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Статті в журналах з теми "Direct nucleic acid detection"
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Zezza, Paola, María Isabel Lucío, Estrella Fernández, Ángel Maquieira, and María-José Bañuls. "Surface Micro-Patterned Biofunctionalized Hydrogel for Direct Nucleic Acid Hybridization Detection." Biosensors 13, no. 3 (February 23, 2023): 312. http://dx.doi.org/10.3390/bios13030312.
Повний текст джерелаАнотація:
The present research is focused on the development of a biofunctionalized hydrogel with a surface diffractive micropattern as a label-free biosensing platform. The biosensors described in this paper were fabricated with a holographic recording of polyethylene terephthalate (PET) surface micro-structures, which were then transferred into a hydrogel material. Acrylamide-based hydrogels were obtained with free radical polymerization, and propargyl acrylate was added as a comonomer, which allowed for covalent immobilization of thiolated oligonucleotide probes into the hydrogel network, via thiol-yne photoclick chemistry. The comonomer was shown to significantly contribute to the immobilization of the probes based on fluorescence imaging. Two different immobilization approaches were demonstrated: during or after hydrogel synthesis. The second approach showed better loading capacity of the bioreceptor groups. Diffraction efficiency measurements of hydrogel gratings at 532 nm showed a selective response reaching a limit of detection in the complementary DNA strand of 2.47 µM. The label-free biosensor as designed could significantly contribute to direct and accurate analysis in medical diagnosis as it is cheap, easy to fabricate, and works without the need for further reagents.
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Ouyang, Wei, and Jongyoon Han. "Universal amplification-free molecular diagnostics by billion-fold hierarchical nanofluidic concentration." Proceedings of the National Academy of Sciences 116, no. 33 (July 29, 2019): 16240–49. http://dx.doi.org/10.1073/pnas.1904513116.
Повний текст джерелаАнотація:
Rapid and reliable detection of ultralow-abundance nucleic acids and proteins in complex biological media may greatly advance clinical diagnostics and biotechnology development. Currently, nucleic acid tests rely on enzymatic processes for target amplification (e.g., PCR), which have many inherent issues restricting their implementation in diagnostics. On the other hand, there exist no protein amplification techniques, greatly limiting the development of protein-based diagnosis. We report a universal biomolecule enrichment technique termed hierarchical nanofluidic molecular enrichment system (HOLMES) for amplification-free molecular diagnostics using massively paralleled and hierarchically cascaded nanofluidic concentrators. HOLMES achieves billion-fold enrichment of both nucleic acids and proteins within 30 min, which not only overcomes many inherent issues of nucleic acid amplification but also provides unprecedented enrichment performance for protein analysis. HOLMES features the ability to selectively enrich target biomolecules and simultaneously deplete nontargets directly in complex crude samples, thereby enormously enhancing the signal-to-noise ratio of detection. We demonstrate the direct detection of attomolar nucleic acids in urine and serum within 35 min and HIV p24 protein in serum within 60 min. The performance of HOLMES is comparable to that of nucleic acid amplification tests and near million-fold improvement over standard enzyme-linked immunosorbent assay (ELISA) for protein detection, being much simpler and faster in both applications. We additionally measured human cardiac troponin I protein in 9 human plasma samples, and showed excellent agreement with ELISA and detection below the limit of ELISA. HOLMES is in an unparalleled position to unleash the potential of protein-based diagnosis.
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Iwanaga, Masanobu. "High-Sensitivity High-Throughput Detection of Nucleic Acid Targets on Metasurface Fluorescence Biosensors." Biosensors 11, no. 2 (January 27, 2021): 33. http://dx.doi.org/10.3390/bios11020033.
Повний текст джерелаАнотація:
Worldwide infection disease due to SARS-CoV-2 is tremendously affecting our daily lives. High-throughput detection methods for nucleic acids are emergently desired. Here, we show high-sensitivity and high-throughput metasurface fluorescence biosensors that are applicable for nucleic acid targets. The all-dielectric metasurface biosensors comprise silicon-on-insulator nanorod array and have prominent electromagnetic resonances enhancing fluorescence emission. For proof-of-concept experiment on the metasurface biosensors, we have conducted fluorescence detection of single-strand oligoDNAs, which model the partial sequences of SARS-CoV-2 RNA indicated by national infection institutes, and succeeded in the high-throughput detection at low concentrations on the order of 100 amol/mL without any amplification technique. As a direct detection method, the metasurface fluorescence biosensors exhibit high performance.
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Knight, Ivor T., Jocelyne DiRuggiero, and Rita R. Colwell. "Direct Detection of Enteropathogenic Bacteria in Estuarine Water Using Nucleic Acid Probes." Water Science and Technology 24, no. 2 (July 1, 1991): 261–66. http://dx.doi.org/10.2166/wst.1991.0070.
Повний текст джерелаАнотація:
Direct detection and enumeration of pathogenic bacteria, rather than indicator organisms, in aquatic environments is desirable but hindered by the difficulties of culturing and identifying specific pathogens from these environments. We have developed a method for concentrating bacteria from water samples and extracting their DNA and RNA for use as targets for pathogen-specific gene probes. The method has been used to detect and enumerate Salmonella spp. in estuarine water samples. The probe binds Salmonella DNA quantitatively, making it possible to estimate relative amounts of target in each sample. Salmonella spp. were detected in samples which yielded no Salmonella spp. using culturing. Since the probe method does not require culturing the target organism, both culturable and non-culturable forms are detected. We have also used polymerase chain reaction to amplify a region of the enterotoxin gene in enterotoxigenic Escherichiacoli and Vibriocholerae (ltx and ctx, respectively). The amplified products are then identified with ctx and ltx probes, making specific, highly sensitive detection possible.
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Kricka, Larry J., and Paolo Fortina. "Analytical Ancestry: “Firsts” in Fluorescent Labeling of Nucleosides, Nucleotides, and Nucleic Acids." Clinical Chemistry 55, no. 4 (April 1, 2009): 670–83. http://dx.doi.org/10.1373/clinchem.2008.116152.
Повний текст джерелаАнотація:
Abstract Background: The inherent fluorescent properties of nucleosides, nucleotides, and nucleic acids are limited, and thus the need has arisen for fluorescent labeling of these molecules for a variety of analytical applications. Content: This review traces the analytical ancestry of fluorescent labeling of nucleosides, nucleotides, and nucleic acids, with an emphasis on the first to publish or patent. The scope of labeling includes (a) direct labeling by covalent labeling of nucleic acids with a fluorescent label or noncovalent binding or intercalation of a fluorescent dye to nucleic acids and (b) indirect labeling via covalent attachment of a secondary label to a nucleic acid, and then binding this to a fluorescently labeled ligand binder. An alternative indirect strategy involves binding of a nucleic acid to a nucleic acid binder molecule (e.g., antibody, antibiotic, histone, antibody, nuclease) that is labeled with a fluorophore. Fluorescent labels for nucleic acids include organic fluorescent dyes, metal chelates, carbon nanotubes, quantum dots, gold particles, and fluorescent minerals. Summary: Fluorescently labeled nucleosides, nucleotides, and nucleic acids are important types of reagents for biological assay methods and underpin current methods of chromosome analysis, gel staining, DNA sequencing and quantitative PCR. Although these methods use predominantly organic fluorophores, new types of particulate fluorophores in the form of nanoparticles, nanorods, and nanotubes may provide the basis of a new generation of fluorescent labels and nucleic acid detection methods.
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Uno, Takeshi, Toshihito Ohtake, Hitoshi Tabata, and Tomoji Kawai. "Direct Deoxyribonucleic Acid Detection Using Ion-Sensitive Field-Effect Transistors Based on Peptide Nucleic Acid." Japanese Journal of Applied Physics 43, No. 12B (November 19, 2004): L1584—L1587. http://dx.doi.org/10.1143/jjap.43.l1584.
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Faron, Matthew L., Nathan A. Ledeboer, Jessica Connolly, Paul A. Granato, Brenda R. Alkins, Jennifer Dien Bard, Judy A. Daly, Stephen Young, and Blake W. Buchan. "Clinical Evaluation and Cost Analysis of Great Basin Shiga Toxin Direct Molecular Assay for Detection of Shiga Toxin-Producing Escherichia coli in Diarrheal Stool Specimens." Journal of Clinical Microbiology 55, no. 2 (December 7, 2016): 519–25. http://dx.doi.org/10.1128/jcm.01939-16.
Повний текст джерелаАнотація:
ABSTRACTThe Shiga Toxin Direct molecular assay (ST Direct) relies on nucleic acid amplification and solid array-based amplicon detection to identify Shiga toxin-producingEscherichia coli(STEC) in preserved stool specimens. Genes encoding Shiga toxin (stx1andstx2), as well as theE. coliserotype O:157-specific markerrfbE, are simultaneously detected within 2 h. ST Direct was evaluated using 1,084 prospectively collected preserved stool specimens across five clinical centers. An additional 55 retrospectively collected, frozen specimens were included to increase the number of positive specimens evaluated. Results were compared to results from routine culture and an enzyme immunoassay (EIA) specific for the recovery and identification of STEC. ST Direct was found to be 93.2% sensitive and 99.3% specific for detection ofstx1andstx2and 95.7% sensitive and 99.3% specific for detection ofE. coliserotype O:157. All specimens with false-positive results were found to containstx1orstx2or were found to be positive for serotype O:157 when analyzed using alternative molecular methods. All 4 false-negativestx1orstx2results were reported for frozen, retrospectively tested specimens. In all cases, the specimens tested positive forstxby an alternative FDA-cleared nucleic acid amplification test (NAAT) but were negative forstx1andstx2following nucleic acid sequence analysis. Based on these data, culture and EIA-based methods for detection of STEC are only 33% sensitive compared to molecular tests. A retrospective cost analysis demonstrated 59% of the cost of routine stool culture to be attributable to the identification of STEC. Taken together, these data suggest that ST Direct may provide a cost-effective, rapid molecular alternative to routine culture for the identification of STEC in preserved stool specimens.
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Ji, Minghui, Yun Xia, Jacky Fong-Chuen Loo, Lang Li, Ho-Pui Ho, Jianan He, and Dayong Gu. "Automated multiplex nucleic acid tests for rapid detection of SARS-CoV-2, influenza A and B infection with direct reverse-transcription quantitative PCR (dirRT-qPCR) assay in a centrifugal microfluidic platform." RSC Advances 10, no. 56 (2020): 34088–98. http://dx.doi.org/10.1039/d0ra04507a.
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Baron, Ellen Jo, Fred C. Tenover, and Devasena Gnanashanmugam. "Direct Detection of Mycobacterium tuberculosis in Clinical Specimens Using Nucleic Acid Amplification Tests." Clinical Microbiology Newsletter 40, no. 13 (July 2018): 107–12. http://dx.doi.org/10.1016/j.clinmicnews.2018.06.003.
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Zhou, Yunying, Fengyan Pei, Mingyu Ji, Li Wang, Huailong Zhao, Huanjie Li, Weihua Yang, Qingxi Wang, Qianqian Zhao, and Yunshan Wang. "Sensitivity evaluation of 2019 novel coronavirus (SARS-CoV-2) RT-PCR detection kits and strategy to reduce false negative." PLOS ONE 15, no. 11 (November 18, 2020): e0241469. http://dx.doi.org/10.1371/journal.pone.0241469.
Повний текст джерелаАнотація:
The early detection and differential diagnosis of respiratory infections increase the chances for successful control of COVID-19 disease. The nucleic acid RT-PCR test is regarded as the current standard for molecular diagnosis. However, the maximal specificity confirmation target ORF1ab gene is considered to be less sensitive than other targets in clinical application. In addition, recent evidence indicated that the initial missed diagnosis of asymptomatic patients with SARS-CoV-2 and discharged patients with “re-examination positive” might be due to low viral load, and the ability of rapid mutation of SARS-CoV-2 also increases the rate of false-negative results. Moreover, the mixed sample nucleic acid detection is helpful in seeking out the early community transmission of SARS-CoV-2 rapidly, but the detection kit needs ultra-high detection sensitivity. Herein, the lowest detection concentration of different nucleic acid detection kits was evaluated and compared to provide direct evidence for the selection of kits for mixed sample detection or make recommendations for the selection of validation kit, which is of great significance for the prevention and control of the current epidemic and the discharge criteria of low viral load patients.
Дисертації з теми "Direct nucleic acid detection"
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Lores, Lareo Pablo. "Nucleic acids and SNP detection via template-directed native chemical ligation and inductively coupled plasma mass spectrometry." Doctoral thesis, Humboldt-Universität zu Berlin, 2019. http://dx.doi.org/10.18452/20133.
Повний текст джерелаАнотація:
In den letzten Jahren gab es rasche Weiterentwicklungen auf dem Gebiet der Nukleinsäure-Erkennung. Von microRNA-Quantifizierung zur Untersuchung von Zelltods, --Teilung und -Regulation bis zur Bewertung genetischer Variabilität in Hinblick auf Krankheitsentstehung und -Behandlung: Die Analyse von Nukleinsäuren wird in der zukünftigen Medizin eine zentrale Rolle zukommen. Vor allem die Erkennung von SNPs als Hauptquelle der genetischen Vielfalt, aber aus Analysesicht auch eine der herausforderndsten Mutationen, stellt in dieser Hinsicht einen wesentlichen Aspekt dar. Methoden zur SNP-Erkennung müssen nicht nur sensibel, selektiv und stabil, sondern auch vielfältig sein und eine der wachsenden Analyseanzahl gerecht werdende hohe Verarbeitungsmenge bieten.
Im Rahmendieser Arbeit wurde ein chemisches Prüfverfahren zur Erkennung von Nukleinsäuren und Einzelnukleotid-Polymorphismen (SNPs) entwickelt. Das Reaktionssystem zur Nukleinsäuren- Erkennung beruht hierbeiauf der Interaktion zweier modifizierter Peptid-Nukleinsäure (PNS) Oligonukleotiden. Das Erste beinhaltet einen C-terminalen Thioester (Donor-Sonde), die zweite einen N-terminalen Cysteinyl-Rest (Akzeptor-Sonde). Zusätzlich ist die Donor-Sonde durch einenmakrocyclischen Metall Chelatkomplex aus 1,4,7,10-tetraazacyclododecan-1,4,7,10-tetraessigsäure(DOTA) mit einem gebundenen lanthanoid-tag funktionalisiert. In die Akzeptor-Sonde wurde, zurReinigung mit magnetischen Streptavidin Partikeln, Biotin integriert. Der Ziel-DNA-Strang bringt beideSonden in räumliche Nähe zueinander und ermöglicht so eine chemische Reaktion. Das so gewonneneLigationsprodukt beinhaltet den Lanthanoid-Tag und Biotin, über welches das Produkt gereinigt wird,bevor die Detektion mittels ICP-MS erfolgt. Die Lanthanoid Konzentration dient als Indikator desLigationsprodukts welches wiederum den Reporter des Ziel-DNS-Strangs darstellt. Die, mithilfe diesesSystems erreichte, methodische Nachweisgrenze lag bei 29 pM mit einem RSD von 6,8% bei 50 pM(n=5).
Zur Erkennung von SNPs wurde das Experiment mit einer Kombination zweier-Sets PNS Sonden mit unterschiedlichen Lanthanoid Tags durchgeführt. Das erste Set zielte auf die SNP beinhaltende Sequenz (Reportersystem) ab, während das zweite an eine benachbarte Sequenz (Kontrollsystem) binden sollte. Zur Erkennung der SNP wurden die Signale bei der Lanthanoide wurden ins Verhältnis gesetzt. Mithilfe dieses Verfahrens konnte durch Messung von sechs Lanthaniden bei einer Konzentration von 5 nM erfolgreich simultan zwischen den Allelen dreier SNPs unterschieden werden.The field of nucleic acid detection has evolved swiftly in recent years. From quantification of micro RNA for the study of cell death, proliferation, and regulation, to the assessment of the influence of genetic variability towards disease development and treatment, the analysis of nucleic acids will play a central role in future medicine. In that regard, the detection of SNPs, as the primary source of genetic variability and the most challenging mutation from the analytical point of view, will be at the forefront of the discussion. Methods for the detection of SNPs not only require sensitivity, selectivity and robustness, but they should also allow multiplexing and offer high throughput in order to face the growing analysis demand In this work an assay for the detection of nucleic acids and single nucleotide polymorphisms (SNPs) was developed. The reaction system for the detection of nucleic acids is based on the interaction between two modified peptide nucleic acid (PNA) oligonucleotides. The first incorporated a C-terminal thioester (donor probe), and the second one a N-terminal cysteinyl residue (acceptor probe). In addition, the donor probe is functionalized with a metal-tag, which consist of a macrocyclic metal chelate complex of 1,4,7,10-tetraazacyclododecane-1,4,7,10-tetraacetic acid (DOTA) with a chelated lanthanoide. A biotin tag for purification by streptavidin magnetic particles was incorporated in the acceptor probe. The target DNA strand brings together the reporter probes allowing the chemical reaction. The resulting ligation product contains the metal-tag and the biotin, which is used to purify the product before measurement in the ICP-MS system. The lanthanoid concentration is used as an indicator of the ligation product, which at the same time serves as reporter of the target template. The methodological limit of detection achieved with this system was 29 pM with RSD of 6.8% at 50 pM (n=5). Detection of SNPs was performed using a combination of two sets of PNA probes labeled with different lanthanoid metal tags. The first probe set targeted the sequence where the SNP was present (reporter probe system), while the second set of probes was designed to bind to a neighboring sequence (control probe system). The signals of both lanthanides were used to establish a ratio that allowed the detection of the SNP. This assay was successfully used to simultaneously differentiate between alleles of 3 SNPs by measuring six lanthanoids at 5 nM concentration.
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Chatwell, Nicola. "Nucleic acid approaches to toxin detection." Thesis, University of Nottingham, 2013. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.606582.
Повний текст джерелаАнотація:
PCR is commonly used for detecting contamination of foods by toxigenic bacteria. However, it is unknown whether it is suitable for detecting toxins in samples which are unlikely to contain bacterial cells, such as purified biological weapons. Quantitative real-time PCR assays were developed for amplification of the genes encoding Clostridium botulinum neurotoxins A to F, Staphylococcal enteroxin B (SEB), ricin, and C. perfringens alpha toxin. Botulinum neurotoxins, alpha toxin, ricin and V antigen from Yersinia pestis were purified at Dstl using methods including precipitation, ion exchange, FPLC, affinity chromatography and gel filtration. Additionally, toxin samples of unknown purity were purchased from a commercial supplier. Q-PCR analysis showed that DNA was present in crudely prepared toxin samples. However, the majority of purified or commercially produced toxins were not detectable by PCR. Therefore, it is unlikely that PCR will serve as a primary toxin detection method in future. Immuno-PCR was investigated as an alternative, more direct method of toxin detection. Several iterations of the method were investigated, each using a different way of labelling the secondary antibody with DNA. It was discovered that the way in which antibodies are labelled with DNA is crucial to the success of the method, as the DNA concentration must be optimised in order to fully take advantage of signal amplification without causing excessive background noise. In general terms immuno-PCR was demonstrated to offer increased sensitivity over conventional ELISA, once fully optimised, making it particularly useful for biological weapons analysis.
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Behrmann, Ole [Verfasser], and Gerald A. [Akademischer Betreuer] Urban. "Methods for rapid nucleic acid extraction and detection." Freiburg : Universität, 2021. http://d-nb.info/1227187289/34.
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Ferrier, David Christopher. "Nucleic acid detection using oligonucleotide cross-linked polymer composites." Thesis, University of Edinburgh, 2017. http://hdl.handle.net/1842/28944.
Повний текст джерелаАнотація:
There has been much interest in recent years about the potential of microRNA as a new source of biomarkers for the diagnosis of disease. The delivery of new diagnostic tools based on this potential has been limited by shortcomings in current microRNA detection techniques. This thesis explores the development of a new method of microRNA detection through the incorporation of conductive particles into oligonucleotide-functionalised polymers to form oligonucleotide cross-linked polymer composites. Such composites could provide a simple, rapid, and low-cost means of microRNA detection that could be easily multiplexed, providing a valuable tool for point-of-care medical diagnostics. This work presents oligonucleotide-functionalised carbon/polyacrylamide composites which demonstrate a selective swelling response in the presence of analyte oligonucleotide sequences and for which the electrical conductivity decreases with swelling. The composites were synthesised via UV-initiated free-radical polymerisation of carbon/- monomer mixtures upon custom electrode devices, consisting of interdigitated platinum electrodes fabricated upon a silicon substrate. The optimal cross-linker density and carbon loading concentration were determined as well as the best means of dispersing the carbon particles within the polymer. Various types of carbon particles, with differing sizes and aspect ratios, were compared and their performances as conductive additives for polymer swelling transduction evaluated. The swelling behaviour of these composites was evaluated by analysing images of composite microdroplets as they swell. The electrical characteristics of the composites were determined by measuring either the two-terminal resistance or the complex impedance of composite microdroplets on the electrode devices. Alternating and direct current measurement techniques were compared to determine the best approach for the transduction of composite swelling. The volumetric and electrical responses of oligonucleotide-functionalised carbon/polyacrylamide composites were analysed in solutions of analyte oligonucleotide and non-complementary controls. It has been demonstrated that, using carbon nanopowder composites and a direct current two-terminal resistance measurement, it is possible to differentiate between analyte and control solutions to concentrations as low as 10 nM, with single-base precision, in less than three minutes. However, the inability to detect at concentrations below this value, difficulties in differentiating between different analyte concentrations and thermal instability mean that, in their current form, oligonucleotide cross-linked polymer composites are unsuitable for the detection of circulating microRNA at clinically relevant concentrations. Potential avenues of work to address these challenges are discussed. Also presented are collaborative results for oligonucleotide-responsive polymers functionalised with morpholino nucleic acid analogues, in what is believed to be the first example of such a material. These morpholino-functionalised polymers offer significant advantages, in terms of stability and sensitivity, over their nucleic acid equivalents for bio-responsive polymer applications.
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Gorgannezhad, Lena. "Advanced Technologies in Rapid and Multiplex Detection of Nucleic acid." Thesis, Griffith University, 2020. http://hdl.handle.net/10072/397045.
Повний текст джерелаАнотація:
Nucleic acids are key macromolecules of living organisms transferring genetic inheritance from one generation to the next. From how a living individual is created to how it interacts with external factors, all and all, can be found in the nucleic acid sequences inside every single cell of every organism. Therefore, the analysis of nucleic acids sequences is a critical capability for cancer and pathogen diagnoses, genotyping, and disease monitoring. To date, numerous methods have been used to detect both characterised and uncharacterised mutations and sequence variations. However, the detection of low amounts of mutant genes in the presence of high levels of wildtype sequences is still a challenge, and existing technologies has room for improvement. The classical approaches for nucleic acid detection and analyses mainly include DNA sequencing and the polymerase chain reaction (PCR). Although these methods with high analytical performance and reliability have facilitated the interrogation of the nucleic acids, some key obstacles such as the need of labelling, high costs for routine clinical use, slow turnaround time for giving results, the complexity of operation, and the inability to dually detect the genetic mutation in one step have limited their applications. To avoid drawbacks of the traditional approaches, a number of chip-based methods leverage electrochemical readouts or microfluidics to identify nucleic acids. However, there is still an unmet need for a less complex, rapid, low-cost, sensitive and accurate method to enable nucleic acid analysis even in resource-poor settings. The overall objective of this PhD thesis is to develop simple, inexpensive and accurate platforms for nucleic acid evaluation.
To achieve the aforementioned goal, the first attempt was to develop a lab-on-a-chip platform for cancer diagnosis by detection of circulating tumor nucleic acids (ctNAs) in plasma samples of cancer patients. ctNAs are fragmented DNA released from cancerous cells and tumours into the bloodstream of patients with cancer. Tumour-specific (epi-)genetic alterations in ctNAs are assumed to reflect tumour burden and could be of high value for cancer diagnosis, prognosis, and management. In the first part of the thesis, I developed a new electrochemical assay for the detection of FGFR2:FAM76A fusion gene in ctNAs extracted from ovarian cancer patients. The assay was based on the high electrocatalytic activity of a new class of superparamagnetic graphene-loaded iron oxide nanoparticles. Electrochemical detection demonstrated a limit of detection (LOD) as low as 1.0 fM, high specificity and excellent reproducibility.
In the second part of the thesis, I designed and developed a real-time and quantitative PCR system for microbial source tracking (MST) in water samples. MST is a DNA-based technology that enables water-quality managers to identify sources of faecal pollution in environmental waters. Most of the MST methodologies typically require specialized and costly equipment, elaborated and time-consuming operations as well as trained personnel. Here, a simple, low-cost, and sensitive platform was implemented on a microfluidic array chip. The array was successfully used for the real-time PCR-based multiplex detection of three human-associated MST markers (H8, Gen bac III, UidA). The PCR mixture was loaded into an array of channels in a single step utilising capillary filling without the need for liquid handling instruments. The array was then integrated with our custom-made thermal cycling and optical detection system. By employing the fabricated platform, the LOD of 71.8 DNA copies/μL was achieved for Gen bac III sequence. In summary, we introduced a sensitive, simple and economical real-time and quantitative PCR system for MST in water samples.
In a further study, I investigated how a nucleic acid amplification setup can be miniaturised. To reach this goal, I utilised liquid marbles as an ideal biochemical microreactors for targeted amplification of the NAs. Liquid marbles are formed by encapsulating microscale volume of liquid with a thin layer of hydrophobic particles. Miniaturization of the nucleic acids (NAs) amplification process inside a liquid droplet provides several advantages upon routine methods, such as reducing reagents consumption and contamination possibility, easy handling of liquids, eliminating the usage of disposable plastic consumables for carrying out biochemical reactions. However, one of the major concerns in liquid marble applications is the high rate of evaporation through the porous walls during the thermal cycling step. To eliminate the evaporation, I used core-shell beads synthesized from a composite liquid marble as a NAs amplification micro reactor comprising two non-miscible liquid droplets forming a spherical shape and a coating of hydrophobic powder. The shell liquid was then polymerised into a solid after exposure to blue light, converting the liquid marble into a core-shell bead. Fabricated core-shell beads were extended to explore their potential as a versatile bioreactor for phylogrouping of the E.coli strains. In general, this platform provided easy manipulation and storage of sample, elimination of the evaporation, and sample protection from possible external contamination. Moreover, this simple and effective method presented a sensitive and inexpensive way to track NAs.
In conclusion, this research endeavour presents a step forward towards the adaption of the selected group of tools and technologies, for the development of assays that can be applied as powerful alternatives to conventional tools used in molecular diagnostic. These technologies have the potential to revolutionise the NAs-based diagnostic approaches, by providing sensitive, rapid, accurate, and inexpensive platforms for point of care devices and in-field tests.Thesis (PhD Doctorate)
Doctor of Philosophy (PhD)
School of Environment and Sc
Science, Environment, Engineering and Technology
Full Text
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Kershaw, David Michael. "Nanoparticle bound nucleic acid probes for DNA detection and gene inactivation." Thesis, University of Birmingham, 2017. http://etheses.bham.ac.uk//id/eprint/7432/.
Повний текст джерелаАнотація:
In this project, a gold nanoparticle system has been developed that is able to detect SNP variations through a DNA based anthracene probe. A second probe is bound to the gold nanoparticle which allows the fluorescent output of the anthracene probe to be normalized. This allows the detection of SNP variations without the need for an initial reading, opening the possibility for using this system for cellular SNP identification. Through this work a new method for coating gold nanoparticles in oligonucleotides has been developed. In further work, the use of gold nanoparticles to deliver siRNA into cells and induce gene inactivation was investigated. Efforts to improve the knockdown efficiency of these siRNA-gold nanoparticles were made by integrating a second probe onto the nanoparticle surface, non-specific effects were observed upon addition of this second probe.
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Saeed, Ibrahim Q. "Optoelectronically active sensitisers for the selective detection of nucleic acid biomarkers." Thesis, Cardiff University, 2016. http://orca.cf.ac.uk/100885/.
Повний текст джерелаАнотація:
This thesis presents biophysical studies of new optoelecronically active DNA-binders. Chapter one gives a brief overview of the importance of DNA in medicine, of DNA structure and of the mode of interactions of small molecules with double-stranded DNA, including electrostatic, intercalation and groove interactions. Various examples of small-molecule binding to DNA are discussed. Additionally, this chapter briefly describes the biophysical techniques which can be exploited to quantify the interaction between small-molecules and duplex DNA. Chapter two describes the results of studies of the interactions of a group of 1,8-naphthalimide derivatives with double-stranded DNA using a variety of techniques viz. spectroscopy, calorimetry, viscosity and molecular docking studies. Additionally, this chapter also presents sequence selectivity studies of this group of compounds for specific sequences (dAdT)12●(dAdT)12 and (dGdC)12●(dGdC)12 through UV-visible spectroscopy. The 1,8-napthtalimide unit is shown to be a useful element for inducing DNA-binding. Chapter three describes studies of the interactions of a family of dendrimeric compounds with double-stranded DNA, again using spectroscopy, calorimetry, viscosity and molecular docking studies. Furthermore, this chapter includes sequence selectivity studies of this group of compounds for (dAdT)12●(dAdT)12 and (dGdC)12●(dGdC)12 via UV-visible spectroscopy. The charge and the length of the dendritic structures is shown to strongly affect nucleic acid affinities of this series of molecules. Chapter four describes the results of studies of the interactions of miscellaneous compounds with double-stranded DNA using variety of techniques viz. spectroscopy, calorimetry, viscosity and molecular docking studies. In addition, this chapter displays sequence selectivity studies of this group of compounds for specific sequences (dAdT)12●(dAdT)12 and (dGdC)12●(dGdC)12 via UV-visible spectroscopy. Chapter five gives an overview and general conclusions about the DNA binding studies presented in Chapters 2, 3 & 4 and finishes with suggestions for future work.
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O'Meara, Deirdre. "Molecular Tools for Nucleic Acid Analysis." Doctoral thesis, Stockholm : Tekniska högsk, 2001. http://urn.kb.se/resolve?urn=urn:nbn:se:kth:diva-3220.
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Khater, Mohga Wagdy Yehia Mohamed. "Nanoparticle-based sensors for pathogen nucleic acid detection with interest for agriculture." Doctoral thesis, Universitat Autònoma de Barcelona, 2019. http://hdl.handle.net/10803/667373.
Повний текст джерелаАнотація:
La presente tesis describe el desarrollo de sensibles, bajo coste y portátiles métodos de sensado basados en nanomateriales aplicados en la detección de ADN de patógenos relacionados a plantas. El trabajo se presenta avances significativos en el campo de los biosensores para el diagnóstico de las enfermedades en las plantas. En el Capítulo I se da una visión general de las aplicaciones llevabas a cabo y mejoras aportadas por parte del uso de nanomateriales diferentes en el campo de los biosensores, y las nuevas aplicaciones en la detección de enfermedad de las plantas en lo punto de atención. En las secciones siguientes se presentan tres estrategias de sensado para la detección de secuencias de ADN específicas para el virus del cidro (citrus tristeza virus (CTV)), un virus modelo. Los sistemas están basados en las técnicas electroquímicas y ópticas.
El Capítulo III se presenta el uso de electrodos serigrafiados de carbono (SPCEs) como plataforma para hibridación y detección del ADN mediante impedancia. Esta plataforma se adaptó mediante la disposición de nanopartículas de oro (AuNPs), con el fin de obtener una estrategia más simple, sin marcas, menos costosa y más rápida.
Del mismo modo, en el Capítulo IV se centra en el desarrollo de nuevos métodos para la amplificación y la detección de ADN de CTV en SPCE modificados con AuNPs. Este biosensor opera en modo libre de marcas y con límite de detección (LOD) conseguido está en el rango de 1000 fg μL-1.
Finalmente, en el Capítulo V se presenta la tercera plataforma utilizada, que fue basada en papel y tiene el formato de un inmunoensayo de flujo lateral (LFIA, del inglés lateral flow immunoassays). En esta plataforma, las nanopartículas de oro se usan como marcas para obtener señal de color rojo, con el fin de realizar nuevas aplicaciones en diagnóstico de plantas, más rápido y simpleThis thesis aims at developing sensitive, affordable and portable biosensors based on nanomaterials for the determination of nucleic acid related to plant pathogens. The work strives to contribute to the keeping up in the advancements of biosensing systems relevant to plant infection diagnostics which would be an essential solution in the future to the issues of plant disease monitoring and food security. Following Chapter I, state-of-the-art on the latest trends in the development of advantageous biosensors based on both antibody and DNA receptors for early plant disease detection, as well as the use of different nanomaterials such as nanochannels and metallic nanoparticles for the development of innovative and sensitive biosensing systems for the detection of pathogens (i.e. bacteria and viruses) at the point-of-care is given. The next sections of this dissertation will describe three diagnostic biosensing strategies for the detection of citrus tristeza virus (CTV) related nucleic acid using electrical and optical transducing techniques. The electrical sensing of CTV through DNA hybridization based approach and the in situ amplified nucleic acid method will be achieved on carbon sensing substrate modified with gold nanoparticles, while paper-based sensors will be operated in lateral flow format for the gold nanoparticle-based optical detection of CTV. Furthermore, all aspects of the developed biosensing systems, from the bioassay and biosensor design to their development and optimization are presented in which will be organized in the following manner: Chapter III will present highly specific DNA hybridization sensor based on AuNP-modified SPCE employing label-free impedance for the detection of the CTV-related nucleic acid, together with dedicating emphasis to the study of electrodeposition time of AuNPs, whose precise particle size and shape will be required for the enhancement of DNA hybridization rate. A set of voltammetric studies of deposited AuNPs will be discussed. Particular attention will be paid for assembling the thiolated DNA probe as sensing layer for biosensor construction. The main sensor design aspects such as AuNPs size, probe DNA concentration and immobilization time together with DNA hybridization time will be optimized, in order to precisely select the best working conditions for this diagnostic platform. Chapter IV will cover the whole process undertaken for preparation of in situ nucleic acid amplification on gold nanoparticle-modified sensor for sensitive and quantitative detection of CTV. Plant disease (Citrus tristeza virus (CTV)) diagnostics was selected as relevant target for the demonstration of the proof-of-concept. This chapter will include two parts, the first one focuses on the design of RPA amplification assay, primers design, optimization of all essential bioassay aspects such as amplification temperature, volume and screening primers and finally the electrophoresis analysis for RPA products. The second part of this chapter will demonstrate label-free highly integrated in situ RPA amplification/detection approach at room temperature that takes advantage of the high sensitivity offered by gold nanoparticle-modified sensing substrates and electrochemical impedance spectroscopic (EIS) detection. Chapter V focuses on the application of isothermal nucleic acid amplification technology in simple lateral flow platform. The preparation of AuNP-based LFA for the highly sensitive direct detection of RPA amplified nucleic acid, the assembling of lateral flow step, the conjugation of AuNPs to the antibodies used for colorimetric detection, as well as the optimization of all working conditions and finally the analytical performance of the bioassay in LF will be explored. Moreover, aiming at truly achieving the point of care requirements of simple and affordable diagnostic technologies, the work here will present the possibility of amplifying nucleic acid without heat source and visual color detection. This approach would be of great potential as point of care diagnostics.
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Baloda, Meenu. "Lateral Flow Nucleic Acid Biosensor for the Detection of Sexually Transmitted Diseases." Diss., North Dakota State University, 2015. https://hdl.handle.net/10365/27596.
Повний текст джерелаАнотація:
Nucleic acid detection is of central importance for the diagnosis and treatment of genetic diseases, infectious agents, bio-warfare agents, and drug discovery. Nucleic acid testing for diseases is exclusively performed in laboratories using high-end instrumentation and personnel. However, this has developed the need for point of care diagnostics which can provide near-patient testing in a clinic, doctor’s office, or home. Such diagnostic tools can prove advantageous when rapid response is required or when suitable facilities are unavailable. Compared to equivalent methods used in laboratories, point of care testing is more affordable, as it eliminates the need for expensive instrumentation and skilled labor. One option involves the use of lateral flow assays. Pre-fabricated strips of dry reagents activated upon fluid application are already used in diagnostics, such as to ascertain pregnancy. Nucleic acid based detection assays on lateral flow offer several advantages over traditional microbiological detection methods. In this work we introduce a lateral flow biosensor that can combine the optical properties of nanoparticles (such as gold nanoparticles) with conventional immunoassay techniques to deliver a simple platform for rapid analysis of DNA with high sensitivity and selectivity. The quick 30 minute assay provides a platform to detect multiple nucleic acids with high efficiency achieved via chromatographic separation sandwich-type DNA hybridization reactions. Captured gold nanoparticles on the device can provide qualitative analysis by observing the color change to red and a semi-quantitative analysis via a strip reader. The biosensor was applied to the detection of human genomic DNA directly with high sensitivity and selectivity. The work was further expanded to detect Chlamydia trachomatis and Neisseria gonorrhoeae samples using nucleic acid amplification to generate large numbers of target copies. Improvements were made in the preparation of the biosensor to enable detection of Human Papilloma Virus Type-16. The clinical samples obtained were amplified using PCR for direct detection on the lateral flow biosensor without interference from other HPV types (e.g. HPV 18). The feasibility of the biosensor shows great potential for further development to assure its use in point of care diagnosis. The promising properties of the biosensor are reported in this dissertation.
Книги з теми "Direct nucleic acid detection"
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Kolpashchikov, Dmitry M., and Yulia V. Gerasimova, eds. Nucleic Acid Detection. Totowa, NJ: Humana Press, 2013. http://dx.doi.org/10.1007/978-1-62703-535-4.
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Astakhova, Kira, and Syeda Atia Bukhari, eds. Nucleic Acid Detection and Structural Investigations. New York, NY: Springer US, 2020. http://dx.doi.org/10.1007/978-1-0716-0138-9.
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Kolpashchikov, Dmitry M., and Yulia V. Gerasimova. Nucleic acid detection: Methods and protocols. New York: Humana Press, 2013.
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Ultrastructural methods for nucleic acid detection by immunocytology. Stuttgart: Gustav Fischer Verlag, 1999.
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Thiry, Marc. Ultrastructural methods for nucleic acid detection by immunocytology. Jena, Germany: Urban & Fischer, 1999.
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Luo, Yunbo. Functional Nucleic Acid Based Biosensors for Food Safety Detection. Singapore: Springer Singapore, 2018. http://dx.doi.org/10.1007/978-981-10-8219-1.
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D, Sobsey Mark, and AWWA Research Foundation, eds. Enteric virus detection in water by nucleic acid methods. Denver, CO: AWWA Research Foundation and American Water Works Association, 1996.
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Li, Tang. Development of liposome-based nucleic acid analyses for rapid detection of listeria monocytogenes. Ithaca, NY: Cornell University, 2003.
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L, Wiedbrauk Danny, and Farkas Daniel H, eds. Molecular methods for virus detection. San Diego: Academic Press, 1995.
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Schillinger, Julia Ann. Detection of human papillomavirus by nucleic acid hybridization as an adjunct to the papanicolaou smear. [New Haven: s.n.], 1990.
Знайти повний текст джерелаЧастини книг з теми "Direct nucleic acid detection"
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Xu, Yao, and Zhi Zheng. "Hybridization Chain Reaction for Direct mRNA Detection Without Nucleic Acid Purification." In Methods in Molecular Biology, 187–96. New York, NY: Springer New York, 2018. http://dx.doi.org/10.1007/978-1-4939-7213-5_12.
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Lehmann, Marc, and Roland P. H. Schmitz. "Nucleic Acid Amplification Techniques." In Modern Techniques for Pathogen Detection, 55–111. Weinheim, Germany: Wiley-VCH Verlag GmbH & Co. KGaA, 2015. http://dx.doi.org/10.1002/9783527687978.ch3.
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Merril, Carl R., Karen M. Washart, and Robert C. Allen. "Ultrasensitive Silver Based Stains for Nucleic Acid Detection." In Nucleic Acid Electrophoresis, 152–61. Berlin, Heidelberg: Springer Berlin Heidelberg, 1998. http://dx.doi.org/10.1007/978-3-642-58924-9_5.
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Dittmann, Elke, Anne Rantala-Ylinen, Vitor Ramos, Vitor Vasconcelos, Guntram Christiansen, and Rainer Kurmayer. "Nucleic Acid Extraction." In Molecular Tools for the Detection and Quantification of Toxigenic Cyanobacteria, 135–61. Chichester, UK: John Wiley & Sons, Ltd, 2017. http://dx.doi.org/10.1002/9781119332169.ch5.
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Wheeler, David. "Detection of DNA Curvature Using Transverse Pore Gradient Polyacrylamide Gel Electrophoresis." In Nucleic Acid Electrophoresis, 311–24. Berlin, Heidelberg: Springer Berlin Heidelberg, 1998. http://dx.doi.org/10.1007/978-3-642-58924-9_13.
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Fried, Michael G., and Mark M. Garner. "The Electrophoretic Mobility Shift Assay (EMSA) for Detection and Analysis of Protein-DNA Interactions." In Nucleic Acid Electrophoresis, 239–71. Berlin, Heidelberg: Springer Berlin Heidelberg, 1998. http://dx.doi.org/10.1007/978-3-642-58924-9_10.
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Sakallah, Sameer A., Robert W. Lanning, and David L. Cooper. "Rapid Detection of Hepatitis C Virus in Plasma and Liver Biopsies by Capillary Electrophoresis." In Nucleic Acid Electrophoresis, 193–201. Berlin, Heidelberg: Springer Berlin Heidelberg, 1998. http://dx.doi.org/10.1007/978-3-642-58924-9_8.
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Xu, Wentao. "Lateral Flow Nucleic Acid Biosensors." In Functional Nucleic Acids Detection in Food Safety, 245–73. Singapore: Springer Singapore, 2016. http://dx.doi.org/10.1007/978-981-10-1618-9_12.
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Karcher, Susan J. "Non-radioactive nucleic acid detection systems." In Plant Molecular Biology Manual, 309–33. Dordrecht: Springer Netherlands, 1994. http://dx.doi.org/10.1007/978-94-011-0511-8_21.
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Xu, Wentao. "Nucleic Acid Biosensors for Food Safety." In Functional Nucleic Acids Detection in Food Safety, 275–322. Singapore: Springer Singapore, 2016. http://dx.doi.org/10.1007/978-981-10-1618-9_13.
Повний текст джерелаТези доповідей конференцій з теми "Direct nucleic acid detection"
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Shen, Chuanjie, Hao Yin, Zhaoduo Tong, Shihui Qiu, Yunxing Lu, Zhenhua Wu, and Hongju Mao. "Digital Microfluidic Chip Based on Direct Ink Writing For Nucleic Acid Multiplex PCR Detection." In 2022 IEEE 35th International Conference on Micro Electro Mechanical Systems Conference (MEMS). IEEE, 2022. http://dx.doi.org/10.1109/mems51670.2022.9699738.
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Uno, Takeshi, Toshihito Ohtake, Hitoshi Tabata, and Tomoji Kawai. "Direct DNA detection using ion-sensitive field effect transistors (IS-FETs) based on peptide nucleic acid." In 2004 International Conference on Solid State Devices and Materials. The Japan Society of Applied Physics, 2004. http://dx.doi.org/10.7567/ssdm.2004.i-4-4.
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Bratcher, Amber R., Laurie B. Connell, and Rosemary L. Smith. "Development of a direct detection method for Alexandrium spp. Using surface plasmon resonance and peptide nucleic acid probes." In 2009 IEEE Sensors. IEEE, 2009. http://dx.doi.org/10.1109/icsens.2009.5398359.
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Bogdanov, Valery L., Yu-Hui Rogers, Guang Lan, and Michael Boyce-Jacino. "Multicolor instrumentation for direct fluorescent detection of nucleic acids in a microchip format." In BiOS '98 International Biomedical Optics Symposium, edited by Gerald E. Cohn. SPIE, 1998. http://dx.doi.org/10.1117/12.307323.
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Fan, Y., X. Chen, J. Kong, and Z. Gao. "Direct Detection of Nucleic Acids by Tagging Phosphates on Their Backbones with Conductive Nanoparticles." In TRANSDUCERS 2007 - 2007 International Solid-State Sensors, Actuators and Microsystems Conference. IEEE, 2007. http://dx.doi.org/10.1109/sensor.2007.4300534.
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Ritzi-Lehnert, Marion, Jan Claussen, Eva Schaeffer, Ole Wiborg, Isabell Wick, Klaus S. Drese, Ralf Himmelreich, et al. "New Lab-on-a-Chip System for Infectious Disease Analysis." In ASME 2010 8th International Conference on Nanochannels, Microchannels, and Minichannels collocated with 3rd Joint US-European Fluids Engineering Summer Meeting. ASMEDC, 2010. http://dx.doi.org/10.1115/fedsm-icnmm2010-31048.
Повний текст джерелаАнотація:
Early diagnosis followed by personalised efficient therapy of infectious diseases (e.g. respiratory diseases, meningitis, sepsis) can lead to considerable reduction of costs in health care. Point-of-care testing (POCT) can provide early detection since this kind of decentralised analysis can be done by unskilled personnel at any time. Other advantages of automated miniaturised Lab-on-a-Chip systems (LoC) are reduction of time and reagents, elimination of cross-contamination and enhanced reproducibility due to enhanced process control. Such Lab-on-a-Chip systems will establish themselves on market only when sensitivity and specificity meet clinical requirements. An integrated cost-efficient lab-on-a-chip system is presented which allows performing all diagnostic process steps for pathogen analysis of respiratory viruses from nasopharyngeal samples. The microfluidic disposable chip comprises structures for lysis of nasopharyngeal swab samples, preparation of total nucleic acids using magnetic silica beads, reverse transcription followed by QIAplex PCR technology and labelling of the nucleic acids by hybridisation with LiquiChip Beads and streptavidin-R-phycoerythrin. Labelled target sequences are transferred for analysis into a QIAGEN LiquiChip 200 workstation. The core of the instrument is a construction based on rotating heating bars allowing for fast cycling. All chemicals needed for performing of 24 analyses are either stored freeze-dried on the single-use disposable microfluidic chip (processing cartridge) or as liquids in a separate reagent cartridge. After introducing the sample into the lysis chamber of the microfluidic chip and inserting the chip into the device all steps are done automatically. To realise these steps, fluidic control in terms of light barriers and turning valves are integrated into the injection moulded disposable chip. This includes metering structures as well as magnetic stir bars for mixing. The functionality was proven by direct comparison of samples processed manually vs. automatically using the “ResPlex Panel II” for detection of respiratory viruses from nasopharyngeal samples. The efficiency of the automated LoC system yields at about 30–60% as compared to the manually performed reference experiments. Comparing the performance of the instrument with commercially available kits and nucleic acid preparation devices showed slightly weaker but clearly positive final signal intensities obtained from the prototype device even without protocol optimization.
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"DNA-nanomachines for nucleic acid detection." In Bioinformatics of Genome Regulation and Structure/Systems Biology (BGRS/SB-2022) :. Institute of Cytology and Genetics, the Siberian Branch of the Russian Academy of Sciences, 2022. http://dx.doi.org/10.18699/sbb-2022-200.
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Lin, Zhihong, Meng Wu, Shu Ren, Michaela Arbter, Martin Boehmer, Vladimir Mirsky, and Otto S. Wolfbeis. "Single- and dual- near-infrared fluorescent labeled nucleic acid conjugate for nucleic acid detection." In International Conference on Sensing units and Sensor Technology, edited by Yikai Zhou and Shunqing Xu. SPIE, 2001. http://dx.doi.org/10.1117/12.440165.
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Liu, Ye, Bo Wu, Sanjida Yeasmin, and Li-Jing Cheng. "Magnetoplasmonic Nanoparticles for Enhanced Nucleic Acid Detection." In CLEO: Applications and Technology. Washington, D.C.: OSA, 2021. http://dx.doi.org/10.1364/cleo_at.2021.am3c.1.
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Gulyaeva, Irina V., Ekaterina V. Efimtseva, Andrei A. Rodionov, Boris S. Ermolinsky, and Sergey N. Mikhailov. "Direct synthesis of 5'-nucleotides using glycosylation reaction." In XIIth Symposium on Chemistry of Nucleic Acid Components. Prague: Institute of Organic Chemistry and Biochemistry, Academy of Sciences of the Czech Republic, 2002. http://dx.doi.org/10.1135/css200205312.
Повний текст джерелаЗвіти організацій з теми "Direct nucleic acid detection"
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Castro, A., and E. B. Shera. Ultrasensitive nucleic acid sequence detection by single-molecule electrophoresis. Office of Scientific and Technical Information (OSTI), September 1996. http://dx.doi.org/10.2172/374265.
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Kingsley, Mark T. Nucleic Acid-Based Detection and Identification of Bacterial and Fungal Plant Pathogens - Final Report. Office of Scientific and Technical Information (OSTI), March 2001. http://dx.doi.org/10.2172/781863.
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Kingsley, Mark T. Nucleic Acid-Based Detection and Identification of Bacterial and Fungal Plant Pathogens - Final Report. Office of Scientific and Technical Information (OSTI), March 2001. http://dx.doi.org/10.2172/965696.
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Lers, Amnon, and Pamela J. Green. LX Senescence-Induced Ribonuclease in Tomato: Function and Regulation. United States Department of Agriculture, September 2003. http://dx.doi.org/10.32747/2003.7586455.bard.
Повний текст джерелаАнотація:
Natural leaf senescence, which occurs even when growth conditions are near optimal, has a negative influence on yield. Postharvest induced senescence contributes to the losses of quality in flowers, foliage, and vegetables. Strategies designed to control the senescence process in crop plants could therefore have great applied significance. However, the successful design of such strategies requires a better insight into the senescence machinery and control in higher plants. A main feature of senescence is the hydrolysis of macromolecules by hydrolases of various types such as ribonucleases (RNases) and proteases. Previously we had identified and characterized the tomato LX RNase gene demonstrating its transcript to be highly and specifically induced during senescence. This reported study was focused on LX but also had broadened our research to other senescence-associated nucleic acids degrading enzymes to learn about their function and the regulation of their encoding genes. Beside tomato we used parsley and Arabidopsis for the study of: the bi-functional nuclease which has a role in senescence. The study of different senescence- associated nucleases in few plant systems will allow a more general view on function and regulation of these enzymes in senescence. The specific original proposed objectives included: 1. Study the consequences of alterations in LX RNase level on tomato leaf senescence and general development; 2. Analyze stimuli which may participate in senescence-specific activation of the LX gene; 3. Clone the senescence-associated BFNI nuclease gene homologue from tomato. 4. Further characterize the sequences required for senescence-specific gene expression. Homozygous transgenic plants in which LX gene was either inhibited or over-expressed were generated. In both of these LX mutated plants no major phenotypic consequences were observed, which may suggests that LX is not essential for plant growth under optimal growth conditions. Lack of any abnormalities in the LX over-expressing lines suggests that special system exist to allow function of the RNase only when needed. Detailed analyses of growth under stress and consequences to RNA metabolism are underway. We have analyzed LX expression on the protein level demonstrating that it is involved also in petal senescing. Our results suggest that LX is responding to complex regulation involving developmental, organ dependent factors and responds differently to hormonal or environmental stimuli in the different plant organs. The cloned 1.4 kb promoter was cloned and its analysis revealed that probably not all required elements for senescence induction are included. Biochemical analysis of senescence-associated be-functional nucleases in the different plants, tomato, parsley and Arabidopsis, suggests they belong to a sub-class within the type I plant nucleases. The parsley PcNUC1/2 nuclease protein was purified from senescing leaves its and activity was studied in vitro revealing endo-, double strand, nucleolytic activity and exo-nucleolytic activity. Its encoding gene was cloned and found to be induced on the mRNA level. The promoter of the related Arabidopsis BFNI nuclease was shown in both tomato and Arabidopsis to be able and direct senescence-specific expression suggesting that, at least part, the gene is regulated on the transcriptional level and that the mechanism for this senescence-specific regulation is conserved between different plants. Few plants in which the BFNI gene is mutated were identified which are subjected now to detailed analysis. Our results suggest that the senescence-related nucleic acid degrading enzymes share similarities in both function and regulation between different plants and possibly have important functions in processes un-related to senescence. Still, the function of these enzymes, at least in some cases is not essential to plant development under optimal growth conditions. We are now at the stage which permits in depth investigation of the specific functions and mode of molecular regulation of senescence-associated nucleases with the aid of the research tools developed. The isolated senescence-specific promoter, shown to be active in heterologous plant system, could be utilized in agricultural-related biotechnological applications for retardation of senescence.
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Lers, Amnon, E. Lomaniec, S. Burd, A. Khalchitski, L. Canetti, and Pamela J. Green. Analysis of Senescence Inducible Ribonuclease in Tomato: Gene Regulation and Function. United States Department of Agriculture, February 2000. http://dx.doi.org/10.32747/2000.7570563.bard.
Повний текст джерелаАнотація:
Natural leaf senescence has a negative influence on yield. Postharvest induced senescence contributes to the losses of quality in flowers, foliage, and vegetables. Strategies designed to control the senescence process in crop plants could therefore have great applied significance. Senescence is regulated by differential gene expression yet, functional characterization of the genes specifically induced and study of their expression control, is still in its infancy. Study of senescence-specific genes is required to allow identification of regulatory elements participating in senescence-induced expression and thus provide insights into the genetic regulation of senescence. A main feature of senescence is the hydrolysis of macromolecules by hydrolases of various types such as RNases and proteases. This study was aimed a analysis of senescence-inducible RNases in tomato with the following objectives: Isolation of senescence-inducible RNase cDNA clones; Expression analyses of RNase genes during senescence; Identification of sequences required for senescence-induced gene expression; Functional analyses of senescence-inducible RNases. We narrowed our aims somewhat to focus on the first three objectives because the budget we were awarded was reduced from that requested. We have expanded our research for identification senescence-related RNase/nuclease activities as we thought it will direct us to new RNase/nuclease genes. We have also carried out research in Arabidopsis and parsley, which enabled us to draw mire general conclusions. We completed the first and second objectives and have made considerable progress on the remaining two. We have defined growth conditions suitable for this research and defined the physiological and biochemical parameters characteristic to the advance of leaf senescence. In tomato and arabidopsis we have focused on natural leaf senescence. Parsley was used mainly for study of postharvest senescence in detached leaves. We have identified a 41-kD a tomato nuclease, LeNUCI, specifically induced during senescence which can degrade both RNA and DNA. This activity could be induced by ethylene in young leaves and was subjected to detailed analysis, which enabled its classification as Nuclease I enzyme. LeNUCI may be involved in nucleic acid metabolism during tomato leaf senescence. In parsley senescing leaves we identified 2 main senescence-related nuclease activities of 41 and 39-kDa. These activities were induced in both naturally or artificially senescing leaves, could degrade both DNA and RNA and were very similar in their characteristics to the LeNUCI. Two senescence-induced RNase cDNAs were cloned from tomato. One RNase cDNA was identical to the tomato LX RNase while the second corresponded to the LE RNase. Both were demonstrated before to be induced following phosphate starvation of tomato cell culture but nothing was known about their expression or function in plants. LX gene expression was much more senescence specific and ethylene could activate it in detached young leaves. LE gene expression, which could be transiently induced by wounding, appeared to be activated by abscisic acid. We suggest that the LX RNase has a role in RNA catabolism in the final stage of senescence, and LE may be a defense-related protein. Transgenic plants were generated for altering LX gene expression. No major visible alterations in the phenotype were observed so far. Detailed analysis of senescence in these plants is performed currently. The LX promoter was cloned and its analysis is performed currently for identification of senescence-specific regulatory elements. In Arabidopsis we have identified and characterized a senescence-associated nuclease 1 gene, BFN1, which is highly expressed during leaf and stem senescence. BFN1, is the first example of a senescence- associated gene encoding a nuclease I enzyme as well as the first nuclease I cloned and characterized from Arabidopsis. Our progress should provide excellent tools for the continued analysis of regulation and function of senescence-inducible ribonucleases and nucleases in plants. The cloned genes can be used in reverse genetic approaches, already initiated, which can yield a more direct evidence for the function of these enzymes. Another contribution of this research will be in respect to the molecular mechanism, which controls senescence. We had already initiated in this project and will continue to identify and characterize regulatory elements involved in senescence-specific expression of the genes isolated in this work.
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Fluhr, Robert, and Maor Bar-Peled. Novel Lectin Controls Wound-responses in Arabidopsis. United States Department of Agriculture, January 2012. http://dx.doi.org/10.32747/2012.7697123.bard.
Повний текст джерелаАнотація:
Innate immune responses in animals and plants involve receptors that recognize microbe-associated molecules. In plants, one set of this defense system is characterized by large families of TIR–nucleotide binding site–leucine-rich repeat (TIR-NBS-LRR) resistance genes. The direct interaction between plant proteins harboring the TIR domain with proteins that transmit and facilitate a signaling pathway has yet to be shown. The Arabidopsis genome encodes TIR-domain containing genes that lack NBS and LRR whose functions are unknown. Here we investigated the functional role of such protein, TLW1 (TIR LECTIN WOUNDRESPONSIVE1). The TLW1 gene encodes a protein with two domains: a TIR domain linked to a lectin-containing domain. Our specific aim in this proposal was to examine the ramifications of the TL1-glycan interaction by; A) The functional characterization of TL1 activity in the context of plant wound response and B) Examine the hypothesis that wounding induced specific polysaccharides and examine them as candidates for TL-1 interactive glycan compounds. The Weizmann group showed TLW1 transcripts are rapidly induced by wounding in a JA-independent pathway and T-DNA-tagged tlw1 mutants that lack TLW1 transcripts, fail to initiate the full systemic wound response. Transcriptome methodology analysis was set up and transcriptome analyses indicates a two-fold reduced level of JA-responsive but not JA-independent transcripts. The TIR domain of TLW1 was found to interact directly with the KAT2/PED1 gene product responsible for the final b-oxidation steps in peroxisomal-basedJA biosynthesis. To identify potential binding target(s) of TL1 in plant wound response, the CCRC group first expressed recombinant TL1 in bacterial cells and optimized conditions for the protein expression. TL1 was most highly expressed in ArcticExpress cell line. Different types of extraction buffers and extraction methods were used to prepare plant extracts for TL1 binding assay. Optimized condition for glycan labeling was determined, and 2-aminobenzamide was used to label plant extracts. Sensitivity of MALDI and LC-MS using standard glycans. THAP (2,4,6- Trihydroxyacetophenone) showed minimal background peaks at positive mode of MALDI, however, it was insensitive with a minimum detection level of 100 ng. Using LC-MS, sensitivity was highly increased enough to detect 30 pmol concentration. However, patterns of total glycans displayed no significant difference between different extraction conditions when samples were separated with Dionex ICS-2000 ion chromatography system. Transgenic plants over-expressing lectin domains were generated to obtain active lectin domain in plant cells. Insertion of the overexpression construct into the plant genome was confirmed by antibiotic selection and genomic DNA PCR. However, RT-PCR analysis was not able to detect increased level of the transcripts. Binding ability of azelaic acid to recombinant TL1. Azelaic acid was detected in GST-TL1 elution fraction, however, DHB matrix has the same mass in background signals, which needs to be further tested on other matrices. The major findings showed the importance of TLW1 in regulating wound response. The findings demonstrate completely novel and unexpected TIR domain interactions and reveal a control nexus and mechanism that contributes to the propagation of wound responses in Arabidopsis. The implications are to our understanding of the function of TIR domains and to the notion that early molecular events occur systemically within minutes of a plant sustaining a wound. A WEB site (http://genome.weizmann.ac.il/hormonometer/) was set up that enables scientists to interact with a collated plant hormone database.
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Delwiche, Michael, Boaz Zion, Robert BonDurant, Judith Rishpon, Ephraim Maltz, and Miriam Rosenberg. Biosensors for On-Line Measurement of Reproductive Hormones and Milk Proteins to Improve Dairy Herd Management. United States Department of Agriculture, February 2001. http://dx.doi.org/10.32747/2001.7573998.bard.
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The original objectives of this research project were to: (1) develop immunoassays, photometric sensors, and electrochemical sensors for real-time measurement of progesterone and estradiol in milk, (2) develop biosensors for measurement of caseins in milk, and (3) integrate and adapt these sensor technologies to create an automated electronic sensing system for operation in dairy parlors during milking. The overall direction of research was not changed, although the work was expanded to include other milk components such as urea and lactose. A second generation biosensor for on-line measurement of bovine progesterone was designed and tested. Anti-progesterone antibody was coated on small disks of nitrocellulose membrane, which were inserted in the reaction chamber prior to testing, and a real-time assay was developed. The biosensor was designed using micropumps and valves under computer control, and assayed fluid volumes on the order of 1 ml. An automated sampler was designed to draw a test volume of milk from the long milk tube using a 4-way pinch valve. The system could execute a measurement cycle in about 10 min. Progesterone could be measured at concentrations low enough to distinguish luteal-phase from follicular-phase cows. The potential of the sensor to detect actual ovulatory events was compared with standard methods of estrus detection, including human observation and an activity monitor. The biosensor correctly identified all ovulatory events during its testperiod, but the variability at low progesterone concentrations triggered some false positives. Direct on-line measurement and intelligent interpretation of reproductive hormone profiles offers the potential for substantial improvement in reproductive management. A simple potentiometric method for measurement of milk protein was developed and tested. The method was based on the fact that proteins bind iodine. When proteins are added to a solution of the redox couple iodine/iodide (I-I2), the concentration of free iodine is changed and, as a consequence, the potential between two electrodes immersed in the solution is changed. The method worked well with analytical casein solutions and accurately measured concentrations of analytical caseins added to fresh milk. When tested with actual milk samples, the correlation between the sensor readings and the reference lab results (of both total proteins and casein content) was inferior to that of analytical casein. A number of different technologies were explored for the analysis of milk urea, and a manometric technique was selected for the final design. In the new sensor, urea in the sample was hydrolyzed to ammonium and carbonate by the enzyme urease, and subsequent shaking of the sample with citric acid in a sealed cell allowed urea to be estimated as a change in partial pressure of carbon dioxide. The pressure change in the cell was measured with a miniature piezoresistive pressure sensor, and effects of background dissolved gases and vapor pressures were corrected for by repeating the measurement of pressure developed in the sample without the addition of urease. Results were accurate in the physiological range of milk, the assay was faster than the typical milking period, and no toxic reagents were required. A sampling device was designed and built to passively draw milk from the long milk tube in the parlor. An electrochemical sensor for lactose was developed starting with a three-cascaded-enzyme sensor, evolving into two enzymes and CO2[Fe (CN)6] as a mediator, and then into a microflow injection system using poly-osmium modified screen-printed electrodes. The sensor was designed to serve multiple milking positions, using a manifold valve, a sampling valve, and two pumps. Disposable screen-printed electrodes with enzymatic membranes were used. The sensor was optimized for electrode coating components, flow rate, pH, and sample size, and the results correlated well (r2= 0.967) with known lactose concentrations.