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In this paper we present the characterization of 3D-printed nanocarbon electrodes (3DnCes) and their application in electrochemical enzyme-linked detection of DNA hybridization. The approach takes advantage of a facile procedure based on adsorption of target DNA on the electrode surface followed by hybridization with a biotinylated probe and binding of streptavidin-alkaline phosphatase conjugate. The alkaline phosphatase converts 1-naphthyl phosphate in the background electrolyte into electrochemically oxidizable 1-naphthol, which is subsequently detected using linear sweep voltammetry. The preparation, characterization, and analytical performance of the 3DnCes are reported. The results show the applicability of such 3DnCes in detection of target DNA hybridization specifically with the complementary biotinylated probe, and indicate the potential of 3D printed electrodes for use in various bioanalytical approaches.

Long-read sequencing technologies have opened up new avenues of research on the mosquito genome biology, enabling scientists to better understand the remarkable abilities of vectors for transmitting pathogens. Although new genome mapping technologies such as Hi-C scaffolding and optical mapping may significantly improve the quality of genomes, only cytogenetic mapping, with the help of fluorescence in situ hybridization (FISH), connects genomic scaffolds to a particular chromosome and chromosome band. This mapping approach is important for creating and validating chromosome-scale genome assemblies for mosquitoes with repeat-rich genomes, which can potentially be misassembled. In this study, we describe a new gene-based physical mapping approach that was optimized using the newly assembled Aedes albopictus genome, which is enriched with transposable elements. To avoid amplification of the repetitive DNA, 15 protein-coding gene transcripts were used for the probe design. Instead of using genomic DNA, complementary DNA was utilized as a template for development of the PCR-amplified probes for FISH. All probes were successfully amplified and mapped to specific chromosome bands. The genome-unique probes allowed to perform unambiguous mapping of genomic scaffolds to chromosome regions. The method described in detail here can be used for physical genome mapping in other insects.

Long-read sequencing technologies have opened up new avenues of research on the mosquito genome biology, enabling scientists to better understand the remarkable abilities of vectors for transmitting pathogens. Although new genome mapping technologies such as Hi-C scaffolding and optical mapping may significantly improve the quality of genomes, only cytogenetic mapping, with the help of fluorescence in situ hybridization (FISH), connects genomic scaffolds to a particular chromosome and chromosome band. This mapping approach is important for creating and validating chromosome-scale genome assemblies for mosquitoes with repeat-rich genomes, which can potentially be misassembled. In this study, we describe a new gene-based physical mapping approach that was optimized using the newly assembled Aedes albopictus genome, which is enriched with transposable elements. To avoid amplification of the repetitive DNA, 15 protein-coding gene transcripts were used for the probe design. Instead of using genomic DNA, complementary DNA was utilized as a template for development of the PCR-amplified probes for FISH. All probes were successfully amplified and mapped to specific chromosome bands. The genome-unique probes allowed to perform unambiguous mapping of genomic scaffolds to chromosome regions. The method described in detail here can be used for physical genome mapping in other insects.

Acinetobacter baumannii is the main cause of nosocomial infections in blood, urinary tract, wounds and in lungs leading to pneumonia. Apart from its strong predilection to be the cause of serious illnesses in intensive care units. Herein, we present a specific and sensitive approach for the monitoring of Acinetobacter baumannii genome based on citrate capped silver nanoparticles (Cit-AgNPs) using spectroscopic methods. In this study, (5' SH-TTG TGA ACT ATT TAC GTC AGC ATG C3') sequence was used as a probe DNA (pDNA) of Acinetobacter baumannii. Then, complementary DNA (cDNA) was used for hybridization. After the hybridization of pDNA with cDNA, target DNA (5' GCA TGC TGA CGT AAA TAGTTC ACA A 3') was recognized and detected using turn-on fluorescence bioassay. After the hybridization of pDNA with cDNA, the target DNA was successfully measured in optimum time of 2 min by spectrophotometric techniques. Moreover, the selectivity of designed bioassay was evaluated in the presence of two mismatch sequences and excellent differentiation was obtained. 1 Zepto-molar (zM) of low limit of quantification (LLOQ) was achieved by this genosensor. The present study paved the way for quick (2 min) and accurate detection of Acinetobacter baumannii, which can be a good alternative to the traditional methods. Current study proposed a novel and significant diagnostic test towards Acinetobacter baumannii detection based on silver nanoparticles aggregation which has the capability of being a good alternative to the traditional methods. Moreover, the proposed genosensor successfully could be applied for the detection of other pathogens., Funding Agency: Tabriz University of Medical Sciences

This chapter details a compendium of protocols that collectively enable the reader to perform a pooled shRNA and/or CRISPR screen-with methods to identify and validate positive controls and subsequent hits; establish a viral titer in the cell line of choice; create and screen libraries, sequence strategies, and bioinformatics resources to analyze outcomes. Collectively, this provides an overarching resource from the start to finish of a screening project, making this technology possible in all laboratories.Copyright © 2018, Springer Science+Business Media, LLC.

We present a droplet PCR workflow for detection of multiple pathogen DNA biomarkers using fluorescent color coded Luminex beads. This strategy enables encoding of multiple singleplex droplet PCRs using a commercially available bead set of several hundred distinguishable fluorescence codes. This workflow provides scalability beyond the limited number offered by fluorescent detection probes such as TaqMan probes, commonly used in current multiplex droplet PCRs. The workflow was validated for three different Luminex bead sets coupled to target specific capture oligos to detect hybridization of three microorganisms infecting poultry: avian influenza, infectious laryngotracheitis virus and Campylobacter jejuni. In this assay, the target DNA was amplified with fluorescently labeled primers by PCR in parallel in monodisperse picoliter droplets, to avoid amplification bias. The color codes of the Luminex detection beads allowed concurrent and accurate classification of the different bead sets used in this assay. The hybridization assay detected target DNA of all three microorganisms with high specificity, from samples with average target concentration of a single DNA template molecule per droplet. This workflow demonstrates the possibility of increasing the droplet PCR assay detection panel to detect large numbers of targets in parallel, utilizing the scalability offered by the color-coded Luminex detection beads., QC 20191028

Based on phenotypic tests, 49 out of 168 isolates of Pseudomonas syringae obtained from various organs of diseased tissue originating from various regions in Poland, were identified as P. syringae pv. morsprunorum race 1 (Psm1), 10 as race 2 of this pathovar (Psm2), 53 as pathovar syringae (Pss) and 56 as an atypical taxon. The pathogenicity test on immature sweet cherry fruits divided the tested strains into two groups: one with isolates causing black brown necrosis and the second containing isolates inducing water soaked superficial lesions. Phenotypic and genetic studies on toxins production by bacteria showed that all Pss produced syringomycin. However, only some Psm1 isolates have the gene for coronatine production. All strains belonging to Psm2 possessed genes encoding yersiniabactin. Results of genetic analyses (rep-PCRs, PCR MP) confirmed the homogeneity of isolates within pathovar morsprunorum and the atypical taxon and revealed diversity within pathovar syringae. A detailed polyphasic approach including phenotypic and genetic characterization applied for eight strains from the atypical taxon showed that the strains represent a novel species of the genus Pseudomonas for which Pseudomonas cerasi sp. nov. (non Griffin, 1911) is proposed.

Nucleotide arrays require controlled surface densities and minimal nucleotide-substrate interactions to enable highly specific and efficient recognition by corresponding targets. We investigated chemical lift-off lithography with hydroxyl- and oligo(ethylene glycol)-terminated alkanethiol self-assembled monolayers as a means to produce substrates optimized for tethered DNA insertion into post-lift-off regions. Residual alkanethiols in the patterned regions after lift-off lithography enabled the formation of patterned DNA monolayers that favored hybridization with target DNA. Nucleotide densities were tunable by altering surface chemistries and alkanethiol ratios prior to lift-off. Lithography-induced conformational changes in oligo(ethylene glycol)-terminated monolayers hindered nucleotide insertion but could be used to advantage via mixed monolayers or double-lift-off lithography. Compared to thiolated DNA self-assembly alone or with alkanethiol backfilling, preparation of functional nucleotide arrays by chemical lift-off lithography enables superior hybridization efficiency and tunability.

High-frequency (40 MHz) and low-frequency (7 MHz) capacitive micromachined ultrasound transducers (CMUT) were fabricated and tested for use in gravimetric detection of biomolecules. The low-frequency CMUT sensors have a gold-coated surface, while the high-frequency sensors have a silicon nitride surface. Both surfaces were functionalized with bovine leukemia virus antigen gp51 acting as the antigen. On addition of an a specific antibody labeled with horseradish peroxidase (HRP), the antigen/antibody complex is formed on the surface and quantified by HRP-catalyzed oxidation of tetramethylbenzidine. It has been found that a considerably smaller quantity of immuno complex is formed on the high frequency sensor surface. In parallel, the loading of the surface of the CMUT was determined via resonance frequency and electromechanical resistance readings. Following the formation of the immuno complexes, the resonance frequencies of the low-frequency and high-frequency sensors decrease by up to 420 and 440 kHz, respectively. Finite element analysis reveals that the loading of the (gold-coated) low frequency sensors is several times larger than that on high frequency sensors. The formation of the protein film with pronounced elasticity and stress on the gold surface case is discussed. We also discuss the adoption of this method for the detection of DNA using a hybridization assay following polymerase chain reaction.

Herein we report an electrochemical DNA biosensor for the rapid detection of sequence (5’ AAT GGA TTT ATC TGC TCT TCG 3’) specific for the breast cancer 1 (BRCA1) gene. The proposed electrochemical genosensor is based on short oligonucleotide DNA probe immobilized onto zinc oxide nanowires (ZnONWs) chemically synthesized onto gold electrode via hydrothermal technique. The morphology studies of the ZnONWs, performed by field emission scanning electron microscopy (FESEM), showed that the ZnO nanowires are uniform, highly dense and oriented perpendicularly to the substrate. Recognition event between the DNA probe and the target was investigated by differential pulse voltammetry (DPV) in 0.1 M acetate buffer solution (ABS), pH 7.00; as a result of the hybridization, an oxidation signal was observed at +0.8 V. The influences of pH, target concentration, and non-complimentary DNA on biosensor performance were examined. The proposed DNA biosensor has the ability to detect the target sequence in the range of concentration between 10.0 and 100.0 µM with a detection limit of 3.32 µM. The experimental results demonstrated that the prepared ZnONWs/Au electrodes are suitable platform for the immobilization of DNA.

Selective covalent immobilization of thiolated oligonucleotides onto an epoxy-functionalized silicon-substrate can be achieved via light radiation (365 nm). Following this approach, thiol-modified oligonucleotide probes were covalently attached as microarrays, reaching an immobilization density of 2.5 pmol·cm -2, with a yield of 53%. The developed method presents the advantages of spatially controlled probe anchoring (by means of using a photomask), direct attachment without using cross-linkers, and short irradiation times (10 min). Hybridization efficiencies up to 65%, with full complementary strands, were reached. The approach was evaluated by scoring single nucleotide polymorphisms with a discrimination ratio around 15. Moreover, sensitive and selective detection of bacterial Escherichia coli was demonstrated. © 2012 American Chemical Society.

The aim of this study was to investigate the effectiveness of tablets containing two probiotic Lactobacillus reuteri strains in inhibiting regrowth of salivary mutans streptococci (MS) after full-mouth disinfection (FMD) with chlorhexidine. The null hypothesis was that the levels of MS would not differ in comparison with a placebo protocol. The study population was comprised of 62 young adults (mean age 23 years) with moderate or high counts of salivary MS who volunteered after informed consent. The study was a double-blinded randomized controlled trial with two parallel groups. After a 3-day chlorhexidine regimen, the subjects were randomly assigned to a test group (n = 32) with probiotic lozenges (2/day) or a placebo group (n = 30). The intervention period was 6 weeks, and stimulated whole saliva was collected at baseline and after 1, 6, and 12 weeks. The samples were processed for MS by a chair-side test and DNA-DNA hybridization as an estimate of 19 bacterial strains associated with oral health and disease. There was no significant difference between the groups at inclusion, and FMD reduced the salivary MS levels significantly in both groups. The MS suppression lasted less than 6 weeks and there were no statistical differences in salivary MS regrowth between the test and control groups at any of the follow-ups. Likewise, there were no major differences in the regrowth patterns of the checkerboard panel between the two groups. We conclude that daily oral administration of L. reuteri did not seem to affect or delay the regrowth of salivary MS after FMD with chlorhexidine. Copyright (c) 2012 S. Karger AG, Basel

[EN] This work addresses the main topics related to the photopolymerization process to develop and to monitor the analytical signal derived from biorecognition assays. We review basic aspects of photopolymerization, together with the nature and the reactivity of the chemicals involved. Focusing on DNA and protein determination and medical applications, we envisage as relevant photopolymers that generate biocompatible hydrogels. We also review the major characterization techniques applied to ascertain the composition of the products formed and to monitor the progress of the reaction. In this sense, we present optical microscopy, fluorescence imaging, ATR-FTIR spectroscopy and profilometry measures.Finally, we discuss analytical examples and applications of protein-to-protein, protein-to-antigen and DNA-hybridization photopolymerization. We demonstrate implementation of CD/DVD player technology to the detection and quantitation of such biointeractions, highlighting the advantages of this powerful methodology in biosensing. © 2012 Elsevier Ltd.

While developing a rapid method to detect carriers of vancomycin-resistant enterococci (VRE), we found the vanB gene by PCR in 13 of 50 human faecal specimens that did not contain culturable VRE. Passaging under antibiotic selection allowed us to isolate two species of anaerobic bacteria that were vanB PCR positive, vancomycin resistant, and teicoplanin sensitive. Sequence analysis of the 16S rRNA genes showed that one isolate resembled Eggerthella lenta (98% identity), and the other Clostridium innocuum (92% identity). Southern hybridisation and nucleotide sequencing showed a vanB locus homologous to that in VRE. We propose that vanB resistance in enterococci might arise from gene transfer in the human bowel.

Isothermal nucleic acid amplification is becoming increasingly important for molecular diagnostics. Therefore, new computational tools are needed to facilitate assay design. In the isothermal EXPonential Amplification Reaction (EXPAR), template sequences with similar thermodynamic characteristics perform very differently. To understand what causes this variability, we characterized the performance of 384 template sequences, and used this data to develop two computational methods to predict EXPAR template performance based on sequence: a position weight matrix approach with support vector machine classifier, and RELIEF attribute evaluation with Nave Bayes classification. The methods identified well and poorly performing EXPAR templates with 6770 sensitivity and 7780 specificity. We combined these methods into a computational tool that can accelerate new assay design by ruling out likely poor performers. Furthermore, our data suggest that variability in template performance is linked to specific sequence motifs. Cytidine, a pyrimidine base, is over-represented in certain positions of well-performing templates. Guanosine and adenosine, both purine bases, are over-represented in similar regions of poorly performing templates, frequently as GA or AG dimers. Since polymerases have a higher affinity for purine oligonucleotides, polymerase binding to GA-rich regions of a single-stranded DNA template may promote non-specific amplification in EXPAR and other nucleic acid amplification reactions. © 2012 The Author(s).

The aim of this study was to investigate the effectiveness of tablets containing two probiotic Lactobacillus reuteri strains in inhibiting regrowth of salivary mutans streptococci (MS) after full-mouth disinfection (FMD) with chlorhexidine. The null hypothesis was that the levels of MS would not differ in comparison with a placebo protocol. The study population was comprised of 62 young adults (mean age 23 years) with moderate or high counts of salivary MS who volunteered after informed consent. The study was a double-blinded randomized controlled trial with two parallel groups. After a 3-day chlorhexidine regimen, the subjects were randomly assigned to a test group (n = 32) with probiotic lozenges (2/day) or a placebo group (n = 30). The intervention period was 6 weeks, and stimulated whole saliva was collected at baseline and after 1, 6, and 12 weeks. The samples were processed for MS by a chair-side test and DNA-DNA hybridization as an estimate of 19 bacterial strains associated with oral health and disease. There was no significant difference between the groups at inclusion, and FMD reduced the salivary MS levels significantly in both groups. The MS suppression lasted less than 6 weeks and there were no statistical differences in salivary MS regrowth between the test and control groups at any of the follow-ups. Likewise, there were no major differences in the regrowth patterns of the checkerboard panel between the two groups. We conclude that daily oral administration of L. reuteri did not seem to affect or delay the regrowth of salivary MS after FMD with chlorhexidine.

The application of photonic biosensor assays to diagnose the category-A select agent Francisella tularensis was investigated. Both interferometric and long period fiber grating sensing structures were successfully demonstrated; both these sensors are capable of detecting the optical changes induced by either immunological binding or DNA hybridization. Detection was made possible by the attachment of DNA probes or immunoglobulins (IgG) directly to the fiber surface via layer-by-layer electrostatic self-assembly. An optical fiber biosensor was tested using a standard transmission mode long period fiber grating of length 15 mm and period 260 µm, and coated with the IgG fraction of antiserum to F. tularensis. The IgG was deposited onto the optical fiber surface in a nanostructured film, and the resulting refractive index change was measured using spectroscopic ellipsometry. The presence of F. tularensis was detected from the decrease of peak wavelength caused by binding of specific antigen. Detection and differentiation of F. tularensis subspecies tularensis (type A strain TI0902) and subspecies holarctica (type B strain LVS) was further accomplished using a single-mode multi-cavity fiber Fabry-Perot interferometric sensor. These sensors were prepared by depositing seven polymer bilayers onto the fiber tip followed by attaching one of two DNA probes: (a) a 101-bp probe from the yhhW gene unique to type-A strains, or (b) a 117-bp probe of the lpnA gene, common to both type-A and type-B strains. The yhhW probe was reactive with the type-A, but not the type-B strain. Probe lpnA was reactive with both type-A and type-B strains. Nanogram quantities of the target DNA could be detected, highlighting the sensitivity of this method for DNA detection without the use of PCR. The DNA probe reacted with 100% homologous target DNA, but did not react with sequences containing 2-bp mismatches, indicating the high specificity of the assay. These assays will fill an important void that e

The application of photonic biosensor assays to diagnose the category-A select agent Francisella tularensis was investigated. Both interferometric and long period fiber grating sensing structures were successfully demonstrated; both these sensors are capable of detecting the optical changes induced by either immunological binding or DNA hybridization. Detection was made possible by the attachment of DNA probes or immunoglobulins (IgG) directly to the fiber surface via layer-by-layer electrostatic self-assembly. An optical fiber biosensor was tested using a standard transmission mode long period fiber grating of length 15 mm and period 260 µm, and coated with the IgG fraction of antiserum to F. tularensis. The IgG was deposited onto the optical fiber surface in a nanostructured film, and the resulting refractive index change was measured using spectroscopic ellipsometry. The presence of F. tularensis was detected from the decrease of peak wavelength caused by binding of specific antigen. Detection and differentiation of F. tularensis subspecies tularensis (type A strain TI0902) and subspecies holarctica (type B strain LVS) was further accomplished using a single-mode multi-cavity fiber Fabry-Perot interferometric sensor. These sensors were prepared by depositing seven polymer bilayers onto the fiber tip followed by attaching one of two DNA probes: (a) a 101-bp probe from the yhhW gene unique to type-A strains, or (b) a 117-bp probe of the lpnA gene, common to both type-A and type-B strains. The yhhW probe was reactive with the type-A, but not the type-B strain. Probe lpnA was reactive with both type-A and type-B strains. Nanogram quantities of the target DNA could be detected, highlighting the sensitivity of this method for DNA detection without the use of PCR. The DNA probe reacted with 100% homologous target DNA, but did not react with sequences containing 2-bp mismatches, indicating the high specificity of the assay. These assays will fill an important void that e

Předložená disertační práce se zabývá detekcí potenciálně aflatoxinogenních plísní rodu Aspergillus. V první části práce jsou optimalizovány a porovnávány metody izolace a imobilizace DNA. Druhá část práce je zaměřena na zavedení elektrochemické detekce hybridizace DNA pro potenciálně aflatoxinogenní plísně rodu Aspergillus., This dissertation deals with the detection of potentially aflatoxigenic Aspergillus species. In the first section the methods of isolation and immobilization of DNA are optimized and compared. The second part focuses on electrochemical detection of DNA hybridization for potentially aflatoxigenic Aspergillus species., Katedra analytické chemie, Před začátkem obhajoby konstatováno, ze oponentka Dr. Horáková se z vážných důvodů nemůže obhajoby zúčastnit. Předseda komise proto nechal hlasovat, přítomní členové komise vyslovili s obhajobou souhlas. Obhajoba tedy měla standardní průběh: - představení doktorandky a tématu disertační práce, seznámení s jejími publikovanými pracemi; - seznámení komise se stanoviskem školitelky a vedoucí KBBV; - doktorandka vyložila podstatný obsah a hlavní výsledky práce; - přítomná oponentka přednesla svůj posudek, za nepřítomnou její posudek přednesl předseda komise v plném znění; - doktorandka se vyjádřila k předneseným materiálům. Poté předseda komise zahájil veřejnou diskusi, při níž se doktorandka vyjádřila k dalším kladeným otázkám. Konstatováno, že zvenčí nepřišly k obhajované práci žádné připomínky. V neveřejné části obhajoby byl zhodnocen vědecký přínos práce Ing. Moťkové. Následovalo tajné hlasování., Dokončená práce s úspěšnou obhajobou

In this study, a gold nanoparticle (Au-NP)-based detection method for sensitive and specific DNA-based diagnostic applications is described. A sandwich format consisting of Au-NPs/DNA/PMP (Streptavidin-coated MagnetSphere Para-Magnetic Particles) was fabricated. PMPs captured and separated target DNA while Au-NPs modified with oligonucleotide detection sequences played a role in recognition and signal production. Due to the much lower stability of mismatched DNA strands caused by unstable duplex structures in solutions of relatively low salt concentration, hybridization efficiency in the presence of different buffers was well investigated, and thus, the optimized salt concentration allowed for discrimination of single-mismatched DNA (MMT) from perfectly matched DNA (PMT). Therefore, quantitative information concerning the target analyte was translated into a colorimetric signal, which could easily and quantitatively measured by low-cost UV–vis spectrophotometric analysis. The results indicated this to be a very simple and economic strategy for detection of single-mismatched DNA strands.

This work describes the synthesis and characterization of a new family of DNA amphiphiles containing modified nucleobases. The hydrophobicity was imparted by the introduction of a dodec-1-yne chain at the 5-position of the uracil base, which allowed precise and simple tuning of the hydrophobic properties through solid-phase DNA synthesis. The micelles formed from these modified DNA sequences were characterized by atomic force microscopy, dynamic light scattering, and polyacrylamide gel electrophoresis. These experiments revealed the role of the quantity and location of the hydrophobic units in determining the morphology and stability of the micelles. The effects of hybridization on the physical characteristics of the DNA micelles were also studied; these results showed potential for the sequence-specific noncovalent functionalization of the self-assembled aggregates.

Tato diplomová práce se zabývá elektrochemickou detekcí hybridizace DNA na povrchu uhlíkových elektrod. Na základě výsledků z předcházející studie, byl pro optimalizaci vybrán povrch uhlíkové pastové elektrody. Práce byla rozšířena pro uhlíkové tištěné elektrody, dále byla použita elektroda ze skelného uhlíku a elektrochemický senzor od firmy BVT. Jednotlivé kroky metody byly optimalizovány tak, aby umožnily lepší citlivost detekce. Kromě adsorpce DNA byla zkoušena hybridizace za vloženého potenciálu, z toho důvodu byla využita i úprava elektrody vloženým potenciálem. Dále bylo testováno několik druhů blokátorů, různá doba hybridizace, přítomnost látek urychlujících hybridizaci a další substráty vhodné pro alkalickou fosfatázu. K celé optimalizaci byly využity syntetické oligonukleotidy a sondy. Optimalizovaná metoda byla použita pro analýzu DNA potenciálně aflatoxinogenních plísní rodu Aspergillus. Vybraný úsek DNA kóduje biosyntézu aflatoxinu B1., The thesis deals with electrochemical detection of DNA hybridization on the surface of carbon paste electrode. Based on the results from the previous studies, the surface of the carbon paste electrode was chosen for better optimalization. The work was extended to the screen-printed carbon electrode, and then another glossy carbon electrode and an electrochemical sensor from the BTV Company were used. The particular steps of the method were optimized in order to allow better sensitivity of detection. In addition to the absorption of DNA, hybridization for the embedded potential was used and therefore special adjustments of the electrode were applied. Furthermore several types of blockers, different times of hybridization, the presence of substances accelerating the hybridization and other substrates suitable for alkaline phosphatase were also tested. In the whole optimalization synthetic oligonucleotides and probes were used. The optimized method was applied for DNA analysis of potentially aflatoxigenic fungi of genus Aspergillus. The selected segment of DNA encodes the biosynthesis of aflatoxin B1., Katedra analytické chemie, Dokončená práce s úspěšnou obhajobou

The technique of Immobilizing DNA in extended nano-space (10-1000 nm) was developed. This technique can be used for rolling circle amplification (RCA) and will finally make single DNA detection and nano-immunoassay possible. Here, we combine UV-mediated immobilization to DNA, examine two different methods for immobilization, and couple to padlock probe and RCA. Detection of fluorescence labeled DNA hybridization to RCA demonstrated that DNA analysis in minute volumes succeeded. Our technique can be served to detect single DNA, and we are developing this technique in extended nano-sapce to define the mechnisms of DNA amplification by record RCA in real time., QC 20150421

A novel anaerobic, thermophilic, Gram-positive, spore-forming, and sugar-fermenting bacterium (strain TLO) was isolated from a geothermal spring in Aya¿, Turkey. The cells were straight to curved rods, 0.4-0.6 mum in diameter and 3.5-10 mum in length. Spores were terminal and round. The temperature range for growth was 40-80 degrees C, with an optimum at 70 degrees C. The pH optimum was between 6.3 and 6.8. Strain TLO has the capability to ferment a wide variety of mono-, di-, and polysaccharides and proteinaceous substrates, producing mainly lactate, next to acetate, ethanol, alanine, H(2), and CO(2). Remarkably, the bacterium was able to grow in an atmosphere of up to 25% of CO as sole electron donor. CO oxidation was coupled to H(2) and CO(2) formation. The G + C content of the genomic DNA was 35.1 mol%. Based on 16S rRNA gene sequence analysis and the DNA-DNA hybridization data, this bacterium is most closely related to Thermoanaerobacter thermohydrosulfuricus and Thermoanaerobacter siderophilus (99% similarity for both). However, strain TLO differs from Thermoanaerobacter thermohydrosulfuricus in important aspects, such as CO-utilization and lipid composition. These differences led us to propose that strain TLO represents a subspecies of Thermoanaerobacter thermohydrosulfuricus, and we therefore name it Thermoanaerobacter thermohydrosulfuricus subsp. carboxydovorans

A screening analysis of DNA hybridization and the presence of DNA mutations using an surface plasmon resonance (SPR) biosensor is shown. The influence of lateral and vertical spacers, as well as several hybridization conditions, was studied to optimize the differentiation between fully complementary and mismatched DNA strands. Our results demonstrated that SPR biosensors were able to detect mismatch sequences related to inherited breast cancer, with high specificity and sensitivity. Using PCR synthetic sequences as targets, mutant sequences were clearly discriminated from fully complementary ones, and detection limits below 50 nM were achieved.

Tato disertační práce se zabývá možnostmi elektrochemické detekce nukleových sekvencí na uhlíkových elektrodách. Byly zde navrženy metody analýzy sekvence DNA (hybridizace DNA, exprese genů,jednonukleotidových polymorfismů a dalších) využívající enzym nebo různé elektroaktivní skupiny jako značky DNA., This dissertation thesis deals with possibilities of electrochemical analysis of nucleotide sequence using carbon electrodes. Methods for DNA sequence analysis (DNA hybridization, gene expression, single nucleotide sequence and others) utilizing enzyme or electroactive groups as DNA labels were proposed., Katedra analytické chemie, Dokončená práce s úspěšnou obhajobou

Ce travail s’inscrit dans le cadre de la recherche relative au développement de biosenseurs à ADN électrochimiques. Des aspects fondamentaux, ainsi que des aspects d’application de la détection d’hybridation d’ADN sont envisagés.Dans un premier temps, le comportement interfacial et le processus d’hybridation d’oligonucléotides d’ADN linéaires et ADN hairpin (structure en épingle à cheveux) nonmarqués sont étudiés en formant des monocouches auto-assemblées mixtes de monobrins d’ADN (ssADN) thiolés et d’un hydroxyalcanethiol (4-mercaptobutan-1-ol) par coadsorption spontanée sur des électrodes d’or polycristallin. L’immobilisation de monocouches mixtes ssADN/MCB est caractérisée par voie électrochimique et par spectroscopie des photoélectrons X. Des mesures de chronocoulométrie, en présence de [Ru(NH3)6]3+ (RuHex), permettent de déterminer la quantité d’ADN dans la monocouche mixte formée. Les résultats montrent que l’excès superficiel d’ADN linéaire est plus important que l’excès superficiel d’ADN hairpin sous des conditions de formation identiques.La réaction de reconnaissance moléculaire d’hybridation est détectée par des mesures d’impédance en présence de [Fe(CN)6]3-/4-. L’hybridation se traduit dans le cas de l’ADN linéaire par une augmentation de la résistance au transfert d’électron Rct tandis que dans le cas de l’ADN hairpin, Rct diminue. Ces différences sont dues au plus faible recouvrement et au changement de conformation des molécules d’ADN hairpin lors de l’hybridation. Des mesures de réflectivité de neutrons nous ont permis de mettre en évidence l’augmentation de l’épaisseur du film d’ADN hairpin et de confirmer le changement conformationel ces sondes lors de la reconnaissance moléculaire.Dans la seconde partie, nous présentons une nouvelle méthode électrochimique de détection d’hybridation, basée sur les interactions électrostatiques entre le complexe cationique RuHex et les groupements phosphates de l’ADN. Afin d’améliorer la détection des moléc, Doctorat en Sciences, info:eu-repo/semantics/nonPublished

For the development of field sensor systems with enough sensitivity and selectivity we develop photonic biosensors based on evanescent wave detection. Two technologies have been implemented in parallel: a plasmonic sensor for multianalyte real-time evaluation and an integrated optical nanosensor fabricated with silicon microelectronics technology. Both devices can be use as portable analyzers and have demonstrated an excellent performance in the immunological determination of chemical pollutants and in the detection of single mutations in DNA strands.

Diplomová práce se zabývá problematikou jednopovrchové metody elektrochemické detekce hybridizace DNA na povrchu uhlíkové pastové elektrody. Konkrétně sleduje tato práce možnost elektrochemické detekce hybridizace vybraného úseku DNA kódujícího biochemickou cestu syntézy aflatoxinu B1 u některých kmenů plísní rodu Aspergillus., The thesis deals with a single-surface electrochemical detection of hybridization DNA on carbon paste electrode. In this particular case it searches a possibility for electrochemical detection of DNA sequence, which codes a biochemical pathway for mycotoxin biosynthetis by some fungi of genus Aspergillus., Katedra analytické chemie, Dokončená práce s úspěšnou obhajobou

Pseudomonas putida KT2440 is the only fully sequenced P. putida strain. Thus, for transcriptomics and proteomics studies with other P. putida strains, the P. putida KT2440 genomic database serves as standard reference. The utility of KT2440 whole-genome, high-density oligonucleotide microarrays for transcriptomics studies of other Pseudomonas strains was investigated. To this end, microarray hybridizations were performed with genomic DNAs of subcultures of P. putida KT2440 (DSM6125), the type strain (DSM291T), plasmid pWW0-containing KT2440-derivative strain mt-2 (DSM3931), the solvent-tolerant P. putida S12, and several other Pseudomonas strains. Depending on the strain tested, 22 to 99% of all genetic elements were identified in the genomic DNAs. The efficacy of these microarrays to study cellular function was determined for all strains included in the study. The vast majority of DSM6125 genes encoding proteins of primary metabolism and genes involved in the catabolism of aromatic compounds were identified in the genomic DNA of strain S12: a prerequisite for reliable transcriptomics analyses. The genomotypic comparisons between Pseudomonas strains were used to construct highly discriminative phylogenetic relationships. DSM6125 and DSM3931 were indistinguishable and clustered together with strain S12 in a separate group, distinct from DSM291T. Pseudomonas monteilii (DSM14164) clustered well with P. putida strains. © 2007 Springer-Verlag.

Spurred by the Human Genome Project, deoxyribonucleic acid (DNA) microarrays are indispensable tools in molecular biology. In particular, they are used in the genetic profiling of human diseases, which includes identifying genes that are expressed in certain cancers. Genotyping allows more accurate identification and consequently, improved treatment. This technique has the potential to revolutionize the diagnosis of many other human ailments and be an important tool in the arsenal of modern medicine. At present however, microarray experiments involve complex protocols, often employing fluorescent labeling as well as sophisticated detection instruments. These systems are thus only affordable by very few large laboratories and pharmaceutical companies. In this work, we propose and demonstrate the feasibility of using a low-cost and improved alternative. We designed and fabricated biochips based on carbon nanotube field effect transistor arrays to detect the presence of specific DNA sequences, e.g. expressed genes, in a solution of DNA or RNA in the same manner as microarrays. The ultimate goal is to optimize the system to make it suitable for point-of-care applications. Our design utilizes CVD-grown carbon nanotube mats on a substrate of silicon oxide and metal contacts patterned using conventional microlithography. The carbon nanotube mats are covered by a thin oxide upon which single stranded DNA 'probe' molecules are immobilized. When exposed to a solution containing the complementary sequence, Watson-Crick hybridization leads to the binding of the complementary 'target' strands. Since DNA's are electrically charged through the excess electron of the phosphate backbone, this process results in the incorporation of additional negative charges at the transistor gate. This effectively causes a change in the conductance of the nanotube channel and a shift in the device threshold voltage. The voltage shift reflects the amount of extra charges deposited on the gate and b

Development of nucleic acid-based detection systems is the main focus of many research groups and high technology companies. The enormous work done in this field is particularly due to the broad versatility and variety of these sensing devices. From optical to electrical systems, from label-dependent to label-free approaches, from single to multi-analyte and array formats, this wide range of possibilities makes the research field very diversified and competitive. New challenges and requirements for an ideal detector suitable for nucleic acid analysis include high sensitivity and high specificity protocol that can be completed in a relatively short time offering at the same time low detection limit. Moreover, systems that can be miniaturized and automated present a significant advantage over conventional technology, especially if detection is needed in the field. Electrical system technology for nucleic acid-based detection is an enabling mode for making miniaturized to micro- and nanometer scale bio-monitoring devices via the fusion of modern micro- and nanofabrication technology and molecular biotechnology. The electrical biosensors that rely on the conversion of the Watson-Crick base-pair recognition event into a useful electrical signal are advancing rapidly, and recently are receiving much attention as a valuable tool for microbial pathogen detection. Pathogens may pose a serious threat to humans, animal and plants, thus their detection and analysis is a significant element of public health. Although different conventional methods for detection of pathogenic microorganisms and their toxins exist and are currently being applied, improvements of molecular-based detection methodologies have changed these traditional detection techniques and introduced a new era of rapid, miniaturized and automated electrical chip detection technologies into pathogen identification sector. In this review some developments and current directions in nucleic acid-based electrical detec, QC 20100525

A novel approach for extracting living cells’ genomic DNA materials utilizing functionalized magnetic particles (MPs) is reported in this investigation. This strategy is amenable to handle bio-samples in a miniaturized environment and it offers a possibility to separate and purify DNA from other cell lysate mixtures “on-chip”, which is known to be a bottle-neck step in an integrated micro-total-analysis-system (TAS). “Species-specific” genomic DNA of interest is captured by the MPs based on the hybridization interaction between the biotinylated probes modified MPs and a complementary region of the targeted genome. The genome DNA anchored on the particles can be separated from the rest of cellular mixtures by a simple buffer washing upon the exertion of external magnetic force. Surface modifications of MPs and hybridization conditions affecting the genome capturing efficiency are investigated. Extraction of genomic DNA from E. coli is demonstrated in a silicon/glass-based micro-reactor patterned with a platinum heater and sensors. On-chip extraction and manipulation of genomic DNAs illustrated in this study is a step forward toward a total integrated bioanalytical microsystem for crude cells/sample analysis. © 2005 Elsevier B.V. All rights reserved.

Two-dimensional micro-bubble actuator arrays were developed and studied in detail to enhance the hybridization kinetics of a DNA micro-biosensor. The hybridization between a molecular beacon, a kind of oligonucleotide probe, and its complement was investigated in a millimeter-sized PDMS based reaction chamber, where various 2D micro-heater arrays were distributed on the bottom for micro-bubble generation. The hybridization assay without the micro-bubble actuation revealed that the fluorescence increased fast at the beginning and slowed down after that. However, a uniform fluorescence increase was observed when periodic micro-bubble agitation was introduced in the static hybridization solution. A comparison of hybridization assays with and without micro-bubble agitation revealed that the hybridization time could be effectively shortened by 33\% with 10 cycles of micro-bubble agitation from a 2 x 1 bubble actuator array, and by 43\% with 10 cycles of micro-bubble agitation from a 2 x 2 bubble actuator array. (c) 2005 Elsevier B.V. All rights reserved.

This paper reports on a microdispensing system for fast, stably and uniformly printing microarrays of DNA or protein on the nylon membrane for biological assay. This pneumatic actuation system is different from other common printing methods and adopts contact spotting technique instead of droplet injection. As a key component in the system, a microdispensing chip is successfully designed and fabricated by employing multilayer SU-8 spin-coating and photolithograph technologies, and the chip can simultaneously produce up to 25 individual fluid spots with the average diameters of 384 mu m. A pressure of 10 kPa is optimal to dispense a 9:1 mixture of DMSO and percolated ink with the coefficient of variation (CV) of diameters as low as 2.6\%, high stability and good reproducibility. A 3:7 mixture of DNA probe and DMSO can be perfectly printed on a 5 x 5 array by our microdispensing system as well as by a conventional computerized robotic system. Comparative study of two hybridized results has proved that our microdispenser is suitable and practical to produce moderate or low density microar-ray for biological assay. (c) 2006 Published by Elsevier B.V.

Themavoordracht over de ontwikkelingen bij de GMO-veredeling: een verschuiving naar 'schone GMO's', zonder antibioticum en/of herbicideresistentiegenen en de beschikbaarheid van meer 'soorteigen genen'. De huidige intepretatie van de regelgeving rond GMO's is de grote bottle neck om nog als volwaardige spelers op het wereldtoneel mee te kunnen blijven doen

We combined microfluidic technology with recently developed algorithms of nucleic acid-based computing to demonstrate a compact, automated nucleotide-based computational device capable of rapid detection of computational output. We explored both experimental and theoretical aspects of the construction of microfluidic nucleic acid computing. Microreactors implementing Boolean operators lent themselves to a relatively simple implementation of DNA computing. On the theoretical side, we simulated the performance of individual reactors and designed small reactor systems (sorters) mimicking AND and OR gates and systems capable of solving complex Boolean expressions. We compared positive vs. negative selection and parallel vs. series arrangements of reactors, considering both the accuracy of and disproportionate representation ( bias ) within the final pool of solutions. We also developed O(P2) software that designs operating instructions for an O(NL) system capable of solving any Boolean expression. We developed software for automated data collection and signal analysis of electronic detection of labeled DNA and explored novel nanocrystal probes that enable detection of DNA for readout in on-chip electrical assays. We focused in the areas of methods to produce nanocrystals bearing precise numbers of oligonucleotides, and development of nanocrystal precise groupings that bear a collective signature., The original document contains color images. Funded in part by DARPA.

We have discovered that the current protocols to assemble Au nanoparticles based on DNA hybridization do not work well with the small metal nanoparticles (e.g. 5 nm Au, 3.6 nm Pt and 3.2 nm Ru particles). Further investigations revealed the presence of strong interaction between the oligonucleotide backbone and the surface of the small metal nanoparticles. The oligonucleotides in this case are recumbent on the particle surface and are therefore not optimally oriented for hybridization. The nonspecific adsorption of oligonucleotides on small metal nanoparticles must be overcome before DNA hybridization can be accepted as a general assembly method. Two methods have been suggested as possible solutions to this problem. One is based on the use of stabilizer molecules which compete with the oligonucleotides for adsorption on the metal nanoparticle surface. Unfortunately, the reported success of this approach in small Au nanoparticles (using K₂BSPP) and Au films (using 6-mercapto-1-hexanol) could not be extended to the assembly of Pt and Ru nanoparticles by DNA hybridization. The second approach is to simply use larger metal particles. Indeed most reports on the DNA hybridization induced assembly of Au nanoparticles have made use of relatively large particles (>10 nm), hinting at a weaker non-specific interaction between the oligonucleotides and large Au nanoparticles. However, most current methods of nanoparticle synthesis are optimized to produce metal nanoparticles only within a narrow size range. We find that core-shell nanoparticles formed by the seeded growth method may be used to artificially enlarge the size of the metal particles to reduce the nonspecific binding of oligonucleotides. We demonstrate herein a core-shell assisted growth method to assemble Pt and Ru nanoparticles by DNA hybridization. This method involves firstly synthesizing approximately 16 nm core-shell Ag-Pt and 21 nm core-shell Au-Ru nanoparticles from 9.6 nm Ag seeds and 17.2 nm Au seeds respec, Singapore-MIT Alliance (SMA)

The distribution of bacterial heterotrophic production and cell concentrations in the world oceans is well documented. Nevertheless, information on the distribution of specific bacterial taxa and how this is influenced by environmental factors in separate sea areas is sparse. Here we report on bacterial community structure across the Skagerrak-Kattegat front. The front separates North Sea water from water with a Baltic Sea origin. We documented community differences across the front using denaturing gradient gel electrophoresis (DGGE) analysis of PCR-amplified bacterial 16S ribosomal DNA and whole-genome DNA hybridization towards community DNA. Analysis of the community 'fingerprints' by DGGE revealed clustering of samples according to side of the front and depth. Consistent with these results, whole-genome DNA hybridization for 28 bacterial species isolated from the sampling region indicated that bacteria related to the Roseobacter clade and Bacteroidetes as well as prosthecate bacteria (e.g. Hyphomonas) showed distinct distribution patterns on each side of the front, and also with depth. Differences in bacterioplankton species composition across the frontal area were paralleled by differences in quantitative variables such as phytoplankton biomass (chl a), dissolved organic carbon, and bacterial production. Furthermore, we observed differences in more descriptive variables such as salinity range, bacterial growth-limiting nutrients and phytoplankton composition. We suggest that not only quantitative but also qualitative differences in variables that affect bacterial growth are required to cause divergence in bacterioplankton community composition between marine regions

An anaerobic, halorespiring bacterium (strain PCE-M2(T) = DSM 13726(T) = ATCC BAA-583(T)) able to reduce tetrachloroethene to cis-dichloroethene was isolated from an anaerobic soil polluted with chlorinated aliphatic compounds. The isolate is assigned to the genus Sulfurospirillum as a novel species, Sulfurospirillum halorespirans sp. nov. Furthermore, on the basis of all available data, a related organism, Dehalospirillum multivorans DSM 12446(T), is reclassified to the genus Sulfurospirillum as Sulfurospirillum multivorans comb. nov.

Branched oligonucleotides with symmetric arms 6-15, which may contain biotin as a second recognition code, were prepared. These molecules are designed to be used for the directed assembly of nanomaterials. The branched structure of the desired oligonucleotides was confirmed by mass spectrometry on small branched oliognucleotides, by gel electrophoresis, and by hybridization with complementary oligonucleotide - nanoparticle conjugates, followed by visualization of the complexes by transmission electron microscopy.

Production-scale synthesis of long DNAs with specific base sequences by conventional methods is generally infeasible due to error rates On the other hand, sticky-ended double-stranded (ds) DNA complexes can be joined into a single dsDNA complex with an enzyme called DNA ligase This idea offers a way to indirectly synthesize very long (>10kb) DNAs-by hybridization of a batch of single-stranded (ss) DNAs followed by ligation-with predetermined base sequences We analyze such synthesis protocols In the natural course of this development, we take a reasonably self-contained tour through much of what is known about the physics of DNA hybridization as it relates to the issues here This paper is therefore a primer on DNA self-assembly, using the staggered ligation model for DNA synthesis as the working example, The original document contains color images.

The Bead ARray Counter (BARC) is a multi-analyte biosensor that uses DNA hybridization, magnetic microbeads, and giant magnetoresistive (GMR) sensors to detect and identify biological warfare agents. The current prototype is a table-top instrument consisting of a microfabricated chip "solid substrate" with an array of GMR sensors, a chip carrier board with electronics for lock-in detection, a fluidics cell and cartridge, and an electromagnet. DNA probes are patterned onto the solid substrate chip directly above the GMR sensors, and sample analyte containing complementary DNA hybridizes with the probes on the surface. Labeled, micron-sized magnetic beads are then injected that specifically bind to the sample DNA. A magnetic field is applied, removing any beads that are not specifically bound to the surface. The beads remaining on the surface are detected by the GMR sensors, and the intensity and location of the signal indicate the concentration and identity of pathogens present in the sample. The current BARC chip contains a 64-element sensor array, however, with recent advances in magnetoresistive technology, chips with millions of these GMR sensors will soon be commercially available, allowing simultaneous detection of thousands of analytes. Because each GMR sensor is capable of detecting a single magnetic bead, in theory, the BARC biosensor should be able to detect the presence of a single analyte molecule., Pub. in Biosensors and Bioelectronics, v14, p805-813, 2000.

QC 20200113

Rice tungro bacilliform virus (RTBV) is one of the two viruses that cause tungro disease. Four RTBV strains maintained in the greenhouse for 4 years, G1, G2, Ic, and L, were differentiated by restriction fragment length polymorphism (RFLP) analysis of the native viral DNA. Although strains G1 and Ic had identical restriction patterns when cleaved with Pst1, BamHI, EcoRI, and EcoRV, they can be differentiated from strains G2 and L by EcoRI and EcoRV digestion. These same endonucleases also differentiate strain G2 from strain L. When total DNA extracts from infected plants were used instead of viral DNA, and digested with EcoRV, identical restriction patterns for each strain (G2 and L) were obtained from roots, leaves, and leaf sheaths of infected plants. The restriction patterns were consistent from plant to plant, in different varieties, and at different times after inoculation. This technique can be used to differentiate RTBV strains and determine the variability of a large number of field samples.

The ability to predict the rates of association of DNA to DNA have been used previously for those reactions occurring in a test tube. This study shows the ability to predict the binding rate of DNA to DNA within a fluid stream. The primary ligand was an oligonucleotide 20 basepairs attached to the dextran matrix in BlAcore. Efficiency of hybridization of this ligand to a secondary ligand of 40 basepairs (containing 20 complementary) was assessed. Association was predictable, based on ssDNA remaining at equilibrium, using second order rate kinetics. Changes in concentration encompassing one order of magnitude had little to no effect on the efficiency of hybridization.