence was reconstructed by overlapping the complementary oligonucleotide probes. We developed a data processing scheme to heighten the discrimination of perfect duplexes from mismatched ones. The procedure was united with a reconstruction of the DNA sequence. The scheme includes the proper definition of a discriminant signal, preprocessing, and the variational principle for the sequence indicator function. The effectiveness of the procedure was confirmed by sequencing, proofreading, and nucleotide polymorphism (mutation) analysis of 13 DNA fragments from 31 to 70 nucleotides long.

  14. High-throughput detection of food-borne pathogenic bacteria using oligonucleotide microarray with quantum dots as fluorescent labels.

    PubMed

    Huang, Aihua; Qiu, Zhigang; Jin, Min; Shen, Zhiqiang; Chen, Zhaoli; Wang, Xinwei; Li, Jun-Wen

    2014-08-18

    Bacterial pathogens are mostly responsible for food-borne diseases, and there is still substantial room for improvement in the effective detection of these organisms. In the present study, we explored a new method to detect target pathogens easily and rapidly with high sensitivity and specificity. This method uses an oligonucleotide microarray combined with quantum dots as fluorescent labels. Oligonucleotide probes targeting the 16SrRNA gene were synthesized to create an oligonucleotide microarray. The PCR products labeled with biotin were subsequently hybridized using an oligonucleotide microarray. Following incubation with CdSe/ZnS quantum dots coated with streptavidin, fluorescent signals were detected with a PerkinElmer Gx Microarray Scanner. The results clearly showed specific hybridization profiles corresponding to the bacterial species assessed. Two hundred and sixteen strains of food-borne bacterial pathogens, including standard strains and isolated strains from food samples, were used to test the specificity, stability, and sensitivity of the microarray system. We found that the oligonucleotide microarray combined with quantum dots used as fluorescent labels can successfully discriminate the bacterial organisms at the genera or species level, with high specificity and stability as well as a sensitivity of 10 colony forming units (CFU)/mL of pure culture. We further tested 105 mock-contaminated food samples and achieved consistent results as those obtained from traditional biochemical methods. Together, these results indicate that the quantum dot-based oligonucleotide microarray has the potential to be a powerful tool in the detection and identification of pathogenic bacteria in foods. Copyright © 2014 Elsevier B.V. All rights reserved.

  15. Antisense Oligonucleotide Therapy in Diabetic Retinopathy

    PubMed Central

    Hnik, Peter; Boyer, David S.; Grillone, Lisa R.; Clement, John G.; Henry, Scott P.; Green, Ellen A.

    2009-01-01

    Diabetic retinopathy is one of the leading causes of blindness in the United States and other parts of the world. Historically, laser photocoagulation and vitrectomy surgery have been used for the treatment of diabetic retinopathy, including diabetic macular edema. Both procedures have proven to be useful under certain conditions but have their limitations. New pathways and processes that promote diabetic retinopathy have been identified, and several new therapeutic approaches are under investigation. These new therapies may be beneficial in the treatment of diabetic retinopathy and include antivascular endothelial growth factor agents, corticosteroids, and therapies that may potentially target a number of additional diabetic retinopathy-related factors and processes, including antisense oligonucleotides. Second-generation antisense oligonucleotides, such as iCo-007, may offer a significant advantage in the treatment of diabetic retinopathy by downregulating the signal pathways of multiple growth factors that seem to play a critical role in the process of ocular angiogenesis and vascular leakage. Benefits of such molecules are expected to include the specificity of the kinase target and an extended half-life, resulting in less frequent intravitreal drug administration, resistance to molecule degradation, and a good safety profile. PMID:20144342

  16. BIOCONJUGATION OF OLIGONUCLEOTIDES FOR TREATING LIVER FIBROSIS

    PubMed Central

    Ye, Zhaoyang; Hajj Houssein, Houssam S.; Mahato, Ram I.

    2009-01-01

    Liver fibrosis results from chronic liver injury due to hepatitis B and C, excessive alcohol ingestion, and metal ion overload. Fibrosis culminates in cirrhosis and results in liver failure. Therefore, a potent antifibrotic therapy is in urgent need to reverse scarring and eliminate progression to cirrhosis. Although activated hepatic stellate cells (HSCs) remains the principle cell type responsible for liver fibrosis, perivascular fibroblasts of portal and central veins as well as periductular fibroblasts are other sources of fibrogenic cells. This review will critically discuss various treatment strategies for liver fibrosis, including prevention of liver injury, reduction of inflammation, inhibition of HSC activation, degradation of scar matrix, and inhibition of aberrant collagen synthesis. Oligonucleotides (ODNs) are short, single-stranded nucleic acids, which disrupt expression of target protein by binding to complementary mRNA or forming triplex with genomic DNA. Triplex forming oligonucleotides (TFOs) provide an attractive strategy for treating liver fibrosis. A series of TFOs have been developed for inhibiting the transcription of α1(I) collagen gene, which opens a new area for antifibrotic drugs. There will be in depth discussion on the use of TFOs and how different bioconjugation strategies can be utilized for their site-specific delivery to HSCs or hepatocytes for enhanced antifibrotic activities. Various insights developed in individual strategy and the need for multipronged approaches will also be discussed. PMID:18154454

  17. Template switching between PNA and RNA oligonucleotides

    NASA Technical Reports Server (NTRS)

    Bohler, C.; Nielsen, P. E.; Orgel, L. E.; Miller, S. L. (Principal Investigator)

    1995-01-01

    The origin of the RNA world is not easily understood, as effective prebiotic syntheses of the components of RNA, the beta-ribofuranoside-5'-phosphates, are hard to envisage. Recognition of this difficulty has led to the proposal that other genetic systems, the components of which are more easily formed, may have preceded RNA. This raises the question of how transitions between one genetic system and another could occur. Peptide nucleic acid (PNA) resembles RNA in its ability to form double-helical complexes stabilized by Watson-Crick hydrogen bonding between adenine and thymine and between cytosine and guanine, but has a backbone that is held together by amide rather than by phosphodiester bonds. Oligonucleotides bases on RNA are known to act as templates that catalyse the non-enzymatic synthesis of their complements from activated mononucleotides, we now show that RNA oligonucleotides facilitate the synthesis of complementary PNA strands and vice versa. This suggests that a transition between different genetic systems can occur without loss of information.

  18. Template switching between PNA and RNA oligonucleotides

    NASA Technical Reports Server (NTRS)

    Bohler, C.; Nielsen, P. E.; Orgel, L. E.; Miller, S. L. (Principal Investigator)

    1995-01-01

    The origin of the RNA world is not easily understood, as effective prebiotic syntheses of the components of RNA, the beta-ribofuranoside-5'-phosphates, are hard to envisage. Recognition of this difficulty has led to the proposal that other genetic systems, the components of which are more easily formed, may have preceded RNA. This raises the question of how transitions between one genetic system and another could occur. Peptide nucleic acid (PNA) resembles RNA in its ability to form double-helical complexes stabilized by Watson-Crick hydrogen bonding between adenine and thymine and between cytosine and guanine, but has a backbone that is held together by amide rather than by phosphodiester bonds. Oligonucleotides bases on RNA are known to act as templates that catalyse the non-enzymatic synthesis of their complements from activated mononucleotides, we now show that RNA oligonucleotides facilitate the synthesis of complementary PNA strands and vice versa. This suggests that a transition between different genetic systems can occur without loss of information.

  19. How is it that microsatellites and random oligonucleotides uncover DNA fingerprint patterns?

    PubMed

    Kashi, Y; Nave, A; Darvasi, A; Gruenbaum, Y; Soller, M; Beckmann, J S

    1994-09-01

    Minisatellites, microsatellites, and short random oligonucleotides all uncover highly polymorphic DNA fingerprint patterns in Southern analysis of genomic DNA that has been digested with a restriction enzyme having a 4-bp specificity. The polymorphic nature of the fragments is attributed to tandem repeat number variation of embedded minisatellite sequences. This explains why DNA fingerprint fragments are uncovered by minisatellite probes, but does not explain how it is that they are also uncovered by microsatellite and random oligonucleotide probes. To clarify this phenomenon, we sequenced a large bovine genomic BamHI restriction fragment hybridizing to the Jeffreys 33.6 minisatellite probe and consisting of small and large Sau3A-resistant subfragments. The large Sau3A subfragment was found to have a complex architecture, consisting of two different minisatellites, flanked and separated by stretches of unique DNA. The three unique sequences were characterized by sequence simplicity, that is, a higher than chance occurrence of tandem or dispersed repetition of simple sequence motifs. This complex repetitive structure explains the absence of Sau3A restriction sites in the large Sau3A subfragment, yet provides this subfragment with the ability to hybridize to a variety of probe sequences. It is proposed that a large class of interspersed tracts sharing this complex yet simplified sequence structure is found in the genome. Each such tract would have a broad ability to hybridize to a variety of probes, yet would exhibit a dearth of restriction sites. For each restriction enzyme having 4-bp specificity, a subclass of such tracts, completely lacking the corresponding restriction sites, will be present. On digestion with the given restriction enzyme, each such tract would form a large fragment.(ABSTRACT TRUNCATED AT 250 WORDS)

  20. Disulfide-linked oligonucleotide phosphorothioates - Novel analogues of nucleic acids

    NASA Technical Reports Server (NTRS)

    Wu, Taifeng; Orgel, Leslie E.

    1991-01-01

    The synthesis of phosphorothioate analogs of oligonucleotides by the oxidation of deoxyadenosine 3',5'-bisphosphorothioate (3) was attempted. Cyclization of 3 is much more efficient than oligomerization under all the conditions investigated. However, a preformed oligonucleotide carrying a 5'-terminal phosphorotioate group undergoes efficient chain-extension when oxidized in the presence of 3.

  1. Oligonucleotide therapies for disorders of the nervous system.

    PubMed

    Khorkova, Olga; Wahlestedt, Claes

    2017-03-01

    Oligonucleotide therapies are currently experiencing a resurgence driven by advances in backbone chemistry and discoveries of novel therapeutic pathways that can be uniquely and efficiently modulated by the oligonucleotide drugs. A quarter of a century has passed since oligonucleotides were first applied in living mammalian brain to modulate gene expression. Despite challenges in delivery to the brain, multiple oligonucleotide-based compounds are now being developed for treatment of human brain disorders by direct delivery inside the blood brain barrier (BBB). Notably, the first new central nervous system (CNS)-targeted oligonucleotide-based drug (nusinersen/Spinraza) was approved by US Food and Drug Administration (FDA) in late 2016 and several other compounds are in advanced clinical trials. Human testing of brain-targeted oligonucleotides has highlighted unusual pharmacokinetic and pharmacodynamic properties of these compounds, including complex active uptake mechanisms, low systemic exposure, extremely long half-lives, accumulation and gradual release from subcellular depots. Further work on oligonucleotide uptake, development of formulations for delivery across the BBB and relevant disease biology studies are required for further optimization of the oligonucleotide drug development process for brain applications.

  2. Voltage-gated calcium channel and antisense oligonucleotides thereto

    NASA Technical Reports Server (NTRS)

    Hruska, Keith A. (Inventor); Friedman, Peter A. (Inventor); Barry, Elizabeth L. R. (Inventor); Duncan, Randall L. (Inventor)

    1998-01-01

    An antisense oligonucleotide of 10 to 35 nucleotides in length that can hybridize with a region of the .alpha..sub.1 subunit of the SA-Cat channel gene DNA or mRNA is provided, together with pharmaceutical compositions containing and methods utilizing such antisense oligonucleotide.

  3. The spherulites™: a promising carrier for oligonucleotide delivery

    PubMed Central

    Mignet, Nathalie; Brun, Armelle; Degert, Corinne; Delord, Brigitte; Roux, Didier; Hélène, Claude; Laversanne, René; François, Jean-Christophe

    2000-01-01

    Concentric multilamellar microvesicles, named spherulites™, were evaluated as an oligonucleotide carrier. Up to 80% oligonucleotide was encapsulated in these vesicles. The study was carried out on two different spherulite™ formulations. The spherulite™ size and stability characteristics are presented. Delivery of encapsulated oligonucleotide was performed on a rat hepatocarcinoma and on a lymphoblastoid T cell line, both expressing the luciferase gene. We showed that spherulites™ were able to transfect both adherent and suspension cell lines and deliver the oligonucleotide to the nucleus. Moreover, 48–62% luciferase inhibition was obtained in the rat hepatocarcinoma cell line when the antisense oligonucleotide targeted to the luciferase coding region was encapsulated at 500 nM concentration in spherulites™ of different compositions. PMID:10931929

  4. 2'-modified nucleosides for site-specific labeling of oligonucleotides

    NASA Technical Reports Server (NTRS)

    Krider, Elizabeth S.; Miller, Jeremiah E.; Meade, Thomas J.

    2002-01-01

    We report the synthesis of 2'-modified nucleosides designed specifically for incorporating labels into oligonucleotides. Conversion of these nucleosides to phosphoramidite and solid support-bound derivatives proceeds in good yield. Large-scale synthesis of 11-mer oligonucleotides possessing the 2'-modified nucleosides is achieved using these derivatives. Thermal denaturation studies indicate that the presence of 2'-modified nucleosides in 11-mer duplexes has minimal destabilizing effects on the duplex structure when the nucleosides are placed at the duplex termini. The powerful combination of phosphoramidite and support-bound derivatives of 2'-modified nucleosides affords the large-scale preparation of an entirely new class of oligonucleotides. The ability to synthesize oligonucleotides containing label attachment sites at 3', intervening, and 5' locations of a duplex is a significant advance in the development of oligonucleotide conjugates.

  5. 2'-modified nucleosides for site-specific labeling of oligonucleotides

    NASA Technical Reports Server (NTRS)

    Krider, Elizabeth S.; Miller, Jeremiah E.; Meade, Thomas J.

    2002-01-01

    We report the synthesis of 2'-modified nucleosides designed specifically for incorporating labels into oligonucleotides. Conversion of these nucleosides to phosphoramidite and solid support-bound derivatives proceeds in good yield. Large-scale synthesis of 11-mer oligonucleotides possessing the 2'-modified nucleosides is achieved using these derivatives. Thermal denaturation studies indicate that the presence of 2'-modified nucleosides in 11-mer duplexes has minimal destabilizing effects on the duplex structure when the nucleosides are placed at the duplex termini. The powerful combination of phosphoramidite and support-bound derivatives of 2'-modified nucleosides affords the large-scale preparation of an entirely new class of oligonucleotides. The ability to synthesize oligonucleotides containing label attachment sites at 3', intervening, and 5' locations of a duplex is a significant advance in the development of oligonucleotide conjugates.

  6. Noncoding oligonucleotides: the belle of the ball in gene therapy.

    PubMed

    Shum, Ka-To; Rossi, John J

    2015-01-01

    Gene therapy carries the promise of cures for many diseases based on manipulating the expression of a person's genes toward the therapeutic goal. The relevance of noncoding oligonucleotides to human disease is attracting widespread attention. Noncoding oligonucleotides are not only involved in gene regulation, but can also be modified into therapeutic tools. There are many strategies that leverage noncoding oligonucleotides for gene therapy, including small interfering RNAs, antisense oligonucleotides, aptamers, ribozymes, decoys, and bacteriophage phi 29 RNAs. In this chapter, we will provide a broad, comprehensive overview of gene therapies that use noncoding oligonucleotides for disease treatment. The mechanism and development of each therapeutic will be described, with a particular focus on its clinical development. Finally, we will discuss the challenges associated with developing nucleic acid therapeutics and the prospects for future success.

  7. The effect of overhanging nucleotides on fluorescence properties of hybridising oligonucleotides labelled with Alexa-488 and FAM fluorophores.

    PubMed

    Noble, J E; Wang, L; Cole, K D; Gaigalas, A K

    2005-03-01

    In order to rationally select and design probes for real-time PCR, we have determined the influence of the overhang region of the complementary strand on the resulting fluorescence from a hybridising probe. A series of target oligonucleotides, each with a unique 3' overhang (4 bases), was hybridised to either 5' fluorescein (FAM)- or Alexa-488-labelled probes, and the changes in fluorescence properties were monitored. We found that the number of guanine bases in the overhang region of the target oligonucleotides was proportional to the amount of fluorescence quenching observed for both the FAM and Alexa-488 dyes. FAM appeared to be more sensitive to guanine-induced quenching with three and four guanine bases resulting in greater than a twofold decrease in the quantum yield of the fluorophore compared to the no-overhang target. In addition, we found that adenine bases caused fluorescence quenching of the Alexa-488-labelled probe, whereas the FAM-labelled probe appeared insensitive. The quenching data, generated with the steady-state fluorescence measurements, displayed a linear correlation with that obtained using a fluorescent thermal cycler, suggesting the applicability to real-time PCR measurements. Anisotropy data from the series of duplexes correlated with the fluorescence quantum yield, suggesting that quenching was accompanied by increased dye mobility.

  8. Mechanosensitive membrane probes.

    PubMed

    Dal Molin, Marta; Verolet, Quentin; Soleimanpour, Saeideh; Matile, Stefan

    2015-04-13

    This article assembles pertinent insights behind the concept of planarizable push-pull probes. As a response to the planarization of their polarized ground state, a red shift of their excitation maximum is expected to report on either the disorder, the tension, or the potential of biomembranes. The combination of chromophore planarization and polarization contributes to various, usually more complex processes in nature. Examples include the color change of crabs or lobsters during cooking or the chemistry of vision, particularly color vision. The summary of lessons from nature is followed by an overview of mechanosensitive organic materials. Although often twisted and sometimes also polarized, their change of color under pressure usually originates from changes in their crystal packing. Intriguing exceptions include the planarization of several elegantly twisted phenylethynyl oligomers and polymers. Also mechanosensitive probes in plastics usually respond to stretching by disassembly. True ground-state planarization in response to molecular recognition is best exemplified with the binding of thoughtfully twisted cationic polythiophenes to single- and double-stranded oligonucleotides. Molecular rotors, en vogue as viscosity sensors in cells, operate by deplanarization of the first excited state. Pertinent recent examples are described, focusing on λ-ratiometry and intracellular targeting. Complementary to planarization of the ground state with twisted push-pull probes, molecular rotors report on environmental changes with quenching or shifts in emission rather than absorption. The labeling of mechanosensitive channels is discussed as a bioengineering approach to bypass the challenge to create molecular mechanosensitivity and use biological systems instead to sense membrane tension. With planarizable push-pull probes, this challenge is met not with twistome screening, but with "fluorescent flippers," a new concept to insert large and bright monomers into oligomeric

  9. Hole hopping rates in single strand oligonucleotides

    NASA Astrophysics Data System (ADS)

    Borrelli, Raffaele; Capobianco, Amedeo; Peluso, Andrea

    2014-08-01

    The rates of hole transfer between guanine and adenine in single strand DNA have been evaluated by using Fermi’s golden rule and Kubo’s generating function approach for the Franck-Condon weighted density of states. The whole sets of the normal modes and vibrational frequencies of the two nucleobases, obtained at DFT/B3LYP level of calculation, have been considered in computations. The results show that in single strand the pyramidalization/planarization mode of the amino groups of both nucleobases plays the major role. At room temperature, the Franck-Condon density of states extends over a wide range of hole site energy difference, 0-1 eV, giving some hints about the design of oligonucleotides of potential technological interest.

  10. Cellular uptake and trafficking of antisense oligonucleotides.

    PubMed

    Crooke, Stanley T; Wang, Shiyu; Vickers, Timothy A; Shen, Wen; Liang, Xue-Hai

    2017-03-01

    Antisense oligonucleotides (ASOs) modified with phosphorothioate (PS) linkages and different 2' modifications can be used either as drugs (e.g., to treat homozygous familial hypercholesterolemia and spinal muscular atrophy) or as research tools to alter gene expression. PS-ASOs can enter cells without additional modification or formulation and can be designed to mediate sequence-specific cleavage of different types of RNA (including mRNA and non-coding RNA) targeted by endogenous RNase H1. Although PS-ASOs function in both the cytoplasm and nucleus, localization to different subcellular regions can affect their therapeutic potency. Cellular uptake and intracellular distribution of PS ASOs are mediated by protein interactions. The main proteins involved in these processes have been identified, and intracellular sites in which PS ASOs are active, or inactive, cataloged.

  11. Abiotic formation of oligonucleotides on basalt surfaces

    NASA Astrophysics Data System (ADS)

    Otroshchenko, V. A.; Vasilyeva, N. V.; Kopilov, A. M.

    1985-06-01

    The complication and further evolution of abiotic syntheses products occurred under environmental influences at the prebiological stage. From this point of view, the influence of some types of irradiation on the organic molecules adsorbed on the surfaces of volcanic rocks, appeared to be of great importance. In this connection, the effect of gamma rays on the AMP molecules adsorbed on mineral surfaces such as cinders and ashes has been studied. It has been shown that they can polymerize with the formation of oligonucleotides. The treatment of oligomers obtained by venom phosphodiesterase has shown that a polymeric product has mainly 3' 5' and 2' 5' bonds between nucleotides. The results obtained have been discussed from the evolutionary aspect.

  12. Antisense Oligonucleotides: Treatment Strategies and Cellular Internalization

    PubMed Central

    Miller, Colton M.; Harris, Edward N.

    2016-01-01

    The clinical applicaton of antisense oligonucleotides (ASOs) is becoming more of a reality as several drugs have been approved for the treatment of human disorders and many others are in various phases in development and clinical trials. ASOs are short DNA/RNA oligos which are heavily modified to increase their stability in biological fluids and retain the properties of creating RNA-RNA and DNA-RNA duplexes that knock-down or correct genetic expression. This review outlines several strategies that ASOs utilize for the treatment of various congenital diseases and syndromes that develop with aging. In addition, we discuss some of the mechanisms for specific non-targeted ASO internalization within cells. PMID:28374018

  13. Conjugation of fluorescent proteins with DNA oligonucleotides.

    PubMed

    Lapiene, Vidmantas; Kukolka, Florian; Kiko, Kathrin; Arndt, Andreas; Niemeyer, Christof M

    2010-05-19

    This work describes the synthesis of covalent ssDNA conjugates of six fluorescent proteins, ECFP, EGFP, E(2)GFP, mDsRed, Dronpa, and mCherry, which were cloned with an accessible C-terminal cystein residue to enable site-selective coupling using a heterobispecific cross-linker. The resulting conjugates revealed similar fluorescence emission intensity to the unconjugated proteins, and the functionality of the tethered oligonucleotide was proven by specific Watson-Crick base pairing to cDNA-modified gold nanoparticles. Fluorescence spectroscopy analysis indicated that the fluorescence of the FP is quenched by the gold particle, and the extent of quenching varied with the intrinsic spectroscopic properties of FP as well as with the configuration of surface attachment. Since this study demonstrates that biological fluorophores can be selectively incorporated into and optically coupled with nanoparticle-based devices, applications in DNA-based nanofabrication can be foreseen.

  14. Direct oligonucleotide-photosensitizer conjugates for photochemical delivery of antisense oligonucleotides.

    PubMed

    Yuan, Ahu; Laing, Brian; Hu, Yiqiao; Ming, Xin

    2015-04-18

    Activation of photosensitizers in endosomes enables release of therapeutic macromolecules into the cytosol of the target cells for pharmacological actions. In this study, we demonstrate that direct conjugation of photosensitizers to oligonucleotides (ONs) allows spatial and temporal co-localization of the two modalities in the target cells, and thus leads to superior functional delivery of ONs. Further, light-activated delivery of an anticancer ON caused cancer cell killing via modulation of an oncogene and photodynamic therapy.

  15. Preparation and application of triple helix forming oligonucleotides and single strand oligonucleotide donors for gene correction.

    PubMed

    Alam, Rowshon; Thazhathveetil, Arun Kalliat; Li, Hong; Seidman, Michael M

    2014-01-01

    Strategies for site-specific modulation of genomic sequences in mammalian cells require two components. One must be capable of recognizing and activating a specific target sequence in vivo, driving that site into an exploitable repair pathway. Information is transferred to the site via participation in the pathway by the second component, a donor nucleic acid, resulting in a permanent change in the target sequence. We have developed biologically active triple helix forming oligonucleotides (TFOs) as site-specific gene targeting reagents. These TFOs, linked to DNA reactive compounds (such as a cross-linking agent), activate pathways that can engage informational donors. We have used the combination of a psoralen-TFO and single strand oligonucleotide donors to generate novel cell lines with directed sequence changes at the target site. Here we describe the synthesis and purification of bioactive psoralen-linked TFOs, their co-introduction into mammalian cells with donor nucleic acids, and the identification of cells with sequence conversion of the target site. We have emphasized details in the synthesis and purification of the oligonucleotides that are essential for preparation of reagents with optimal activity.

  16. The "Clickable" Method for Oligonucleotide Immobilization Onto Azide-Functionalized Microarrays.

    PubMed

    Ratajczak, Tomasz; Uszczyńska, Barbara; Frydrych-Tomczak, Emilia; Chmielewski, Marcin K

    2016-01-01

    The DNA microarray technique was supposed to help identifying and analyzing the expression level of tens of thousands of genes in the whole genome. But there is a serious problem concerning fabrication of the microarrays by chemical synthesis, such as specific and efficient linking of probes to a solid support. Therefore, we reckon that applying "click" chemistry to covalently anchor oligonucleotides on chemically modified supports may help construct microarrays in applications such as gene identification. Silanization of the glass support with organofunctional silane makes it possible to link azide groups on glass surface and the nucleic acid probe that is equipped with a pentynyl group. This is followed by direct spotting of the nucleic acid on the azide-modified glass support in the presence of copper ions, and this is a frequently applied method of "click" chemistry.

  17. Electrochemical Detection of a Dengue-related Oligonucleotide Sequence Using Ferrocenium as a Hybridization Indicator

    PubMed Central

    Ribeiro Teles, Fernando Rodrigues; França dos Prazeres, Duarte Miguel; de Lima-Filho, José Luiz

    2007-01-01

    A simple method for electrochemical detection of a synthetic 20-bp oligonucleotide sequence related with dengue virus genome was developed. A complimentary DNA probe sequence was electrostatically immobilized onto a glassy carbon electrode modified with chitosan. Electrochemical detection of hybridization between probe and target was performed by cyclic voltammetry, using ferrocene (Fc+) as a hybridization label. After hybridization, the peak current response of Fc+ oxidation increased around 26%. A higher voltammetric decay rate constant (kd) and a lower half-life period (t1/2) for the interaction of Fc+ with dsDNA compared to those with ssDNA quantitatively characterize the different strengths of interaction with both types of DNA. By combining the simplicity of DNA immobilization onto a chitosan film and suitable voltammetric detection of hybridization concomitant with ferrocene attachment, a good discrimination between ssDNA and dsDNA was obtained.

  18. An Oligonucleotide Microarray for High-Throughput Sequencing of the Mitochondrial Genome

    PubMed Central

    Zhou, Shaoyu; Kassauei, Keyaunoosh; Cutler, David J.; Kennedy, Giulia C.; Sidransky, David; Maitra, Anirban; Califano, Joseph

    2006-01-01

    Previously we developed an oligonucleotide sequencing microarray (MitoChip) as an array-based sequencing platform for rapid and high-throughput analysis of mitochondrial DNA. The first generation MitoChip, however, was not tiled with probes for the noncoding D-loop region, a site frequently mutated in human cancers. Here we report the development of a second-generation MitoChip (v2.0) with oligonucleotide probes to sequence the entire mitochondrial genome. In addition, the MitoChip v2.0 contains redundant tiling of sequences for 500 of the most common haplotypes including single-nucleotide changes, insertions, and deletions. Sequencing results from 14 primary head and neck tumor tissues demonstrated that the v2.0 MitoChips detected a larger number of variants than the original version. Multiple coding region variants detected only in the second generation MitoChips, but not the earlier chip version, were further confirmed with conventional sequencing. Moreover, 31 variations in noncoding region were identified using MitoChips v2.0. Replicate experiments demonstrated >99.99% reproducibility in the second generation MitoChip. In seven head and neck cancer samples with matched lymphocyte DNA, the MitoChip v2.0 detected at least one cancer-associated mitochondrial mutation in four (57%) samples. These results indicate that the second generation MitoChip is a high-throughput platform for identification of mitochondrial DNA mutations in primary tumors. PMID:16931588

  19. Brilliant cresyl blue as electroactive indicator in electrochemical DNA oligonucleotide sensors.

    PubMed

    Hejazi, Mohammad Saeid; Raoof, Jahan-Bakhsh; Ojani, Reza; Golabi, Seyd Mehdi; Asl, Ezat Hamidi

    2010-06-01

    A new electrochemical DNA biosensor is presented based on carbon past electrode (CPE) for immobilization and detection of short DNA sequences with brilliant cresyl blue (BCB) as electroactive label. The interaction of BCB with DNA is electrochemically detected and BCB displays different signals in the interaction to ssDNA and dsDNA and variation in the BCB signal represents the extent of hybridization at the electrode surface. The effect of solution pH on electrochemical behavior of BCB was investigated. Additionally, the effect of solution pH on BCB accumulation on the CPE was studied. Furthermore, experiments showed that the solution pH could influence the differential pulse voltammetry (DPV) signal of BCB accumulated on the electrode and the highest BCB signal was obtained in pH 7.00. The effect of electrochemical pretreatment of CPE on the ability of electrode in probe adsorption, BCB accumulation and conditions of probe immobilization including potential and time was investigated and optimum conditions were suggested. The peak currents of BCB were linearly related to the concentration of the target oligonucleotide sequence in the range of 1.0x10(-8) to 5.0x10(-6)M. The detection limit of this approach was 9.00nM. The selectivity of the biosensor was studied using noncomplementary oligonucleotide. Copyright 2009 Elsevier B.V. All rights reserved.

  20. Advanced Molecular Probes for Sequence-Specific DNA Recognition

    NASA Astrophysics Data System (ADS)

    Bertucci, Alessandro; Manicardi, Alex; Corradini, Roberto

    DNA detection can be achieved using the Watson-Crick base pairing with oligonucleotides or oligonucleotide analogs, followed by generation of a physical or chemical signal coupled with a transducer device. The nature of the probe is an essential feature which determines the performances of the sensing device. Many synthetic processes are presently available for "molecular engineering" of DNA probes, enabling label-free and PCR-free detection to be performed. Furthermore, many DNA analogs with improved performances are available and are under development; locked nucleic acids (LNA), peptide nucleic acids (PNA) and their analogs, morpholino oligonucleotides (MO) and other modified probes have shown improved properties of affinity and selectivity in target recognition compared to those of simple DNA probes. The performances of these probes in sensing devices, and the requirements for detection of unamplified DNA will be discussed in this chapter. Chemistry and architectures for conjugation of probes to reporter units, surfaces and nanostructures will also be discussed. Examples of probes used in ultrasensitive detection of unamplified DNA are listed.

  1. Nucleic acid sequence detection using multiplexed oligonucleotide PCR

    DOEpatents

    Nolan, John P.; White, P. Scott

    2006-12-26

    Methods for rapidly detecting single or multiple sequence alleles in a sample nucleic acid are described. Provided are all of the oligonucleotide pairs capable of annealing specifically to a target allele and discriminating among possible sequences thereof, and ligating to each other to form an oligonucleotide complex when a particular sequence feature is present (or, alternatively, absent) in the sample nucleic acid. The design of each oligonucleotide pair permits the subsequent high-level PCR amplification of a specific amplicon when the oligonucleotide complex is formed, but not when the oligonucleotide complex is not formed. The presence or absence of the specific amplicon is used to detect the allele. Detection of the specific amplicon may be achieved using a variety of methods well known in the art, including without limitation, oligonucleotide capture onto DNA chips or microarrays, oligonucleotide capture onto beads or microspheres, electrophoresis, and mass spectrometry. Various labels and address-capture tags may be employed in the amplicon detection step of multiplexed assays, as further described herein.

  2. Regulation of Gene Expression with Double-Stranded Phosphorothioate Oligonucleotides

    NASA Astrophysics Data System (ADS)

    Bielinska, Anna; Shivdasani, Ramesh A.; Zhang, Liquan; Nabel, Gary J.

    1990-11-01

    Alteration of gene transcription by inhibition of specific transcriptional regulatory proteins is necessary for determining how these factors participate in cellular differentiation. The functions of these proteins can be antagonized by several methods, each with specific limitations. Inhibition of sequence-specific DNA-binding proteins was achieved with double-stranded (ds) phosphorothioate oligonucleotides that contained octamer or kappaB consensus sequences. The phosphorothioate oligonucleotides specifically bound either octamer transcription factor or nuclear factor (NF)-kappaB. The modified oligonucleotides accumulated in cells more effectively than standard ds oligonucleotides and modulated gene expression in a specific manner. Octamer-dependent activation of a reporter plasmid or NF-kappaB-dependent activation of the human immunodeficiency virus (HIV) enhancer was inhibited when the appropriate phosphorothioate oligonucleotide was added to a transiently transfected B cell line. Addition of phosphorothioate oligonucleotides that contained the octamer consensus to Jurkat T leukemia cells inhibited interleukin-2 (IL-2) secretion to a degree similar to that observed with a mutated octamer site in the IL-2 enhancer. The ds phosphorothioate oligonucleotides probably compete for binding of specific transcription factors and may provide anti-viral, immunosuppressive, or other therapeutic effects.

  3. Optimization of single-base-pair mismatch discrimination in oligonucleotide microarrays

    NASA Technical Reports Server (NTRS)

    Urakawa, Hidetoshi; El Fantroussi, Said; Smidt, Hauke; Smoot, James C.; Tribou, Erik H.; Kelly, John J.; Noble, Peter A.; Stahl, David A.

    2003-01-01

    The discrimination between perfect-match and single-base-pair-mismatched nucleic acid duplexes was investigated by using oligonucleotide DNA microarrays and nonequilibrium dissociation rates (melting profiles). DNA and RNA versions of two synthetic targets corresponding to the 16S rRNA sequences of Staphylococcus epidermidis (38 nucleotides) and Nitrosomonas eutropha (39 nucleotides) were hybridized to perfect-match probes (18-mer and 19-mer) and to a set of probes having all possible single-base-pair mismatches. The melting profiles of all probe-target duplexes were determined in parallel by using an imposed temperature step gradient. We derived an optimum wash temperature for each probe and target by using a simple formula to calculate a discrimination index for each temperature of the step gradient. This optimum corresponded to the output of an independent analysis using a customized neural network program. These results together provide an experimental and analytical framework for optimizing mismatch discrimination among all probes on a DNA microarray.

  4. Optimization of single-base-pair mismatch discrimination in oligonucleotide microarrays

    NASA Technical Reports Server (NTRS)

    Urakawa, Hidetoshi; El Fantroussi, Said; Smidt, Hauke; Smoot, James C.; Tribou, Erik H.; Kelly, John J.; Noble, Peter A.; Stahl, David A.

    2003-01-01

    The discrimination between perfect-match and single-base-pair-mismatched nucleic acid duplexes was investigated by using oligonucleotide DNA microarrays and nonequilibrium dissociation rates (melting profiles). DNA and RNA versions of two synthetic targets corresponding to the 16S rRNA sequences of Staphylococcus epidermidis (38 nucleotides) and Nitrosomonas eutropha (39 nucleotides) were hybridized to perfect-match probes (18-mer and 19-mer) and to a set of probes having all possible single-base-pair mismatches. The melting profiles of all probe-target duplexes were determined in parallel by using an imposed temperature step gradient. We derived an optimum wash temperature for each probe and target by using a simple formula to calculate a discrimination index for each temperature of the step gradient. This optimum corresponded to the output of an independent analysis using a customized neural network program. These results together provide an experimental and analytical framework for optimizing mismatch discrimination among all probes on a DNA microarray.

  5. Optical characterization of oligonucleotide DNA influenced by magnetic fields.

    PubMed

    Banihashemian, Seyedeh Maryam; Periasamy, Vengadesh; Mohammadi, Seyed Mohammad Hossein Mousa Kazemi; Ritikos, Richard; Rahman, Saadah Abdul

    2013-09-25

    UV-VIS spectroscopic analysis of oligonucleotide DNA exposed to different magnetic fields was performed in order to investigate the relationship between DNA extinction coefficients and optical parameters according to magnetic-field strength. The results with the oligonucleotides adenine-thymine 100 mer (AT-100 DNA) and cytosine-guanine 100 mer (CG-100 DNA) indicate that the magnetic field influences DNA molar extinction coefficients and refractive indexes. The imaginary parts of the refractive index and molar extinction coefficients of the AT-100 and CG-100 DNA decreased after exposure to a magnetic field of 750 mT due to cleavage of the DNA oligonucleotides into smaller segments.

  6. Therapeutic oligonucleotides and delivery technologies: Research topics in Japan.

    PubMed

    Murakami, Masahiro

    Oligonucleotides have been gaining considerable attention as promising and effective candidate therapeutics against various diseases. This special issue is aimed at providing a better understanding of the recent progress in the development of oligonucleotide-based therapeutics to encourage further research and innovation in this field to achieve these advancements. Several Japanese scientists have been invited to contribute to this issue by describing their recent findings, overviews, insights, or commentaries on rational designing of therapeutic oligonucleotide molecules and their novel delivery technologies, especially nanocarrier systems.

  7. Method for the preparation of size marker for synthetic oligonucleotides

    SciTech Connect

    Jing, G.Z.; Liu, A.; Leung, W.C.

    1986-01-01

    Terminal deoxynucleotidyltransferase was used for the addition of (..cap alpha..-/sup 32/P)dCTP to the 3'-OH termini of oligo(dT)/sub 12-18/. A collection of oligonucleotides with chain lengths ranging continuously from 13-mer to over 100-mer was generated. The reaction mixture was then mixed with oligo(dT)/sub 12-18/ labeled with (..gamma..-/sup 32/P)ATP by T/sub 4/ polynucleotide kinase. A sequence ladder with the bottom base as 12-mer was then formed. These oligonucleotides served as size marker for the purification and identification of oligonucleotides on polyacrylamide gel.

  8. Synthesis and characterization of oligonucleotide conjugates bearing electroactive labels.

    PubMed

    Moreau, Julie; Dendane, Nabil; Schöllhorn, Bernd; Spinelli, Nicolas; Fave, Claire; Defrancq, Eric

    2013-02-15

    Oxime bond formation has been applied to the preparation of oligonucleotides labeled with electrochemical ferrocene and viologen labels. Aminooxy functionalized ferrocene and viologen derivatives were prepared by a straightforward route and efficiently conjugated with aldehyde containing oligonucleotides either at 3' or 5' end. Both labels were found to not disturb the recognition properties of the oligonucleotide. The versatility of the method was further demonstrated by preparing bi-functionalized conjugates with a disulfide at 3' end and an electrochemical label at 5' end. Copyright © 2012 Elsevier Ltd. All rights reserved.

  9. Selection and application of strand displacement probes for a fumonisin B1 aptamer

    USDA-ARS?s Scientific Manuscript database

    Fumonisin B1 (FB1) is a toxin produced by Fusarium moniliforme, mainly on contaminated maize and maize products. In this study a solid surface chain displacement strategy was used to isolate oligonucleotide displacement probes for a FB1 aptamer. The probes were used as the basis for the development ...

  10. Polyphosphorylation and non-enzymatic template-directed ligation of oligonucleotides

    NASA Technical Reports Server (NTRS)

    Gao, K.; Orgel, L. E.; Bada, J. L. (Principal Investigator)

    2000-01-01

    Oligonucleotide 5'-polyphosphates are formed under potentially prebiotic conditions from oligonucleotide 5'-phosphates and sodium trimetaphosphate. Oligonucleotides activated as polyphosphates undergo template-directed ligation. We believe that these reactions could have produced longer oligonucleotide products from shorter substrates under prebiotic conditions.

  11. Oligonucleotides complementary to the Oxytricha nova telomerase RNA delineate the template domain and uncover a novel mode of primer utilization.

    PubMed Central

    Melek, M; Davis, B T; Shippen, D E

    1994-01-01

    The telomerase reverse transcriptase uses an essential RNA subunit as a template to direct telomeric DNA synthesis. The 190-nucleotide Oxytricha nova telomerase RNA was identified by using an oligonucleotide probe complementary to the predicted CCCCAAAA template. This RNA displays extensive sequence similarity to the Euplotes crassus telomerase RNA and carries the same 5' CAAAACCCCAAAACC 3' telomeric domain. Antisense oligonucleotides were used to map the boundaries of the functional template and to investigate the mechanism of primer recognition and elongation. On the basis of their ability to inhibit or to prime telomerase, oligonucleotides were classified into three categories. Category 1 oligonucleotides, which extended 5' of residue 42 in the RNA, abolished elongation of (T4G4)3 and (G4T4)3 primers in vitro. In contrast, oligonucleotides terminating between residues 42 and 50 (categories 2 and 3), served as efficient telomerase primers. We conclude that the O. nova template comprises residues 42 to 50 in the 190-nucleotide RNA, a different set of nucleotides than are used by the E. crassus enzyme. Category 2 primer reactions amassed short products, and their abundance could be decreased by altering the 5' sequence of the primer, consistent with the two-primer-binding-site model for telomerase. Category 3 primers generated a bimodal distribution of short and long products, each having a unique elongation profile. The long-product profile is inconsistent with sequence-specific primer alignment. Rather, each primer was extended by the same register of TTTTGGGG repeats, suggesting shuttling to a default position within the template. The parallels between telomerase and RNA polymerase elongation mechanisms are discussed. Images PMID:7969123

  12. Oligonucleotide-Functionalized Anisotropic Gold Nanoparticles

    NASA Astrophysics Data System (ADS)

    Jones, Matthew Robert

    In this thesis, we describe the properties of oligonucleotide-functionalized gold colloids under the unique set of conditions where the particles are geometrically anisotropic and have nanometer-scale dimensions. While nearly two decades of previous work elucidated numerous unexpected and emergent phenomena arising from the combination of inorganic nanoparticles with surface-bound DNA strands, virtually nothing was known about how these properties are altered when the shape of the nanoparticle core is chosen to be non-spherical. In particular, we are interested in understanding, and ultimately controlling, the ways in which these DNA-conjugated anisotropic nanostructures interact when their attraction is governed by programmable DNA hybridization events. Chapter 1 introduces the field of DNA-based materials assembly by discussing how nanoscale building blocks which present rigid, directional interactions can be thought of as possessing artificial versions of the familiar chemical principles of "bonds" and "valency". In chapter 2 we explore the fundamental interparticle binding thermodynamics of DNA-functionalized spherical and anisotropic nanoparticles, which reveals enormous preferences for collective ligand interactions occurring between flat surfaces over those that occur between curved surfaces. Using these insights, chapter 3 demonstrates that when syntheses produce mixtures of different nanoparticle shapes, the tailorable nature of DNA-mediated interparticle association can be used to selectively crystallize and purify the desired anisotropic nanostructure products, leaving spherical impurity particles behind. Chapter 4 leverages the principle that the flat facets of anisotropic particles generate directional DNA-based hybridization interactions to assemble a variety of tailorable nanoparticle superlattices whose symmetry and dimensionality are a direct consequence of the shape of the nanoparticle building block used in their construction. Chapter 5 explores

  13. Biotin-labeled synthetic oligodeoxyribonucleotides: chemical synthesis and uses as hybridization probes.

    PubMed Central

    Chollet, A; Kawashima, E H

    1985-01-01

    Oligodeoxynucleotides have been selectively labeled with biotin at their 5'-termini through an aminoalkylphosphoramide linker arm by an efficient chemical method. The reactions were performed in aqueous solution on unprotected oligonucleotides and were insensitive of the sequence and length of the oligonucleotide. 5'-biotin-labeled oligonucleotides were hybridized to dot, Southern and genomic blots of target plasmid DNA immobilized on nitrocellulose filters. Detection level is about 2 fmole. There is no noticeable disturbance of the strength and selectivity of hybridization of the 5'-biotin-labeled probes in comparison with non-modified DNA. Images PMID:4000941

  14. Sequence-dependent theory of oligonucleotide hybridization kinetics

    SciTech Connect

    Marimuthu, Karthikeyan; Chakrabarti, Raj E-mail: rajc@andrew.cmu.edu

    2014-05-07

    A theoretical approach to the prediction of the sequence and temperature-dependent rate constants for oligonucleotide hybridization reactions has been developed based on the theory of relaxation kinetics. One-sided and two-sided melting reaction mechanisms for oligonucleotide hybridization reactions have been considered, analyzed, modified, and compared to select a physically consistent as well as robust model for prediction of the relaxation times of DNA hybridization reactions that agrees with the experimental evidence. The temperature- and sequence-dependent parameters of the proposed model have been estimated using available experimental data. The relaxation time model that we developed has been combined with the nearest neighbor model of hybridization thermodynamics to estimate the temperature- and sequence-dependent rate constants of an oligonucleotide hybridization reaction. The model-predicted rate constants are compared to experimentally determined rate constants for the same oligonucleotide hybridization reactions. Finally, we consider a few important applications of kinetically controlled DNA hybridization reactions.

  15. PRACTICAL STRATEGIES FOR PROCESSING AND ANALYZING SPOTTED OLIGONUCLEOTIDE MICROARRAY DATA

    EPA Science Inventory

    Thoughtful data analysis is as important as experimental design, biological sample quality, and appropriate experimental procedures for making microarrays a useful supplement to traditional toxicology. In the present study, spotted oligonucleotide microarrays were used to profile...

  16. Methods to Characterize the Oligonucleotide Functionalization of Quantum Dots.

    PubMed

    Weichelt, Richard; Leubner, Susanne; Henning-Knechtel, Anja; Mertig, Michael; Gaponik, Nikolai; Schmidt, Thorsten-Lars; Eychmüller, Alexander

    2016-09-01

    Currently, DNA nanotechnology offers the most programmable, scalable, and accurate route for the self-assembly of matter with nanometer precision into 1, 2, or 3D structures. One example is DNA origami that is well suited to serve as a molecularly defined "breadboard", and thus, to organize various nanomaterials such as nanoparticles into hybrid systems. Since the controlled assembly of quantum dots (QDs) is of high interest in the field of photonics and other optoelectronic applications, a more detailed view on the functionalization of QDs with oligonucleotides shall be achieved. In this work, four different methods are presented to characterize the functionalization of thiol-capped cadmium telluride QDs with oligonucleotides and for the precise quantification of the number of oligonucleotides bound to the QD surface. This study enables applications requiring the self-assembly of semiconductor-oligonucleotide hybrid materials and proves the conjugation success in a simple and straightforward manner.

  17. Micro- and nano-structure based oligonucleotide sensors.

    PubMed

    Ferrier, David C; Shaver, Michael P; Hands, Philip J W

    2015-06-15

    This paper presents a review of micro- and nano-structure based oligonucleotide detection and quantification techniques. The characteristics of such devices make them very attractive for Point-of-Care or On-Site-Testing biosensing applications. Their small scale means that they can be robust and portable, their compatibility with modern CMOS electronics means that they can easily be incorporated into hand-held devices and their suitability for mass production means that, out of the different approaches to oligonucleotide detection, they are the most suitable for commercialisation. This review discusses the advantages of micro- and nano-structure based sensors and covers the various oligonucleotide detection techniques that have been developed to date. These include: Bulk Acoustic Wave and Surface Acoustic Wave devices, micro- and nano-cantilever sensors, gene Field Effect Transistors, and nanowire and nanopore based sensors. Oligonucleotide immobilisation techniques are also discussed.

  18. Reagents for the preparation of two oligonucleotides per synthesis (TOPS).

    PubMed Central

    Hardy, P M; Holland, D; Scott, S; Garman, A J; Newton, C R; McLean, M J

    1994-01-01

    In order to increase the efficiency of use of automated DNA synthesizers (i.e. the number of oligomers prepared per day), we have devised and prepared novel phosphoramidite reagents that contain a linking group which, while stable under the normal synthesis conditions, is cleaved under basic conditions. When one of these linkers is introduced at the desired position in the synthesis of an oligonucleotide, subsequent detritylation enables the synthesis of a second oligonucleotides sequence upon the first. During deprotection of the oligonucleotide with ammonium hydroxide, the chain is cleaved at either side of the points of introduction of the novel reagent, generating two oligonucleotides free in solution. These reagents are of particular use in applications where oligomers are used in pairs (such as PCR, chemical synthesis of genes etc.) and means that an automated synthesis facility can be used more efficiently, without the need for operator intervention, after the working day is over. Images PMID:8065912

  19. Highly parallel oligonucleotide purification and functionalization using reversible chemistry.

    PubMed

    York, Kerri T; Smith, Ryan C; Yang, Rob; Melnyk, Peter C; Wiley, Melissa M; Turk, Casey M; Ronaghi, Mostafa; Gunderson, Kevin L; Steemers, Frank J

    2012-01-01

    We have developed a cost-effective, highly parallel method for purification and functionalization of 5'-labeled oligonucleotides. The approach is based on 5'-hexa-His phase tag purification, followed by exchange of the hexa-His tag for a functional group using reversible reaction chemistry. These methods are suitable for large-scale (micromole to millimole) production of oligonucleotides and are amenable to highly parallel processing of many oligonucleotides individually or in high complexity pools. Examples of the preparation of 5'-biotin, 95-mer, oligonucleotide pools of >40K complexity at micromole scale are shown. These pools are prepared in up to ~16% yield and 90-99% purity. Approaches for using this method in other applications are also discussed.

  20. Highly parallel oligonucleotide purification and functionalization using reversible chemistry

    PubMed Central

    York, Kerri T.; Smith, Ryan C.; Yang, Rob; Melnyk, Peter C.; Wiley, Melissa M.; Turk, Casey M.; Ronaghi, Mostafa; Gunderson, Kevin L.; Steemers, Frank J.

    2012-01-01

    We have developed a cost-effective, highly parallel method for purification and functionalization of 5′-labeled oligonucleotides. The approach is based on 5′-hexa-His phase tag purification, followed by exchange of the hexa-His tag for a functional group using reversible reaction chemistry. These methods are suitable for large-scale (micromole to millimole) production of oligonucleotides and are amenable to highly parallel processing of many oligonucleotides individually or in high complexity pools. Examples of the preparation of 5′-biotin, 95-mer, oligonucleotide pools of >40K complexity at micromole scale are shown. These pools are prepared in up to ~16% yield and 90–99% purity. Approaches for using this method in other applications are also discussed. PMID:22039155

  1. Oligonucleotide-based theranostic nanoparticles in cancer therapy

    PubMed Central

    Shahbazi, Reza; Ozpolat, Bulent; Ulubayram, Kezban

    2016-01-01

    Theranostic approaches, combining the functionality of both therapy and imaging, have shown potential in cancer nanomedicine. Oligonucleotides such as small interfering RNA and microRNA, which are powerful therapeutic agents, have been effectively employed in theranostic systems against various cancers. Nanoparticles are used to deliver oligonucleotides into tumors by passive or active targeting while protecting the oligonucleotides from nucleases in the extracellular environment. The use of quantum dots, iron oxide nanoparticles and gold nanoparticles and tagging with contrast agents, like fluorescent dyes, optical or magnetic agents and various radioisotopes, has facilitated early detection of tumors and evaluation of therapeutic efficacy. In this article, we review the advantages of theranostic applications in cancer therapy and imaging, with special attention to oligonucleotide-based therapeutics. PMID:27102380

  2. Modification of end phosphate gruops in mono- and oligonucleotides.

    PubMed Central

    Shumyantzeva, V V; Sokolova, N I; Shabarova, Z A

    1976-01-01

    A method is described for selective activation of phosphomonoester end groups of oligonucleotides and nucleosidedi-(tri) phosphates via mixed anhydrides with mesitoic acid. Mixed anhydrides are synthesized in high yield and isolated by paper or DEAE-cellulose column chromatography. The ability of such anhydrides to phosphorylate different nucleophilic agents was used for synthesis of amidates, imidazolidates, esters, thioesters and pyrophosphates of mono- and oligonucleotides. Mixed anhydrides mono-, oligonucleotides and nucleosidedi-(tri)phosphates and mesitoic acid were also applied to achieve immobilization of the mono- and oligonucleotides via their end groups on hexamethylenediamine - Sepharose support. Mixed anhydrides studied may be efficiently used for affinity labeling of proteins and nucleic acids and also as material for preparating reagents for template reactions. PMID:1272808

  3. PRACTICAL STRATEGIES FOR PROCESSING AND ANALYZING SPOTTED OLIGONUCLEOTIDE MICROARRAY DATA

    EPA Science Inventory

    Thoughtful data analysis is as important as experimental design, biological sample quality, and appropriate experimental procedures for making microarrays a useful supplement to traditional toxicology. In the present study, spotted oligonucleotide microarrays were used to profile...

  4. Tritium labeling of antisense oligonucleotides by exchange with tritiated water.

    PubMed Central

    Graham, M J; Freier, S M; Crooke, R M; Ecker, D J; Maslova, R N; Lesnik, E A

    1993-01-01

    We describe a simple, efficient, procedure for labeling oligonucleotides to high specific activity (< 1 x 10(8) cpm/mumol) by hydrogen exchange with tritiated water at the C8 positions of purines in the presence of beta-mercaptoethanol, an effective radical scavenger. Approximately 90% of the starting material is recovered as intact, labeled oligonucleotide. The radiolabeled compounds are stable in biological systems; greater than 90% of the specific activity is retained after 72 hr incubation at 37 degrees C in serum-containing media. Data obtained from in vitro cellular uptake experiments using oligonucleotides labeled by this method are similar to those obtained using 35S or 14C-labeled compounds. Because this protocol is solely dependent upon the existence of purine residues, it should be useful for radiolabeling modified as well as unmodified phosphodiester oligonucleotides. Images PMID:8367289

  5. Antisense oligonucleotide therapeutics for human leukemia.

    PubMed

    Gewirtz, A M

    1998-01-01

    The development of reliable gene disruption strategies, and their application in living cells, has proven to be an extraordinary important advance for cell and molecular biologists. Using the various available approaches, the specific functions of any given gene may now be investigated directly in the relevant cell type. Application of similar experimental tools in a clinical setting might prove to be equally valuable and could well form the basis of a monumental advance in the practice of clinical medicine. This seems particularly true at the present time because much progress has been made in understanding the molecular pathogenesis of many diseases, including cancer. For these reasons a tremendous amount of interest has been generated in the use of oligodeoxynucleotides to modify gene expression. However, in spite of some notable successes which are detailed in this review, oligonucleotides have generated controversy in regard to their mechanism of action, reliability, and ultimate therapeutic utility. Nevertheless, the potential power of the "antisense" approach remains undisputed, and its ultimate therapeutic utility is far reaching. Accordingly, the problems associated with the use of these compounds are clearly worth solving. It remains the hope of many laboratories that the day will soon come when these techniques will make an important contribution to the management of chronic myelogenous leukemia and other neoplastic disorders.

  6. Antisense oligonucleotide therapeutics for human leukemia.

    PubMed

    Gewirtz, A M

    1997-01-01

    The development of reliable gene disruption strategies, and their application in living cells, has proven to be an extraordinarily important advance for cell and molecular biologists. Using the various available approaches, the specific functions of any given gene may now be investigated directly in the relevant cell type. Application of similar experimental tools in a clinical setting might prove to be equally valuable and could well form the basis of a monumental advance in the practice of clinical medicine. This seems particularly true at the present time since much progress has been made in understanding the molecular pathogenesis of many diseases, including cancer. For these reasons a tremendous amount of interest has been generated in the use of oligodeoxynucleotides to modify gene expression. However, in spite of some notable successes which are detailed in this review, oligonucleotides have generated controversy in regards to their mechanism of action, reliability, and ultimate therapeutic utility. Nevertheless, the potential power of the "antisense" approach remains undisputed, and its ultimate therapeutic utility is far reaching. Accordingly, the problems associated with the use of these compounds are clearly worth solving. It remains the hope of many laboratories that the day will soon come when these techniques will make an important contribution to the management of CML and other neoplastic disorders.

  7. Antisense Oligonucleotide Therapy for Inherited Retinal Dystrophies.

    PubMed

    Gerard, Xavier; Garanto, Alejandro; Rozet, Jean-Michel; Collin, Rob W J

    2016-01-01

    Inherited retinal dystrophies (IRDs) are an extremely heterogeneous group of genetic diseases for which currently no effective treatment strategies exist. Over the last decade, significant progress has been made utilizing gene augmentation therapy for a few genetic subtypes of IRD, although several technical challenges so far prevent a broad clinical application of this approach for other forms of IRD. Many of the mutations leading to these retinal diseases affect pre-mRNA splicing of the mutated genes . Antisense oligonucleotide (AON)-mediated splice modulation appears to be a powerful approach to correct the consequences of such mutations at the pre-mRNA level , as demonstrated by promising results in clinical trials for several inherited disorders like Duchenne muscular dystrophy, hypercholesterolemia and various types of cancer. In this mini-review, we summarize ongoing pre-clinical research on AON-based therapy for a few genetic subtypes of IRD , speculate on other potential therapeutic targets, and discuss the opportunities and challenges that lie ahead to translate splice modulation therapy for retinal disorders to the clinic.

  8. Oligonucleotide conjugates - Candidates for gene silencing therapeutics.

    PubMed

    Gooding, Matt; Malhotra, Meenakshi; Evans, James C; Darcy, Raphael; O'Driscoll, Caitriona M

    2016-10-01

    The potential therapeutic and diagnostic applications of oligonucleotides (ONs) have attracted great attention in recent years. The capability of ONs to selectively inhibit target genes through antisense and RNA interference mechanisms, without causing un-intended sideeffects has led them to be investigated for various biomedical applications, especially for the treatment of viral diseases and cancer. In recent years, many researchers have focused on enhancing the stability and target specificity of ONs by encapsulating/complexing them with polymers or lipid chains to formulate nanoparticles/nanocomplexes/micelles. Also, chemical modification of nucleic acids has emerged as an alternative to impart stability to ONs against nucleases and other degrading enzymes and proteins found in blood. In addition to chemically modifying the nucleic acids directly, another strategy that has emerged, involves conjugating polymers/peptide/aptamers/antibodies/proteins, preferably to the sense strand (3'end) of siRNAs. Conjugation to the siRNA not only enhances the stability and targeting specificity of the siRNA, but also allows for the development of self-administering siRNA formulations, with a much smaller size than what is usually observed for nanoparticle (∼200nm). This review concentrates mainly on approaches and studies involving ON-conjugates for biomedical applications.

  9. Photophysical deactivation pathways in adenine oligonucleotides.

    PubMed

    Spata, Vincent A; Matsika, Spiridoula

    2015-12-14

    In this work we study deactivation processes in adenine oligomers after absorption of UV radiation using Quantum Mechanics combined with Molecular Mechanics (QM/MM). Correlated electronic structure methods appropriate for describing the excited states are used to describe a π-stacked dimer of adenine bases incorporated into (dA)20(dT)20. The results of these calculations reveal three different types of excited state minima which play a role in deactivation processes. Within this set of minima there are minima where the excited state is localized on one adenine (monomer-like) as well as minima where the excited state is delocalized on two adenines, forming different types of excimers and bonded excimers of varying but inter-related character. The proximity of their energies reveals that the minima can decay into one another along a flat potential energy surface dependent on the interbase separation. Additionally, analysis of the emissive energies and other physical properties, including theoretical anisotropy calculations, and comparison with fluorescence experiments, provides evidence that excimers play an important role in long-lived signals in adenine oligonucleotides while the subpicosecond decay is attributed to monomer-like minima. The necessity for a close approach of the nucleobases reveals that the deactivation mechanism is tied to macro-molecular motion.

  10. Oligonucleotide and Long Polymeric DNA Encoding

    SciTech Connect

    Miller, E; Mariella Jr., R P; Christian, A T; Gardner, S N; Williams, J M

    2003-11-24

    This report summarizes the work done at Lawrence Livermore National Laboratory for the Oligonucleotide and Long Polymeric DNA Encoding project, part of the Microelectronic Bioprocesses Program at DARPA. The goal of the project was to develop a process by which long (circa 10,000 base-pair) synthetic DNA molecules could be synthesized in a timely and economic manner. During construction of the long molecule, errors in DNA sequence occur during hybridization and/or the subsequent enzymatic process. The work done on this project has resulted in a novel synthesis scheme that we call the parallel pyramid synthesis protocol, the development of a suit of computational tools to minimize and quantify errors in the synthesized DNA sequence, and experimental proof of this technique. The modeling consists of three interrelated modules: the bioinformatics code which determines the specifics of parallel pyramid synthesis for a given chain of long DNA, the thermodynamics code which tracks the products of DNA hybridization and polymerase extension during the later steps in the process, and the kinetics model which examines the temporal and spatial processes during one thermocycle. Most importantly, we conducted the first successful syntheses of a gene using small starting oligomers (tetramers). The synthesized sequence, 813 base pairs long, contained a 725 base pair gene, modified green fluorescent protein (mGFP), which has been shown to be a functional gene by cloning into cells and observing its green fluorescent product.

  11. Syntheses of oligonucleotide derivatives with P(V) porphyrin and their properties.

    PubMed

    Shimidzu, T; Segawa, H; Kitamura, M; Nimura, A

    1992-01-01

    Two types of oligonucleotide derivatives which are substituted by P(V) porphyrin at the phosphorus atom of an internucleotidic linkage and at the 5'-terminal internucleotidic linkage via a spacer were synthesized (Fig. 1), and hybridization capabilities of them with complementary oligonucleotides were evaluated. A novel method for a sensing of oligonucleotide by the fluorescence quenching via photo-induced electron transfer between the P(V) porphyrin labeled oligonucleotide and pyrene-labeled one on the oligonucleotide template is reported.

  12. 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.

    PubMed

    Hakala, H; Virta, P; Salo, H; Lönnberg, H

    1998-12-15

    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.

  13. 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.

    PubMed Central

    Hakala, H; Virta, P; Salo, H; Lönnberg, H

    1998-01-01

    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. PMID:9837986

  14. Spectrophotometric probe

    DOEpatents

    Prather, William S.; O'Rourke, Patrick E.

    1994-01-01

    A support structure bearing at least one probe for making spectrophotometric measurements of a fluid using a source of light and a spectrophotometer. The probe includes a housing with two optical fibers and a planoconvex lens. A sleeve bearing a mirror surrounds the housing. The lens is separated from the mirror by a fixed distance, defining an interior space for receiving a volume of the fluid sample. A plurality of throughholes extending through the sleeve communicate between the sample volume and the exterior of the probe, all but one hole bearing a screen. A protective jacket surrounds the probe. A hollow conduit bearing a tube is formed in the wall of the probe for venting any air in the interior space when fluid enters. The probe is held at an acute angle so the optic fibers carrying the light to and from the probe are not bent severely on emergence from the probe.

  15. Spectrophotometric probe

    DOEpatents

    Prather, W.S.; O'Rourke, P.E.

    1994-08-02

    A support structure is described bearing at least one probe for making spectrophotometric measurements of a fluid using a source of light and a spectrophotometer. The probe includes a housing with two optical fibers and a planoconvex lens. A sleeve bearing a mirror surrounds the housing. The lens is separated from the mirror by a fixed distance, defining an interior space for receiving a volume of the fluid sample. A plurality of throughholes extending through the sleeve communicate between the sample volume and the exterior of the probe, all but one hole bearing a screen. A protective jacket surrounds the probe. A hollow conduit bearing a tube is formed in the wall of the probe for venting any air in the interior space when fluid enters. The probe is held at an acute angle so the optic fibers carrying the light to and from the probe are not bent severely on emergence from the probe. 3 figs.

  16. Simplified Paper Format for Detecting HIV Drug Resistance in Clinical Specimens by Oligonucleotide Ligation

    PubMed Central

    Panpradist, Nuttada; Beck, Ingrid A.; Chung, Michael H.; Kiarie, James N.; Frenkel, Lisa M.; Lutz, Barry R.

    2016-01-01

    Human immunodeficiency virus (HIV) is a chronic infection that can be managed by antiretroviral treatment (ART). However, periods of suboptimal viral suppression during lifelong ART can select for HIV drug resistant (DR) variants. Transmission of drug resistant virus can lessen or abrogate ART efficacy. Therefore, testing of individuals for drug resistance prior to initiation of treatment is recommended to ensure effective ART. Sensitive and inexpensive HIV genotyping methods are needed in low-resource settings where most HIV infections occur. The oligonucleotide ligation assay (OLA) is a sensitive point mutation assay for detection of drug resistance mutations in HIV pol. The current OLA involves four main steps from sample to analysis: (1) lysis and/or nucleic acid extraction, (2) amplification of HIV RNA or DNA, (3) ligation of oligonucleotide probes designed to detect single nucleotide mutations that confer HIV drug resistance, and (4) analysis via oligonucleotide surface capture, denaturation, and detection (CDD). The relative complexity of these steps has limited its adoption in resource-limited laboratories. Here we describe a simplification of the 2.5-hour plate-format CDD to a 45-minute paper-format CDD that eliminates the need for a plate reader. Analysis of mutations at four HIV-1 DR codons (K103N, Y181C, M184V, and G190A) in 26 blood specimens showed a strong correlation of the ratios of mutant signal to total signal between the paper CDD and the plate CDD. The assay described makes the OLA easier to perform in low resource laboratories. PMID:26751207

  17. Development and validation of an oligonucleotide microarray to characterise ectomycorrhizal fungal communities

    PubMed Central

    2009-01-01

    Background In forest ecosystems, communities of ectomycorrhizal fungi (ECM) are influenced by several biotic and abiotic factors. To understand their underlying dynamics, ECM communities have been surveyed with ribosomal DNA-based sequencing methods. However, most identification methods are both time-consuming and limited by the number of samples that can be treated in a realistic time frame. As a result of ongoing implementation, the array technique has gained throughput capacity in terms of the number of samples and the capacity for parallel identification of several species. Thus far, although phylochips (microarrays that are used to detect species) have been mostly developed to trace bacterial communities or groups of specific fungi, no phylochip has been developed to carry oligonucleotides for several ectomycorrhizal species that belong to different genera. Results We have constructed a custom ribosomal DNA phylochip to identify ECM fungi. Specific oligonucleotide probes were targeted to the nuclear internal transcribed spacer (ITS) regions from 95 fungal species belonging to 21 ECM fungal genera. The phylochip was first validated using PCR amplicons of reference species. Ninety-nine percent of the tested oligonucleotides generated positive hybridisation signals with their corresponding amplicons. Cross-hybridisation was mainly restricted at the genus level, particularly for Cortinarius and Lactarius species. The phylochip was subsequently tested with environmental samples that were composed of ECM fungal DNA from spruce and beech plantation fungal communities. The results were in concordance with the ITS sequencing of morphotypes and the ITS clone library sequencing results that were obtained using the same PCR products. Conclusion For the first time, we developed a custom phylochip that is specific for several ectomycorrhizal fungi. To overcome cross-hybridisation problems, specific filter and evaluation strategies that used spot signal intensity were

  18. Nanogels for Oligonucleotide Delivery to the Brain

    PubMed Central

    Vinogradov, Serguei V.; Batrakova, Elena V.; Kabanov, Alexander V.

    2009-01-01

    Systemic delivery of oligonucleotides (ODN) to the central nervous system is needed for development of therapeutic and diagnostic modalities for treatment of neurodegenerative disorders. Macromolecules injected in blood are poorly transported across the blood–brain barrier (BBB) and rapidly cleared from circulation. In this work we propose a novel system for ODN delivery to the brain based on nanoscale network of cross-linked poly(ethylene glycol) and polyethylenimine (“nanogel”). The methods of synthesis of nanogel and its modification with specific targeting molecules are described. Nanogels can bind and encapsulate spontaneously negatively charged ODN, resulting in formation of stable aqueous dispersion of polyelectrolyte complex with particle sizes less than 100 nm. Using polarized monolayers of bovine brain microvessel endothelial cells as an in vitro model this study demonstrates that ODN incorporated in nanogel formulations can be effectively transported across the BBB. The transport efficacy is further increased when the surface of the nanogel is modified with transferrin or insulin. Importantly the ODN is transported across the brain microvessel cells through the transcellular pathway; after transport, ODN remains mostly incorporated in the nanogel and ODN displays little degradation compared to the free ODN. Using mouse model for biodistribution studies in vivo, this work demonstrated that as a result of incorporation into nanogel 1 h after intravenous injection the accumulation of a phosphorothioate ODN in the brain increases by over 15 fold while in liver and spleen decreases by 2-fold compared to the free ODN. Overall, this study suggests that nanogel is a promising system for delivery of ODN to the brain. PMID:14733583

  19. Fluorescent probes for nucleic Acid visualization in fixed and live cells.

    PubMed

    Boutorine, Alexandre S; Novopashina, Darya S; Krasheninina, Olga A; Nozeret, Karine; Venyaminova, Alya G

    2013-12-11

    This review analyses the literature concerning non-fluorescent and fluorescent probes for nucleic acid imaging in fixed and living cells from the point of view of their suitability for imaging intracellular native RNA and DNA. Attention is mainly paid to fluorescent probes for fluorescence microscopy imaging. Requirements for the target-binding part and the fluorophore making up the probe are formulated. In the case of native double-stranded DNA, structure-specific and sequence-specific probes are discussed. Among the latest, three classes of dsDNA-targeting molecules are described: (i) sequence-specific peptides and proteins; (ii) triplex-forming oligonucleotides and (iii) polyamide oligo(N-methylpyrrole/N-methylimidazole) minor groove binders. Polyamides seem to be the most promising targeting agents for fluorescent probe design, however, some technical problems remain to be solved, such as the relatively low sequence specificity and the high background fluorescence inside the cells. Several examples of fluorescent probe applications for DNA imaging in fixed and living cells are cited. In the case of intracellular RNA, only modified oligonucleotides can provide such sequence-specific imaging. Several approaches for designing fluorescent probes are considered: linear fluorescent probes based on modified oligonucleotide analogs, molecular beacons, binary fluorescent probes and template-directed reactions with fluorescence probe formation, FRET donor-acceptor pairs, pyrene excimers, aptamers and others. The suitability of all these methods for living cell applications is discussed.

  20. Oligodeoxynucleotide Probes for Detecting Intact Cells

    NASA Technical Reports Server (NTRS)

    Rosson, Reinhardt A.; Maurina-Brunker, Julie; Langley, Kim; Pynnonen, Christine M.

    2004-01-01

    A rapid, sensitive test using chemiluminescent oligodeoxynucleotide probes has been developed for detecting, identifying, and enumerating intact cells. The test is intended especially for use in detecting and enumerating bacteria and yeasts in potable water. As in related tests that have been developed recently for similar purposes, the oligodeoxynucleotide probes used in this test are typically targeted at either singlecopy deoxyribonucleic acid (DNA) genes (such as virulence genes) or the multiple copies (10,000 to 50,000 copies per cell) of 16S ribosomal ribonucleic acids (rRNAs). Some of those tests involve radioisotope or fluorescent labeling of the probes for reporting hybridization of probes to target nucleic acids. Others of those tests involve labeling with enzymes plus the use of chemiluminescent or chromogenic substrates to report hybridization via color or the emission of light, respectively. The present test is of the last-mentioned type. The chemiluminescence in the present test can be detected easily with relatively simple instrumentation. In developing the present test, the hybridization approach was chosen because hybridization techniques are very specific. Hybridization detects stable, inheritable genetic targets within microorganisms. These targets are not dependent on products of gene expression that can vary with growth conditions or physiological states of organisms in test samples. Therefore, unique probes can be designed to detect and identify specific genera or species of bacteria or yeast (in terms of rRNA target sequences) or can be designed to detect and identify virulence genes (genomic target sequences). Because of the inherent specificity of this system, there are few problems of cross-reactivity. Hybridization tests are rapid, but hybridization tests now available commercially lack sensitivity; typically, between 10(exp 6) and 10(exp 7) cells of the target organism are needed to ensure a reliable test. Consequently, the numbers of

  1. Biominetic High Density Lipoproteins for the Delivery of Therapeutic Oligonucleotides

    NASA Astrophysics Data System (ADS)

    Tripathy, Sushant

    Advances in nanotechnology have brought about novel inorganic and hybrid nanoparticles with unique physico-chemical properties that make them suitable for a broad range of applications---from nano-circuitry to drug delivery. A significant part of those advancements have led to ground-breaking discoveries that have changed the approaches to formulation of therapeutics against diseases, such as cancer. Now-a-days the focus does not lie solely on finding a candidate small-molecule therapeutic with minimal adverse effects, but researchers are looking up to nanoparticles to improve biodistribution and biocompatibility profile of clinically proven therapeutics. The plethora of conjugation chemistries offered by currently extant inorganic nanoparticles have, in recent years, led to great leaps in the field of biomimicry---a modality that promises high biocompatibility. Further, in the pursuit of highly specific therapeutic molecules, researchers have turned to silencing oligonucleotides and some have already brought together the strengths of nanoparticles and silencing oligonucleotides in search of an efficacious therapy for cancer with minimal adverse effects. This dissertation work focuses on such a biomimetic platform---a gold nanoparticle based high density lipoprotein biomimetic (HDL NP), for the delivery of therapeutic oligonucleotides. The first chapter of this body of work introduces the molecular target of the silencing oligonucleotides---VEGFR2, and its role in the progression of solid tumor cancers. The background information also covers important aspects of natural high density lipoproteins (HDL), especially their innate capacity to bind and deliver exogenous and endogenous silencing oligonucleotides to tissues that express their high affinity receptor SRB1. We subsequently describe the synthesis of the biomimetic HDL NP and its oligonucleotide conjugates, and establish their biocompatibility. Further on, experimental data demonstrate the efficacy of silencing

  2. Detection of adenosine 5'-triphosphate by fluorescence variation of oligonucleotide-templated silver nanoclusters.

    PubMed

    Lee, Jennifer Daneen; Cang, Jinshun; Chen, Ying-Chieh; Chen, Wei-Yu; Ou, Chung-Mao; Chang, Huan-Tsung

    2014-08-15

    Oligonucleotide-templated Ag nanoclusters (DNA-Ag NCs) prepared from AgNO3 using an oligonucleotide (5'-TAACCCCTAACCCCT-3') as a template and NaBH4 as a reducing agent have been used for sensing of adenosine 5'-triphosphate (ATP). The fluorescence intensity and emission wavelength of DNA-Ag NCs are dependent on the pH value and ATP concentration. At pH 3.0 and 11.0, ATP shows greater effects on fluorescence of the DNA-Ag NCs. Upon increasing ATP concentration from 10 to 50μM, their emission wavelength at pH 3.0 shifts from 525 to 585nm. At pH 11.0, their fluorescence intensity (510nm) increases upon increasing ATP concentration. The circular dichroism (CD), electrospray ionization-mass spectrometry (ESI-MS), absorption, and fluorescence results indicate that ATP and pH affect the interactions between DNAs and Ag atoms, resulting in changes in their fluorescence. The DNA-Ag NCs allow detection of ATP over a concentration range of 0.1-10μM, with a limit of detection 33nM. Practicality of the DNA-Ag NCs probe has been validated with the determination of ATP concentrations in the lysate of MDA-MB-231 breast carcinoma cells.

  3. Universality in the Timescales of Internal Loop Formation in Unfolded Proteins and Single-Stranded Oligonucleotides

    PubMed Central

    Cheng, Ryan R.; Uzawa, Takanori; Plaxco, Kevin W.; Makarov, Dmitrii E.

    2010-01-01

    Understanding the rate at which various parts of a molecular chain come together to facilitate the folding of a biopolymer (e.g., a protein or RNA) into its functional form remains an elusive goal. Here we use experiments, simulations, and theory to study the kinetics of internal loop closure in disordered biopolymers such as single-stranded oligonucleotides and unfolded proteins. We present theoretical arguments and computer simulation data to show that the relationship between the timescale of internal loop formation and the positions of the monomers enclosing the loop can be recast in a form of a universal master dependence. We also perform experimental measurements of the loop closure times of single-stranded oligonucleotides and show that both these and previously reported internal loop closure kinetics of unfolded proteins are well described by this theoretically predicted dependence. Finally, we propose that experimental deviations from the master dependence can then be used as a sensitive probe of dynamical and structural order in unfolded proteins and other biopolymers. PMID:21156138

  4. Development of a gold nanoparticle-based oligonucleotide microarray for simultaneous detection of seven swine viruses.

    PubMed

    Wang, Xiaoqiang; Dang, Erle; Gao, Jinzhuai; Guo, Sen; Li, Zheng

    2013-07-01

    A gold nanoparticle (GNP) based oligonucleotide microarray assay (GNMA) that combined GNP-based multiplex asymmetric PCR with silver enhancement detection, was developed for simultaneous detection of seven important swine viruses in intensive swine production. Multiplex PCR was first performed to enable the target fragments of seven viruses containing a universal sequence, which were labeled simultaneously with GNPs by multiplex asymmetric PCR in the presence of excess GNP-conjugated primer. Target labeled products were captured by virus-specific oligonucleotide probes immobilized on microarrays, followed by silver staining for signal enhancement. Black image of microarray spots was easily detected by the naked eye or a simple flatbed scanner and quantified. The results for purified plasmid constructs indicated that the assay was highly specific for detecting the seven viruses in single or multiple infections, and as few as 6-80 copies/μl of specific viral target fragments were detected successfully. Fifty-seven archived samples were tested by this assay, and the results were 100% consistent with previous results based on real-time PCR and those obtained by conventional PCR/RT-PCR and sequencing. The assay is appropriate for the routine diagnosis of viral infections in pigs due to its simplicity, low-cost, high specificity and sensitivity. Copyright © 2013 Elsevier B.V. All rights reserved.

  5. DNA Probes Using Fluorescence Resonance Energy Transfer (FRET): Designs and Applications

    PubMed Central

    Didenko, Vladimir V.

    2007-01-01

    Fluorescence resonance energy transfer (FRET) is widely used in biomedical research as a reporter method. Oligonucleotides with a DNA backbone and one or several chromophore tags have found multiple applications as FRET probes. They are especially advantageous for the real-time monitoring of biochemical reactions and in vivo studies. This paper reviews the design and applications of various DNA-based probes that use FRET. The approaches used in the design of new DNA FRET probes are discussed. PMID:11730017

  6. Target mRNA inhibition by oligonucleotide drugs in man

    PubMed Central

    Lightfoot, Helen L.; Hall, Jonathan

    2012-01-01

    Oligonucleotide delivery in vivo is commonly seen as the principal hurdle to the successful development of oligonucleotide drugs. In an analysis of 26 oligonucleotide drugs recently evaluated in late-stage clinical trials we found that to date at least half have demonstrated suppression of the target mRNA and/or protein levels in the relevant cell types in man, including those present in liver, muscle, bone marrow, lung, blood and solid tumors. Overall, this strongly implies that the drugs are being delivered to the appropriate disease tissues. Strikingly we also found that the majority of the drug targets of the oligonucleotides lie outside of the drugable genome and represent new mechanisms of action not previously investigated in a clinical setting. Despite the high risk of failure of novel mechanisms of action in the clinic, a subset of the targets has been validated by the drugs. While not wishing to downplay the technical challenges of oligonucleotide delivery in vivo, here we demonstrate that target selection and validation are of equal importance for the success of this field. PMID:22989709

  7. Predicting oligonucleotide-directed mutagenesis failures in protein engineering

    PubMed Central

    Wassman, Christopher D.; Tam, Phillip Y.; Lathrop, Richard H.; Weiss, Gregory A.

    2004-01-01

    Protein engineering uses oligonucleotide-directed mutagenesis to modify DNA sequences through a two-step process of hybridization and enzymatic synthesis. Inefficient reactions confound attempts to introduce mutations, especially for the construction of vast combinatorial protein libraries. This paper applied computational approaches to the problem of inefficient mutagenesis. Several results implicated oligonucleotide annealing to non-target sites, termed ‘cross-hybridization’, as a significant contributor to mutagenesis reaction failures. Test oligonucleotides demonstrated control over reaction outcomes. A novel cross-hybridization score, quickly computable for any plasmid and oligonucleotide mixture, directly correlated with yields of deleterious mutagenesis side products. Cross-hybridization was confirmed conclusively by partial incorporation of an oligonucleotide at a predicted cross-hybridization site, and by modification of putative template secondary structure to control cross-hybridization. Even in low concentrations, cross-hybridizing species in mixtures poisoned reactions. These results provide a basis for improved mutagenesis efficiencies and increased diversities of cognate protein libraries. PMID:15585664

  8. Characteristic archaebacterial 16S rRNA oligonucleotides

    NASA Technical Reports Server (NTRS)

    McGill, T. J.; Jurka, J.; Sobieski, J. M.; Pickett, M. H.; Woese, C. R.; Fox, G. E.

    1986-01-01

    A method of analyzing 16S rRNA catalog data has been developed in which groupings at various taxonomic levels can be characterized in terms of specific "signature" oligonucleotides. This approach provides an alternative means for evaluating higher order branching possibilities and can be used to assess the phylogenetic position of isolates that are poorly placed by the usual clustering procedures. This signature approach has been applied to forty archaebacterial catalogs and every oligonucleotide with significant signature value has been identified. Sets of specific oligonucleotides were identified for every major group on a dendrogram produced by cluster analysis procedures. Signatures that would establish between group relationships were also sought and found. In the case of the Methanobacteriaceae the clustering methods suggest a specific relationship to the Methanococcaceae. This inclusion is in fact supported by six strong signature oligonucleotides. However there are also significant numbers of signature oligonucleotides supporting a specific relationship of the Methanobacteriaceae to either the Halobacteriaceae or the Methanomicrobiaceae. Thus the placement of the Methanobacteriaceae is less certain than the usual dendrograms imply. The signature approach also was used to assess the phylogenetic position of Thermoplasma acidophilum which is found to be more closely related to the methanogen/halophile Division than to the sulfur dependent Division of the archaebacteria. This does not imply however that Thermoplasma acidophilum is properly regarded as being in the methanogen/halophile Division.

  9. Characteristic archaebacterial 16S rRNA oligonucleotides.

    PubMed

    McGill, T J; Jurka, J; Sobieski, J M; Pickett, M H; Woese, C R; Fox, G E

    1986-01-01

    A method of analyzing 16S rRNA catalog data has been developed in which groupings at various taxonomic levels can be characterized in terms of specific "signature" oligonucleotides. This approach provides an alternative means for evaluating higher order branching possibilities and can be used to assess the phylogenetic position of isolates that are poorly placed by the usual clustering procedures. This signature approach has been applied to forty archaebacterial catalogs and every oligonucleotide with significant signature value has been identified. Sets of specific oligonucleotides were identified for every major group on a dendrogram produced by cluster analysis procedures. Signatures that would establish between group relationships were also sought and found. In the case of the Methanobacteriaceae the clustering methods suggest a specific relationship to the Methanococcaceae. This inclusion is in fact supported by six strong signature oligonucleotides. However there are also significant numbers of signature oligonucleotides supporting a specific relationship of the Methanobacteriaceae to either the Halobacteriaceae or the Methanomicrobiaceae. Thus the placement of the Methanobacteriaceae is less certain than the usual dendrograms imply. The signature approach also was used to assess the phylogenetic position of Thermoplasma acidophilum which is found to be more closely related to the methanogen/halophile Division than to the sulfur dependent Division of the archaebacteria. This does not imply however that Thermoplasma acidophilum is properly regarded as being in the methanogen/halophile Division.

  10. A PELDOR-based nanometer distance ruler for oligonucleotides.

    PubMed

    Schiemann, Olav; Piton, Nelly; Mu, Yuguang; Stock, Gerhard; Engels, Joachim W; Prisner, Thomas F

    2004-05-12

    A pulsed electron paramagnetic resonance (EPR) spectroscopic ruler for oligonucleotides was developed using a series of duplex DNAs. The spin-labeling is accomplished during solid-phase synthesis of the oligonucleotides utilizing a palladium-catalyzed cross-coupling reaction between 5-iodo-2'-deoxyuridine and the rigid spin-label 2,2,5,5-tetramethyl-pyrrolin-1-yloxyl-3-acetylene (TPA). 4-Pulse electron double resonance (PELDOR) was then used to measure the intramolecular spin-spin distances via the dipolar coupling, yielding spin-spin distances of 19.2, 23.3, 34.7, 44.8, and 52.5 A. Employing a full-atom force field with explicit water, molecular dynamic (MD) simulations on the same spin-labeled oligonucleotides in their duplex B-form gave spin-spin distances of 19.6, 21.4, 33.0, 43.3, and 52.5 A, respectively, in very good agreement with the measured distances. This shows that the oligonucleotides adopt a B-form duplex structure also in frozen aqueous buffer solution. It also demonstrates that the combined use of site-directed spin-labeling, PELDOR experiments, and MD simulations can yield a microscopic picture about the overall structure of oligonucleotides. The technique is also applicable to more complex systems, like ribozymes or DNA/RNA-protein complexes, which are difficult to access by NMR or X-ray crystallography.

  11. Event-specific detection of seven genetically modified soybean and maizes using multiplex-PCR coupled with oligonucleotide microarray.

    PubMed

    Xu, Jia; Zhu, Shuifang; Miao, Haizhen; Huang, Wensheng; Qiu, Minyan; Huang, Yan; Fu, Xuping; Li, Yao

    2007-07-11

    With the increasing development of genetically modified organism (GMO) detection techniques, the polymerase chain reaction (PCR) technique has been the mainstay for GMO detection. An oligonucleotide microarray is a glass chip to the surface of which an array of oligonucleotides was fixed as spots, each containing numerous copies of a sequence-specific probe that is complementary to a gene of interest. So it is used to detect ten or more targets synchronously. In this research, an event-specific detection strategy based on the unique and specific integration junction sequences between the host plant genome DNA and the integrated gene is being developed for its high specificity using multiplex-PCR together with oligonucleotide microarray. A commercial GM soybean (GTS 40-3-2) and six GM maize events (MON810, MON863, Bt176, Bt11, GA21, and T25) were detected by this method. The results indicate that it is a suitable method for the identification of these GM soybean and maizes.

  12. Fast large scale oligonucleotide selection using the longest common factor approach.

    PubMed

    Rahmann, Sven

    2003-07-01

    We present a fast method that selects oligonucleotide probes (such as DNA 25-mers) for microarray experiments on a truly large scale. For example, reliable oligos for human genes can be found within four days, a speedup of one to two orders of magnitude compared to previous approaches. This speed is attained by using the longest common substring as a specificity measure for candidate oligos. We present a space- and time-efficient algorithm, based on a suffix array with additional information, to compute matching statistics (lengths of longest matches) between all candidate oligos and all remaining sequences. With the matching statistics available, we show how to incorporate constraints such as oligo length, melting temperature, and self-complementarity into the selection process at a postprocessing stage. As a result, we can now design custom oligos for any sequenced genome, just as the technology for on-site chip synthesis is becoming increasingly mature.

  13. [The second generation universal oligonucleotide microarray for subtyping of influenza virus A].

    PubMed

    Kostina, E V; Riabinin, V A; Maksakova, G A; Siniakov, A N

    2012-01-01

    The microchip for influenza A subtyping was developed, functioning on a principle "one spot--one subtype". Each spot contains the set of oligonucleotide probes, specific for a particular subtype of hemagglutinin, neuraminidase or matrix gene. Reliability of the proposed chip version is the same as for earlier created in our group full-size microchip for separate hemagglutinin and neuraminidase subtyping. To visualize the image, analyzed DNA can be labeled by either fluorescent dye or biotin with the further fixation in system streptavidin-gold nanoparticles and image development by silver precipitation. In the second case common version of scanner can be used for the image analysis, that essentially simplifies procedure of influenza A subtyping.

  14. Automated synthesis of new ferrocenyl-modified oligonucleotides: study of their properties in solution

    PubMed Central

    Navarro, Aude-Emmanuelle; Spinelli, Nicolas; Moustrou, Corinne; Chaix, Carole; Mandrand, Bernard; Brisset, Hugues

    2004-01-01

    We have developed new ferrocenyl-modified oligonucleotide (ODN) probes for electrochemical DNA sensors. A monofunctional ferrocene containing phosphoramidite group has been prepared, and a new bisfunctional ferrocene containing phosphoramidite and dimethoxytrityl (DMT) groups has been developed. These ferrocenyl-phosphoramidites have been directly employed in an automated solid-phase DNA synthesizer using phosphoramidite chemistry. The advantages of this method are that it allows a non-specialist in nucleotide chemistry to access labeled ODNs and that it has demonstrated good results. ODNs modified at the 3′ and/or 5′ extremities have been prepared, with the incorporation of the ferrocenyl group into the chain. The 5′ position appears to be more important due to its particular behavior. The thermal stability and electrochemical properties of these new ODN ferrocenes were analyzed before and after hybridization with different ODNs. The feasibility of using these new ferrocenyl-labeled ODNs in DNA sensors has been demonstrated. PMID:15466597

  15. Array of nucleic acid probes on biological chips for diagnosis of HIV and methods of using the same

    DOEpatents

    Chee, Mark; Gingeras, Thomas R.; Fodor, Stephen P. A.; Hubble, Earl A.; Morris, MacDonald S.

    1999-01-19

    The invention provides an array of oligonucleotide probes immobilized on a solid support for analysis of a target sequence from a human immunodeficiency virus. The array comprises at least four sets of oligonucleotide probes 9 to 21 nucleotides in length. A first probe set has a probe corresponding to each nucleotide in a reference sequence from a human immunodeficiency virus. A probe is related to its corresponding nucleotide by being exactly complementary to a subsequence of the reference sequence that includes the corresponding nucleotide. Thus, each probe has a position, designated an interrogation position, that is occupied by a complementary nucleotide to the corresponding nucleotide. The three additional probe sets each have a corresponding probe for each probe in the first probe set. Thus, for each nucleotide in the reference sequence, there are four corresponding probes, one from each of the probe sets. The three corresponding probes in the three additional probe sets are identical to the corresponding probe from the first probe or a subsequence thereof that includes the interrogation position, except that the interrogation position is occupied by a different nucleotide in each of the four corresponding probes.

  16. Safety of antisense oligonucleotide and siRNA-based therapeutics.

    PubMed

    Chi, Xuan; Gatti, Philip; Papoian, Thomas

    2017-01-31

    Oligonucleotide-based therapy is an active area of drug development designed to treat a variety of gene-specific diseases. Two of the more promising platforms are the antisense oligonucleotides (ASOs) and short interfering RNAs (siRNAs), both of which are often directed against similar targets. In light of recent reports on clinical trials of severe thrombocytopenia with two different ASO drugs and increased peripheral neuropathy with an siRNA drug, we compared and contrasted the specific safety characteristics of these two classes of oligonucleotide therapeutic. The objectives were to assess factors that could contribute to the specific toxicities observed with these two classes of promising drugs, and get a better understanding of the potential mechanism(s) responsible for these rare, but serious, adverse events.

  17. Retro-1 Analogues Differentially Affect Oligonucleotide Delivery and Toxin Trafficking.

    PubMed

    Yang, Bing; Ming, Xin; Abdelkafi, Hajer; Pons, Valerie; Michau, Aurelien; Gillet, Daniel; Cintrat, Jean-Christophe; Barbier, Julien; Juliano, Rudy

    2016-11-21

    Retro-1 is a small molecule that displays two important biological activities: First, it blocks the actions of certain toxins by altering their intracellular trafficking. Second, it enhances the activity of oligonucleotides by releasing them from entrapment in endosomes. This raises the question of whether the two actions involve the same cellular target. Herein we report the effects of several Retro-1 analogues on both toxins and oligonucleotides. We found analogues that affect toxins but not oligonucleotides and vice-versa, while Retro-1 is the only compound that affects both. This indicates that the molecular target(s) involved in the two processes are distinct. © 2016 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim.

  18. OligoFaktory: a visual tool for interactive oligonucleotide design.

    PubMed

    Schretter, Colas; Milinkovitch, Michel C

    2006-01-01

    The OligoFaktory is a set of tools for the design, on an arbitrary number of target sequences, of high-quality long oligonucleotide for micro-array, of primer pair for PCR, of siRNA and more. The user-centered interface exists in two flavours: a web portal and a standalone software for Mac OS X Tiger. A unified presentation of results provides overviews with distribution charts and relative location bar graphs, as well as detailed features for each oligonucleotide. Input and output files conform to a common XML interchange file format to allow both automatic generation of input data, archiving, and post-processing of results. The design pipeline can use BLAST servers to evaluate specificity of selected oligonucleotides. The web portal http://ueg.ulb.ac.be/oligofaktory/; the software for Macintosh: http://www.oligofaktory.org/

  19. Current progress on aptamer-targeted oligonucleotide therapeutics

    PubMed Central

    Dassie, Justin P; Giangrande, Paloma H

    2014-01-01

    Exploiting the power of the RNAi pathway through the use of therapeutic siRNA drugs has remarkable potential for treating a vast array of human disease conditions. However, difficulties in delivery of these and similar nucleic acid-based pharmacological agents to appropriate organs or tissues, remains a major impediment to their broad clinical application. Synthetic nucleic acid ligands (aptamers) have emerged as effective delivery vehicles for therapeutic oligonucleotides, including siRNAs. In this review, we summarize recent attractive developments in creatively employing cell-internalizing aptamers to deliver therapeutic oligonucleotides (e.g., siRNAs, miRNAs, anti-miRs and antisense oligos) to target cells. We also discuss advancements in aptamer-siRNA chimera technology, as well as, aptamer-functionalized nanoparticles for siRNA delivery. In addition, the challenges and future prospects of aptamer-targeted oligonucleotide drugs for clinical translation are further highlighted. PMID:24304250

  20. Discrimination of single base mismatched oligonucleotides related to the rpoB gene of Mycobacterium tuberculosis using a surface plasmon resonance biosensor.

    PubMed

    Rachkov, A; Patskovsky, S; Soldatkin, A; Meunier, M

    2013-01-01

    Single base mismatched oligonucleotides related to the rpoB gene of Mycobacterium tuberculosis, the mutations of which cause drug resistance of the infectious agent, were detected and discriminated using a surface plasmon resonance biosensor system. Thiol-modified oligonucleotides of the selected sequence (the probe) and 1-mercapto-6-hexanol were immobilized on a gold sensor surface. Hybridization between immobilized probe P2 and perfectly matched target T2 as well as a single base mismatched target TN was investigated in buffer solutions of various stringencies. Discrimination of perfectly matched and single base mismatched targets is achieved due to a difference in the level of their hybridization with the immobilized probe depending on stringency of the buffer solution. In 0.5×SSC buffer solution (7.5 mM sodium citrate, pH 7, containing 75 mM NaCl), sensor response at T2 injection into the measuring sensor cell was 16 times that at TN injection. The experimental results on surface hybridization between the studied oligonucleotides demonstrated a good correlation with theoretical calculations of thermodynamic parameters of these interactions in the solution. The described approach could be proposed as a basis for creating a biosensor for real-time label-free diagnostics of drug-resistant tuberculosis. © 2013 International Union of Biochemistry and Molecular Biology, Inc.

  1. Bulged Invader probes: activated duplexes for mixed-sequence dsDNA recognition with improved thermodynamic and kinetic profiles.

    PubMed

    Guenther, Dale C; Karmakar, Saswata; Hrdlicka, Patrick J

    2015-10-18

    Double-stranded oligonucleotides with +1 interstrand zipper arrangements of intercalator-functionalized nucleotides are energetically activated for recognition of mixed-sequence double-stranded DNA. Incorporation of nonyl (C9) bulges at specific positions of these probes, results in more highly affine (>5-fold), faster (>4-fold) and more persistent dsDNA recognition relative to conventional Invader probes.

  2. Dissecting tBHQ induced ARE-driven gene expression through long and short oligonucleotide arrays.

    PubMed

    Li, Jiang; Spletter, Maria L; Johnson, Jeffrey A

    2005-03-21

    This paper compares the gene expression profiles identified by short (Affymetrix U95AV2) or long (Agilent Hu1A) oligonucleotide arrays on a model for upregulation of a cluster of antioxidant responsive element-driven genes by treatment with tert-butylhydroquinone. MAS 5.0, dCHIP, and RMA were applied to normalize the Affymetrix data, and Lowess regression was considered for Agilent data. SAM was used to identify the differential gene expression. A set of biological markers and housekeeping genes were chosen to evaluate the performance of multiple normalization approaches. Both arrays illustrated a definite set of overlapping genes between the data sets regardless of data mining tools used. However, unique gene expression profiles based on the platform used were also revealed and confirmed by quantitative RT-PCR. Further analysis of the data revealed by alternative approaches suggested that alternative splicing, multiple vs. single probe(s) measurement, and use or nonuse of mismatch probes may account for the discrepant data. Therefore, these two microarray technologies offer relatively reliable data. Integration of the gene expression profiles from different array platforms may not only help for cross-validation but also provide a more complete view of the transcriptional scenario.

  3. Identification of Upper Respiratory Tract Pathogens Using Electrochemical Detection on an Oligonucleotide Microarray

    PubMed Central

    Lodes, Michael J.; Suciu, Dominic; Wilmoth, Jodi L.; Ross, Marty; Munro, Sandra; Dix, Kim; Bernards, Karen; Stöver, Axel G.; Quintana, Miguel; Iihoshi, Naomi; Lyon, Wanda J.; Danley, David L.; McShea, Andrew

    2007-01-01

    Bacterial and viral upper respiratory infections (URI) produce highly variable clinical symptoms that cannot be used to identify the etiologic agent. Proper treatment, however, depends on correct identification of the pathogen involved as antibiotics provide little or no benefit with viral infections. Here we describe a rapid and sensitive genotyping assay and microarray for URI identification using standard amplification and hybridization techniques, with electrochemical detection (ECD) on a semiconductor-based oligonucleotide microarray. The assay was developed to detect four bacterial pathogens (Bordetella pertussis, Streptococcus pyogenes, Chlamydia pneumoniae and Mycoplasma pneumoniae) and 9 viral pathogens (adenovirus 4, coronavirus OC43, 229E and HK, influenza A and B, parainfluinza types 1, 2, and 3 and respiratory syncytial virus. This new platform forms the basis for a fully automated diagnostics system that is very flexible and can be customized to suit different or additional pathogens. Multiple probes on a flexible platform allow one to test probes empirically and then select highly reactive probes for further iterative evaluation. Because ECD uses an enzymatic reaction to create electrical signals that can be read directly from the array, there is no need for image analysis or for expensive and delicate optical scanning equipment. We show assay sensitivity and specificity that are excellent for a multiplexed format. PMID:17895966

  4. Development of a short oligonucleotide microarray for the detection and identification of multiple potyviruses.

    PubMed

    Wei, Ting; Pearson, Michael N; Blohm, Dietmar; Nölte, Manfred; Armstrong, Karen

    2009-12-01

    The genus Potyvirus is the largest and one of the most economically important virus genera infecting plants. However, current diagnostic techniques are limited in their ability to identify multiple potyvirus infections. An assay that can identify multiple potyviruses simultaneously, with good specificity and sensitivity, is therefore highly desirable. To determine the feasibility of simultaneous detection of multiple potyviruses a 25-mer oligonucleotide microarray was developed targeting four distinct potyviruses: Dasheen mosaic virus (DsMV), Leek yellow stripe virus (LYSV), Potato virus Y (PVY) and Zucchini yellow mosaic virus (ZYMV). A total of 85 probes including 33 perfect-match and 52 mismatch probes were designed from conserved and variable sequence regions of the nuclear inclusion b (NIb) gene, RNA-dependent RNA polymerase (RdRp) gene, coat protein (CP) gene and the 3' untranslated region (UTR), representing the four targeted potyviruses at both species and strain levels. Each probe was synthesized with spacers of either 6 or 12 poly-cytosine or poly-thymine at the 5' terminus. The array showed high specificity when tested with nineteen different geographically diverse potyvirus isolates of the four target species, four distinct but closely related potyviruses, and four healthy plant species. The approaches and protocols developed in this study form a useful basis for developing other potyviruses arrays and the results also provide useful insights into generic issues for the development of arrays for detecting other pathogens.

  5. Versatile functionalization of nanoelectrodes by oligonucleotides via pyrrole electrochemistry.

    PubMed

    Descamps, Emeline; Nguyen, Khoa; Bouchain-Gautier, Christelle; Filoramo, Arianna; Goux-Capes, Laurence; Goffman, Marcello; Bourgoin, Jean-Philippe; Mailley, Pascal; Livache, Thierry

    2010-11-15

    Surface modification at the nanometer scale is a challenge for the future of molecular electronics. In particular, the precise anchoring and electrical addressing of biological scaffolds such as complex DNA nanonetworks is of importance for generating bio-directed assemblies of nano-objects for nanocircuit purposes. Herein, we consider the individual modification of nanoelectrodes with different oligonucleotide sequences by an electrochemically driven co-polymerization process of pyrrole and modified oligonucleotide sequences bearing pyrrole monomers. We demonstrate that this one-step technique presents the advantages of simplicity, localization of surface modification, mechanical, biological and chemical stability of the coatings, and high lateral resolution.

  6. Inhibition of microRNA with antisense oligonucleotides.

    PubMed

    Esau, Christine C

    2008-01-01

    Antisense inhibition of microRNA (miRNA) function has been an important tool for uncovering miRNA biology. Chemical modification of anti-miRNA oligonucleotides (AMOs) is necessary to improve affinity for target miRNA, stabilize the AMO to nuclease degradation, and to promote tissue uptake for in vivo delivery. Here I summarize the work done to evaluate the effectiveness of various chemically modified AMOs for use in cultured cells and rodent models, and outline important issues to consider when inhibiting miRNAs with antisense oligonucleotides.

  7. Emission Anisotropy of Fluorescein Covalently Linked to Oligonucleotides

    NASA Astrophysics Data System (ADS)

    Blokhin, A. P.; Kvach, M. V.; Povedailo, V. A.; Shmanai, V. V.; Yakovlev, D. L.

    2017-03-01

    Rotational depolarization of fluorescence from fluorescein covalently linked to oligonucleotides was studied. Fluorescence anisotropy of two nucleic acids was measured as a function of the temperature-to-viscosity ratio in buffer solutions with various glycerin concentrations. It was shown that the experimental results could be satisfactorily explained by a diffusion model of an elongated molecular top with internal rotation. It was found that the coefficient of internal rotational diffusion in all instances was 1.5-2 times greater than that for rotation around the oligonucleotide axis.

  8. Single-base-pair discrimination of terminal mismatches by using oligonucleotide microarrays and neural network analyses

    NASA Technical Reports Server (NTRS)

    Urakawa, Hidetoshi; Noble, Peter A.; El Fantroussi, Said; Kelly, John J.; Stahl, David A.

    2002-01-01

    The effects of single-base-pair near-terminal and terminal mismatches on the dissociation temperature (T(d)) and signal intensity of short DNA duplexes were determined by using oligonucleotide microarrays and neural network (NN) analyses. Two perfect-match probes and 29 probes having a single-base-pair mismatch at positions 1 to 5 from the 5' terminus of the probe were designed to target one of two short sequences representing 16S rRNA. Nonequilibrium dissociation rates (i.e., melting profiles) of all probe-target duplexes were determined simultaneously. Analysis of variance revealed that position of the mismatch, type of mismatch, and formamide concentration significantly affected the T(d) and signal intensity. Increasing the concentration of formamide in the washing buffer decreased the T(d) and signal intensity, and it decreased the variability of the signal. Although T(d)s of probe-target duplexes with mismatches in the first or second position were not significantly different from one another, duplexes with mismatches in the third to fifth positions had significantly lower T(d)s than those with mismatches in the first or second position. The trained NNs predicted the T(d) with high accuracies (R(2) = 0.93). However, the NNs predicted the signal intensity only moderately accurately (R(2) = 0.67), presumably due to increased noise in the signal intensity at low formamide concentrations. Sensitivity analysis revealed that the concentration of formamide explained most (75%) of the variability in T(d)s, followed by position of the mismatch (19%) and type of mismatch (6%). The results suggest that position of the mismatch at or near the 5' terminus plays a greater role in determining the T(d) and signal intensity of duplexes than the type of mismatch.

  9. Single-base-pair discrimination of terminal mismatches by using oligonucleotide microarrays and neural network analyses

    NASA Technical Reports Server (NTRS)

    Urakawa, Hidetoshi; Noble, Peter A.; El Fantroussi, Said; Kelly, John J.; Stahl, David A.

    2002-01-01

    The effects of single-base-pair near-terminal and terminal mismatches on the dissociation temperature (T(d)) and signal intensity of short DNA duplexes were determined by using oligonucleotide microarrays and neural network (NN) analyses. Two perfect-match probes and 29 probes having a single-base-pair mismatch at positions 1 to 5 from the 5' terminus of the probe were designed to target one of two short sequences representing 16S rRNA. Nonequilibrium dissociation rates (i.e., melting profiles) of all probe-target duplexes were determined simultaneously. Analysis of variance revealed that position of the mismatch, type of mismatch, and formamide concentration significantly affected the T(d) and signal intensity. Increasing the concentration of formamide in the washing buffer decreased the T(d) and signal intensity, and it decreased the variability of the signal. Although T(d)s of probe-target duplexes with mismatches in the first or second position were not significantly different from one another, duplexes with mismatches in the third to fifth positions had significantly lower T(d)s than those with mismatches in the first or second position. The trained NNs predicted the T(d) with high accuracies (R(2) = 0.93). However, the NNs predicted the signal intensity only moderately accurately (R(2) = 0.67), presumably due to increased noise in the signal intensity at low formamide concentrations. Sensitivity analysis revealed that the concentration of formamide explained most (75%) of the variability in T(d)s, followed by position of the mismatch (19%) and type of mismatch (6%). The results suggest that position of the mismatch at or near the 5' terminus plays a greater role in determining the T(d) and signal intensity of duplexes than the type of mismatch.

  10. Hybridization probe pairs and single-labeled probes: an alternative approach for genotyping and quantification.

    PubMed

    Froehlich, Thomas; Geulen, Oliver

    2008-01-01

    Real-time polymerase chain reaction (PCR) has become a standard tool in both quantitative gene expression and genetic variation analysis. Data collection is performed throughout the PCR process, thus combining amplification and detection into a single step. This can be achieved by combining a variety of different fluorescent chemistries that correlate the concentration of an amplified PCR product to changes in fluorescence intensity. Hybridization probe pairs and single-labeled probes are sequence-specific, dye-labeled oligonucleotides, used in real-time PCR approaches, in particular for genotyping of single nucleotide polymorphisms (SNPs). In that case, a detector probe is designed to cover the polymorphism. Allelic variants are identified and differentiated via post-PCR melting curve analysis. A single melting curve can distinguish different T (m)s, and differently labeled probes may be used, theoretically allowing multiplexed genotyping of several SNPs.

  11. Oligonucleotide labelling using a fluorogenic "click" reaction with a hemicarboxonium salt.

    PubMed

    Maether, Marie-Pierre; Lapin, Kristie; Muntean, Andreea; Payrastre, Corinne; Escudier, Jean-Marc

    2013-10-17

    Two fluorescent streptocyanine labelled oligonucleotides have been synthesized by a simple "click" reaction between a non-fluorescent hemicarboxonium salt and aminoalkyl functionalized thymidines within the oligonucleotide and their spectrophotometric properties have been studied.

  12. Construction and Evaluation of Desulfovibrio vulgaris Whole-Genome Oligonucleotide Microarrays

    SciTech Connect

    Z. He; Q. He; L. Wu; M.E. Clark; J.D. Wall; Jizhong Zhou; Matthew W. Fields

    2004-03-17

    Desulfovibrio vulgaris Hildenborough has been the focus of biochemical and physiological studies in the laboratory, and the metabolic versatility of this organism has been largely recognized, particularly the reduction of sulfate, fumarate, iron, uranium and chromium. In addition, a Desulfovibrio sp. has been shown to utilize uranium as the sole electron acceptor. D. vulgaris is a d-Proteobacterium with a genome size of 3.6 Mb and 3584 ORFs. The whole-genome microarrays of D. vulgaris have been constructed using 70mer oligonucleotides. All ORFs in the genome were represented with 3471 (97.1%) unique probes and 103 (2.9%) non-specific probes that may have cross-hybridization with other ORFs. In preparation for use of the experimental microarrays, artificial probes and targets were designed to assess specificity and sensitivity and identify optimal hybridization conditions for oligonucleotide microarrays. The results indicated that for 50mer and 70mer oligonucleotide arrays, hybridization at 45 C to 50 C, washing at 37 C and a wash time of 2.5 to 5 minutes obtained specific and strong hybridization signals. In order to evaluate the performance of the experimental microarrays, growth conditions were selected that were expected to give significant hybridization differences for different sets of genes. The initial evaluations were performed using D. vulgaris cells grown at logarithmic and stationary phases. Transcriptional analysis of D. vulgaris cells sampled during logarithmic phase growth indicated that 25% of annotated ORFs were up-regulated and 3% of annotated ORFs were downregulated compared to stationary phase cells. The up-regulated genes included ORFs predicted to be involved with acyl chain biosynthesis, amino acid ABC transporter, translational initiation factors, and ribosomal proteins. In the stationary phase growth cells, the two most up-regulated ORFs (70-fold) were annotated as a carboxynorspermidine decarboxylase and a 2C-methyl-D-erythritol-2

  13. Synthesis and properties of double-stranded antisense oligonucleotides connected with a pentaerythritol linker.

    PubMed

    Shibata, Aya; Ueno, Yoshihito; Matsuda, Akira; Kitade, Yukio

    2006-01-01

    This paper describes the synthesis and properties of double-stranded antisense oligonucleotides connected with a pentaerythritol linker. We found that double-stranded antisense oligonucleotides with aminomethyl residues have high affinity for single-stranded DNA or RNA in buffer solutions both with and without MgCl(2). Thus, these oligonucleotides would be useful as antisense oligonucleotides for targeting single-stranded RNA through triplex formation.

  14. Modeling Formamide Denaturation of Probe-Target Hybrids for Improved Microarray Probe Design in Microbial Diagnostics

    PubMed Central

    Yilmaz, L. Safak; Loy, Alexander; Wright, Erik S.; Wagner, Michael; Noguera, Daniel R.

    2012-01-01

    Application of high-density microarrays to the diagnostic analysis of microbial communities is challenged by the optimization of oligonucleotide probe sensitivity and specificity, as it is generally unfeasible to experimentally test thousands of probes. This study investigated the adjustment of hybridization stringency using formamide with the idea that sensitivity and specificity can be optimized during probe design if the hybridization efficiency of oligonucleotides with target and non-target molecules can be predicted as a function of formamide concentration. Sigmoidal denaturation profiles were obtained using fluorescently labeled and fragmented 16S rRNA gene amplicon of Escherichia coli as the target with increasing concentrations of formamide in the hybridization buffer. A linear free energy model (LFEM) was developed and microarray-specific nearest neighbor rules were derived. The model simulated formamide melting with a denaturant m-value that increased hybridization free energy (ΔG°) by 0.173 kcal/mol per percent of formamide added (v/v). Using the LFEM and specific probe sets, free energy rules were systematically established to predict the stability of single and double mismatches, including bulged and tandem mismatches. The absolute error in predicting the position of experimental denaturation profiles was less than 5% formamide for more than 90 percent of probes, enabling a practical level of accuracy in probe design. The potential of the modeling approach for probe design and optimization is demonstrated using a dataset including the 16S rRNA gene of Rhodobacter sphaeroides as an additional target molecule. The LFEM and thermodynamic databases were incorporated into a computational tool (ProbeMelt) that is freely available at http://DECIPHER.cee.wisc.edu. PMID:22952791

  15. Synthesis and anti-HIV activity of thiocholesteryl-coupled phosphodiester antisense oligonucleotides incorporated into immunoliposomes.

    PubMed

    Zelphati, O; Wagner, E; Leserman, L

    1994-09-01

    Encapsulation of oligonucleotides in antibody-targeted liposomes (immunoliposomes) which bind to target cells permits intracellular delivery of the oligonucleotides. This approach circumvents problems of extracellular degradation by nucleases and poor membrane permeability which free phosphodiester oligonucleotides are subject to, but leaves unresolved the inefficiency of encapsulation of oligonucleotides in liposomes. We have coupled oligonucleotides to cholesterol via a reversible disulfide bond. This modification of oligonucleotides improved their association with immunoliposomes by a factor of about 10 in comparison to unmodified oligonucleotides. The presence of cholesteryl-modified oligonucleotides incorporated in the bilayer of liposomes did not interfere with the coupling of the targeting protein to the liposome surface. Free or cholesterol coupled oligonucleotides associated with liposomes and directed against the tat gene of HIV-1 were tested for inhibition of HIV-1 proliferation in acutely infected cells. We demonstrate that the cholesteryl-modified as well as unmodified oligonucleotides acquire the target specificity of the antibody on the liposome. Their antiviral activity when delivered into cells is sequence-specific. The activity of these modified or unmodified oligonucleotides to inhibit the replication of HIV was the same on an equimolar basis (EC50 around 0.1 microM). Cholesterol coupled oligonucleotides thus offer increased liposome association without loss of antiviral activity.

  16. Effects of fluid flow on the oligonucleotide folding in single-walled carbon nanotubes.

    PubMed

    Lim, M C G; Zhong, Z W

    2009-10-01

    This paper presents molecular-dynamics (MD) simulations of DNA oligonucleotide and water molecules translocating through carbon nanotube (CNT) channels. An induced pressure difference is applied to the system by pushing a layer of water molecules toward the flow direction to drive the oligonucleotide and other molecules. This MD simulation investigates the changes that occur in the conformation of the oligonucleotide due to water molecules in nanochannels while controlling the temperature and volume of the system in a canonical ensemble. The results show that the oligonucleotide in the (8,8)-(12,12) CNT channel forms a folded state at a lower pressure, whereas the oligonucleotide in the (10,10)-(14,14) CNT channel forms a folded state at a higher pressure instead. The van der Waals forces between the water molecules and the oligonucleotide suggest that the attraction between these two types of molecules results in the linear arrangements of the bases of the oligonucleotide. For a larger nanotube channel, the folding of the oligonucleotide is mainly dependent on the solvent (water molecules), whereas pressure, the size of the nanotube junction, and water molecules are the considering factors of the folding of the oligonucleotide at a smaller nanotube channel. For a folded oligonucleotide, the water distribution around the oligonucleotide is concentrated at a smaller range than that for the distribution around an unfolded oligonucleotide.

  17. Ratiometric detection of oligonucleotide stoichiometry on multifunctional gold nanoparticles by whispering gallery mode biosensing.

    PubMed

    Wu, F C; Wu, Y; Niu, Z; Vollmer, F

    2015-05-07

    A label-free method is developed to ratiometrically determine the stoichiometry of oligonucleotides attached to the surface of gold nanoparticle (GNP) by whispering gallery mode biosensing. Utilizing this scheme, it is furthermore shown that the stoichiometric ratio of GNP attached oligonucleotide species can be controlled by varying the concentration ratio of thiolated oligonucleotides that are used to modify the GNP.

  18. Oligonucleotide-directed mutagenesis for precision gene editing.

    PubMed

    Sauer, Noel J; Mozoruk, Jerry; Miller, Ryan B; Warburg, Zachary J; Walker, Keith A; Beetham, Peter R; Schöpke, Christian R; Gocal, Greg F W

    2016-02-01

    Differences in gene sequences, many of which are single nucleotide polymorphisms, underlie some of the most important traits in plants. With humanity facing significant challenges to increase global agricultural productivity, there is an urgent need to accelerate the development of these traits in plants. oligonucleotide-directed mutagenesis (ODM), one of the many tools of Cibus' Rapid Trait Development System (RTDS(™) ) technology, offers a rapid, precise and non-transgenic breeding alternative for trait improvement in agriculture to address this urgent need. This review explores the application of ODM as a precision genome editing technology, with emphasis on using oligonucleotides to make targeted edits in plasmid, episomal and chromosomal DNA of bacterial, fungal, mammalian and plant systems. The process of employing ODM by way of RTDS technology has been improved in many ways by utilizing a fluorescence conversion system wherein a blue fluorescent protein (BFP) can be changed to a green fluorescent protein (GFP) by editing a single nucleotide of the BFP gene (CAC→TAC; H66 to Y66). For example, dependent on oligonucleotide length, applying oligonucleotide-mediated technology to target the BFP transgene in Arabidopsis thaliana protoplasts resulted in up to 0.05% precisely edited GFP loci. Here, the development of traits in commercially relevant plant varieties to improve crop performance by genome editing technologies such as ODM, and by extension RTDS, is reviewed.

  19. Chromosome-specific painting in Cucumis species using bulked oligonucleotides

    USDA-ARS?s Scientific Manuscript database

    Chromosome-specific painting is a powerful technique in molecular cytogenetic and genome research. We developed an oligonucleotide (oligo)-based chromosome painting technique in cucumber (Cucumis sativus) that will be applicable in any plant species with a sequenced genome. Oligos specific to a sing...

  20. Construction and validation of a 45K rice oligonucleotide array

    USDA-ARS?s Scientific Manuscript database

    We have constructed and validated a 43,311 oligonucleotide (oligos, 50-70-mers) gene array (NSF 45K) based on 45,116 gene models from release 3 of The Institute for Genomic Research's rice pseudomolecules. To test the array, we generated expression profiles on light- and dark-grown rice leaf tissue...

  1. Validation of the Swine Protein-Annotated Oligonucleotide Microarray

    USDA-ARS?s Scientific Manuscript database

    The specificity and utility of the Swine Protein-Annotated Oligonucleotide Microarray, or Pigoligoarray (www.pigoligoarray.org), has been evaluated by profiling the expression of transcripts from four porcine tissues. Tools for comparative analyses of expression on the Pigoligoarray were developed i...

  2. Solid-phase-supported synthesis of morpholinoglycine oligonucleotide mimics

    PubMed Central

    Belov, Sergey S; Tarasenko, Yulia V; Silnikov, Vladimir N

    2014-01-01

    Summary An efficient solid-phase-supported peptide synthesis (SPPS) of morpholinoglycine oligonucleotide (MorGly) mimics has been developed. The proposed strategy includes a novel specially designed labile linker group containing the oxalyl residue and the 2-aminomethylmorpholino nucleoside analogues as first subunits. PMID:24991266

  3. Oligonucleotide N-alkylphosphoramidates: Synthesis and binding to polynucleotides

    SciTech Connect

    Jaeger, A.; Levy, M.J.; Hecht, S.M. )

    1988-09-20

    A few different methods for the preparation of oligonucleotide N-alkylphosphoramidates were compared directly. One of these, involving the use of protected nucleoside phosphites as building blocks, provided the requisite N-alkylphosphoramidates via oxidation of the intermediate dinucleoside methyl phosphites with iodine in the presence of the appropriate alkylamine. This method was found to have several attractive features, including the use of building blocks identical with those employed for the synthesis of DNA and compatibility with procedures and instruments employed for the stepwise synthesis of oligonucleotides by solution and solid-phase methods. This procedure was used to make several di-, tri-, and tetranucleotide N-alkylphosphoramidates derived from deoxyadenosine and thymidine; alkyl substituents included N,N-dimethyl, N-butyl, N-octyl, N-dodecyl, and N-(5-aminopentyl). The oligonucleotide N-alkylphosphoramidates were separated into their component diastereomers and characterized structurally by a number of techniques including circular dichroism, high-field {sup 1}H NMR spectroscopy, FAB mass spectrometry, and enzymatic digestion to authentic nucleosides and nucleotides. Physicochemical characterization of several di- and trinucleotide alkylphosphoramidates revealed that the adenine nucleotide analogues formed stable complexes with poly(thymidylic acid). The stabilities of these complexes were found to increase with increasing chain length of the N-alkylphosphoramidate substituents. The finding that N-alkylphosphoramidate substituents can enhance the binding of certain oligonucleotides to their complementary polynucleotides suggests the existence of a novel source of polynucleotide affinity.

  4. Differential oligonucleotide activity in cell culture versus mouse models.

    PubMed

    Wickstrom, E; Tyson, F L

    1997-01-01

    The usual course of drug discovery begins with the demonstration of compound activity in cells and, usually, a lower level of activity in animals. Successive rounds of drug design may result in a compound with sufficient activity in animals to justify clinical trials. The basic endpoints of therapeutic oligonucleotide experiments include target antigen reduction, target messenger reduction and inhibition of transformed cell proliferation or viral replication. However, one should expect oligonucleotides to exhibit pleiotropic behaviour, as do all other drugs. In an animal oligonucleotides will necessarily bind to and dissociate from all macromolecules encountered in the blood, in tissues, on cell surfaces and within cellular compartments. Contrary to expectations, oligonucleotides designed to be complementary to certain transcripts have sometimes been found moderately effective in cell-free extracts, more effective in cell culture and most effective in animal models. If greater potency against standard endpoints is reported in mouse models than was observed in cell culture, critical examination must consider alternate modes of action in animals that may not apply in cell culture. This counterintuitive paradox will be examined, based on studies of Ha-ras expression in bladder cancer, Ki-ras expression in pancreatic cancer, erbB2 expression in ovarian cancer and c-myc expression in B cell lymphoma.

  5. Regioselective immobilization of short oligonucleotides to acrylic copolymer gels.

    PubMed Central

    Timofeev, E; Kochetkova, S V; Mirzabekov, A D; Florentiev, V L

    1996-01-01

    Four types of polyacrylamide or polydimethyl-acrylamide gels for regioselective (by immobilization at the 3' end) of short oligonucleotides have been designed for use in manufacturing oligonucleotide microchips. Two of these supports contain amino or aldehyde groups in the gel, allowing coupling with oligonucleotides bearing aldehyde or amino groups, respectively, in the presence of a reducing agent. The aldehyde gel support showed a higher immobilization efficiency relative to the amino gel. Of all reducing agents tested, the best results were obtained with a pyridine-borane complex. The other supports are based on an acrylamide gel activated with glutaraldehyde or a hydroxyalkyl-functionalized gel treated with mesyl chloride. The use of dimethylacrylamide instead of acrylamide allows subsequent gel modifications in organic solvents. All the immobilization methods are easy and simple to perform, give high and reproducible yields, allow long durations of storage of the activated support, and provide high stability of attachment and low non-specific binding. Although these gel supports have been developed for preparing oligonucleotide microchips, they may be used for other purposes as well. PMID:8774893

  6. Gene expression profiling in peanut using oligonucleotide microarrays

    USDA-ARS?s Scientific Manuscript database

    Transcriptome expression analysis in peanut to date has been limited to a relatively small set of genes and only recently have a moderately significant number of ESTs been released into the public domain. Utilization of these ESTs for the oligonucleotide microarrays provides a means to investigate l...

  7. Development of an oligonucleotide-based DNA microarray for transcriptional analysis of Choristoneura fumiferana nucleopolyhedrovirus (CfMNPV) genes.

    PubMed

    Yang, Dan-Hui; Barari, Mehrnoosh; Arif, Basil M; Krell, Peter J

    2007-08-01

    A modified oligonucleotide-based two-channel DNA microarray was developed for characterization of temporal expression profiles of select Choristoneura fumiferana nucleopolyhedrovirus (CfMNPV) ORFs including its 7 unique ORFs. The microarray chip contained oligonucleotide probes for 23 CfMNPV ORFs and their complements as well as five host genes. Total RNA was isolated at different times post infection from Cf203 insect cells infected with CfMNPV. The cDNA was synthesized, fluorescent labelled with Cy3, and co-hybridized to the microarray chips along with Cy5-labelled viral genomic DNA, which served as equimolar reference standards for each probe. Transcription of the 7 CfMNPV unique ORFs was detected using DNA microarray analysis and their temporal expression profiles suggest that they are functional genes. The expression levels of three host genes varied throughout virus infection and therefore were unsuitable for normalization between microarrays. The DNA microarray results were compared to quantitative RT-PCR (qRT-PCR). Transcription of the non-coding (antisense) strands of some of the CfMNPV select genes including the polyhedrin gene, was also detected by array analysis and confirmed by qRT-PCR. The polyhedrin antisense transcript, based on long-range RT-PCR analysis, appeared to be a read-through product of an adjacent ORF in the same orientation as the antisense transcript.

  8. Electrochemical biosensor for the detection of cauliflower mosaic virus 35 S gene sequences using lead sulfide nanoparticles as oligonucleotide labels.

    PubMed

    Sun, Wei; Zhong, Jianghua; Qin, Peng; Jiao, Kui

    2008-06-15

    Lead sulfide (PbS) nanoparticles were synthesized in aqueous solution and used as oligonucleotide labels for electrochemical detection of the 35 S promoter from cauliflower mosaic virus (CaMV) sequence. The PbS nanoparticles were modified with mercaptoacetic acid and could easily be linked with CaMV 35 S oligonucleotide probe. Target DNA sequences were covalently linked on a mercaptoacetic acid self-assembled gold electrode, and DNA hybridization of target DNA with probe DNA was completed on the electrode surface. PbS nanoparticles anchored on the hybrids were dissolved in the solution by oxidation of HNO3 and detected using a sensitive differential pulse anodic stripping voltammetric method. The detection results can be used to monitor the hybridization reaction. The CaMV 35 S target sequence was satisfactorily detected with the detection limit as 4.38 x 10(-12)mol/L (3sigma). The established method extends nanoparticle-labeled electrochemical DNA analysis to specific sequences from genetically modified organisms with higher sensitivity and selectivity.

  9. Specific detection of very low concentrations of DNA oligonucleotides with DNA-coated long-period grating biosensor

    NASA Astrophysics Data System (ADS)

    Czarnecka, Karolina H.; Dominik, Magdalena; Janczuk-Richter, Marta; Niedziółka-Jönsson, Joanna; Bock, Wojtek J.; Śmietana, Mateusz

    2017-04-01

    Label-free biosensing using optical long-period gratings (LPGs) induced in optical fiber and coated with biological compound enables for detection and monitoring the kinetics of reactions taking places on the sensor's surface. The labelfree detection effect for the LPG biosensor working near the dispersion turning point (DTP) of higher order cladding modes can be observed as resonance wavelength shift induced by binding of biomolecules to fiber surface. In this study we analyzed shift of the resonance wavelength for LPG after its functionalization with DNA layer and during DNA hybridization. It has seen found that the LPG is able to be functionalized with DNA oligonucleotide, acting as a probe and after the functionalization can selectively bind specific complementary DNA oligonucleotides in a very low concentration. DNA-coated LPG can be used as a probe capturing specific DNA sequences with wide ranges of concentrations from 0.1 to 10 pM. Hybridization of the DNA on the fiber surface has also been verified with Midorii Green florescent marker.

  10. Pentopyranosyl Oligonucleotide Systems. Part 11: Systems with Shortened Backbones: D)-beta-Ribopyranosyl-(4 yields 3 )- and (L)-alpha - Lyxopyranosyl-(4 yields 3 )-oligonucleotides

    NASA Technical Reports Server (NTRS)

    Wippo, Harald; Reck, Folkert; Kudick, Rene; Ramaseshan, Mahesh; Ceulemans, Griet; Bolli, Martin; Krishnamurthy, Ramanarayanan; Eschenmoser, Albert

    2001-01-01

    The (L)-a-lyxopyranosyl-(4'yields 3')-oligonucleotide system-a member of a pentopyranosyl oligonucleotide family containing a shortened backbone-is capable of cooperative base-pairing and of cross-pairing with DNA and RNA. In contrast, corresponding (D)-beta-ribopyransoyl-(4' yields 3')-oligonucleotides do not show base-pairing under similar conditions. We conclude that oligonucleotide systems can violate the six-bonds-per-backbone-unit rule by having five bonds instead, if their vicinally bound phosphodiester bridges can assume an antiperiplanar conformation. An additional structural feature that seems relevant to the cross-pairing capability of the (L)-a-lyxopyranosyl-(4' yields 3')-oligonucleotide system is its (small) backbone/basepair axes inclination. An inclination which is similar to that in B-DNA seems to be a prerequisite for an oligonucleotide system s capability to cross-pair with DNA.

  11. Glycoclusters on oligonucleotide and PNA scaffolds: synthesis and applications.

    PubMed

    Spinelli, Nicolas; Defrancq, Eric; Morvan, François

    2013-06-07

    Conjugation of oligonucleotides (ONs) to a variety of reporter groups has been the subject of intensive research during the last decade. Conjugation is indeed of great interest because it can be used not only to improve the existing ONs properties but also to impart new ones. In this context tremendous efforts have been made to conjugate carbohydrate moieties to ONs. Indeed carbohydrates play an important role in biological processes such as signal transduction and cell adhesion through the recognition with sugar-binding proteins (i.e. lectins) located on the surface of cells. For this reason, carbohydrate-oligonucleotide conjugates (COCs) have been first developed for improving the poor cellular uptake or tissue specific delivery of ONs through receptor-mediated endocytosis. Besides the targeted ONs delivery, carbohydrate-oligonucleotide conjugates (COCs) are also evaluated in the context of carbohydrate biochips in which surface coating with carbohydrates is achieved by using the DNA-directed immobilization strategy (DDI). Peptide nucleic acids (PNAs) have also been extensively investigated as a surrogate of DNA for diverse applications. Therefore attachment of carbohydrate moieties to this class of molecules has been studied. The aforementioned applications of COCs require mimicking of the natural processes, in which the weak individual protein-carbohydrate binding is overcome by using multivalent interactions. This tutorial review focuses on the recent advances in carbohydrate-oligonucleotide conjugates and describes the major synthetic approaches available. In addition, an overview of applications that have been developed using various scaffolds allowing multivalent interactions is provided. Finally recent results on the use of peptide nucleic acids as oligonucleotides surrogate are described.

  12. Intracellular distribution of microinjected antisense oligonucleotides.

    PubMed

    Leonetti, J P; Mechti, N; Degols, G; Gagnor, C; Lebleu, B

    1991-04-01

    Antisense oligomers constitute an attractive class of specific tools for genetic analysis and for potential therapeutic applications. Targets with different cellular locations have been described, such as mRNA translation initiation sites, pre-mRNA splicing sites, or the genes themselves. However the mechanism(s) of action and the intracellular distribution of antisense oligomers remain poorly understood. Antisense oligomers conjugated with various fluorochromes or with BrdUrd were microinjected into the cytoplasm of somatic cells, and their cellular distribution was monitored by fluorescence microscopy in fixed and nonfixed cells. A fast translocation in the nuclei and a concentration on nuclear structures were observed whatever probe was used. Nuclear transport occurs by diffusion since it is not affected by depletion of the intracellular ATP pool, temperature, or excess unlabeled oligomer. Accumulation of the oligomers in the nuclei essentially takes place on a set of proteins preferentially extracted between 0.2 M and 0.4 M NaCl as revealed by crosslinking of photosensitive oligomers. The relationship between nuclear location of antisense oligomers and their mechanism of action remains to be ascertained and could be of major interest in the design of more efficient antisense molecules.

  13. Intracellular distribution of microinjected antisense oligonucleotides.

    PubMed Central

    Leonetti, J P; Mechti, N; Degols, G; Gagnor, C; Lebleu, B

    1991-01-01

    Antisense oligomers constitute an attractive class of specific tools for genetic analysis and for potential therapeutic applications. Targets with different cellular locations have been described, such as mRNA translation initiation sites, pre-mRNA splicing sites, or the genes themselves. However the mechanism(s) of action and the intracellular distribution of antisense oligomers remain poorly understood. Antisense oligomers conjugated with various fluorochromes or with BrdUrd were microinjected into the cytoplasm of somatic cells, and their cellular distribution was monitored by fluorescence microscopy in fixed and nonfixed cells. A fast translocation in the nuclei and a concentration on nuclear structures were observed whatever probe was used. Nuclear transport occurs by diffusion since it is not affected by depletion of the intracellular ATP pool, temperature, or excess unlabeled oligomer. Accumulation of the oligomers in the nuclei essentially takes place on a set of proteins preferentially extracted between 0.2 M and 0.4 M NaCl as revealed by crosslinking of photosensitive oligomers. The relationship between nuclear location of antisense oligomers and their mechanism of action remains to be ascertained and could be of major interest in the design of more efficient antisense molecules. Images PMID:1849273

  14. Optical probe

    DOEpatents

    Hencken, Kenneth; Flower, William L.

    1999-01-01

    A compact optical probe is disclosed particularly useful for analysis of emissions in industrial environments. The instant invention provides a geometry for optically-based measurements that allows all optical components (source, detector, rely optics, etc.) to be located in proximity to one another. The geometry of the probe disclosed herein provides a means for making optical measurements in environments where it is difficult and/or expensive to gain access to the vicinity of a flow stream to be measured. Significantly, the lens geometry of the optical probe allows the analysis location within a flow stream being monitored to be moved while maintaining optical alignment of all components even when the optical probe is focused on a plurality of different analysis points within the flow stream.

  15. A label-free electrochemiluminescence aptasensor for thrombin based on novel assembly strategy of oligonucleotide and luminol functionalized gold nanoparticles.

    PubMed

    Li, Fang; Cui, Hua

    2013-01-15

    In the work, a label-free electrochemiluminescence (ECL) aptasensor for the sensitive and selective detection of thrombin was constructed based on target-induced direct ECL signal change by virtue of a novel assembly strategy of oligonucleotide and luminol functionalized gold nanoparticles (luminol-AuNPs). It is the first label-free ECL biosensor based on luminol and its analogs functionalized AuNPs. Streptavidin AuNPs coated with biotinylated DNA capture probe 1 (AuNPs-probe 1) were firstly assembled onto an gold electrode through 1,3-propanedithiol. Then luminol-AuNPs co-loaded with thiolated DNA capture probe 2 and thiolated thrombin binding aptamer (TBA) (luminol-AuNPs-probe 2/TBA) were assembled onto AuNPs-probe 1 modified electrode through the hybridization between capture probes 1 and 2. The luminol-AuNPs-probe 2/TBA acted as both molecule recognition probe and sensing interface. An Au/AuNPs/ds-DNA/luminol-AuNPs/TBA multilayer architecture was obtained. In the presence of target thrombin, TBA on the luminol-AuNPs could capture the thrombin onto the electrode surface, which produced a barrier for electro-transfer and influenced the electro-oxidation reaction of luminol, leading to a decrease in ECL intensity. The change of ECL intensity indirectly reflected the concentration of thrombin. Thus, the approach showed a high sensitivity and a wider linearity for the detection of thrombin in the range of 0.005-50nM with a detection limit of 1.7pM. This work reveals that luminol-AuNPs are ideal platform for label-free ECL bioassays. Copyright © 2012 Elsevier B.V. All rights reserved.

  16. Droplet Digital Enzyme-Linked Oligonucleotide Hybridization Assay for Absolute RNA Quantification

    NASA Astrophysics Data System (ADS)

    Guan, Weihua; Chen, Liben; Rane, Tushar D.; Wang, Tza-Huei

    2015-09-01

    We present a continuous-flow droplet-based digital Enzyme-Linked Oligonucleotide Hybridization Assay (droplet digital ELOHA) for sensitive detection and absolute quantification of RNA molecules. Droplet digital ELOHA incorporates direct hybridization and single enzyme reaction via the formation of single probe-RNA-probe (enzyme) complex on magnetic beads. It enables RNA detection without reverse transcription and PCR amplification processes. The magnetic beads are subsequently encapsulated into a large number of picoliter-sized droplets with enzyme substrates in a continuous-flow device. This device is capable of generating droplets at high-throughput. It also integrates in-line enzymatic incubation and detection of fluorescent products. Our droplet digital ELOHA is able to accurately quantify (differentiate 40% difference) as few as ~600 RNA molecules in a 1 mL sample (equivalent to 1 aM or lower) without molecular replication. The absolute quantification ability of droplet digital ELOHA is demonstrated with the analysis of clinical Neisseria gonorrhoeae 16S rRNA to show its potential value in real complex samples.

  17. Direct Detection of 16S rRNA in Soil Extracts by Using Oligonucleotide Microarrays

    PubMed Central

    Small, Jack; Call, Douglas R.; Brockman, Fred J.; Straub, Timothy M.; Chandler, Darrell P.

    2001-01-01

    We report on the development and validation of a simple microarray method for the direct detection of intact 16S rRNA from unpurified soil extracts. Total RNAs from Geobacter chapellei and Desulfovibrio desulfuricans were hybridized to an oligonucleotide array consisting of universal and species-specific 16S rRNA probes. PCR-amplified products from Geobacter and Desulfovibrio were easily and specifically detected under a range of hybridization times, temperatures, and buffers. However, reproducible, specific hybridization and detection of intact rRNA could be accomplished only by using a chaperone-detector probe strategy. With this knowledge, assay conditions were developed for rRNA detection using a 2-h hybridization time at room temperature. Hybridization specificity and signal intensity were enhanced using fragmented RNA. Formamide was required in the hybridization buffer in order to achieve species-specific detection of intact rRNA. With the chaperone detection strategy, we were able to specifically hybridize and detect G. chapellei 16S rRNA directly from a total-RNA soil extract, without further purification or removal of soluble soil constituents. The detection sensitivity for G. chapellei 16S rRNA in soil extracts was at least 0.5 μg of total RNA, representing approximately 7.5 × 106 Geobacter cell equivalents of RNA. These results suggest that it is now possible to apply microarray technology to the direct detection of microorganisms in environmental samples, without using PCR. PMID:11571176

  18. Detection of ≥1Mb microdeletions and microduplications in a single cell using custom oligonucleotide arrays.

    PubMed

    Bi, Weimin; Breman, Amy; Shaw, Chad A; Stankiewicz, Pawel; Gambin, Tomasz; Lu, Xinyan; Cheung, Sau Wai; Jackson, Laird G; Lupski, James R; Van den Veyver, Ignatia B; Beaudet, Arthur L

    2012-01-01

    High resolution detection of genomic copy number abnormalities in a single cell is relevant to preimplantation genetic diagnosis and potentially to noninvasive prenatal diagnosis. Our objective is to develop a reliable array comparative genomic hybridization (CGH) platform to detect genomic imbalances as small as ~1Mb ina single cell. We empirically optimized the conditions for oligonucleotide-based array CGH using single cells from multiple lymphoblastoid cell lines with known copy number abnormalities. To improve resolution, we designed custom arrays with high density probes covering clinically relevant genomic regions. The detection of megabase-sized copy number variations (CNVs) in a single cell was influenced by the number of probes clustered in the interrogated region. Using our custom array, we reproducibly detected multiple chromosome abnormalities including trisomy 21, a 1.2Mb Williams syndrome deletion, and a 1.3Mb CMT1A duplication. Replicate analyses yielded consistent results. Aneuploidy and genomic imbalances with CNVs as small as 1.2Mb in a single cell are detectable by array CGH using arrays with high-density coverage in the targeted regions. This approach has the potential to be applied for preimplantation genetic diagnosis to detect aneuploidy and common microdeletion/duplication syndromes and for noninvasive prenatal diagnosis if single fetal cells can be isolated. © 2012 John Wiley & Sons, Ltd.

  19. Oligonucleotide-stabilized fluorescent silver nanoclusters for the specific and sensitive detection of biotin.

    PubMed

    Xiong, Xiaoli; Tang, Yan; Zhao, Jingjin; Zhao, Shulin

    2016-02-21

    A novel biotin fluorescent probe based on oligonucleotide-stabilized silver nanoclusters (DNA-AgNCs) was synthesized by employing a biotinylated cytosine-rich sequence as a synthesized template. The fluorescence properties of the DNA-AgNCs are related to the modified position of the DNA. When biotin is linked to the middle thymine base of the DNA sequence, the DNA-AgNCs emit the strongest fluorescence. Moreover, the stability of the DNA-AgNCs was affected by avidin through biotin-avidin binding, quenching the fluorescence of the DNA-AgNCs. In contrast, if free biotin is further introduced into this system, the quenching is apparently weakened by competition, leading to the restoration of fluorescence. This phenomenon can be utilized for the detection of biotin. Under the optimal conditions, the fluorescence recovery is linearly proportional to the concentration of biotin in the range of 10 nM-1.0 μM with a detection limit of 6.0 nM. This DNA-AgNCs probe with excellent fluorescent properties is sensitive and selective for the detection of biotin and has been applied for the determination of biotin in wheat flour.

  20. Combining ligation reaction and capillary gel electrophoresis to obtain reliable long DNA probes.

    PubMed

    García-Cañas, Virginia; Mondello, Monica; Cifuentes, Alejandro

    2011-05-01

    New DNA amplification methods are continuously developed for sensitive detection and quantification of specific DNA target sequences for, e.g. clinical, environmental or food applications. These new applications often require the use of long DNA oligonucleotides as probes for target sequences hybridization. Depending on the molecular technique, the length of DNA probes ranges from 40 to 450 nucleotides, solid-phase chemical synthesis being the strategy generally used for their production. However, the fidelity of chemical synthesis of DNA decreases for larger DNA probes. Defects in the oligonucleotide sequence result in the loss of hybridization efficiency, affecting the sensitivity and selectivity of the amplification method. In this work, an enzymatic procedure has been developed as an alternative to solid-phase chemical synthesis for the production of long oligonucleotides. The enzymatic procedure for probe production was based on ligation of short DNA sequences. Long DNA probes were obtained from smaller oligonucleotides together with a short sequence that acts as bridge stabilizing the molecular complex for DNA ligation. The ligation reactions were monitored by capillary gel electrophoresis with laser-induced fluorescence detection (CGE-LIF) using a bare fused-silica capillary. The capillary gel electrophoresis-LIF method demonstrated to be very useful and informative for the characterization of the ligation reaction, providing important information about the nature of some impurities, as well as for the fine optimization of the ligation conditions (i.e. ligation cycles, oligonucleotide and enzyme concentration). As a result, the yield and quality of the ligation product were highly improved. The in-lab prepared DNA probes were used in a novel multiplex ligation-dependent genome amplification (MLGA) method for the detection of genetically modified maize in samples. The great possibilities of the whole approach were demonstrated by the specific and sensitive

  1. Oligonucleotide Microarray for the Study of Functional Gene Diversity in the Nitrogen Cycle in the Environment

    PubMed Central

    Taroncher-Oldenburg, Gaspar; Griner, Erin M.; Francis, Chris A.; Ward, Bess B.

    2003-01-01

    The analysis of functional diversity and its dynamics in the environment is essential for understanding the microbial ecology and biogeochemistry of aquatic systems. Here we describe the development and optimization of a DNA microarray method for the detection and quantification of functional genes in the environment and report on their preliminary application to the study of the denitrification gene nirS in the Choptank River-Chesapeake Bay system. Intergenic and intragenic resolution constraints were determined by an oligonucleotide (70-mer) microarray approach. Complete signal separation was achieved when comparing unrelated genes within the nitrogen cycle (amoA, nifH, nirK, and nirS) and detecting different variants of the same gene, nirK, corresponding to organisms with two different physiological modes, ammonia oxidizers and denitrifying halobenzoate degraders. The limits of intragenic resolution were investigated with a microarray containing 64 nirS sequences comprising 14 cultured organisms and 50 clones obtained from the Choptank River in Maryland. The nirS oligonucleotides covered a range of sequence identities from approximately 40 to 100%. The threshold values for specificity were determined to be 87% sequence identity and a target-to-probe perfect match-to-mismatch binding free-energy ratio of 0.56. The lower detection limit was 10 pg of DNA (equivalent to approximately 107 copies) per target per microarray. Hybridization patterns on the microarray differed between sediment samples from two stations in the Choptank River, implying important differences in the composition of the denitirifer community along an environmental gradient of salinity, inorganic nitrogen, and dissolved organic carbon. This work establishes a useful set of design constraints (independent of the target gene) for the implementation of functional gene microarrays for environmental applications. PMID:12571043

  2. A new oligonucleotide microarray for detection of pathogenic and non-pathogenic Legionella spp.

    PubMed

    Cao, Boyang; Liu, Xiangqian; Yu, Xiang; Chen, Min; Feng, Lu; Wang, Lei

    2014-01-01

    Legionella pneumophila has been recognized as the major cause of legionellosis since the discovery of the deadly disease. Legionella spp. other than L. pneumophila were later found to be responsible to many non-pneumophila infections. The non-L. pneumophila infections are likely under-detected because of a lack of effective diagnosis. In this report, we have sequenced the 16S-23S rRNA gene internal transcribed spacer (ITS) of 10 Legionella species and subspecies, including L. anisa, L. bozemanii, L. dumoffii, L. fairfieldensis, L. gormanii, L. jordanis, L. maceachernii, L. micdadei, L. pneumophila subspp. fraseri and L. pneumophila subspp. pasculleii, and developed a rapid oligonucleotide microarray detection technique accordingly to identify 12 most common Legionella spp., which consist of 11 pathogenic species of L. anisa, L. bozemanii, L. dumoffii, L. gormanii, L. jordanis, L. longbeachae, L. maceachernii, L. micdadei, and L. pneumophila (including subspp. pneumophila, subspp. fraseri, and subspp. pasculleii) and one non-pathogenic species, L. fairfieldensis. Twenty-nine probes that reproducibly detected multiple Legionella species with high specificity were included in the array. A total of 52 strains, including 30 target pathogens and 22 non-target bacteria, were used to verify the oligonucleotide microarray assay. The sensitivity of the detection was at 1.0 ng with genomic DNA or 13 CFU/100 mL with Legionella cultures. The microarray detected seven samples of air conditioner-condensed water with 100% accuracy, validating the technique as a promising method for applications in basic microbiology, clinical diagnosis, food safety, and epidemiological surveillance. The phylogenetic study based on the ITS has also revealed that the non-pathogenic L. fairfieldensis is the closest to L. pneumophila than the nine other pathogenic Legionella spp.

  3. Simultaneous detection and differentiation of Newcastle disease and avian influenza viruses using oligonucleotide microarrays.

    PubMed

    Wang, Lih-Chiann; Pan, Chu-Hsiang; Severinghaus, Lucia Liu; Liu, Lu-Yuan; Chen, Chi-Tsong; Pu, Chang-En; Huang, Dean; Lir, Jihn-Tsair; Chin, Shih-Chien; Cheng, Ming-Chu; Lee, Shu-Hwae; Wang, Ching-Ho

    2008-03-18

    Newcastle disease (ND) and avian influenza (AI) are two of the most important zoonotic viral diseases of birds throughout the world. These two viruses often have a great impact upon the poultry industry. Both viruses are associated with transmission from wild to domestic birds, and often display similar signs that need to be differentiated. A rapid surveillance among wild and domestic birds is important for early disease detection and intervention, and is the basis for what measures should be taken. The surveillance, thus, should be able to differentiate the diseases and provide a detailed analysis of the virus strains. Here, we described a fast, simultaneous and inexpensive approach to the detection of Newcastle disease virus (NDV) and avian influenza virus (AIV) using oligonucleotide microarrays. The NDV pathotypes and the AIV haemagglutinin subtypes H5 and H7 were determined at the same time. Different probes on a microarray targeting the same gene were implemented in order to encompass the diversified virus strains or provide multiple confirmations of the genotype. This ensures good sensitivity and specificity among divergent viruses. Twenty-four virus isolates and twenty-four various combinations of the viruses were tested in this study. All viruses were successfully detected and typed. The hybridization results on microarrays were clearly identified with the naked eyes, with no further imaging equipment needed. The results demonstrate that the detection and typing of multiple viruses can be performed simultaneously and easily using oligonucleotide microarrays. The proposed method may provide potential for rapid surveillance and differential diagnosis of these two important zoonoses in both wild and domestic birds.

  4. Hybridization probe for femtomolar quantification of selected nucleic acid sequences on a disposable electrode.

    PubMed

    Jenkins, Daniel M; Chami, Bilal; Kreuzer, Matthias; Presting, Gernot; Alvarez, Anne M; Liaw, Bor Yann

    2006-04-01

    Mixed monolayers of electroactive hybridization probes on gold surfaces of a disposable electrode were investigated as a technology for simple, sensitive, selective, and rapid gene identification. Hybridization to the ferrocene-labeled hairpin probes reproducibly diminished cyclic redox currents, presumably due to a displacement of the label from the electrode. Observed peak current densities were roughly 1000x greater than those observed in previous studies, such that results could easily be interpreted without the use of algorithms to correct for background polarization currents. Probes were sensitive to hybridization with a number of oligonucleotide sequences with varying homology, but target oligonucleotides could be distinguished from competing nontarget sequences based on unique "melting" profiles from the probe. Detection limits were demonstrated down to nearly 100 fM, which may be low enough to identify certain genetic conditions or infections without amplification. This technology has rich potential for use in field devices for gene identification as well as in gene microarrays.

  5. Time-Resolved Sequence Analysis on High Density Fiberoptic DNA Probe

    SciTech Connect

    Walt, D. R.; Lee, K-H

    2002-11-19

    A universal array format has been developed in which all possible n-mers of a particular oligonucleotide sequence can be represented. The ability to determine the sequence of the probes at every position in the array should enable unbiased gene expression as well as arrays for de novo sequencing.

  6. Inhibition of HTLV-III by exogenous oligonucleotides

    SciTech Connect

    Goodchild, J.; Zamecnik, P.C.

    1989-02-21

    A method is described of detecting the presence of HTLV-III virus in a sample by demonstrating inhibition of replication of the virus in cells which are normally killed by the HTLV-III virus after the cells have been (a) combined with the sample and an oligonucleotide complementary to at least one highly conserved region of the HTLV-III genome necessary for HTLV-III replication and capable of hybridizing with at least the highly conserved region, the highly conserved region of the HTLV-III genome being a nucleotide sequence present in the genomes of HTLV-III isolates and the oligonucleotide complementary to at least one highly conserved region of the HTLV-III genome necessary for HTLV-III replication being complementary to a region of the HTLV-III genome.

  7. Structure and dynamics of oligonucleotides in the gas phase.

    PubMed

    Arcella, Annalisa; Dreyer, Jens; Ippoliti, Emiliano; Ivani, Ivan; Portella, Guillem; Gabelica, Valérie; Carloni, Paolo; Orozco, Modesto

    2015-01-07

    By combining ion-mobility mass spectrometry experiments with sub-millisecond classical and ab initio molecular dynamics we fully characterized, for the first time, the dynamic ensemble of a model nucleic acid in the gas phase under electrospray ionization conditions. The studied oligonucleotide unfolds upon vaporization, loses memory of the solution structure, and explores true gas-phase conformational space. Contrary to our original expectations, the oligonucleotide shows very rich dynamics in three different timescales (multi-picosecond, nanosecond, and sub-millisecond). The shorter timescale dynamics has a quantum mechanical nature and leads to changes in the covalent structure, whereas the other two are of classical origin. Overall, this study suggests that a re-evaluation on our view of the physics of nucleic acids upon vaporization is needed. © 2015 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

  8. Detection of high-resolution Raman spectra in short oligonucleotides

    NASA Astrophysics Data System (ADS)

    Bairamov, F. B.; Poloskin, E. D.; Chernev, A. L.; Toporov, V. V.; Dubina, M. V.; Lahderanta, E.; Lipsanen, H.; Bairamov, B. Kh.

    2014-06-01

    High-resolution spectra of single-chain short oligonucleotides d(20G, 20T), where d is a deoxyribonucleoside, G is guanine, and T is thymine, have been obtained by the highly sensitive nonresonant Raman scattering method of biomacromolecules. In addition to their own multifunctional significance, short oligonucleotides attract interest as ideal model objects for revealing poorly studied peculiarities of tertiary and quaternary structures of DNA. The detection of narrow spectral lines has allowed determining the characteristic time scale and makes it possible to study the dynamics of fast relaxation processes of vibrational motions of atoms in biomacromolecules. It has been found that the FWHM of the narrowest 1355.4 cm-1 spectral line attributed to the vibrations of the dT methyl group is 14.6 cm-1. The corresponding lifetime is 0.38 ps.

  9. Mechanism of Oligonucleotide Uptake by Cells: Involvement of Specific receptors?

    NASA Astrophysics Data System (ADS)

    Yakubov, Leonid A.; Deeva, Elena A.; Zarytova, Valentina F.; Ivanova, Eugenia M.; Ryte, Antonina S.; Yurchenko, Lyudmila V.; Vlassov, Valentin V.

    1989-09-01

    We have investigated the interaction of oligonucleotides and their alkylating derivatives with mammalian cells. In experiments with L929 mouse fibroblast and Krebs 2 ascites carcinoma cells, it was found that cellular uptake of oligodeoxynucleotide derivatives is achieved by an endocytosis mechanism. Uptake is considerably more efficient at low oligomer concentration (< 1 μ M), because at this concentration a significant percentage of the total oligomer pool is absorbed on the cell surface and internalized by a more efficient absorptive endocytosis process. Two modified proteins were detected in mouse fibroblasts that were treated with the alkylating oligonucleotide derivatives. The binding of the oligomers to the proteins is inhibited by other oligodeoxynucleotides, single- and double-stranded DNA, and RNA. The polyanions heparin and chondroitin sulfates A and B do not inhibit binding. These observations suggest the involvement of specific receptor proteins in binding of oligomers to mammalian cells.

  10. Recursive DNA Assembly Using Protected Oligonucleotide Duplex Assisted Cloning (PODAC).

    PubMed

    Van Hove, Bob; Guidi, Chiara; De Wannemaeker, Lien; Maertens, Jo; De Mey, Marjan

    2017-06-16

    A problem rarely tackled by current DNA assembly methods is the issue of cloning additional parts into an already assembled construct. Costly PCR workflows are often hindered by repeated sequences, and restriction based strategies impose design constraints for each enzyme used. Here we present Protected Oligonucleotide Duplex Assisted Cloning (PODAC), a novel technique that makes use of an oligonucleotide duplex for iterative Golden Gate cloning using only one restriction enzyme. Methylated bases confer protection from digestion during the assembly reaction and are removed during replication in vivo, unveiling a new cloning site in the process. We used this method to efficiently and accurately assemble a biosynthetic pathway and demonstrated its robustness toward sequence repeats by constructing artificial CRISPR arrays. As PODAC is readily amenable to standardization, it would make a useful addition to the synthetic biology toolkit.

  11. The Design of Oligonucleotides Which Attack Specific Gene Targets

    DTIC Science & Technology

    1989-12-08

    identify by block number) FIELD GROUP SUB-GROUP ’" DNA Recognition; 06 03 Triplet helix formation, <r: " 19 ABSTRACT (Continue on reverse if necessary and...Such local triplet bonding schemes give rise to H bonding between the triplex forming oligonucleotide and the purine of the underlying Watson Crick ...identify by block number) During the first year of Navy support, we have refined our understanding of triple helix formation and in the process, have

  12. Cationic carbosilane dendrimers and oligonucleotide binding: an energetic affair

    NASA Astrophysics Data System (ADS)

    Marson, D.; Laurini, E.; Posocco, P.; Fermeglia, M.; Pricl, S.

    2015-02-01

    Generation 2 cationic carbosilane dendrimers hold great promise as internalizing agents for gene therapy as they present low toxicity and retain and internalize the genetic material as an oligonucleotide or siRNA. In this work we carried out complete in silico structural and energetical characterization of the interactions of a set of G2 carbosilane dendrimers, showing different affinity towards two single strand oligonucleotide (ODN) sequences in vitro. Our simulations predict that these four dendrimers and the relevant ODN complexes are characterized by similar size and shape, and that the molecule-specific ODN binding ability can be rationalized only by considering a critical molecular design parameter: the normalized effective binding energy ΔGbind,eff/Neff, i.e. the performance of each active individual dendrimer branch directly involved in a binding interaction.Generation 2 cationic carbosilane dendrimers hold great promise as internalizing agents for gene therapy as they present low toxicity and retain and internalize the genetic material as an oligonucleotide or siRNA. In this work we carried out complete in silico structural and energetical characterization of the interactions of a set of G2 carbosilane dendrimers, showing different affinity towards two single strand oligonucleotide (ODN) sequences in vitro. Our simulations predict that these four dendrimers and the relevant ODN complexes are characterized by similar size and shape, and that the molecule-specific ODN binding ability can be rationalized only by considering a critical molecular design parameter: the normalized effective binding energy ΔGbind,eff/Neff, i.e. the performance of each active individual dendrimer branch directly involved in a binding interaction. Electronic supplementary information (ESI) available: Additional figures and tables. See DOI: 10.1039/c4nr04510f

  13. Oligonucleotide switches and nanomaterials for intracellular mRNA sensing

    NASA Astrophysics Data System (ADS)

    Tombelli, S.; Ballestri, M.; Giambastiani, G.; Giannetti, A.; Guerrini, A.; Sotgiu, G.; Trono, C.; Tuci, G.; Varchi, G.; Baldini, F.

    2013-06-01

    We describe here the conjugation of polymethylmethacrylate nanoparticles to particular oligonucleotide switches, termed molecular beacons (MBs), as potential intracellular nanosensors. Survivin mRNA targeting MBs have been used with Atto647N and Blackberry 650 as fluorophore/quencher pair. The nanosensors have been characterized in vitro by investigating the analytical performances of the chosen molecular beacon and its functionalities after conjugation to the nanoparticles.

  14. Functionalization of silicon oxide using supercritical fluid deposition of 3,4-epoxybutyltrimethoxysilane for the immobilization of amino-modified oligonucleotide

    NASA Astrophysics Data System (ADS)

    Rull, Jordi; Nonglaton, Guillaume; Costa, Guillaume; Fontelaye, Caroline; Marchi-Delapierre, Caroline; Ménage, Stéphane; Marchand, Gilles

    2015-11-01

    The functionalization of silicon oxide based substrates using silanes is generally performed through liquid phase methodologies. These processes involve a huge quantity of potentially toxic solvents and present some important disadvantages for the functionalization of microdevices or porous materials, for example the low diffusion. To overcome this drawback, solvent-free methodologies like molecular vapor deposition (MVD) or supercritical fluid deposition (SFD) have been developed. In this paper, the deposition process of 3,4-epoxybutyltrimethoxysilane (EBTMOS) on silicon oxide using supercritical carbon dioxide (scCO2) as a solvent is studied for the first time. The oxirane ring of epoxy silanes readily reacts with amine group and is of particular interest for the grafting of amino-modified oligonucleotides or antibodies for diagnostic application. Then the ability of this specific EBTMOS layer to react with amine functions has been evaluated using the immobilization of amino-modified oligonucleotide probes. The presence of the probes is revealed by fluorescence using hybridization with a fluorescent target oligonucleotide. The performances of SFD of EBTMOS have been optimized and then compared with the dip coating and molecular vapor deposition methods, evidencing a better grafting efficiency and homogeneity, a lower reaction time in addition to the eco-friendly properties of the supercritical carbon dioxide. The epoxysilane layers have been characterized by surface enhanced ellipsometric contrast optical technique, atomic force microscopy, multiple internal reflection infrared spectroscopy and X-ray photoelectron spectroscopy. The shelf life of the 3,4-epoxybutyltrimethoxysilane coating layer has also been studied. Finally, two different strategies of NH2-oligonucleotide grafting on EBTMOS coating layer have been compared, i.e. reductive amination and nucleophilic substitution, SN2. This EBTMOS based coating layer can be used for a wide range of applications

  15. G-Quadruplex Forming Oligonucleotides as Anti-HIV Agents.

    PubMed

    Musumeci, Domenica; Riccardi, Claudia; Montesarchio, Daniela

    2015-09-22

    Though a variety of different non-canonical nucleic acids conformations have been recognized, G-quadruplex structures are probably the structural motifs most commonly found within known oligonucleotide-based aptamers. This could be ascribed to several factors, as their large conformational diversity, marked responsiveness of their folding/unfolding processes to external stimuli, high structural compactness and chemo-enzymatic and thermodynamic stability. A number of G-quadruplex-forming oligonucleotides having relevant in vitro anti-HIV activity have been discovered in the last two decades through either SELEX or rational design approaches. Improved aptamers have been obtained by chemical modifications of natural oligonucleotides, as terminal conjugations with large hydrophobic groups, replacement of phosphodiester linkages with phosphorothioate bonds or other surrogates, insertion of base-modified monomers, etc. In turn, detailed structural studies have elucidated the peculiar architectures adopted by many G-quadruplex-based aptamers and provided insight into their mechanism of action. An overview of the state-of-the-art knowledge of the relevance of putative G-quadruplex forming sequences within the viral genome and of the most studied G-quadruplex-forming aptamers, selectively targeting HIV proteins, is here presented.

  16. Recursive construction of perfect DNA molecules from imperfect oligonucleotides.

    PubMed

    Linshiz, Gregory; Yehezkel, Tuval Ben; Kaplan, Shai; Gronau, Ilan; Ravid, Sivan; Adar, Rivka; Shapiro, Ehud

    2008-01-01

    Making faultless complex objects from potentially faulty building blocks is a fundamental challenge in computer engineering, nanotechnology and synthetic biology. Here, we show for the first time how recursion can be used to address this challenge and demonstrate a recursive procedure that constructs error-free DNA molecules and their libraries from error-prone oligonucleotides. Divide and Conquer (D&C), the quintessential recursive problem-solving technique, is applied in silico to divide the target DNA sequence into overlapping oligonucleotides short enough to be synthesized directly, albeit with errors; error-prone oligonucleotides are recursively combined in vitro, forming error-prone DNA molecules; error-free fragments of these molecules are then identified, extracted and used as new, typically longer and more accurate, inputs to another iteration of the recursive construction procedure; the entire process repeats until an error-free target molecule is formed. Our recursive construction procedure surpasses existing methods for de novo DNA synthesis in speed, precision, amenability to automation, ease of combining synthetic and natural DNA fragments, and ability to construct designer DNA libraries. It thus provides a novel and robust foundation for the design and construction of synthetic biological molecules and organisms.

  17. Therapeutic Antisense Oligonucleotides against Cancer: Hurdling to the Clinic

    NASA Astrophysics Data System (ADS)

    Moreno, Pedro; Pêgo, Ana

    2014-10-01

    Under clinical development since the early 90’s and with two successfully approved drugs (Fomivirsen and Mipomersen), oligonucleotide-based therapeutics have not yet delivered a clinical drug to the market in the cancer field. Whilst many pre-clinical data has been generated, a lack of understanding still exists on how to efficiently tackle all the different challenges presented for cancer targeting in a clinical setting. Namely, effective drug vectorization, careful choice of target gene or synergistic multi-gene targeting are surely decisive, while caution must be exerted to avoid potential toxic, often misleading off-target-effects. Here a brief overview will be given on the nucleic acid chemistry advances that established oligonucleotide technologies as a promising therapeutic alternative and ongoing cancer related clinical trials. Special attention will be given towards a perspective on the hurdles encountered specifically in the cancer field by this class of therapeutic oligonucleotides and a view on possible avenues for success is presented, with particular focus on the contribution from nanotechnology to the field.

  18. Oligonucleotide Frequencies of Barcoding Loci Can Discriminate Species across Kingdoms

    PubMed Central

    Shukla, Virendra; Tuli, Rakesh

    2010-01-01

    Background DNA barcoding refers to the use of short DNA sequences for rapid identification of species. Genetic distance or character attributes of a particular barcode locus discriminate the species. We report an efficient approach to analyze short sequence data for discrimination between species. Methodology and Principal Findings A new approach, Oligonucleotide Frequency Range (OFR) of barcode loci for species discrimination is proposed. OFR of the loci that discriminates between species was characteristic of a species, i.e., the maxima and minima within a species did not overlap with that of other species. We compared the species resolution ability of different barcode loci using p-distance, Euclidean distance of oligonucleotide frequencies, nucleotide-character based approach and OFR method. The species resolution by OFR was either higher or comparable to the other methods. A short fragment of 126 bp of internal transcribed spacer region in ribosomal RNA gene was sufficient to discriminate a majority of the species using OFR. Conclusions/Significance Oligonucleotide frequency range of a barcode locus can discriminate between species. Ability to discriminate species using very short DNA fragments may have wider applications in forensic and conservation studies. PMID:20808837

  19. Gene expression profiling in peanut using high density oligonucleotide microarrays

    PubMed Central

    Payton, Paxton; Kottapalli, Kameswara Rao; Rowland, Diane; Faircloth, Wilson; Guo, Baozhu; Burow, Mark; Puppala, Naveen; Gallo, Maria

    2009-01-01

    Background Transcriptome expression analysis in peanut to date has been limited to a relatively small set of genes and only recently has a significant number of ESTs been released into the public domain. Utilization of these ESTs for oligonucleotide microarrays provides a means to investigate large-scale transcript responses to a variety of developmental and environmental signals, ultimately improving our understanding of plant biology. Results We have developed a high-density oligonucleotide microarray for peanut using 49,205 publicly available ESTs and tested the utility of this array for expression profiling in a variety of peanut tissues. To identify putatively tissue-specific genes and demonstrate the utility of this array for expression profiling in a variety of peanut tissues, we compared transcript levels in pod, peg, leaf, stem, and root tissues. Results from this experiment showed 108 putatively pod-specific/abundant genes, as well as transcripts whose expression was low or undetected in pod compared to peg, leaf, stem, or root. The transcripts significantly over-represented in pod include genes responsible for seed storage proteins and desiccation (e.g., late-embryogenesis abundant proteins, aquaporins, legumin B), oil production, and cellular defense. Additionally, almost half of the pod-abundant genes represent unknown genes allowing for the possibility of associating putative function to these previously uncharacterized genes. Conclusion The peanut oligonucleotide array represents the majority of publicly available peanut ESTs and can be used as a tool for expression profiling studies in diverse tissues. PMID:19523230

  20. DNA/RNA heteroduplex oligonucleotide for highly efficient gene silencing

    PubMed Central

    Nishina, Kazutaka; Piao, Wenying; Yoshida-Tanaka, Kie; Sujino, Yumiko; Nishina, Tomoko; Yamamoto, Tsuyoshi; Nitta, Keiko; Yoshioka, Kotaro; Kuwahara, Hiroya; Yasuhara, Hidenori; Baba, Takeshi; Ono, Fumiko; Miyata, Kanjiro; Miyake, Koichi; Seth, Punit P.; Low, Audrey; Yoshida, Masayuki; Bennett, C. Frank; Kataoka, Kazunori; Mizusawa, Hidehiro; Obika, Satoshi; Yokota, Takanori

    2015-01-01

    Antisense oligonucleotides (ASOs) are recognized therapeutic agents for the modulation of specific genes at the post-transcriptional level. Similar to any medical drugs, there are opportunities to improve their efficacy and safety. Here we develop a short DNA/RNA heteroduplex oligonucleotide (HDO) with a structure different from double-stranded RNA used for short interfering RNA and single-stranded DNA used for ASO. A DNA/locked nucleotide acid gapmer duplex with an α-tocopherol-conjugated complementary RNA (Toc-HDO) is significantly more potent at reducing the expression of the targeted mRNA in liver compared with the parent single-stranded gapmer ASO. Toc-HDO also improves the phenotype in disease models more effectively. In addition, the high potency of Toc-HDO results in a reduction of liver dysfunction observed in the parent ASO at a similar silencing effect. HDO technology offers a novel concept of therapeutic oligonucleotides, and the development of this molecular design opens a new therapeutic field. PMID:26258894

  1. NanoCluster Beacon - A New Molecular Probe for Homogeneous Detection of Nucleic Acid Targets

    DTIC Science & Technology

    2011-02-01

    requires only a single preparation step (i.e. nanocluster formation on NC probes), but because there is no need to remove excess silver ions or...Oligonucleotide-templated nanoclusters consisting of a few atoms of silver (DNA/Ag NCs) have been made into a new molecular probe that “lights up...upon target DNA binding, termed a NanoCluster Beacon (NCB). We discovered that interactions between silver nanoclusters and a proximal, guanine- rich

  2. Exclusive formation of monovalent quantum dot imaging probes by steric exclusion

    PubMed Central

    Farlow, Justin; Seo, Daeha; Broaders, Kyle E.; Taylor, Marcus; Gartner, Zev J.; Jun, Young-wook

    2013-01-01

    Precise control over interfacial chemistry between nanoparticles and other materials remains a significant challenge limiting the broad application of nanotechnology in biology. To address this challenge, we use “Steric Exclusion” to completely convert commercial quantum dots (QDs) into monovalent imaging probes by wrapping the QD with a functionalized oligonucleotide. We demonstrate the utility of these QDs as modular and non-perturbing imaging probes by tracking individual Notch receptors on live cells. PMID:24122039

  3. Synthesis of triazole-linked oligonucleotides with high affinity to DNA complements and an analysis of their compatibility with biosystems.

    PubMed

    Varizhuk, Anna M; Kaluzhny, Dmitry N; Novikov, Roman A; Chizhov, Alexandr O; Smirnov, Igor P; Chuvilin, Andrey N; Tatarinova, Olga N; Fisunov, Gleb Y; Pozmogova, Galina E; Florentiev, Vladimir L

    2013-06-21

    New oligonucleotide analogues with triazole internucleotide linkages were synthesized, and their hybridization properties were studied. The analogues demonstrated DNA binding affinities similar to those of unmodified oligonucleotides. The modification was shown to protect the oligonucleotides from nuclease hydrolysis. The modified oligonucleotides were tested as PCR primers. Modifications remote from the 3'-terminus were tolerated by polymerases. Our results suggest that these new oligonucleotide analogues are among the most promising triazole DNA mimics characterized to date.

  4. Influence of the length of target DNA overhang proximal to the array surface on discrimination of single-base mismatches on a 25-mer oligonucleotide array

    PubMed Central

    2014-01-01

    Background The performance of probes on an oligonucleotide microarray can be characterised in terms of hybridisation signal strength and the ability to discriminate sequence mismatches between the probe and the hybridising target strand, such as those resulting from SNPs. Various properties of the probe affect mismatch discrimination, such as probe length and the position of mismatched bases, and the effects of these factors have been well characterised in a variety of array formats. Results A low-density microarray was developed to systematically investigate the effect of a probe’s position within hybridised target PCR products on the tolerance and discrimination of single-nucleotide mismatches between the probe and target. In line with previous reports, hybridisation signals were attenuated by different degrees depending on the identity of the mismatch, the position of the mismatch within the probe, and the length of the PCR product. However, the same mismatch caused different degrees of attenuation depending on the position of the probe within the hybridising product, such that improved mismatch discrimination was observed for PCR products where a greater proportion of the total length was proximal to the array surface. Conclusions These results suggest that the degree of mismatch discrimination can be influenced by the choice of PCR primers, providing a means by which array performance could be fine-tuned in addition to manipulation of the properties of the probes themselves. PMID:24742004

  5. Static magnetic field reduced exogenous oligonucleotide uptake by spermatozoa using magnetic nanoparticle gene delivery system

    NASA Astrophysics Data System (ADS)

    Katebi, Samira; Esmaeili, Abolghasem; Ghaedi, Kamran

    2016-03-01

    Spermatozoa could introduce exogenous oligonucleotides of interest to the oocyte. The most important reason of low efficiency of sperm mediated gene transfer (SMGT) is low uptake of exogenous DNA by spermatozoa. The aim of this study was to evaluate the effects of static magnetic field on exogenous oligonucleotide uptake of spermatozoa using magnetofection method. Magnetic nanoparticles (MNPs) associated with the labeled oligonucleotides were used to increase the efficiency of exogenous oligonucleotide uptake by rooster spermatozoa. We used high-field/high-gradient magnet (NdFeB) to enhance and accelerate exogenous DNA sedimentation at the spermatozoa surface. Flow cytometry analysis was performed to measure viability and percentage of exogenous oligonucleotide uptake by sperm. Flow cytometry analysis showed a significant increase in exogenous oligonucleotide uptake by rooster spermatozoa (P<0.001) when spermatozoa were incubated in exogenous oligonucleotide solution and MNPs. However, by applying static magnetic field during magnetofection method, a significant decrease in exogenous oligonucleotide uptake was observed (P<0.05). Findings of this study showed that MNPs were effective to increase exogenous oligonucleotide uptake by rooster spermatozoa; however unlike others studies, static magnetic field, was not only ineffective to enhance exogenous oligonucleotide uptake by rooster spermatozoa but also led to reduction in efficiency of magnetic nanoparticles in gene transfer.

  6. Oligonucleotide-modified screen-printed gold electrodes for enzyme-amplified sensing of nucleic acids.

    PubMed

    Carpini, Guido; Lucarelli, Fausto; Marrazza, Giovanna; Mascini, Marco

    2004-09-15

    An electrochemical genosensor for the detection of specific sequences of DNA has been developed using disposable screen-printed gold electrodes. Screen-printed gold electrodes were firstly modified with a mixed monolayer of a 25-mer thiol-tethered DNA probe and a spacer thiol, 6-mercapto-1-hexanol (MCH). The DNA probe sequence was internal to the sequence of the 35S promoter, which sequence is inserted in the genome of GMOs regulating the transgene expression. An enzyme-amplified detection scheme, based on the coupling of a streptavidin-alkaline phosphatase conjugate and biotinylated target sequences was then applied. The enzyme catalysed the hydrolysis of the electroinactive alpha-naphthyl phosphate to alpha-naphthol; this product is electroactive and has been detected by means of differential pulse voltammetry. The assay was, firstly, characterised using synthetic oligonucleotides. Relevant parameters, such as the probe concentration and the immobilisation time, the use of the MCH and different enzymatic conjugates, were investigated and optimised. The genosensor response was found to be linearly related to the target concentration between 0 and 25 nmol/L; the detection limit was 0.25 nmol/L. The analytical procedure was then applied for the detection of the 35S promoter sequence, which was amplified from the pBI121 plasmid by polymerase chain reaction (PCR). Hybridisation conditions (i.e., hybridisation buffer and hybridisation time) were further optimised. The selectivity of the assay was confirmed using biotinylated non-complementary amplicons and PCR blanks. The results showed that the genosensor enabled sensitive (detection limit: 1 nmol/L) and specific detection of GMO-related sequences, thus providing a useful tool for the screening analysis of bioengineered food samples.

  7. Array-based electrical detection of DNA with nanoparticle probes.

    PubMed

    Park, So-Jung; Taton, T Andrew; Mirkin, Chad A

    2002-02-22

    A DNA array detection method is reported in which the binding of oligonucleotides functionalized with gold nanoparticles leads to conductivity changes associated with target-probe binding events. The binding events localize gold nanoparticles in an electrode gap; silver deposition facilitated by these nanoparticles bridges the gap and leads to readily measurable conductivity changes. An unusual salt concentration-dependent hybridization behavior associated with these nanoparticle probes was exploited to achieve selectivity without a thermal-stringency wash. Using this method, we have detected target DNA at concentrations as low as 500 femtomolar with a point mutation selectivity factor of approximately 100,000:1.

  8. Structured oligonucleotides for target indexing to allow single-vessel PCR amplification and solid support microarray hybridization.

    PubMed

    Girard, Laurie D; Boissinot, Karel; Peytavi, Régis; Boissinot, Maurice; Bergeron, Michel G

    2015-02-07

    The combination of molecular diagnostic technologies is increasingly used to overcome limitations on sensitivity, specificity or multiplexing capabilities, and provide efficient lab-on-chip devices. Two such techniques, PCR amplification and microarray hybridization are used serially to take advantage of the high sensitivity and specificity of the former combined with high multiplexing capacities of the latter. These methods are usually performed in different buffers and reaction chambers. However, these elaborate methods have high complexity and cost related to reagent requirements, liquid storage and the number of reaction chambers to integrate into automated devices. Furthermore, microarray hybridizations have a sequence dependent efficiency not always predictable. In this work, we have developed the concept of a structured oligonucleotide probe which is activated by cleavage from polymerase exonuclease activity. This technology is called SCISSOHR for Structured Cleavage Induced Single-Stranded Oligonucleotide Hybridization Reaction. The SCISSOHR probes enable indexing the target sequence to a tag sequence. The SCISSOHR technology also allows the combination of nucleic acid amplification and microarray hybridization in a single vessel in presence of the PCR buffer only. The SCISSOHR technology uses an amplification probe that is irreversibly modified in presence of the target, releasing a single-stranded DNA tag for microarray hybridization. Each tag is composed of a 3-nucleotide sequence-dependent segment and a unique "target sequence-independent" 14-nucleotide segment allowing for optimal hybridization with minimal cross-hybridization. We evaluated the performance of five (5) PCR buffers to support microarray hybridization, compared to a conventional hybridization buffer. Finally, as a proof of concept, we developed a multiplexed assay for the amplification, detection, and identification of three (3) DNA targets. This new technology will facilitate the design

  9. Structured oligonucleotides for target indexing to allow single-vessel PCR amplification and solid support microarray hybridization

    PubMed Central

    Girard, Laurie D.; Boissinot, Karel; Peytavi, Régis; Boissinot, Maurice; Bergeron, Michel G.

    2014-01-01

    The combination of molecular diagnostic technologies is increasingly used to overcome limitations on sensitivity, specificity or multiplexing capabilities, and provide efficient lab-on-chip devices. Two such techniques, PCR amplification and microarray hybridization are used serially to take advantage of the high sensitivity and specificity of the former combined with high multiplexing capacities of the latter. These methods are usually performed in different buffers and reaction chambers. However, these elaborate methods have a high complexity cost related to reagent requirements, liquid storage and the number of reaction chambers to integrate into automated devices. Furthermore, microarray hybridizations have a sequence dependent efficiency not always predictable. In this work, we have developed the concept of a structured oligonucleotide probe which is activated by cleavage from polymerase exonuclease activity. This technology is called SCISSOHR for Structured Cleavage Induced Single-Stranded Oligonucleotide Hybridization Reaction. The SCISSOHR probes enable indexing the target sequence to a tag sequence. The SCISSOHR technology also allows the combination of nucleic acid amplification and microarray hybridization in a single vessel in presence of the PCR buffer only. The SCISSOHR technology uses an amplification probe that is irreversibly modified in presence of the target, releasing a single-stranded DNA tag for microarray hybridization. Each tag is composed of a 3-nucleotidesequence-dependent segment and a unique “target sequence-independent” 14-nucleotide segment allowing for optimal hybridization with minimal cross-hybridization. We evaluated the performance of five (5) PCR buffers to support microarray hybridization, compared to a conventional hybridization buffer. Finally, as a proof of concept, we developed a multiplexed assay for the amplification, detection, and identification of three (3) DNA targets. This new technology will facilitate the design

  10. Biocompatible core-shell nanoparticle-based surface-enhanced Raman scattering probes for detection of DNA related to HIV gene using silica-coated magnetic nanoparticles as separation tools.

    PubMed

    Liang, Yi; Gong, Ji-Lai; Huang, Yong; Zheng, Yue; Jiang, Jian-Hui; Shen, Guo-Li; Yu, Ru-Qin

    2007-04-30

    A novel, highly selective DNA hybridization assay has been developed based on surface-enhanced Raman scattering (SERS) for DNA sequences related to HIV. This strategy employs the Ag/SiO(2) core-shell nanoparticle-based Raman tags and the amino group modified silica-coated magnetic nanoparticles as immobilization matrix and separation tool. The hybridization reaction was performed between Raman tags functionalized with 3'-amino-labeled oligonucleotides as detection probes and the amino group modified silica-coated magnetic nanoparticles functionalized with 5'-amino-labeled oligonucleotides as capture probes. The Raman spectra of Raman tags can be used to monitor the presence of target oligonucleotides. The utilization of silica-coated magnetic nanoparticles not only avoided time-consuming washing, but also amplified the signal of hybridization assay. Additionally, the results of control experiments show that no or very low signal would be obtained if the hybridization assay is conducted in the presence of DNA sequences other than complementary oligonucleotides related to HIV gene such as non-complementary oligonucleotides, four bases mismatch oligonucleotides, two bases mismatch oligonucleotides and even single base mismatch oligonucleotides. It was demonstrated that the method developed in this work has high selectivity and sensitivity for DNA detection related to HIV gene.

  11. A study of oligonucleotide occurrence distributions in DNA coding segments.

    PubMed

    Castrignanò, T; Colosimo, A; Morante, S; Parisi, V; Rossi, G C

    1997-02-21

    In this paper we present a general strategy designed to study the occurrence frequency distributions of oligonucleotides in DNA coding segments and to deal with the problem of detecting possible patterns of genomic compositional inhomogeneities and disuniformities. Identifying specific tendencies or peculiar deviations in the distributions of the effective occurrence frequencies of oligonucleotides, with respect to what can be a priori expected, is of the greatest importance in biology. Differences between expected and actual distributions may in fact suggest or confirm the existence of specific biological mechanisms related to them. Similarly, a marked deviation in the occurrence frequency of an oligonucleotide may suggest that it belongs to the class of so-called "DNA signal (target) sequences". The approach we have elaborated is innovative in various aspects. Firstly, the analysis of the genomic data is carried out in the light of the observation that the distribution of the four nucleotides along the coding regions of the genoma is biased by the existence of a well-defined "reading frame". Secondly, the "experimental" numbers found by counting the occurrences of the various oligonucleotide sequences are appropriately corrected for the many kinds of mistakes and redundancies present in the available genetic Data Bases. A methodologically significant further improvement of our approach over the existing searching strategies is represented by the fact that, in order to decide whether or not the (corrected) "experimental" value of the occurrence frequency of a given oligonucleotide is within statistical expectations, a measure of the strength of the selective pressure, having acted on it in the course of the evolution, is assigned to the sequence, in a way that takes into account both the value of the "experimental" occurrence frequency of the sequence and the magnitude of the probability that this number might be the result of statistical fluctuations. If the

  12. Development of the Large-Scale Oligonucleotide Chip for the Diagnosis of Plant Viruses and its Practical Use

    PubMed Central

    Nam, Moon; Kim, Jeong-Seon; Lim, Seungmo; Park, Chung Youl; Kim, Jeong-Gyu; Choi, Hong-Soo; Lim, Hyoun-Sub; Moon, Jae Sun; Lee, Su-Heon

    2014-01-01

    A large-scale oligonucleotide (LSON) chip was developed for the detection of the plant viruses with known genetic information. The LSON chip contains two sets of 3,978 probes for 538 species of targets including plant viruses, satellite RNAs and viroids. A hundred forty thousand probes, consisting of isolate-, species- and genus-specific probes respectively, are designed from 20,000 of independent nucleotide sequence of plant viruses. Based on the economic importance, the amount of genome information, and the number of strains and/or isolates, one to fifty-one probes for each target virus are selected and spotted on the chip. The standard and field samples for the analysis of the LSON chip have been prepared and tested by RT-PCR. The probe’s specific and/or nonspecific reaction patterns by LSON chip allow us to diagnose the unidentified viruses. Thus, the LSON chip in this study could be highly useful for the detection of unexpected plant viruses, the monitoring of emerging viruses and the fluctuation of the population of major viruses in each plant. PMID:25288985

  13. Pollution Probe.

    ERIC Educational Resources Information Center

    Chant, Donald A.

    This book is written as a statement of concern about pollution by members of Pollution Probe, a citizens' anti-pollution group in Canada. Its purpose is to create public awareness and pressure for the eventual solution to pollution problems. The need for effective government policies to control the population explosion, conserve natural resources,…

  14. Pollution Probe.

    ERIC Educational Resources Information Center

    Chant, Donald A.

    This book is written as a statement of concern about pollution by members of Pollution Probe, a citizens' anti-pollution group in Canada. Its purpose is to create public awareness and pressure for the eventual solution to pollution problems. The need for effective government policies to control the population explosion, conserve natural resources,…

  15. Edesign: Primer and Enhanced Internal Probe Design Tool for Quantitative PCR Experiments and Genotyping Assays

    PubMed Central

    Kasahara, Naoko; Delobel, Diane; Hanami, Takeshi; Tanaka, Yuki; de Hoon, Michiel J. L.; Hayashizaki, Yoshihide; Usui, Kengo; Harbers, Matthias

    2016-01-01

    Analytical PCR experiments preferably use internal probes for monitoring the amplification reaction and specific detection of the amplicon. Such internal probes have to be designed in close context with the amplification primers, and may require additional considerations for the detection of genetic variations. Here we describe Edesign, a new online and stand-alone tool for designing sets of PCR primers together with an internal probe for conducting quantitative real-time PCR (qPCR) and genotypic experiments. Edesign can be used for selecting standard DNA oligonucleotides like for instance TaqMan probes, but has been further extended with new functions and enhanced design features for Eprobes. Eprobes, with their single thiazole orange-labelled nucleotide, allow for highly sensitive genotypic assays because of their higher DNA binding affinity as compared to standard DNA oligonucleotides. Using new thermodynamic parameters, Edesign considers unique features of Eprobes during primer and probe design for establishing qPCR experiments and genotyping by melting curve analysis. Additional functions in Edesign allow probe design for effective discrimination between wild-type sequences and genetic variations either using standard DNA oligonucleotides or Eprobes. Edesign can be freely accessed online at http://www.dnaform.com/edesign2/, and the source code is available for download. PMID:26863543

  16. Edesign: Primer and Enhanced Internal Probe Design Tool for Quantitative PCR Experiments and Genotyping Assays.

    PubMed

    Kimura, Yasumasa; Soma, Takahiro; Kasahara, Naoko; Delobel, Diane; Hanami, Takeshi; Tanaka, Yuki; de Hoon, Michiel J L; Hayashizaki, Yoshihide; Usui, Kengo; Harbers, Matthias

    2016-01-01

    Analytical PCR experiments preferably use internal probes for monitoring the amplification reaction and specific detection of the amplicon. Such internal probes have to be designed in close context with the amplification primers, and may require additional considerations for the detection of genetic variations. Here we describe Edesign, a new online and stand-alone tool for designing sets of PCR primers together with an internal probe for conducting quantitative real-time PCR (qPCR) and genotypic experiments. Edesign can be used for selecting standard DNA oligonucleotides like for instance TaqMan probes, but has been further extended with new functions and enhanced design features for Eprobes. Eprobes, with their single thiazole orange-labelled nucleotide, allow for highly sensitive genotypic assays because of their higher DNA binding affinity as compared to standard DNA oligonucleotides. Using new thermodynamic parameters, Edesign considers unique features of Eprobes during primer and probe design for establishing qPCR experiments and genotyping by melting curve analysis. Additional functions in Edesign allow probe design for effective discrimination between wild-type sequences and genetic variations either using standard DNA oligonucleotides or Eprobes. Edesign can be freely accessed online at http://www.dnaform.com/edesign2/, and the source code is available for download.

  17. Oligonucleotide fingerprinting using simple repeat motifs: a convenient, ubiquitously applicable method to detect hypervariability for multiple purposes.

    PubMed

    Epplen, J T; Ammer, H; Epplen, C; Kammerbauer, C; Mitreiter, R; Roewer, L; Schwaiger, W; Steimle, V; Zischler, H; Albert, E

    1991-01-01

    A panel of simple repetitive oligonucleotide probes has been designed and tested for multilocus DNA fingerprinting in some 200 fungal, plant and animal species as well as man. To date at least one of the probes has been found to be informative in each species. The human genome, however, has been the major target of many fingerprinting studies. Using the probe (CAC)5 or (GTG)5, individualization of all humans is possible except for monozygotic twins. Paternity analyses are now performed on a routine basis by the use of multilocus fingerprints, including also cases of deficiency, i.e. where one of the parents is not available for analysis. In forensic science stain analysis is feasible in all tissue remains containing nucleated cells. Depending on the degree of DNA degradation a variety of oligonucleotides are informative, and they have been proven useful in actual case work. Advantages in comparison to other methods including enzymatic DNA amplification techniques (PCR) are evident. Fingerprint patterns of tumors may be changed due to the gain or loss of chromosomes and/or intrachromosomal deletion and amplification events. Locus-specific probes were isolated from the human (CAC)5/(GTG)5 fingerprint with a varying degree of informativeness (monomorphic versus truly hypervariable markers). The feasibility of three different approaches for the isolation of hypervariable mono-locus probes was evaluated. Finally, one particular mixed simple (gt)n(ga)m repeat locus in the second intron of the HLA-DRB genes has been scrutinized to allow comparison of the extent of exon-encoded (protein-) polymorphisms versus intronic hypervariability of simple repeats: adjacent to a single gene sequence (e.g. HLA-DRB1*0401) many different length alleles were found. Group-specific structures of basic repeats were identified within the evolutionarily related DRB alleles. As a further application it is suggested here that due to the ubiquitous interspersion of their targets, short probes for

  18. Discrimination of oligonucleotides of different lengths with a wild-type aerolysin nanopore

    NASA Astrophysics Data System (ADS)

    Cao, Chan; Ying, Yi-Lun; Hu, Zheng-Li; Liao, Dong-Fang; Tian, He; Long, Yi-Tao

    2016-08-01

    Protein nanopores offer an inexpensive, label-free method of analysing single oligonucleotides. The sensitivity of the approach is largely determined by the characteristics of the pore-forming protein employed, and typically relies on nanopores that have been chemically modified or incorporate molecular motors. Effective, high-resolution discrimination of oligonucleotides using wild-type biological nanopores remains difficult to achieve. Here, we show that a wild-type aerolysin nanopore can resolve individual short oligonucleotides that are 2 to 10 bases long. The sensing capabilities are attributed to the geometry of aerolysin and the electrostatic interactions between the nanopore and the oligonucleotides. We also show that the wild-type aerolysin nanopores can distinguish individual oligonucleotides from mixtures and can monitor the stepwise cleavage of oligonucleotides by exonuclease I.

  19. Nanoparticle-bridge assay for amplification-free electrical detection of oligonucleotides

    NASA Astrophysics Data System (ADS)

    Teimouri, Manouchehr

    The aim of this research is to investigate a highly sensitive, fast, inexpensive, and field-applicable amplification-free nanoparticle-based oligonucleotide detection method which does not rely on any enzymatic or signal amplification process. In this approach, target oligonucleotide strands are detected through the formation of nanoparticle satellites which make an electrical path between two electrodes. This method enables an extremely sensitive oligonucleotide detection because even a few oligonucleotide strands can form a single nanoparticle satellite which can solely generates an electrical output signal. Results showed that this oligonucleotide detection method can detect oligonucleotide single strands at concentrations as low as 50 femtomolar without any amplification process. This detection method can be implemented in many fields such as biodefense, food safety, clinical research, and forensics.

  20. Investigation of the structural organization of cationic nanoemulsion/antisense oligonucleotide complexes.

    PubMed

    Bruxel, Fernanda; Vilela, José Mario Carneiro; Andrade, Margareth Spangler; Malachias, Ângelo; Perez, Carlos A; Magalhães-Paniago, Rogério; Oliveira, Mônica Cristina; Teixeira, Helder F

    2013-12-01

    Atomic force microscopy image analysis and energy dispersive X-ray diffraction experiments were used to investigate the structural organization of cationic nanoemulsion/oligonucleotide complexes. Oligonucleotides targeting topoisomerase II gene were adsorbed on cationic nanoemulsions obtained by means of spontaneous emulsification procedure. Topographical analysis by atomic force microscopy allowed the observation of the nanoemulsion/oligonucleotide complexes through three-dimensional high-resolution images. Flattening of the oil droplets was observed, which was reduced in the complexes obtained at high amount of adsorbed oligonucleotides. In such conditions, complexes exhibit droplet size in the 600nm range. The oligonucleotides molecules were detected on the surface of the droplets, preventing their fusion during aggregation. A lamellar structure organization was identified by energy dispersive X-ray diffraction experiments. The presence of the nucleic acid molecules led to a disorganization of the lipid arrangement and an expansion in the lattice spacing, which was proportional to the amount of oligonucleotides added.

  1. [Isolation of oligonucleotides from partial hydrolysates of DNA using template chromatography].

    PubMed

    Schott, H; Schrade, H; Watzlawick, H

    1984-03-02

    Purine oligonucleotides are adsorbed at 0 degree C on poly(vinyl alcohol)-p(dC)n-DEAE-cellulose, and pyrimidine oligonucleotides on oligo(guanylic acid) gel according to the base-pairing mechanism, if their sequences contain at least three or more homologous, consecutive guanylic or cytidylic moieties. By increasing the temperature all base-paired oligonucleotides are desorbed. With this template chromatography mixtures of defined purine- or pyrimidine oligonucleotides could be isolated from fractionated partial hydrolyzates of herring sperm DNA. Afterwards these mixtures are rechromatographed on QAE-Sephadex and/or Nucleosil C18. Using this approach oligonucleotides up to nine monomer units can be isolated either as single substances or as mixtures with defined composition on a preparative scale, which would be not possible with the already existing separation procedures when partial hydrolyzates of herring sperm DNA as starting material are used. Purity and sequence of the isolated oligonucleotides are determined by the "fingerprint" method.

  2. Polyamine-oligonucleotide conjugates: a promising direction for nucleic acid tools and therapeutics.

    PubMed

    Menzi, Mirjam; Lightfoot, Helen L; Hall, Jonathan

    2015-01-01

    Chemical modification and/or the conjugation of small functional molecules to oligonucleotides have significantly improved their biological and biophysical properties, addressing issues such as poor cell penetration, stability to nucleases and low affinity for their targets. Here, the authors review the literature reporting on the biophysical, biochemical and biological properties of one particular class of modification - polyamine-oligonucleotide conjugates. Naturally derived and synthetic polyamines have been grafted onto a variety of oligonucleotide formats, including antisense oligonucleotides and siRNAs. In many cases this has had beneficial effects on their properties such as target hybridization, nuclease resistance, cellular uptake and activity. Polyamine-oligonucleotide conjugation, therefore, represents a promising direction for the further development of oligonucleotide-based therapeutics and tools.

  3. Specific discrimination of three pathogenic Salmonella enterica subsp. enterica serotypes by carB-based oligonucleotide microarray.

    PubMed

    Shin, Hwa Hui; Hwang, Byeong Hee; Seo, Jeong Hyun; Cha, Hyung Joon

    2014-01-01

    It is important to rapidly and selectively detect and analyze pathogenic Salmonella enterica subsp. enterica in contaminated food to reduce the morbidity and mortality of Salmonella infection and to guarantee food safety. In the present work, we developed an oligonucleotide microarray containing duplicate specific capture probes based on the carB gene, which encodes the carbamoyl phosphate synthetase large subunit, as a competent biomarker evaluated by genetic analysis to selectively and efficiently detect and discriminate three S. enterica subsp. enterica serotypes: Choleraesuis, Enteritidis, and Typhimurium. Using the developed microarray system, three serotype targets were successfully analyzed in a range as low as 1.6 to 3.1 nM and were specifically discriminated from each other without nonspecific signals. In addition, the constructed microarray did not have cross-reactivity with other common pathogenic bacteria and even enabled the clear discrimination of the target Salmonella serotype from a bacterial mixture. Therefore, these results demonstrated that our novel carB-based oligonucleotide microarray can be used as an effective and specific detection system for S. enterica subsp. enterica serotypes.

  4. Specific Discrimination of Three Pathogenic Salmonella enterica subsp. enterica Serotypes by carB-Based Oligonucleotide Microarray

    PubMed Central

    Shin, Hwa Hui; Hwang, Byeong Hee; Seo, Jeong Hyun

    2014-01-01

    It is important to rapidly and selectively detect and analyze pathogenic Salmonella enterica subsp. enterica in contaminated food to reduce the morbidity and mortality of Salmonella infection and to guarantee food safety. In the present work, we developed an oligonucleotide microarray containing duplicate specific capture probes based on the carB gene, which encodes the carbamoyl phosphate synthetase large subunit, as a competent biomarker evaluated by genetic analysis to selectively and efficiently detect and discriminate three S. enterica subsp. enterica serotypes: Choleraesuis, Enteritidis, and Typhimurium. Using the developed microarray system, three serotype targets were successfully analyzed in a range as low as 1.6 to 3.1 nM and were specifically discriminated from each other without nonspecific signals. In addition, the constructed microarray did not have cross-reactivity with other common pathogenic bacteria and even enabled the clear discrimination of the target Salmonella serotype from a bacterial mixture. Therefore, these results demonstrated that our novel carB-based oligonucleotide microarray can be used as an effective and specific detection system for S. enterica subsp. enterica serotypes. PMID:24185846

  5. Synthesis, hybridization characteristics, and fluorescence properties of oligonucleotides modified with nucleobase-functionalized locked nucleic acid adenosine and cytidine monomers.

    PubMed

    Kaura, Mamta; Kumar, Pawan; Hrdlicka, Patrick J

    2014-07-03

    Conformationally restricted nucleotides such as locked nucleic acid (LNA) are very popular as affinity-, specificity-, and stability-enhancing modifications in oligonucleotide chemistry to produce probes for nucleic acid targeting applications in molecular biology, biotechnology, and medicinal chemistry. Considerable efforts have been devoted in recent years to optimize the biophysical properties of LNA through additional modification of the sugar skeleton. We recently introduced C5-functionalization of LNA uridines as an alternative and synthetically more straightforward approach to improve the biophysical properties of LNA. In the present work, we set out to test the generality of this concept by studying the characteristics of oligonucleotides modified with four different C5-functionalized LNA cytidine and C8-functionalized LNA adenosine monomers. The results strongly suggest that C5-functionalization of LNA pyrimidines is indeed a viable approach for improving the binding affinity, target specificity, and/or enzymatic stability of LNA-modified ONs, whereas C8-functionalization of LNA adenosines is detrimental to binding affinity and specificity. These insights will impact the future design of conformationally restricted nucleotides for nucleic acid targeting applications.

  6. Synthesis and characterization of a material derived from 4-mercaptobenzoic acid: A novel platform for oligonucleotide immobilization.

    PubMed

    Alves, Rafael da Fonseca; da Silva, Amanda Gonçalves; Ferreira, Lucas Franco; Franco, Diego Leoni

    2017-04-01

    This paper reports the electrochemical modification of pencil carbon graphite electrodes with a polymeric material derived from 4-mercaptobenzoic acid. Acidic solutions (pH 0 and 5.02) yielded an insulating polymeric film with anionic permselective properties. Scanning Electron Microscopy (SEM) analysis showed a complete coverage of the carbon graphite electrodes with a laminar-like polymeric structure. Different characterization studies indicate that the carboxyl group remained unchanged since the absorbance peak and oxidation potential did not change with the increase in pH at the pKa accounting for the carboxyl/carboxylate redox transition. The functionalized matrix was activated using carbodiimide, succinimide and an amine-modified oligonucleotide. The immobilization and hybridization processes were successfully verified using the redox electroactive indicator methylene blue, where better electrochemical signals were obtained when compared with the traditional self-assembled monolayer system. The selectivity of the system was verified using a noncomplementary target where no significant difference in electric current was observed when compared to the system containing only the probe. The method showed a good linear correlation coefficient (r(2)=0.9915), low limit of detection (1.17nmolL(-1)), and an acceptable precision (RSD=2.75%). The proposed method is suitable for further studies using different sequences of oligonucleotides. Copyright © 2016 Elsevier B.V. All rights reserved.

  7. Identification of candidate downstream genes for the homeodomain transcription factor Labial in Drosophila through oligonucleotide-array transcript imaging

    PubMed Central

    Leemans, Ronny; Loop, Thomas; Egger, Boris; He, Haiqiong; Kammermeier, Lars; Hartmann, Beate; Certa, Ullrich; Reichert, Heinrich; Hirth, Frank

    2001-01-01

    Background: Homeotic genes are key developmental regulators that are highly conserved throughout evolution. Their encoded homeoproteins function as transcription factors to control a wide range of developmental processes. Although much is known about homeodomain-DNA interactions, only a small number of genes acting downstream of homeoproteins have been identified. Here we use a functional genomic approach to identify candidate target genes of the Drosophila homeodomain transcription factor Labial. Results: High-density oligonucleotide arrays with probe sets representing 1,513 identified and sequenced genes were used to analyze differential gene expression following labial overexpression in Drosophila embryos. We find significant expression level changes for 96 genes belonging to all functional classes represented on the array. In accordance with our experimental procedure, we expect that these genes are either direct or indirect targets of labial gene action. Among these genes, 48 were upregulated and 48 were downregulated following labial overexpression. This corresponds to 6.3% of the genes represented on the array. For a selection of these genes, we show that the data obtained with the oligonucleotide arrays are consistent with data obtained using quantitative RT-PCR. Conclusions: Our results identify a number of novel candidate downstream target genes for Labial, suggesting that this homeoprotein differentially regulates a limited and distinct set of embryonically expressed Drosophila genes. PMID:11387036

  8. Fluorescent cyanine probe for DNA detection and cellular imaging

    NASA Astrophysics Data System (ADS)

    Zheng, Yong-Chao; Zheng, Mei-Ling; Zhao, Zhen-Sheng; Duan, Xuan-Ming

    2014-03-01

    In our study, two carbazole-based cyanines, 3,6-bis[2-(1-methylpyridinium)vinyl]-9-methyl carbazole diiodide (A) and 6,6'-bis[2-(1-methylpyridinium)vinyl]-bis(9-methyl-carbazol-3yl)methane diiodide (B) were synthesized and employed as light-up probes for DNA and cell imaging. Both of the cyanine probes possess a symmetric structure and bis-cationic center. The obvious induced circular dichroism signals in circular dichroism spectra reveal that the molecules can specifically interact with DNA. Strong fluorescence enhancement is observed when these two cyanines are bound to DNA. These cyanine probes show high binding affinity to oligonucleotides but different binding preferences to various secondary structures. Confocal microscopy images of fixed cell stained by the probes exhibit strong brightness and high contrast in nucleus with a very low cytoplasmic background.

  9. Identification of sequence motifs in oligonucleotides whose presence is correlated with antisense activity.

    PubMed

    Matveeva, O V; Tsodikov, A D; Giddings, M; Freier, S M; Wyatt, J R; Spiridonov, A N; Shabalina, S A; Gesteland, R F; Atkins, J F

    2000-08-01

    Design of antisense oligonucleotides targeting any mRNA can be much more efficient when several activity-enhancing motifs are included and activity-decreasing motifs are avoided. This conclusion was made after statistical analysis of data collected from >1000 experiments with phosphorothioate-modified oligonucleotides. Highly significant positive correlation between the presence of motifs CCAC, TCCC, ACTC, GCCA and CTCT in the oligonucleotide and its antisense efficiency was demonstrated. In addition, negative correlation was revealed for the motifs GGGG, ACTG, AAA and TAA. It was found that the likelihood of activity of an oligonucleotide against a desired mRNA target is sequence motif content dependent.

  10. Identification of sequence motifs in oligonucleotides whose presence is correlated with antisense activity

    PubMed Central

    Matveeva, O. V.; Tsodikov, A. D.; Giddings, M.; Freier, S. M.; Wyatt, J. R.; Spiridonov, A. N.; Shabalina, S. A.; Gesteland, R. F.; Atkins, J. F.

    2000-01-01

    Design of antisense oligonucleotides targeting any mRNA can be much more efficient when several activity-enhancing motifs are included and activity-decreasing motifs are avoided. This conclusion was made after statistical analysis of data collected from >1000 experiments with phosphorothioate-modified oligonucleotides. Highly significant positive correlation between the presence of motifs CCAC, TCCC, ACTC, GCCA and CTCT in the oligonucleotide and its antisense efficiency was demonstrated. In addition, negative correlation was revealed for the motifs GGGG, ACTG, AAA and TAA. It was found that the likelihood of activity of an oligonucleotide against a desired mRNA target is sequence motif content dependent. PMID:10908347

  11. New strategies for cyclization and bicyclization of oligonucleotides by click chemistry assisted by microwaves.

    PubMed

    Lietard, Jory; Meyer, Albert; Vasseur, Jean-Jacques; Morvan, François

    2008-01-04

    The synthesis of cyclic, branched, and bicyclic oligonucleotides was performed by copper-catalyzed azide-alkyne cycloaddition assisted by microwaves in solution and on solid support. For that purpose, new phosphoramidite building blocks and new solid supports were designed to introduce alkyne and bromo functions into the same oligonucleotide by solid-phase synthesis on a DNA synthesizer. The bromine atom was then substituted by sodium azide to yield azide oligonucleotides. Cyclizations were found to be more efficient in solution than on solid support. This method allowed the efficient preparation of cyclic (6- to 20-mers), branched (with one or two dangling sequences), and bicyclic (2 x 10-mers) oligonucleotides.

  12. Use of inherently conducting polymers and pulsed amperometry in flow injection analysis to detect oligonucleotides.

    PubMed

    Wu, Yanzhe; Moulton, Simon E; Too, Chee O; Wallace, Gordon G; Zhou, Dezhi

    2004-07-01

    Pulsed amperometry has been used to detect and discriminate between different base-pair oligonucleotides using flow injection analysis with polypyrrole modified electrodes. Oligonucleotides with 20-repeat units were immobilized as the sole counter anion during polymerisation of pyrrole onto a glassy carbon (GC) electrode. The modified electrode was then used in a flow injection analysis (FIA) set-up to selectively detect complementary oligonucleotides. Results showed that complementary oligo(dG)(20) and non-complementary oligonucleotides can be distinguished by using the oligo(dC)(20) modified electrode.

  13. Inhibition of DNA Binding Proteins by Oligonucleotide-Directed Triple Helix Formation

    NASA Astrophysics Data System (ADS)

    Maher, Louis J.; Wold, Barbara; Dervan, Peter B.

    1989-08-01

    Oligonucleotides that bind to duplex DNA in a sequence-specific manner by triple helix formation offer an approach to the experimental manipulation of sequence-specific protein binding. Micromolar concentrations of pyrimidine oligodeoxyribonucleotides are shown to block recognition of double helical DNA by prokaryotic modifying enzymes and a eukaryotic transcription factor at a homopurine target site. Inhibition is sequence-specific. Oligonucleotides containing 5-methylcytosine provide substantially more efficient inhibition than oligonucleotides containing cytosine. The results have implications for gene-specific repression by oligonucleotides or their analogs.

  14. Diels-Alder cycloadditions in water for the straightforward preparation of peptide–oligonucleotide conjugates

    PubMed Central

    Marchán, Vicente; Ortega, Samuel; Pulido, Daniel; Pedroso, Enrique; Grandas, Anna

    2006-01-01

    The Diels-Alder reaction between diene-modified oligonucleotides and maleimide-derivatized peptides afforded peptide–oligonucleotide conjugates with high purity and yield. Synthesis of the reagents was easily accomplished by on-column derivatization of the corresponding peptides and oligonucleotides. The cycloaddition reaction was carried out in mild conditions, in aqueous solution at 37°C. The speed of the reaction was found to vary depending on the size of the reagents, but it can be completed in 8–10 h by reacting the diene-oligonucleotide with a small excess of maleimide-peptide. PMID:16478710

  15. Developing mixed films of immobilized oligonucleotides and quantum dots for the multiplexed detection of nucleic acid hybridization using a combination of fluorescence resonance energy transfer and direct excitation of fluorescence.

    PubMed

    Algar, W Russ; Krull, Ulrich J

    2010-04-20

    Methods have been developed for the simultaneous and selective detection of three target nucleic acid sequences based on mixed films of immobilized quantum dots (QDs) and oligonucleotide probes. CdSe/ZnS QDs were immobilized on optical fibers and conjugated with mixtures of different probe oligonucleotides. Hybridization events were detected using a combination of fluorescence from direct excitation and fluorescence sensitized by resonance energy transfer (FRET). A sandwich assay format was used to associate dye labeled reporter oligonucleotides with probe-target hybrids formed at the surface of the optical fiber. One detection channel utilized direct excitation of Pacific Blue and the two other detection channels were based on FRET. In one strategy, green emitting QDs were used as donors with Cy3 and Rhodamine Red-X acceptors. In a second strategy, green and red emitting QDs were coimmobilized and used as donors with Cy3 and Alexa Fluor 647 acceptors, respectively. Selective three-plex detection was demonstrated with both strategies. Several key design criteria that were explored to optimize the relative signal magnitude between channels included: the ratio of probe associated with direct excitation versus probes associated with FRET; the relative amounts of each FRET probe and corresponding spectral overlap; and the photoluminescence ratio between immobilized green and red emitting QDs (where applicable). Careful selection of probe sequences and lengths were important for the discrimination of single nucleotide polymorphisms in one channel without suppressing binding of target in the other two channels. This work provides a basis for the development of multiplexed biosensors that are ensemble compatible and do not require discrete sensor elements, spatial registration, sorting technology, or single molecule spectroscopy.

  16. Toward an on-chip multiplexed nucleic acid hybridization assay using immobilized quantum dot-oligonucleotide conjugates and fluorescence resonance energy transfer

    NASA Astrophysics Data System (ADS)

    Tavares, Anthony J.; Noor, M. Omair; Algar, W. Russ; Vannoy, Charles H.; Chen, Lu; Krull, Ulrich J.

    2011-03-01

    Semiconductor quantum dots (QD) are a class of NP with photophysical properties that are ideally suited for optical multiplexing and use as donors in fluorescence resonance energy transfer (FRET). A new strategy is presented for the development of multiplexed DNA hybridization assays using immobilized QDs in a microfluidic system. Green- or red-emitting QDs were immobilized via self-assembly with a multidentate-thiol-derivatized glass slide, and subsequently conjugated with amine-terminated probe oligonucleotides using carbodiimide activation. Immobilized QD-probe conjugates were then passivated with adsorbed non-complementary oligonucleotides to achieve selectivity in microfluidic assays. Target nucleic acid sequences hybridized with QD-probe conjugates and were labeled with Cy3 or Alexa Fluor 647 as acceptor dyes for the QD donors, where FRET-sensitized dye emission provided a signal for the detection of picomolar quantities of target. The simultaneous immobilization of green- and red-emitting QDs at different ratios within a microfluidic channel was demonstrated as a step toward multiplexed assays.

  17. An oligonucleotide prokaryotic acidophile microarray: its validation and its use to monitor seasonal variations in extreme acidic environments with total environmental RNA.

    PubMed

    Garrido, Patricia; González-Toril, Elena; García-Moyano, Antonio; Moreno-Paz, Mercedes; Amils, Ricardo; Parro, Víctor

    2008-04-01

    An oligonucleotide microarray that monitors prokaryotic diversity in extremely acidic environments has been developed. The oligonucleotide probes target most known acidophilic microorganisms, including members of the Nitrospira phylum, Acidithiobacillus genus, acidobacteria, sulfur reducing bacteria, Actinobacteria and Archaea of the Ferroplasma and Thermoplasma genera. The probes were tested for their specificity against the corresponding type strain by microarray hybridization using PCR-amplified fluorescent DNA of the 16S rRNA genes. The microarray was tested and validated against well-established molecular ecology techniques such as molecular cloning and sequencing and FISH by using samples obtained from a natural extremely acidic environment, the Río Tinto (SW Spain). Also, fluorescent labelled total environmental RNA from Río Tinto samples were used as targets for microarray hybridizations. This approach allowed the detection of the most metabolically active prokaryotes of the ecosystem by simultaneously checking probes against 16S and 23S rRNAs as well as other functional genes. Seasonal and spatial variations in the relative expression of specific rRNA genes have been detected between two sampling sites that differ in several physicochemical parameters, mainly iron and sulfur content.

  18. New oligonucleotide microarray for rapid diagnosis of avian viral diseases.

    PubMed

    Sultankulova, Kulyaisan T; Kozhabergenov, Nurlan S; Strochkov, Vitaliy M; Burashev, Yerbol D; Shorayeva, Kamshat A; Chervyakova, Olga V; Rametov, Nurkuisa M; Sandybayev, Nurlan T; Sansyzbay, Abylay R; Orynbayev, Mukhit B

    2017-04-05

    We developed a new oligonucleotide microarray comprising 16 identical subarrays for simultaneous rapid detection of avian viruses: avian influenza virus (AIV), Newcastle disease virus (NDV), infection bronchitis virus (IBV), and infectious bursal disease virus (IBDV) in single- and mixed-virus infections. The objective of the study was to develop an oligonucleotide microarray for rapid diagnosis of avian diseases that would be used in the course of mass analysis for routine epidemiological surveillance owing to its ability to test one specimen for several infections. The paper describes the technique for rapid and simultaneous diagnosis of avian diseases such as avian influenza, Newcastle disease, infectious bronchitis and infectious bursal disease with use of oligonucleotide microarray, conditions for hybridization of fluorescent-labelled viral cDNA on the microarray and its specificity tested with use of AIV, NDV, IBV, IBDV strains as well as biomaterials from poultry. Sensitivity and specificity of the developed microarray was evaluated with use of 122 specimens of biological material: 44 cloacal swabs from sick birds and 78 tissue specimens from dead wild and domestic birds, as well as with use of 15 AIV, NDV, IBV and IBDV strains, different in their origin, epidemiological and biological characteristics (RIBSP Microbial Collection). This microarray demonstrates high diagnostic sensitivity (99.16% within 95% CI limits 97.36-100%) and specificity (100%). Specificity of the developed technique was confirmed by direct sequencing of NP and M (AIV), VP2 (IBDV), S1 (IBV), NP (NDV) gene fragments. Diagnostic effectiveness of the developed DNA microarray is 99.18% and therefore it can be used in mass survey for specific detection of AIV, NDV, IBV and IBDV circulating in the region in the course of epidemiological surveillance. Rather simple method for rapid diagnosis of avian viral diseases that several times shortens duration of assay versus classical diagnostic

  19. In vivo site-directed mutagenesis using oligonucleotides.

    PubMed

    Storici, F; Lewis, L K; Resnick, M A

    2001-08-01

    Functional characterization of the genes of higher eukaryotes has been aided by their expression in model organisms and by analyzing site-specific changes in homologous genes in model systems such as the yeast Saccharomyces cerevisiae. Modifying sequences in yeast or other organisms such that no heterologous material is retained requires in vitro mutagenesis together with subcloning. PCR-based procedures that do not involve cloning are inefficient or require multistep reactions that increase the risk of additional mutations. An alternative approach, demonstrated in yeast, relies on transformation with an oligonucleotide, but the method is restricted to the generation of mutants with a selectable phenotype. Oligonucleotides, when combined with gap repair, have also been used to modify plasmids in yeast; however, this approach is limited by restriction-site availability. We have developed a mutagenesis approach in yeast based on transformation by unpurified oligonucleotides that allows the rapid creation of site-specific DNA mutations in vivo. A two-step, cloning-free process, referred to as delitto perfetto, generates products having only the desired mutation, such as a single or multiple base change, an insertion, a small or a large deletion, or even random mutations. The system provides for multiple rounds of mutation in a window up to 200 base pairs. The process is RAD52 dependent, is not constrained by the distribution of naturally occurring restriction sites, and requires minimal DNA sequencing. Because yeast is commonly used for random and selective cloning of genomic DNA from higher eukaryotes such as yeast artificial chromosomes, the delitto perfetto strategy also provides an efficient way to create precise changes in mammalian or other DNA sequences.

  20. Mediator probe PCR: detection of real-time PCR by label-free probes and a universal fluorogenic reporter.

    PubMed

    Wadle, Simon; Rubenwolf, Stefanie; Lehnert, Michael; Faltin, Bernd; Weidmann, Manfred; Hufert, Frank; Zengerle, Roland; von Stetten, Felix

    2014-01-01

    Mediator probe PCR (MP PCR) is a novel detection format for real-time nucleic acid analysis. Label-free mediator probes (MP) and fluorogenic universal reporter (UR) oligonucleotides are combined to accomplish signal generation. Compared to conventional hydrolysis probe PCRs costs can thus be saved by using the same fluorogenic UR for signal generation in different assays. This tutorial provides a practical guideline to MP and UR design. MP design rules are very similar to those of hydrolysis probes. The major difference is in the replacement of the fluorophore and quencher by one UR-specific sequence tag, the mediator. Further protocols for the setup of reactions, to detect either DNA or RNA targets with clinical diagnostic target detection as models, are explained. Ready to use designs for URs are suggested and guidelines for their de novo design are provided as well, including a protocol for UR signal generation characterization.

  1. Oligonucleotide primers for PCR amplification of coelomate introns.

    PubMed

    Jarman, Simon N; Ward, Robert D; Elliott, Nicholas G

    2002-09-01

    Abstract Seven novel oligonucleotide primer pairs for polymerase chain reaction amplification of introns from nuclear genes in coelomates were designed and tested. Each pair bound to adjacent exons that are separated by a single intron in most coelomate species. The primer sets amplified introns in species as widely separated by the course of evolution as oysters (Mollusca: Protostoma) and salmon (Chordata: Deuterostoma). Each primer set was tested on a further 6 coelomate species and found to amplify introns in most cases. These primer sets may therefore be useful tools for developing nuclear DNA markers in diverse coelomate species for studies of population genetics, phylogenetics, or genome mapping.

  2. Cardiovascular and Metabolic Effects of ANGPTL3 Antisense Oligonucleotides.

    PubMed

    Graham, Mark J; Lee, Richard G; Brandt, Teresa A; Tai, Li-Jung; Fu, Wuxia; Peralta, Raechel; Yu, Rosie; Hurh, Eunju; Paz, Erika; McEvoy, Bradley W; Baker, Brenda F; Pham, Nguyen C; Digenio, Andres; Hughes, Steven G; Geary, Richard S; Witztum, Joseph L; Crooke, Rosanne M; Tsimikas, Sotirios

    2017-07-20

    Epidemiologic and genomewide association studies have linked loss-of-function variants in ANGPTL3, encoding angiopoietin-like 3, with low levels of plasma lipoproteins. We evaluated antisense oligonucleotides (ASOs) targeting Angptl3 messenger RNA (mRNA) for effects on plasma lipid levels, triglyceride clearance, liver triglyceride content, insulin sensitivity, and atherosclerosis in mice. Subsequently, 44 human participants (with triglyceride levels of either 90 to 150 mg per deciliter [1.0 to 1.7 mmol per liter] or >150 mg per deciliter, depending on the dose group) were randomly assigned to receive subcutaneous injections of placebo or an antisense oligonucleotide targeting ANGPTL3 mRNA in a single dose (20, 40, or 80 mg) or multiple doses (10, 20, 40, or 60 mg per week for 6 weeks). The main end points were safety, side-effect profile, pharmacokinetic and pharmacodynamic measures, and changes in levels of lipids and lipoproteins. The treated mice had dose-dependent reductions in levels of hepatic Angptl3 mRNA, Angptl3 protein, triglycerides, and low-density lipoprotein (LDL) cholesterol, as well as reductions in liver triglyceride content and atherosclerosis progression and increases in insulin sensitivity. After 6 weeks of treatment, persons in the multiple-dose groups had reductions in levels of ANGPTL3 protein (reductions of 46.6 to 84.5% from baseline, P<0.01 for all doses vs. placebo) and in levels of triglycerides (reductions of 33.2 to 63.1%), LDL cholesterol (1.3 to 32.9%), very-low-density lipoprotein cholesterol (27.9 to 60.0%), non-high-density lipoprotein cholesterol (10.0 to 36.6%), apolipoprotein B (3.4 to 25.7%), and apolipoprotein C-III (18.9 to 58.8%). Three participants who received the antisense oligonucleotide and three who received placebo reported dizziness or headache. There were no serious adverse events. Oligonucleotides targeting mouse Angptl3 retarded the progression of atherosclerosis and reduced levels of atherogenic lipoproteins in

  3. Oligonucleotide microarray for subtyping of influenza A viruses

    NASA Astrophysics Data System (ADS)

    Klotchenko, S. A.; Vasin, A. V.; Sandybaev, N. T.; Plotnikova, M. A.; Chervyakova, O. V.; Smirnova, E. A.; Kushnareva, E. V.; Strochkov, V. M.; Taylakova, E. T.; Egorov, V. V.; Koshemetov, J. K.; Kiselev, O. I.; Sansyzbay, A. R.

    2012-02-01

    Influenza is one of the most widespread respiratory viral diseases, infecting humans, horses, pigs, poultry and some other animal populations. Influenza A viruses (IAV) are classified into subtypes on the basis of the surface hemagglutinin (H1 to H16) and neuraminidase (N1 to N9) glycoproteins. The correct determination of IAV subtype is necessary for clinical and epidemiological studies. In this article we propose an oligonucleotide microarray for subtyping of IAV using universal one-step multisegment RT-PCR fluorescent labeling of viral gene segments. It showed to be an advanced approach for fast detection and identification of IAV.

  4. PCR amplification on microarrays of gel immobilized oligonucleotides

    DOEpatents

    Strizhkov, Boris; Tillib, Sergei; Mikhailovich, Vladimir; Mirzabekov, Andrei

    2003-11-04

    The invention relates two general methods for performing PCR amplification, combined with the detection and analysis of the PCR products on a microchip. In the first method, the amplification occurs both outside and within a plurality of gel pads on a microchip, with at least one oligonucleotide primer immobilized in a gel pad. In the second method, PCR amplification also takes place within gel pads on a microchip, but the pads are surrounded by a hydrophobic liquid such as that which separates the individual gel pads into environments which resemble micro-miniaturized test tubes.

  5. Inhibition Of Molecular And Biological Processes Using Modified Oligonucleotides

    DOEpatents

    Kozyavkin, Sergei A.; Malykh, Andrei G.; Polouchine, Nikolai N.; Slesarev, Alexei I.

    2003-04-15

    A method of inhibiting at least one molecular process in a sample, comprising administering to the sample an oligonucleotide or polynucleotide containing at least one monomeric unit having formula (I): wherein A is an organic moiety, n is at least 1, and each X is independently selected from the group consisting of --NRCOCONu, --NHCOCR.sub.2 CR.sub.2 CONu, --NHCOCR.dbd.CRCONu, and --NHCOSSCONu, wherein each R independently represents H or a substituted or unsubstituted alkyl group, and Nu represents a nucleophile, or a salt of the compound.

  6. Aptamer oligonucleotides as potential therapeutics in hematologic diseases.

    PubMed

    Li, Weibin; Zhao, Meng; Wang, Kaiyu; Yan, Huihui; Lan, XIaopeng

    2017-10-02

    Aptamers are single-stranded DNA or RNA oligonucleotides generated by a novel in vitro selection technique termed Systematic Evolution of Ligands by Exponential Enrichment (SELEX). During the past two decades, various aptamer drugs have been developed and many of them have entered into clinical trials. In the present review, we focus on aptamers as potential therapeutics for hematological diseases, including anemia of chronic inflammation (ACI) and anemia of chronic disease (ACD), hemophilia, thrombotic thrombocytopenic purpura (TTP) or VWD type-2B, and sickle cell disease (SCD), in particular, those that have entered into clinical trials. Copyright© Bentham Science Publishers; For any queries, please email at epub@benthamscience.org.

  7. Chemically programmed cell adhesion with membrane-anchored oligonucleotides

    PubMed Central

    Selden, Nicholas S.; Todhunter, Michael E.; Jee, Noel Y.; Liu, Jennifer S.; Broaders, Kyle E.; Gartner, Zev J.

    2012-01-01

    Cell adhesion organizes the structures of tissues and mediates their mechanical, chemical, and electrical integration with their surroundings. Here, we describe a strategy for chemically controlling cell adhesion using membrane anchored single-stranded DNA oligonucleotides. The reagents are pure chemical species prepared from phosphoramidites synthesized in a single chemical step from commercially available starting materials. The approach enables rapid, efficient, tunable cell adhesion, independent of proteins or glycans, by facilitating interactions with complementary labeled surfaces or other cells. We demonstrate the utility of this approach by imaging drug-induced changes in the membrane dynamics of non-adherent human cells while chemically immobilized on a passivated glass surface. PMID:22176556

  8. On the rapid deprotection of synthetic oligonucleotides and analogs.

    PubMed Central

    Polushin, N N; Morocho, A M; Chen, B C; Cohen, J S

    1994-01-01

    The efficiency of oligodeoxynucleotide deprotection is greatly enhanced using a combination of: (a) ethanolamine, and especially a mixture of hydrazine, ethanolamine and methanol, in place of the usual aqueous ammonia; (b) tert-butylphenoxyacetyl amino protecting groups, and (c) oxalyl link between the first nucleotide and the polymeric support. The extent of base modification, particularly of C, is shown to be extremely low, and the quality of deprotected oligonucleotides is as high as in the case of ammonia deprotection. This method is also shown to be applicable to the preparation of phosphorothioate and methylphosphonate oligodeoxynucleotides an