62 results
Search Results
2. Efficient transfer of small DNA fragments from polyacrylamide gels to diazo or nitrocellulose paper and hybridization.
- Author
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Frei E, Levy A, Gowland P, and Noll M
- Subjects
- Base Composition, Collodion, DNA isolation & purification, Diazonium Compounds, Electrophoresis, Polyacrylamide Gel methods, Filtration methods, Indicators and Reagents, Molecular Weight, Paper, Base Sequence, DNA genetics, Nucleic Acid Hybridization
- Published
- 1983
- Full Text
- View/download PDF
3. Plasmid screening at high colony density.
- Author
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Hanahan D and Meselson M
- Subjects
- Autoradiography, Chloramphenicol pharmacology, DNA genetics, Escherichia coli genetics, Filtration methods, Gene Amplification drug effects, Paper, Radioisotopes, Base Sequence, DNA isolation & purification, Nucleic Acid Hybridization, Plasmids, RNA isolation & purification
- Published
- 1983
- Full Text
- View/download PDF
4. Rapid Visual Authentication Based on DNA Strand Displacement
- Author
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Alvin T. Liem, Kimberly L. Berk, In-Young Yang, Aleksandr E. Miklos, Matthew W. Lux, Steven M Blum, Yuhua Sun, Michael E. Hogan, Pierce A. Roth, Peter A. Emanuel, Vanessa L Funk, and Mark V. Gostomski
- Subjects
Paper ,0303 health sciences ,Authentication ,Time Factors ,Materials science ,Base Sequence ,Oligonucleotide ,Base pair ,Flashlight ,Reproducibility of Results ,DNA ,010402 general chemistry ,01 natural sciences ,Signal ,0104 chemical sciences ,Taggant ,03 medical and health sciences ,chemistry.chemical_compound ,chemistry ,DNA nanotechnology ,Nanotechnology ,General Materials Science ,Biological system ,030304 developmental biology - Abstract
Novel ways to track and verify items of a high value or security is an ever-present need. Taggants made from deoxyribonucleic acid (DNA) have several advantageous properties, such as high information density and robust synthesis; however, existing methods require laboratory techniques to verify, limiting applications. Here, we leverage DNA nanotechnology to create DNA taggants that can be validated in the field in seconds to minutes with a simple equipment. The system is driven by toehold-mediated strand-displacement reactions where matching oligonucleotide sequences drive the generation of a fluorescent signal through the potential energy of base pairing. By pooling different "input" oligonucleotide sequences in a taggant and spatially separating "reporter" oligonucleotide sequences on a paper ticket, unique, sequence-driven patterns emerge for different taggant formulations. Algorithmically generated oligonucleotide sequences show no crosstalk and ink-embedded taggants maintain activity for at least 99 days at 60 °C (equivalent to nearly 2 years at room temperature). The resulting fluorescent signals can be analyzed by the eye or a smartphone when paired with a UV flashlight and filtered glasses.
- Published
- 2021
5. Folding Paper-Based Aptasensor Platform Coated with Novel Nanoassemblies for Instant and Highly Sensitive Detection of 17β-Estradiol
- Author
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Jinping Luo, Yang Wang, Yu Xing, Tao Ming, Hongyan Jin, Xinxia Cai, Shuai Sun, Juntao Liu, and Guihua Xiao
- Subjects
Paper ,Working electrode ,Materials science ,Aptamer ,Metal Nanoparticles ,Bioengineering ,Nanotechnology ,Biosensing Techniques ,02 engineering and technology ,01 natural sciences ,chemistry.chemical_compound ,Limit of Detection ,Lab-On-A-Chip Devices ,Humans ,Electrodes ,Instrumentation ,Fluid Flow and Transfer Processes ,Detection limit ,Base Sequence ,Estradiol ,Nanotubes, Carbon ,Process Chemistry and Technology ,010401 analytical chemistry ,New methylene blue ,Electrochemical Techniques ,Folding (DSP implementation) ,Aptamers, Nucleotide ,Microfluidic Analytical Techniques ,021001 nanoscience & nanotechnology ,0104 chemical sciences ,Methylene Blue ,Linear range ,chemistry ,Point-of-Care Testing ,Colloidal gold ,Gold ,0210 nano-technology ,Luminescence - Abstract
Owing to its critical role in the development of female reproductive tissues and as a clinical biomarker, there is an urgent need to develop a rapid and cost-effective method to sensitively detect 17β-estradiol (E2). In this work, a folding aptasensor platform with microfluidic channels for the label-free electrochemical detection of E2 is described. The platform, designed with a delicate folding structure, integrating filter holes, microfluidic channels, reaction chambers, and a three-electrode system, is extremely easy to use. To increase the detection sensitivity and immobilize the aptamer, we synthesized a novel nanoassembly consisting of amine-functionalized single-walled carbon nanotube/new methylene blue/gold nanoparticles (AuNPs) and modified the working electrode with this nanoassembly. The calibration curve obtained from the experimental results exhibited a linear range between 10 pg mL-1 and 500 ng mL-1 (R2 = 0.993), and a detection limit of 5 pg mL-1 was achieved (S/N = 3). Furthermore, experiments to detect E2 in clinical serum were conducted, and the results were highly similar to those obtained using a large electrochemical luminescence apparatus. By integrating multiple functional components, adopting novel nanoassemblies, and using a folding structure, this paper-based platform not only has great potential as a simple and convenient integrated device for point-of-care testing of E2, but also as a portable, low-cost, and highly sensitive aptasensor platform capable of detecting many diagnostic biomarkers with the appropriate aptamers.
- Published
- 2019
6. Enzymatic amplification of oligonucleotides in paper substrates
- Author
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Abootaleb Sedighi and Ulrich J. Krull
- Subjects
Paper ,Oligonucleotides ,Loop-mediated isothermal amplification ,Nanoparticle ,010402 general chemistry ,01 natural sciences ,7. Clean energy ,Analytical Chemistry ,Matrix (chemical analysis) ,Endonuclease ,Quantum Dots ,Fluorescence Resonance Energy Transfer ,Exonuclease III ,Base Sequence ,biology ,Chemistry ,Oligonucleotide ,010401 analytical chemistry ,Endonucleases ,0104 chemical sciences ,Exodeoxyribonucleases ,Förster resonance energy transfer ,Quantum dot ,biology.protein ,Biophysics ,Nucleic Acid Amplification Techniques - Abstract
Several solution-based methods have recently been adapted for use in paper substrates for enzymatic amplification to increase the number of copies of DNA sequences. There is limited information available about the impact of a paper matrix on DNA amplification by enzymatic processes, and about how to optimize conditions to maximize yields. The work reported herein provides insights about the impact of physicochemical properties of a paper matrix, using nuclease-assisted amplification by exonuclease III and nicking endonuclease Nt. Bbv, and a quantum dot (QD) - based Forster Resonance Energy Transfer (FRET) assay to monitor the extent of amplification. The influence of several properties of paper on amplification efficiency and kinetics were investigated, such as surface adsorption of reactants, and pore size. Additional factors that impact amplification processes such as target length and the packing density of oligonucleotide probes on the nanoparticle surfaces were also studied. The work provides guidance for development of more efficient enzymatic target-recycling DNA amplification methods in paper substrates.
- Published
- 2018
7. An electrochemical paper based nano-genosensor modified with reduced graphene oxide-gold nanostructure for determination of glycated hemoglobin in blood
- Author
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Ali Ghaffarinejad, Seyedeh Yasaman Shajaripour Jaberi, and Eskandar Omidinia
- Subjects
Paper ,Aptamer ,Metal Nanoparticles ,Nanotechnology ,02 engineering and technology ,Biosensing Techniques ,01 natural sciences ,Biochemistry ,Analytical Chemistry ,law.invention ,Nanocomposites ,law ,Limit of Detection ,Environmental Chemistry ,Humans ,Electrodes ,Spectroscopy ,Detection limit ,Glycated Hemoglobin ,Nanocomposite ,Base Sequence ,Chemistry ,Graphene ,010401 analytical chemistry ,Reproducibility of Results ,DNA ,Electrochemical Techniques ,Aptamers, Nucleotide ,021001 nanoscience & nanotechnology ,0104 chemical sciences ,Linear range ,Electrode ,Graphite ,Differential pulse voltammetry ,Gold ,0210 nano-technology ,Biosensor - Abstract
Hemoglobin A1c (HbA1c) is a standard biomarker to measure long-term average glucose concentration for diagnosis and monitoring of diabetes. Various methods have been reported for measuring HbA1c, however, portable and precise determination is still challenging. Herein, a new highly sensitive electrochemical nanobiosensor is developed for the specific determination of HbA1c. A nanocomposite of reduced graphene oxide (rGO) and gold with hierarchical architecture structure was electrochemically deposited on a cheap and flexible graphite sheet (GS) electrode. The nanocomposite increased the surface area, improved the electron transfer on the electrode surface and augmented the signal. It also provided a suitable substrate for linkage of thiolated DNA aptamer as a bioreceptor on the electrode surface by strong covalent bonding. The quantitative label free detection was carried out by differential pulse voltammetry (DPV) in a phosphate-buffered saline (PBS) solution containing redox probe Fe(CN)63−/4-. The detection is based on insulating the surface in presence of HbA1c and decreasing the current, which is directly related to the HbA1c concentration. The nanobiosensor demonstrated high sensitivity of 269.2 μA. cm−2, wide linear range of 1 nM–13.83 μM with a low detection limit of 1 nM. The biosensor was successfully used for measuring HbA1c in blood real sample. Furthermore, it is promising to use it as a part of a point of care device for low-invasive screening and management of diabetes.
- Published
- 2019
8. Development of a fluorescence-based cellular apoptosis reporter
- Author
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Balderstone, Lucy A, Dawson, John C, Welman, Arkadiusz, Serrels, Alan, Wedge, Stephen R, and Brunton, Valerie G
- Subjects
Paper ,Cell Nucleus ,Microscopy, Confocal ,Base Sequence ,Caspase 3 ,caspase ,Recombinant Fusion Proteins ,Cell Membrane ,Green Fluorescent Proteins ,apoptosis ,Membrane Proteins ,Nuclear Proteins ,Fluorescence ,Mice ,Microscopy, Fluorescence ,Genes, Reporter ,fluorescent probe ,Cell Line, Tumor ,Animals ,fluorescence ,Single-Cell Analysis - Abstract
Evasion of apoptosis is a hallmark of human cancer, and a desired endpoint of many anticancer agents is the induction of cell death. With the heterogeneity of cancer becoming increasingly apparent, to understand drug mechanisms of action and identify combination therapies in cell populations, the development of tools to assess drug effects at the single cell level is a necessity for future preclinical drug development. Herein we describe the development of pCasFSwitch, a genetically encoded reporter construct designed to identify cells undergoing caspase-3 mediated apoptosis, by a translocation of a GFP signal from the cell membrane into the nucleus. Anticipated cellular distribution was demonstrated by use of confocal microscopy and cleavage by caspase-3 was shown to be required for the translocation of the GFP signal seen in apoptotic cells. Quantification of apoptosis using the construct revealed similar levels to that obtained with a commercially available apoptosis imaging agent (22.6% versus 20.3%). Moreover, we demonstrated its capacity for use in a high-throughput setting making it a powerful tool for drug development pipelines.
- Published
- 2018
9. A paper-based resonance energy transfer nucleic acid hybridization assay using upconversion nanoparticles as donors and quantum dots as acceptors
- Author
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Ulrich J. Krull, Uvaraj Uddayasankar, and Samer Doughan
- Subjects
Paper ,Hypoxanthine Phosphoribosyltransferase ,Analytical chemistry ,Conjugated system ,7. Clean energy ,Biochemistry ,Analytical Chemistry ,Fluorides ,Quantum Dots ,Animals ,Humans ,Environmental Chemistry ,Yttrium ,Ytterbium ,Spectroscopy ,Detection limit ,Genes, Essential ,Luminescent Agents ,Base Sequence ,Chemistry ,Oligonucleotide ,Nucleic Acid Hybridization ,DNA ,Combinatorial chemistry ,Photon upconversion ,Covalent bond ,Quantum dot ,Luminescent Measurements ,Nanoparticles ,Oligonucleotide Probes ,Selectivity ,Luminescence - Abstract
Monodisperse aqueous upconverting nanoparticles (UCNPs) were covalently immobilized on aldehyde modified cellulose paper via reduction amination to develop a luminescence resonance energy transfer (LRET)-based nucleic acid hybridization assay. This first account of covalent immobilization of UCNPs on paper for a bioassay reports an optically responsive method that is sensitive, reproducible and robust. The immobilized UCNPs were decorated with oligonucleotide probes to capture HPRT1 housekeeping gene fragments, which in turn brought reporter conjugated quantum dots (QDs) in close proximity to the UCNPs for LRET. This sandwich assay could detect unlabeled oligonucleotide target, and had a limit of detection of 13 fmol and a dynamic range spanning nearly 3 orders of magnitude. The use of QDs, which are excellent LRET acceptors, demonstrated improved sensitivity, limit of detection, dynamic range and selectivity compared to similar assays that have used molecular fluorophores as acceptors. The selectivity of the assay was attributed to the decoration of the QDs with polyethylene glycol to eliminate non-specific adsorption. The kinetics of hybridization were determined to be diffusion limited and full signal development occurred within 3 min.
- Published
- 2015
10. Linkage inversion assembled nano-aptasensors (LIANAs) for turn-on fluorescence detection
- Author
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Maria C. DeRosa, Nadine R. Frost, and Ranganathan Velu
- Subjects
Crops, Agricultural ,Paper ,Specific detection ,Aptamer ,Molecular Sequence Data ,Nanotechnology ,Biosensing Techniques ,Fluorescence ,Catalysis ,Nano ,Materials Chemistry ,Base sequence ,Base Sequence ,Chemistry ,Metals and Alloys ,General Chemistry ,Aptamers, Nucleotide ,Mycotoxins ,Ochratoxins ,Surfaces, Coatings and Films ,Electronic, Optical and Magnetic Materials ,Solutions ,Liana ,Ceramics and Composites ,Biosensor - Abstract
A strategy for aptamer-based biosensing termed linkage inversion assembled nano-aptasensors (LIANAs) is shown to be a generally applicable approach to the sensitive and specific detection of a target molecule in turn-on fluorescence solution-based and paper-based tests.
- Published
- 2015
11. Scalable Low-Cost Fabrication of Disposable Paper Sensors for DNA Detection
- Author
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Dennis Nordlund, M. Meyyappan, Vivek Jayan, Jessica E. Koehne, and Ram P. Gandhiraman
- Subjects
Paper ,Materials science ,Fabrication ,Molecular Sequence Data ,Nanotechnology ,Biosensing Techniques ,02 engineering and technology ,Chemical vapor deposition ,010402 general chemistry ,Sensitivity and Specificity ,01 natural sciences ,X-ray absorption ,NEXAFS ,Deposition (phase transition) ,General Materials Science ,Sensitivity (control systems) ,Disposable Equipment ,Absorption (electromagnetic radiation) ,Oligonucleotide Array Sequence Analysis ,DNA detection ,cellulose functionalization ,Base Sequence ,Reproducibility of Results ,DNA ,Equipment Design ,021001 nanoscience & nanotechnology ,Chip ,paper sensors ,0104 chemical sciences ,Equipment Failure Analysis ,Systems Integration ,Surface modification ,0210 nano-technology ,Biosensor ,Research Article - Abstract
Controlled integration of features that enhance the analytical performance of a sensor chip is a challenging task in the development of paper sensors. A critical issue in the fabrication of low-cost biosensor chips is the activation of the device surface in a reliable and controllable manner compatible with large-scale production. Here, we report stable, well-adherent, and repeatable site-selective deposition of bioreactive amine functionalities and biorepellant polyethylene glycol-like (PEG) functionalities on paper sensors by aerosol-assisted, atmospheric-pressure, plasma-enhanced chemical vapor deposition. This approach requires only 20 s of deposition time, compared to previous reports on cellulose functionalization, which takes hours. A detailed analysis of the near-edge X-ray absorption fine structure (NEXAFS) and its sensitivity to the local electronic structure of the carbon and nitrogen functionalities. σ*, π*, and Rydberg transitions in C and N K-edges are presented. Application of the plasma-processed paper sensors in DNA detection is also demonstrated.
- Published
- 2014
12. Multiplex Paper-Based Colorimetric DNA Sensor Using Pyrrolidinyl Peptide Nucleic Acid-Induced AgNPs Aggregation for Detecting MERS-CoV, MTB, and HPV Oligonucleotides
- Author
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Charles S. Henry, Orawon Chailapakul, Weena Siangproh, Tirayut Vilaivan, Prinjaporn Teengam, and Adisorn Tuantranont
- Subjects
DNA, Bacterial ,Paper ,Peptide Nucleic Acids ,Silver ,Oligonucleotides ,Metal Nanoparticles ,02 engineering and technology ,01 natural sciences ,Silver nanoparticle ,Analytical Chemistry ,chemistry.chemical_compound ,Nucleic acid thermodynamics ,Limit of Detection ,Complementary DNA ,Image Processing, Computer-Assisted ,Humans ,Multiplex ,Papillomaviridae ,Peptide nucleic acid ,Base Sequence ,Chemistry ,Oligonucleotide ,010401 analytical chemistry ,RNA ,Nucleic Acid Hybridization ,DNA ,Mycobacterium tuberculosis ,021001 nanoscience & nanotechnology ,0104 chemical sciences ,Biochemistry ,DNA, Viral ,Middle East Respiratory Syndrome Coronavirus ,Colorimetry ,0210 nano-technology - Abstract
The development of simple fluorescent and colorimetric assays that enable point-of-care DNA and RNA detection has been a topic of significant research because of the utility of such assays in resource limited settings. The most common motifs utilize hybridization to a complementary detection strand coupled with a sensitive reporter molecule. Here, a paper-based colorimetric assay for DNA detection based on pyrrolidinyl peptide nucleic acid (acpcPNA)-induced nanoparticle aggregation is reported as an alternative to traditional colorimetric approaches. PNA probes are an attractive alternative to DNA and RNA probes because they are chemically and biologically stable, easily synthesized, and hybridize efficiently with the complementary DNA strands. The acpcPNA probe contains a single positive charge from the lysine at C-terminus and causes aggregation of citrate anion-stabilized silver nanoparticles (AgNPs) in the absence of complementary DNA. In the presence of target DNA, formation of the anionic DNA-acpcPNA duplex results in dispersion of the AgNPs as a result of electrostatic repulsion, giving rise to a detectable color change. Factors affecting the sensitivity and selectivity of this assay were investigated, including ionic strength, AgNP concentration, PNA concentration, and DNA strand mismatches. The method was used for screening of synthetic Middle East respiratory syndrome coronavirus (MERS-CoV), Mycobacterium tuberculosis (MTB), and human papillomavirus (HPV) DNA based on a colorimetric paper-based analytical device developed using the aforementioned principle. The oligonucleotide targets were detected by measuring the color change of AgNPs, giving detection limits of 1.53 (MERS-CoV), 1.27 (MTB), and 1.03 nM (HPV). The acpcPNA probe exhibited high selectivity for the complementary oligonucleotides over single-base-mismatch, two-base-mismatch, and noncomplementary DNA targets. The proposed paper-based colorimetric DNA sensor has potential to be an alternative approach for simple, rapid, sensitive, and selective DNA detection.
- Published
- 2017
13. Capture and Detection of DNA Hybrids on Paper via the Anchoring of Antibodies with Fusions of Carbohydrate Binding Modules and ZZ-Domains
- Author
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Joao Inacio, Sofia Martins, Ana Margarida Azevedo, Ana Rosa, Samir Martins, Duarte Miguel Prazeres, and Ana Filipa Louro
- Subjects
Paper ,Binding Sites ,Base Sequence ,biology ,Carbohydrates ,Nucleic Acid Hybridization ,DNA ,biology.organism_classification ,Fusion protein ,Antibodies ,Analytical Chemistry ,chemistry.chemical_compound ,Biochemistry ,Biotin ,chemistry ,biology.protein ,Clostridium thermocellum ,Carbohydrate-binding module ,Bioactive paper ,Antibody ,Protein A - Abstract
Microfluidic paper-based analytical devices (μPADs) fabricated by wax-printing are suitable platforms for the development of simple and affordable molecular diagnostic assays for infectious diseases, especially in resource-limited settings. Paper devices can be modified for biological assays by adding appropriate reagents to the test areas. For this purpose, the use of affinity immobilization strategies can be a good solution for bioactive paper fabrication. This paper describes a methodology to capture labeled-DNA strands and hybrids on paper via the anchoring of antibodies with a fusion protein that combines a family 3 carbohydrate binding module (CBM) from Clostridium thermocellum, with high affinity to cellulose, and the ZZ fragment of the staphyloccocal protein A, which recognizes IgG antibodies via their Fc portion. Antibodies immobilized via CBM-ZZ were able to capture appropriately labeled (biotin, fluorescein) DNA strands and DNA hybrids. The ability of an antibody specific to biotin to discriminate complementary from noncomplementary, biotin-labeled targets was demonstrated in both spot and microchannel assays. Hybridization was detected by fluorescence emission of the fluorescein-labeled DNA probe. The efficiency of the capture of labeled-DNA by antibodies immobilized on paper via the CBM-ZZ construct was significantly higher when compared with a physical adsorption method where antibodies were simply spotted on paper without the intermediation of other molecules. The experimental proof of concept of wax-printed μPADs functionalized with CBM-ZZ for DNA detection at room temperature presented in this study constitutes an important step toward the development of easy to use and affordable molecular diagnostic tests.
- Published
- 2014
14. DNA Detection Using Origami Paper Analytical Devices
- Author
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Bingling Li, Andrew D. Ellington, Richard M. Crooks, and Karen Scida
- Subjects
Paper ,Detection limit ,Analyte ,Base Sequence ,Logic ,Molecular Sequence Data ,Relative standard deviation ,Analytical chemistry ,DNA, Single-Stranded ,Nucleic Acid Hybridization ,Substrate (chemistry) ,Fluorescence ,Article ,Chemistry Techniques, Analytical ,Analytical Chemistry ,Dna detection ,Computers, Molecular ,Nucleic acid thermodynamics ,chemistry.chemical_compound ,chemistry ,DNA ,Fluorescent Dyes - Abstract
We demonstrate the hybridization-induced fluorescence detection of DNA on an origami-based paper analytical device (oPAD). The paper substrate was patterned by wax printing and controlled heating to construct hydrophilic channels and hydrophobic barriers in a three-dimensional fashion. A competitive assay was developed where the analyte, a single-stranded DNA (ssDNA), and a quencher-labeled ssDNA competed for hybridization with a fluorophore-labeled ssDNA probe. Upon hybridization of the analyte with the fluorophore-labeled ssDNA, a linear response of fluorescence vs. analyte concentration was observed with an extrapolated limit of detection < 5 nM and a sensitivity relative standard deviation as low as 3%. The oPAD setup was also tested against OR/AND logic gates, proving to be successful in both detection systems.
- Published
- 2013
15. Paper-Based Solid-Phase Multiplexed Nucleic Acid Hybridization Assay with Tunable Dynamic Range Using Immobilized Quantum Dots As Donors in Fluorescence Resonance Energy Transfer
- Author
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M. Omair Noor and Ulrich J. Krull
- Subjects
Models, Molecular ,Paper ,Detection limit ,Base Sequence ,Oligonucleotide ,Chemistry ,Dynamic range ,Oligonucleotides ,Analytical chemistry ,Color ,Nucleic Acid Hybridization ,Polymorphism, Single Nucleotide ,Acceptor ,Combinatorial chemistry ,Analytical Chemistry ,Nucleic acid thermodynamics ,Förster resonance energy transfer ,Quantum dot ,Quantum Dots ,Fluorescence Resonance Energy Transfer ,Nucleic Acid Conformation ,Adsorption ,Alexa Fluor - Abstract
A multiplexed solid-phase nucleic acid hybridization assay on a paper-based platform is presented using multicolor immobilized quantum dots (QDs) as donors in fluorescence resonance energy transfer (FRET). The surface of paper was modified with imidazole groups to immobilize two types of QD-probe oligonucleotide conjugates that were assembled in solution. Green-emitting QDs (gQDs) and red-emitting QDs (rQDs) served as donors with Cy3 and Alexa Fluor 647 (A647) acceptors. The gQD/Cy3 FRET pair served as an internal standard, while the rQD/A647 FRET pair served as a detection channel, combining the control and analytical test zones in one physical location. Hybridization of dye-labeled oligonucleotide targets provided the proximity for FRET sensitized emission from the acceptor dyes, which served as an analytical signal. Hybridization assays in the multicolor format provided a limit of detection of 90 fmol and an upper limit of dynamic range of 3.5 pmol. The use of an array of detection zones was designed to provide improved analytical figures of merit compared to that which could be achieved on one type of array design in terms of relative concentration of multicolor QDs. The hybridization assays showed excellent resistance to nonspecific adsorption of oligonucleotides. Selectivity of the two-plex hybridization assay was demonstrated by single nucleotide polymorphism (SNP) detection at a contrast ratio of 50:1. Additionally, it is shown that the use of preformed QD-probe oligonucleotide conjugates and consideration of the relative number density of the two types of QD-probe conjugates in the two-color assay format is advantageous to maximize assay sensitivity and the upper limit of dynamic range.
- Published
- 2013
16. Determination of cocaine on banknotes through an aptamer-based electrochemiluminescence biosensor
- Author
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Zhenyu Lin, Guonan Chen, Lifen Chen, Bin Qiu, Qihong Cai, and Fang Luo
- Subjects
Paper ,Detection limit ,Luminescence ,Chromatography ,Base Sequence ,Chemistry ,Aptamer ,chemistry.chemical_element ,Biosensing Techniques ,Biochemistry ,Analytical Chemistry ,Ruthenium ,Cocaine ,Reagent ,Electrode ,Electrochemistry ,Nanoparticles ,Electrochemiluminescence ,DNA Probes ,Biosensor - Abstract
A novel electrochemiluminescence (ECL) "sandwich" biosensor has been developed to detect cocaine. The sandwich biosensor was fabricated on the basis of the fact that a single aptamer could be split into two fragments and the two dissociated parts could form a folded, associated complex in the presence of targets. One of these (capture probe), which had hexane-thiol at its 5'-terminus, was immobilized on a gold electrode via thiol-gold binding. The other one (detection probe) was labeled with the ECL reagent tris(2,2'-bipyridyl)ruthenium(II)-doped silica nanoparticles (RuSiNPs) at its 3'-terminus. Owing to the weak interaction between the two fragments, the sensor exhibited a low ECL signal in the absence of cocaine. After the target cocaine had been added to the solution, it induced association of the two fragments and stabilized the associated complexes, leading to immobilization of RuSiNPs on the electrode surface, and the ECL detected on the electrode surface was enhanced. The enhanced ECL intensity was directly proportional to the logarithm of the cocaine concentration in the range from 1.0 × 10(-9) to 1.0 × 10(-11) mol/L, with a detection limit of 3.7 × 10(-12) mol/L. The biosensor was applied to detect trace amounts of cocaine on banknotes with satisfactory results.
- Published
- 2011
17. Genetic and functional diversity of Bacillus strains in the soils long-term irrigated with paper and pulp mill effluent
- Author
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Dilip K. Arora, Anil Kumar Saxena, Subhash Yadav, and Rajeev Kaushik
- Subjects
Paper ,Irrigation ,Veterinary medicine ,Agricultural Irrigation ,Molecular Sequence Data ,India ,Industrial Waste ,Bacillus ,Fresh Water ,Waste Disposal, Fluid ,Applied Microbiology and Biotechnology ,Microbiology ,Soil ,Paenibacillus ,Diversity index ,RNA, Ribosomal, 16S ,Botany ,Cluster Analysis ,Soil Pollutants ,Bacillaceae ,Effluent ,Phylogeny ,Soil Microbiology ,Base Sequence ,biology ,business.industry ,Genetic Variation ,Paper mill ,Sequence Analysis, DNA ,Hydrogen-Ion Concentration ,biology.organism_classification ,Amplified Ribosomal DNA Restriction Analysis ,Phenotype ,Soil water ,Species richness ,business ,Water Pollutants, Chemical - Abstract
The genetic and functional diversity of Bacillus and Bacillus-derived genera was analyzed in soil samples collected from three different fields near Century Paper Mill, Lal Kuan, Uttarakhand, India. Two had been subjected to concentrated and diluted effluent irrigation for the past 25 years and were designated as a concentrated effluent irrigated field (CEIF) and a dilute effluent irrigated field (DEIF), respectively. The field irrigated with fresh water was designated as a water irrigated field (WIF). Increase in pH, Na and Zn content and decrease in Fe content was observed due to effluent irrigation. The population count of Bacillus and Bacillus-derived was maximum in DEIF followed by WIF and CEIF. Variations in plant growth-promoting traits and extracellular enzymes were observed among the isolates from the three different field soils. Based on the amplified ribosomal DNA restriction analysis (ARDRA) with three restriction enzymes, all the selected 104 isolates were clustered into 14 groups. The sequencing of the representative isolates revealed that the majority belonged to the genus Bacillus, while three isolates belonged to Paenibacillus, Lysinibacillus and Orthinibacillus. There were a few species like Orthinibacillus contaminans, B. oleronius, B. safensis, B. methylotrophicus, B. stratosphericus, B. aryabhattai, B. asahii and B. bataviensis that were prevalent only in DEIF and CEIF but not in WIF field soil. The diversity index parameters like the Shannon Index, indices of species richness and species evenness based on biochemical profiling and ARDRA profiling revealed that Bacillaceae members in the fresh water irrigated field were diverse.
- Published
- 2011
18. Use of metabolic and molecular methods for the identification of a Bacillus strain isolated from paper affected by foxing
- Author
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Maria Rita De Paolis and D. Lippi
- Subjects
Bacillus (shape) ,DNA, Bacterial ,Paper ,biology ,Base Sequence ,Strain (biology) ,Foxing ,Molecular Sequence Data ,Bacillus ,Sequence Analysis, DNA ,biology.organism_classification ,16S ribosomal RNA ,DNA, Ribosomal ,Microbiology ,Bacterial Typing Techniques ,Random Amplified Polymorphic DNA Technique ,Paenibacillus ,RNA, Ribosomal, 16S ,Bacillus circulans ,Paenibacillus polymyxa ,Sequence Alignment ,Bacteria ,Phylogeny - Abstract
Foxing of paper is a deterioration phenomenon occurring in the form of brown-yellowish spots, the abiotic and/or biotic causes of which are not yet completely understood. Nevertheless, microbiological infection has been recognized that may contribute to paper damage and therefore it becomes important to know the taxonomic position and the degradative activity of the potential infectious biological agents which mostly are fungi, but also bacteria and yeasts. A cellulolytic bacterial strain isolated from a foxed paper sample exhibited morphological and physiological characteristics of the Bacillus genus. To study its taxonomic position, different identification methods were used: the Biolog system, the direct amplified polymorphic DNA-polymerase chain reaction analysis (DAPD-PCR) and the partial sequencing of the 16S rDNA gene. Biolog system and partial sequencing of 16S rDNA gene assigned the strain to the Paenibacillus polymyxa species. DAPD-PCR analysis indicated a high similarity with Bacillus circulans, by comparing the isolated strain with some closely related Bacillus species.
- Published
- 2008
- Full Text
- View/download PDF
19. The feasibility of storing ovarian tumor cells on databasing paper: establishing a library of ovarian cancer DNA
- Author
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L. Allcroft, Richard J. Edmondson, Khadra Galaal, R. Hussain, M. Meirovitz, Alberto Lopes, and N. Sullivan
- Subjects
Paper ,Molecular Sequence Data ,Biology ,Specimen Handling ,chemistry.chemical_compound ,Ovarian tumor ,Hemocytometer ,Complementary DNA ,Humans ,Genomic library ,RNA, Neoplasm ,Gene Library ,Ovarian Neoplasms ,Base Sequence ,Reverse Transcriptase Polymerase Chain Reaction ,Obstetrics and Gynecology ,RNA ,DNA, Neoplasm ,Molecular biology ,Real-time polymerase chain reaction ,Oncology ,chemistry ,Nucleic acid ,Feasibility Studies ,Female ,DNA - Abstract
The purpose of this study was to assess the feasibility of establishing a library of ovarian cancer nucleic acids using paper matrix by: 1) confirming the stability of DNA stored on paper matrix over a prolonged period of time, 2) determining the amount of genetic material required for storage, and 3) establishing the stability of RNA. Tumor tissue from 66 patients with ovarian cancer was collected intraoperatively, frozen, and dissociated with collagenase and trypsin. A cell suspension was then prepared and spotted onto the paper. The numbers of cells that were stored on the paper were counted using a hemocytometer. The cell suspension was serially diluted and spotted on the paper matrix until the minimum cell number that can be stored and produce a PCR product was determined. PCR, STR genotyping and direct sequencing were performed on tissue stored on the paper matrix. FTA® paper was used as RNA template, and RT PCR converted the RNA to cDNA. Ten to 50 mg of ovarian cancer tissue was stored on FTA® paper. We stored 7 × 104cells on ISOcode® paper and 18 × 104cells on FTA® and obtained extractable DNA. PCR analysis on cards with DNA stored 18 months ago enabled us to establish the stability of DNA after storage. RNA was stable for 6 months when stored on FTA® cards. Since genetic material is extractable from the paper matrices after passage of time, it could be a suitable medium for the storage of genetic material in cancer tissue banks.
- Published
- 2007
20. Potentiality of Eisenia fetida to degrade disposable paper cups-an ecofriendly solution to solid waste pollution
- Author
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Vivekanadhan Munusamy, Susila Sugumar, Karthika Arumugam, Swabna Vivek, Vasanthy Muthunarayanan, and Seethadevi Ganesan
- Subjects
Paper ,Eisenia fetida ,Municipal solid waste ,business.product_category ,Health, Toxicology and Mutagenesis ,Molecular Sequence Data ,Bacillus cereus ,India ,Paper cup ,engineering.material ,Feces ,Soil ,RNA, Ribosomal, 16S ,Spectroscopy, Fourier Transform Infrared ,Environmental Chemistry ,Animals ,Food science ,Oligochaeta ,Soil Microbiology ,Waste management ,biology ,Bacteria ,Base Sequence ,Chemistry ,Compost ,Sequence Analysis, RNA ,General Medicine ,biology.organism_classification ,Pollution ,Manure ,Refuse Disposal ,Biodegradation, Environmental ,engineering ,Cattle ,Electrophoresis, Polyacrylamide Gel ,business ,Vermicompost ,Cow dung - Abstract
The aim of the present study was to subject the post-consumer waste, namely paper cups for vermicomposting along with cow dung in three different ratios for a period of 90–140 days employing Eisenia fetida. The post-consumer wastes are a menace in many developing countries including India. This waste was provided as feed for earthworms and was converted to vermicompost. Vermicompost prepared with paper cup waste was analyzed for their physicochemical properties. Based on the physicochemical properties, it was evident that the best manure is obtained from type A (paper cup/cow dung in the ratio 1:1) than type B (paper cup/cow dung in the ratio 1.5:0.5) and type C (paper cup/cow dung in the ratio 0.5:1.5). The results showed that earthworms accelerated the rate of mineralization and converted the wastes into compost with needed elements which could support the growth of crop plants. The predominant bacterial strains in the vermicompost were characterized biochemically as well as by 16S ribosomal RNA (rRNA) gene sequencing. The bacterial strains like Bacillus anthracis (KM289159), Bacillus endophyticus (KM289167), Bacillus funiculus (KM289165), Virigibacillius chiquenigi (KM289163), Bacillus thuringiensis (KM289164), Bacillus cereus (KM289160), Bacillus toyonensis (KM289161), Acinetobacter baumanni (KM289162), and Lactobacillus pantheries (KM289166) were isolated and identified from the final compost. The total protein content of E. fetida involved in vermicomposting was extracted, and the banding pattern was analyzed. During final stages of vermicomposting, it was observed that the earthworm did not act on the plastic material coated inside the paper cups and stagnated it around the rim of the tub. Further, the degradation of paper cup waste was confirmed by Fourier transform infrared spectroscopy analysis. Hence, vermicomposting was found to be an effective technology for the conversion of the paper cup waste material into a nutrient-rich manure, a value-added product.
- Published
- 2014
21. Bioaugmentation with resin-acid-degrading bacteria enhances resin acid removal in sequencing batch reactors treating pulp mill effluents
- Author
-
William W. Mohn and Zhongtang Yu
- Subjects
Paper ,Bioaugmentation ,Environmental Engineering ,Ribosomal Intergenic Spacer analysis ,Sequencing batch reactor ,Polymerase Chain Reaction ,Waste Disposal, Fluid ,Water Purification ,Bioreactors ,Pseudomonas ,Waste Management and Disposal ,DNA Primers ,Water Science and Technology ,Civil and Structural Engineering ,Base Sequence ,biology ,Waste management ,Ecological Modeling ,technology, industry, and agriculture ,Betaproteobacteria ,Hydrogen-Ion Concentration ,Biodegradation ,biology.organism_classification ,Pulp and paper industry ,Pollution ,Biodegradation, Environmental ,Activated sludge ,Abietanes ,Resin acid ,Diterpenes ,Water Pollutants, Chemical ,Bacteria ,Pseudomonas abietaniphila - Abstract
Resin acids are the major toxicants in pulp and paper mill effluents (PPMEs), and they form pitch interfering with papermaking. Efficient and reliable resin acid removal is critically important to prevent toxicity discharge and ensure proper functioning of paper machines. Two resin-acid-degrading bacteria, Pseudomonas abietaniphila BKME-9 and Zoogloea resiniphila DhA-35, were tested in laboratory sequencing batch reactors (SBRs) for their ability to enhance resin acid removal by biomass from a full-scale biotreatment system treating PPMEs. Both bacteria enhanced resin acid removal but not removal of total organic carbon (TOC) by either pH-shocked or starved activated sludge. These two bacteria also increased resin acid removal when the sludge was given high concentration (200 μM) of resin acid. A most-probable-number polymerase chain reaction (MPN-PCR) assay showed that these two bacteria were initially not detectable (detection limit: 102 bacterial cells/ml) in the sludge community and were persistent after inoculation. Both bacteria did not substantially change the indigenous microbial community composition, as assayed by ribosomal intergenic spacer analysis (RISA). Our results suggest that it is feasible and potentially useful to enhance resin acid removal by bioaugmentation using resin-acid-degrading bacteria such as BKME-9 and DhA-35.
- Published
- 2001
22. Coliform Bacteria and Nitrogen Fixation in Pulp and Paper Mill Effluent Treatment Systems
- Author
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Frederick Archibald, Brian T. Driscoll, Francis Gauthier, and Josh D. Neufeld
- Subjects
Paper ,Microorganism ,engineering.material ,Biology ,complex mixtures ,Waste Disposal, Fluid ,Applied Microbiology and Biotechnology ,Microbial Ecology ,Microbiology ,Bioreactors ,Enterobacteriaceae ,Klebsiella ,Nitrogen Fixation ,Effluent ,Ecosystem ,DNA Primers ,Base Sequence ,Ecology ,business.industry ,Pulp (paper) ,technology, industry, and agriculture ,food and beverages ,Paper mill ,Pulp and paper industry ,Coliform bacteria ,Biodegradation, Environmental ,Activated sludge ,Genes, Bacterial ,Nitrogen fixation ,engineering ,Water Microbiology ,business ,Food Science ,Biotechnology ,Waste disposal - Abstract
The majority of pulp and paper mills now biotreat their combined effluents using activated sludge. On the assumption that their wood-based effluents have negligible fixed N, and that activated-sludge microorganisms will not fix significant N, these mills routinely spend large amounts adding ammonia or urea to their aeration tanks (bioreactors) to permit normal biomass growth. N 2 fixation in seven Eastern Canadian pulp and paper mill effluent treatment systems was analyzed using acetylene reduction assays, quantitative nitrogenase ( nifH ) gene probing, and bacterial isolations. In situ N 2 fixation was undetectable in all seven bioreactors but was present in six associated primary clarifiers. One primary clarifier was studied in greater detail. Approximately 50% of all culturable cells in the clarifier contained nifH , of which >90% were Klebsiella strains. All primary-clarifier coliform bacteria growing on MacConkey agar were identified as klebsiellas, and all those probed contained nifH . In contrast, analysis of 48 random coliform isolates from other mill water system locations showed that only 24 (50%) possessed the nifH gene, and only 13 (27%) showed inducible N 2 -fixing activity. Thus, all the pulp and paper mill primary clarifiers tested appeared to be sites of active N 2 fixation (0.87 to 4.90 mg of N liter −1 day −1 ) and a microbial community strongly biased toward this activity. This may also explain why coliform bacteria, especially klebsiellas, are indigenous in pulp and paper mill water systems.
- Published
- 2000
23. Analysis of the Epstein-Barr virus (EBV) latent membrane protein 1 (LMP-1) gene and promoter in Hodgkin's disease isolates: selection against EBV variants with mutations in the LMP-1 promoter ATF-1/CREB-1 binding site
- Author
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Niels Henrik Gregersen, Xiao-Ge Zhou, Stephen Hamilton-Dutoit, Kristian Sandvej, and Brage S. Andresen
- Subjects
Paper ,Genetic Markers ,Herpesvirus 4, Human ,Molecular Sequence Data ,Response element ,Epitopes, T-Lymphocyte ,medicine.disease_cause ,Polymerase Chain Reaction ,Virus ,Pathology and Forensic Medicine ,Viral Matrix Proteins ,hemic and lymphatic diseases ,medicine ,Humans ,Gammaherpesvirinae ,Infectious Mononucleosis ,Promoter Regions, Genetic ,Gene ,Activating Transcription Factor 1 ,Mutation ,Base Sequence ,biology ,NF-kappa B ,Nuclear Proteins ,Promoter ,Sequence Analysis, DNA ,biology.organism_classification ,CREB-Binding Protein ,Hodgkin Disease ,Epstein–Barr virus ,Virology ,DNA-Binding Proteins ,Trans-Activators ,Carcinogenesis ,T-Lymphocytes, Cytotoxic ,Transcription Factors - Abstract
Aims—To study the distribution of Epstein-Barr virus (EBV) variants containing mutations in the latent membrane protein 1 (LMP-1) oncogene and promoter in EBV associated Hodgkin's disease and infectious mononucleosis compared with previous findings in asymptomatic EBV carriers. Methods—Sequence analysis of the EBV LMP-1 promoter and gene in isolates from Danish patients with Hodgkin's disease (n = 61) and infectious mononucleosis (n = 10). Results—Viruses (previously designated group D) that contain two mutations in the activating transcription factor/cAMP response element (ATF/CRE) in the LMP-1 promoter, which are known to decrease promoter activity greatly, were significantly less frequent in Hodgkin's disease than in both infectious mononucleosis (p = 0.0081) and asymptomatic EBV carriers (p = 0.0084). In some cases, the LMP-1 gene contained mutations in a recently identified cytotoxic T cell (CTL) epitope. Most viral isolates contained mutations shown to increase nuclear factor κB (NF-κB) activation and had one of two newly identified C-terminal activation regions 3 (CTAR-3) deleted. The exon 1 Xho-I restriction site in the LMP-1 gene could be lost through a range of different mutations. Conclusions—These findings indicate selection pressure against EBV strains with weak LMP-1 promoter activity in Hodgkin's disease and thus provide further strong circumstantial evidence for the pathogenic role of EBV (and LMP-1) in this disease. Mutation of the CTL epitope suggests immune selection of EBV strains. Many EBV isolates contain functionally important mutations in the LMP-1 gene. Loss of the Xho-I restriction site should not be used as a marker of specific LMP-1 variants.
- Published
- 2000
24. A Two-Dimensional Support for Selective Binding of Polyhistidine-Tagged Proteins: Identification of a Proliferating Cell Nuclear Antigen Point Mutant with Altered Functionin Vitro
- Author
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Dmitry Ju. Mozzherin, Alex Zaika, Paul A. Fisher, Kathleen M. Downey, and Cheng Keat Tan
- Subjects
Paper ,DNA polymerase ,Biophysics ,Thymus Gland ,In Vitro Techniques ,medicine.disease_cause ,Biochemistry ,DNA polymerase delta ,law.invention ,chemistry.chemical_compound ,Transformation, Genetic ,law ,Proliferating Cell Nuclear Antigen ,Escherichia coli ,medicine ,Animals ,Point Mutation ,Histidine ,Binding site ,Molecular Biology ,DNA Polymerase III ,DNA Primers ,Binding Sites ,Base Sequence ,DNA synthesis ,biology ,DNA ,Cell Biology ,Molecular biology ,Recombinant Proteins ,Proliferating cell nuclear antigen ,chemistry ,biology.protein ,Recombinant DNA ,Cattle ,Drosophila - Abstract
Whatman 3MM paper was chemically modified to generate nickel-charged iminodiacetic acid paper (Ni2+-IDA paper). Bacteria were transformed with Escherichia coli expression plasmids coding for either unmodified proliferating cell nuclear antigen (PCNA) or PCNA containing a genetically engineered polyhistidine tract (his-tag) located at its NH2 terminus. They were then grown, induced, and lysed, and macromolecules were transferred to Ni2+-IDA paper. After exhaustive washing, his-tagged PCNA but not unmodified PCNA remained bound to the paper. Moreover, bound his-tagged PCNA was biochemically active in an in situ DNA synthesis assay with exogenous template-primer and purified calf thymus DNA polymerase delta (pol delta). Ni2+-IDA paper was used to identify a PCNA- point mutant that, relative to wild-type PCNA, promotes increased DNA synthesis by pol delta beyond a model abasic template site. In addition, metal-charged IDA paper promises to be generally useful for functional screening of cells expressing cloned proteins.
- Published
- 1999
25. Morphological and Biochemical Properties of a Sphaerotilus sp. Isolated From Paper Mill Slimes
- Author
-
Michael Wagner, Stefan Juretschko, Véronique Pellegrin, and Gilles Cottenceau
- Subjects
DNA, Bacterial ,Paper ,Sequence analysis ,Segmented filamentous bacteria ,Molecular Sequence Data ,DNA, Ribosomal ,Polymerase Chain Reaction ,Applied Microbiology and Biotechnology ,Microbiology ,Botany ,Sphaerotilus ,Ribosomal DNA ,Phylogeny ,DNA Primers ,Base Sequence ,Gram-Negative Aerobic Bacteria ,Ecology ,biology ,Sphaerotilus natans ,business.industry ,Paper mill ,Hydrogen-Ion Concentration ,Physiology and Biotechnology ,biology.organism_classification ,16S ribosomal RNA ,Microscopy, Electron, Scanning ,business ,Bacteria ,Food Science ,Biotechnology - Abstract
Four strains of filamentous bacteria were isolated from slimes collected in different paper mill factories. Morphological and physiological characterization of the isolates indicated an affiliation with the genus Sphaerotilus . However, while the physiological properties of the isolates were almost identical, pronounced physiological differences between the isolates and Sphaerotilus natans DSM 6575 T , DSM 565, and DSM 566 with respect to their ability to metabolize complex polysaccharides, sugars, polyalcohols, or organic acids as carbon sources were detected. In contrast to the analyzed culture collection strains of S. natans , all paper mill isolates were able to grow at elevated temperatures of up to 40°C. Comparative sequence analysis of nearly complete 16S ribosomal DNA (rDNA) sequences from the four new isolates demonstrated that the retrieved sequences were highly similar to each other (99.6 to 99.8% similarity) and to previously published partial 16S rDNA sequences of S. natans DSM 6575 T and ATCC 15291. Polyphasic characterization of the isolated Sphaerotilus strains revealed interesting adaptations of the strains to the environmental paper mill conditions with regard to temperature tolerance and utilization of cellulose and starch.
- Published
- 1999
26. Dried blood spots collected on filter paper: an international resource for the diagnosis and genetic characterization of human immunodeficiency virus Type-1
- Author
-
P G Babu, N Lapointe, P Gomez, C.-Y. Ou, S Cassol, S Read, and Bruce G. Weniger
- Subjects
Paper ,Microbiology (medical) ,lcsh:Arctic medicine. Tropical medicine ,Resource (biology) ,lcsh:RC955-962 ,diagnosis ,Population ,lcsh:QR1-502 ,Drug Resistance ,Human immunodeficiency virus (HIV) ,Biology ,medicine.disease_cause ,Polymerase Chain Reaction ,lcsh:Microbiology ,Genetic Evolution ,Genetic variation ,medicine ,Screening programs ,Humans ,Amino Acid Sequence ,Dried blood ,education ,DNA Primers ,AIDS Vaccines ,Acquired Immunodeficiency Syndrome ,Blood Specimen Collection ,dried blood ,education.field_of_study ,Base Sequence ,Infant, Newborn ,AIDS Serodiagnosis ,Genetic Variation ,Infant ,vaccines ,Early infancy ,Virology ,Mutation ,genetic variation ,HIV-1 ,Sequence Analysis ,Zidovudine ,Filtration - Abstract
The collection of dried blood spots (DBS) on filter paper provides a powerful approach for the development of large-scale, population-based screening programs. DBS methods are particularly valuable in developing countries and isolated rural regions where resources are limited. Large numbers of field specimens can be economically collected and shipped to centralized reference laboratories for genetic and (or) serological analysis. Alternatively, the dried blood can be stored and used as an archival resource to rapidly establish the frequency and distribution of newly recognized mutations, confirm patient identity or track the origins and emergence of newly identified pathogens. In this report, we describe how PCR-based technologies are beginning to interface with international screening programmes for the diagnosis and genetic characterization of human immunodeficiency virus type 1 (HIV-1). In particular, we review recent progress using DBS specimens to resolve the HIV-1 infection status of neonates, monitor the genetic evolution of HIV-1 during early infancy and establish a sentinel surveillance system for the systematic monitoring of HIV-1 genetic variation in Asia.
- Published
- 1996
27. Sequence and expression of a xylanase gene from the hyperthermophile Thermotoga sp. strain FjSS3-B.1 and characterization of the recombinant enzyme and its activity on kraft pulp
- Author
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Moreland D. Gibbs, Peter L. Bergquist, David J. Saul, Rosalind A. Reeves, and Liam Williams
- Subjects
DNA, Bacterial ,Paper ,Molecular Sequence Data ,Gene Expression ,Applied Microbiology and Biotechnology ,law.invention ,law ,Enzyme Stability ,Genomic library ,Amino Acid Sequence ,Peptide sequence ,Gram-Negative Anaerobic Bacteria ,Base Sequence ,Ecology ,biology ,Temperature ,Nucleic acid sequence ,Hydrogen-Ion Concentration ,Thermotoga ,biology.organism_classification ,Recombinant Proteins ,Hyperthermophile ,Xylan Endo-1,3-beta-Xylosidase ,Xylosidases ,Biochemistry ,Genes, Bacterial ,Xylanase ,Recombinant DNA ,Kraft paper ,Research Article ,Food Science ,Biotechnology - Abstract
A gene expressing xylanase activity was isolated from a genomic library of Thermotoga sp. strain FjSS3-B.1. The sequence of the gene shows that it encodes a single domain, family 10 xylanase. The recombinant enzyme has extremely high thermal stability, activity over a relatively broad pH range, and activity on Pinus radiata kraft pulp.
- Published
- 1995
28. Two neutral thermostable cellulases from Phialophora sp. G5 act synergistically in the hydrolysis of filter paper
- Author
-
Bin Yao, Zhongyuan Li, Pengjun Shi, Huiying Luo, Yingguo Bai, Junqi Zhao, Yaru Wang, and Peilong Yang
- Subjects
Paper ,Environmental Engineering ,Molecular Sequence Data ,Bioengineering ,Cellulase ,Pichia ,Pichia pastoris ,Substrate Specificity ,Hydrolysis ,chemistry.chemical_compound ,Hydrolase ,Cellulases ,Glycosyl ,Cloning, Molecular ,Waste Management and Disposal ,Thermostability ,chemistry.chemical_classification ,biology ,Base Sequence ,Renewable Energy, Sustainability and the Environment ,Reverse Transcriptase Polymerase Chain Reaction ,Phialophora ,Temperature ,Computational Biology ,General Medicine ,Sequence Analysis, DNA ,Hydrogen-Ion Concentration ,biology.organism_classification ,Protein Structure, Tertiary ,Kinetics ,Enzyme ,chemistry ,Biochemistry ,biology.protein ,Electrophoresis, Polyacrylamide Gel ,Filtration - Abstract
Two novel cellulase genes, cbh6A and egGH45, were cloned from Phialophora sp. G5 and successfully expressed in Pichia pastoris. The putative polypeptide of CBH6A consists of a family 1 CBM and a catalytic domain of glycosyl hydrolase family 6 cellobiohydrolases, while deduced EgGH45 only contains a catalytic domain of family 45 endoglucanases. CBH6A and EgGH45 were optimally active at pH 7.0 and 65°C, and pH 6.0 and 60°C, respectively. Both enzymes exhibited high activities and stabilities over a wide pH range and had good thermostability at 70°C. CBH6A and EgGH45 had significant resistance to SDS (10mM), remaining 35% and 54% activities, respectively. These enzymes had synergic effect on the hydrolysis of filter paper, showing the highest efficiency in the ratio of CBH6A to EgGH45 at 80:20. The properties make this enzyme combination potential for application in textile and detergents industries.
- Published
- 2012
29. Detection of Human Immunodeficiency Virus-1 Nucleic Acid on Inactivated Filter Paper Disks by Polymerase Chain Reaction and Microtiter Plate Assay
- Author
-
Wolfgang Röder, Kunihiro Saito, Werner E.G. Müller, Hiroshi Ushijima, Michael Kruse, Shuji Ando, Takao Kunisada, and Yuki Eshita
- Subjects
Paper ,Hot Temperature ,Molecular Sequence Data ,Immunology ,HIV Infections ,Biology ,Polymerase Chain Reaction ,Sensitivity and Specificity ,Microbiology ,Virus ,law.invention ,Immunoenzyme Techniques ,chemistry.chemical_compound ,Microtiter plate ,law ,Virology ,Humans ,False Positive Reactions ,Cells, Cultured ,Polymerase chain reaction ,Base Sequence ,Filter paper ,RNA ,Genes, gag ,Molecular biology ,chemistry ,DNA, Viral ,HIV-1 ,Nucleic acid ,RNA, Viral ,Nested polymerase chain reaction ,Filtration ,DNA - Abstract
Human immunodeficiency virus type 1 (HIV-1) in cultured cells, peripheral blood samples and sera were adsorbed on filter paper disks and inactivated by heat or ethanol. Two procedures, the polymerase chain reaction (PCR) and microtiter plate assay (HMPA) were used to detect the nucleic acid. The sensitivity after different heat treatments with nested PCR for HIV-1 DNA (or nested reverse transcription-PCR for HIV-1 RNA) was identical regardless of whether the samples were examined immediately or one month later. Inactivation by ethanol treatment resulted in a slight loss of sensitivity. The HMPA proved to be as reliable and specific as the conventional PCR technique. We conclude that the heat-treated filter paper disk assay is suitable for identifying HIV nucleic acid in clinical samples sent to the laboratory from a distance, e.g. in an envelope.
- Published
- 1994
30. Immobilization of oligonucleotides onto zirconia-modified filter paper and specific molecular recognition
- Author
-
Jianguo Huang and Wei Xiao
- Subjects
Paper ,Materials science ,Surface Properties ,Molecular Sequence Data ,Oligonucleotides ,Biosensing Techniques ,chemistry.chemical_compound ,Molecular recognition ,Electrochemistry ,General Materials Science ,Cubic zirconia ,Cellulose ,Spectroscopy ,Filter paper ,Base Sequence ,Molecular Structure ,Oligonucleotide ,Substrate (chemistry) ,Nucleic Acid Hybridization ,Surfaces and Interfaces ,DNA ,Condensed Matter Physics ,chemistry ,Chemical engineering ,Nanofiber ,Zirconium - Abstract
A morphologically complex cellulosic substance (e.g., commercial filter paper) was employed as a substrate for DNA immobilization and successive recognition. A uniform ultrathin zirconia gel film was first deposited on each cellulose nanofiber in bulk filter paper by a facile sol-gel process. Relying on the large surface area of filter paper and the strong affinity of zirconia for the phosphate group, terminal-phosphate probe DNA was abundantly immobilized on the zirconia-modified filter paper so as to convert the composite to a biofunctional material for the sensitive and repetitive recognition of the corresponding complementary target DNA on the nanomolar level. By contrast, in spite of the viability of the immobilization of the probe DNA and the recognition of target DNA on the quartz plate, the amount of captured probe DNA or recognized target DNA on such a flat substrate was much less than that captured or recognized on filter paper, resulting in a relatively insensitive recognition event. Moreover, control experiments on bare filter paper (without a zirconia nanocoating) suggested that the zirconia gel film was essential to probe DNA immobilization and subsequent target DNA recognition.
- Published
- 2011
31. A simple non-enzymatic method for the preparation of white spot syndrome virus (WSSV) DNA from the haemolymph of Marsupenaeus japonicus using FTA matrix cards
- Author
-
Tomoya Kono, Kidchakan Supamattaya, Masahiro Sakai, T. Mekata, Raja Sudhakaran, Yoshihiro Suzuki, Toshiaki Itami, and N T H Linh
- Subjects
Paper ,Veterinary (miscellaneous) ,White spot syndrome ,Molecular Sequence Data ,Aquatic Science ,Specimen Handling ,Matrix (chemical analysis) ,chemistry.chemical_compound ,Penaeidae ,Animals ,DNA Primers ,Chromatography ,biology ,Base Sequence ,Densovirinae ,Sequence Analysis, DNA ,biology.organism_classification ,Virology ,DNA extraction ,Shrimp ,Real-time polymerase chain reaction ,chemistry ,DNA, Viral ,Nucleic acid ,Sample collection ,DNA ,Filtration - Abstract
White spot syndrome virus (WSSV) is an important shrimp pathogen responsible for large economic losses for the shrimp culture industry worldwide. The nucleic acids of the virus must be adequately preserved and transported from the field to the laboratory before molecular diagnostic analysis is performed. Here, we developed a new method to isolate WSSV-DNA using Flinders Technology Associates filter paper (FTA matrix card; Whatman) without centrifugation or hazardous steps involved. FTA technology is a new method allowing the simple collection, shipment and archiving of nucleic acids from haemolymph samples providing DNA protection against nucleases, oxidation, UV damage, microbial and fungal attack. DNA samples prepared from 10-fold dilutions of moribund shrimp haemolymph using FTA matrix cards were analysed using semi-quantitative and quantitative polymerase chain reaction (PCR) and were compared with two commercially available DNA isolation methods, the blood GenomicPrep Mini Spin Kit (GE Healthcare) and the DNAzol (Invitrogen). Sequence analysis was performed for the DNA samples prepared using the various isolation procedures and no differences in the sequence among these methods were identified. Results based on the initial copy number of DNA prepared from the GenomicPrep Mini Spin Kit are a little more sensitive than the DNA prepared from FTA matrix cards, whereas the DNAzol method is not suitable for blood samples. Our data shows the efficiency of retention capacity of WSSV-DNA samples from impregnated FTA matrix cards. Matrix cards were easy to store and ship for long periods of time. They provide ease of handling and are a reliable alternative for sample collection and for molecular detection and characterization of WSSV isolates.
- Published
- 2009
32. Paper-based bioassays using gold nanoparticle colorimetric probes
- Author
-
Yingfu Li, Sergio D. Aguirre, Weian Zhao, M. Monsur Ali, and Michael A. Brook
- Subjects
Paper ,Vinyl alcohol ,Adenosine ,Aptamer ,Nanoparticle ,Color ,Metal Nanoparticles ,Nanotechnology ,Biosensing Techniques ,Analytical Chemistry ,chemistry.chemical_compound ,Deoxyribonuclease I ,Colorimetry ,Reagent Strips ,chemistry.chemical_classification ,Chromatography ,Base Sequence ,Biomolecule ,Temperature ,DNA ,Aptamers, Nucleotide ,Cross-Linking Reagents ,chemistry ,Reagent ,Biological Assay ,Gold ,Bioactive paper ,Biosensor ,Hydrophobic and Hydrophilic Interactions - Abstract
The majority of bioassays utilize thermosensitive reagents (e.g., biomolecules) and laboratory conditions for analysis. The developing world, however, requires inexpensive, simple-to-perform tests that do not require refrigeration or access to highly trained technicians. To address this need, paper-based bioassays using gold nanoparticle (AuNP) colorimetric probes have been developed. In the two prototype DNase I and adenosine-sensing assays, blue (or black)-colored DNA-cross-linked AuNP aggregates were spotted on paper substrates. The addition of target DNase I (or adenosine) solution dissociated the gold aggregates into dispersed AuNPs, which generated an intense red color on paper within one minute. Both hydrophobic and (poly(vinyl alcohol)-coated) hydrophilic paper substrates were suitable for this biosensing platform; by contrast, uncoated hydrophilic paper caused "bleeding" and premature cessation of the assay due to surface drying. The assays are surprisingly thermally stable. During preparation, AuNP aggregate-coated papers can be dried at elevated temperatures (e.g., 90 degrees C) without significant loss of biosensing performance, which suggests the paper substrate protects AuNP aggregate probes from external nonspecific stimuli (e.g., heat). Moreover, the dried AuNP aggregate-coated papers can be stored for at least several weeks without loss of the biosensing function. The combination of paper substrates and AuNP colorimetric probes makes the final products inexpensive, low-volume, portable, disposable, and easy-to-use. We believe this simple, practical bioassay platform will be of interest for use in areas such as disease diagnostics, pathogen detection, and quality monitoring of food and water.
- Published
- 2008
33. Microbial community dynamics in a chemolithotrophic denitrification reactor inoculated with methanogenic granular sludge
- Author
-
Ricardo Amils, Jose Luis Sanz, Jim A. Field, Reyes Sierra-Alvarez, and Nuria Fernández
- Subjects
DNA, Bacterial ,Paper ,Environmental Engineering ,Denitrification ,Firmicutes ,Health, Toxicology and Mutagenesis ,Microorganism ,Molecular Sequence Data ,Thiosulfates ,Industrial Waste ,Methanosaeta ,Microbiology ,Denitrifying bacteria ,Bioreactors ,RNA, Ribosomal, 16S ,Environmental Chemistry ,Phylogeny ,Nitrates ,biology ,Bacteria ,Base Sequence ,Sequence Analysis, RNA ,Sulfates ,Public Health, Environmental and Occupational Health ,General Medicine ,General Chemistry ,biology.organism_classification ,Pollution ,Archaea ,DNA, Archaeal ,Microbial population biology ,Proteobacteria ,Methane ,Sequence Alignment - Abstract
Denitrification is applied in the tertiary treatment of wastewater to reduce nitrogen pollution. Fluorescence in situ hybridization (FISH), catalyzed reporter deposition (CARD)-FISH, cloning, and scanning electron microscopy (SEM) were applied to follow the evolution of the microbial composition and structure of granular sludge in chemolithotrophic denitrifying bioreactors fed with nitrate and thiosulfate. FISH oligonucleotide probes for the chemolitoautotrophic denitrifiers Thiobacillus denitrificans and Thiomicrospira denitrificans were designed and their utility tested. CARD-FISH and cloning data showed that bacterial diversity in the biofilms changed during the reactor operation. Chemoorganotrophic fermentative Gram-positive strains in the phyla, Actinobacteria and Firmicutes, were dominant in the methanogenic inoculum, both in terms of biodiversity and in number. Other significant phyla were Bacteroidetes and Chloroflexi. After 6 months of operation, Proteobacteria became dominant (83% of the clones). The diversity of Gram-positive bacteria was partially maintained although their abundance decreased notably. After 110 d of operation, the abundance of Tb. denitrificans cells increased considerably, from 1% to 35% of total DAPI-stained cells and from no isolated clones to 15% of the total clones. Tm. denitrificans only represented a minor fraction of the microorganisms in the sludge (1-4% of the DAPI-stained cells). These findings confirm that Tb. denitrificans was the dominant chemolitoautotrophic denitrifying microorganism in the bioreactors. The Archaeal diversity remained almost unchanged and it was represented mostly by Methanosaeta soehngenii. SEM results indicated a considerable loss in the integrity of the sludge granules during the operation, with risk of sludge buoyancy.
- Published
- 2007
34. Simple and rapid detection of the porcine reproductive and respiratory syndrome virus from pig whole blood using filter paper
- Author
-
Takamitsu Tsukahara, Ryo Inoue, Kazunari Ushida, Chinatsu Sunaba, and Mitsugi Itoh
- Subjects
Paper ,Time Factors ,biology ,Filter paper ,Base Sequence ,animal diseases ,Molecular Sequence Data ,Sus scrofa ,Porcine reproductive and respiratory syndrome virus ,biology.organism_classification ,Virology ,Polymerase Chain Reaction ,Virus ,Reverse transcription polymerase chain reaction ,Arterivirus ,Nidovirales ,Sequence Homology, Nucleic Acid ,Animals ,RNA, Viral ,Porcine respiratory and reproductive syndrome virus ,RNA extraction ,Nucleic Acid Amplification Techniques ,Filtration ,Whole blood - Abstract
The combination of Flinders Technology Associates filter papers (FTA® cards) and real-time PCR was examined to establish a simple and rapid technique for the detection of porcine reproductive and respiratory syndrome virus (PRRSV) from whole pig blood. A modified live PRRS vaccine was diluted with either sterilised saline or pig whole blood, and the suspensions were applied onto the FTA® cards. The real-time RT-PCR detection of PRRSV was performed directly with the samples applied to the FTA® card without the RNA extraction step. Six whole blood samples from at random selected piglets in the PRRSV infected farm were also assayed in this study. The expected PCR product was successfully amplified from either saline diluted or pig whole blood diluted vaccine. The same PCR ampliocon was detected from all blood samples assayed in this study. This study suggested that the combination of an FTA® card and real-time PCR is a rapid and easy technique for the detection of PRRSV. This technique can remarkably shorten the time required for PRRSV detection from whole blood and makes the procedure much easier.
- Published
- 2006
35. Use of filter paper (FTA) technology for sampling, recovery and molecular characterisation of rabies viruses
- Author
-
Florence Cliquet, Evelyne Picard-Meyer, and J. Barrat
- Subjects
Paper ,Time Factors ,Molecular Sequence Data ,Foxes ,Biology ,medicine.disease_cause ,Sensitivity and Specificity ,Virus ,Microbiology ,Cell Line ,Specimen Handling ,Dogs ,Virology ,Cricetinae ,Sequence Homology, Nucleic Acid ,medicine ,Animals ,Cloning, Molecular ,Lyssavirus ,DNA Primers ,Base Sequence ,Reverse Transcriptase Polymerase Chain Reaction ,Rabies virus ,RNA ,Nucleic acid amplification technique ,Rhabdoviridae ,biology.organism_classification ,Reverse transcriptase ,Nucleoprotein ,Evaluation Studies as Topic ,Feasibility Studies ,RNA, Viral ,Nucleic Acid Amplification Techniques ,Filtration ,Biotechnology - Abstract
This study evaluates the feasibility of the use of the FTA® Gene Guard System (a commercial product consisting of filter paper impregnated with patented chemicals supplied by the Whatman company) for the shipment, storage and detection of RNA rabies viruses by a simplified hemi-nested reverse transcriptase polymerase chain reaction. HnRT-PCR of the rabies virus nucleoprotein gene with specific primers showed that viral RNA extracted from crude infected tissues remained stable after fixation on the filter paper under diverse environmental conditions for at least 35 days. The sequence analysis of the products amplified from five out of the seven known genotypes of Lyssaviruses showed the stability of viral RNA viruses after fixation on the filter paper. Furthermore, the sensitivity of the hnRT-PCR following RNA fixation on the filter paper was equivalent to that of standard hnRT-PCR. In conclusion, the stability of viral RNA and the inactivation of infectivity make the FTA® technology useful for the storage, transport, collection and subsequent molecular analysis of viral rabies RNA, facilitating epidemiological investigations in the field.
- Published
- 2006
36. Methylated DNA labels for marking objects
- Author
-
Lindsey J, Cook and Jonathan P L, Cox
- Subjects
Paper ,Base Sequence ,Archaeal Proteins ,Molecular Sequence Data ,Reproducibility of Results ,DNA ,Methyltransferases ,Templates, Genetic ,DNA Methylation ,Product Labeling ,Polymerase Chain Reaction ,Sensitivity and Specificity ,Sequence Alignment - Abstract
We recently described a method for digitally labelling objects with DNA. Here we show that, using DNA methyltransferases to create polymorphic DNA templates, it is possible to significantly increase the number of labels that can be generated by this method. Nine double-stranded DNA templates of different length were methylated with either M.HaeIII or M.AluI methyltransferase, or both. Different mixtures of methylated and unmethylated versions of this template set were used to 'invisibly' label paper. The mixtures were eluted from the paper and the methylated status of the templates in each mixture successfully determined, and the labels read, by digestion with the complementary restriction endonuclease, followed by a polymerase chain reaction and agarose gel electrophoresis. One methylated DNA label was read after it had been left on paper for two months.
- Published
- 2003
37. CelI, a noncellulosomal family 9 enzyme from Clostridium thermocellum, is a processive endoglucanase that degrades crystalline cellulose
- Author
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Larisa Rabinovich, Sima Yaron, Harry J. Gilbert, Edward A. Bayer, Raphael Lamed, Yuval Shoham, and Rachel Gilad
- Subjects
Paper ,Molecular Sequence Data ,Cellulase ,Cleavage (embryo) ,Microbiology ,law.invention ,Frameshift mutation ,chemistry.chemical_compound ,law ,Glycoside hydrolase ,Amino Acid Sequence ,Cellulose ,Cloning, Molecular ,Molecular Biology ,chemistry.chemical_classification ,Clostridium ,biology ,Base Sequence ,Sequence Analysis, DNA ,biology.organism_classification ,Enzymes and Proteins ,Recombinant Proteins ,Enzyme ,Biochemistry ,chemistry ,biology.protein ,Recombinant DNA ,Clostridium thermocellum ,Crystallization - Abstract
The family 9 cellulase gene celI of Clostridium thermocellum , was previously cloned, expressed, and characterized (G. P. Hazlewood, K. Davidson, J. I. Laurie, N. S. Huskisson, and H. J. Gilbert, J. Gen. Microbiol. 139:307-316, 1993). We have recloned and sequenced the entire celI gene and found that the published sequence contained a 53-bp deletion that generated a frameshift mutation, resulting in a truncated and modified C-terminal segment of the protein. The enzymatic properties of the wild-type protein were characterized and found to conform to those of other family 9 glycoside hydrolases with a so-called theme B architecture, where the catalytic module is fused to a family 3c carbohydrate-binding module (CBM3c); CelI also contains a C-terminal CBM3b. The intact recombinant CelI exhibited high levels of activity on all cellulosic substrates tested, with pH and temperature optima of 5.5 and 70°C, respectively, using carboxymethylcellulose as a substrate. Native CelI was capable of solubilizing filter paper, and the distribution of reducing sugar between the soluble and insoluble fractions suggests that the enzyme acts as a processive cellulase. A truncated form of the enzyme, lacking the C terminal CBM3b, failed to bind to crystalline cellulose and displayed reduced activity toward insoluble substrates. A truncated form of the enzyme, in which both the cellulose-binding CBM3b and the fused CBM3c were removed, failed to exhibit significant levels of activity on any of the substrates examined. This study underscores the general nature of this type of enzymatic theme, whereby the fused CBM3c plays a critical accessory role for the family 9 catalytic domain and changes its character to facilitate processive cleavage of recalcitrant cellulose substrates.
- Published
- 2003
38. HPV-16 E2 gene disruption and sequence variation in CIN 3 lesions and invasive squamous cell carcinomas of the cervix: relation to numerical chromosome abnormalities
- Author
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C S Herrington and D A Graham
- Subjects
Paper ,Pathology ,medicine.medical_specialty ,Molecular Sequence Data ,Uterine Cervical Neoplasms ,medicine.disease_cause ,Cervical intraepithelial neoplasia ,Pathology and Forensic Medicine ,law.invention ,law ,medicine ,Humans ,Neoplasm Invasiveness ,Amino Acid Sequence ,Papillomaviridae ,Gene ,Polymerase chain reaction ,Chromosome Aberrations ,biology ,Base Sequence ,Chromosome ,Oncogene Proteins, Viral ,biology.organism_classification ,medicine.disease ,Uterine Cervical Dysplasia ,DNA-Binding Proteins ,Epidermoid carcinoma ,DNA, Viral ,Chromosome abnormality ,Carcinoma, Squamous Cell ,Female ,Carcinogenesis - Abstract
Aim—To test the hypothesis that, because the human papillomavirus (HPV) E2 protein represses viral early gene transcription, E2 gene sequence variation or disruption could play a part in the induction of the numerical chromosome abnormalities that have been described in squamous cervical lesions. Methods—The integrity and sequence of the E2 gene from 11 cervical intraepithelial neoplasia (CIN) grade 3 lesions and 14 invasive squamous cell carcinomas, all of which contained HPV-16, were analysed by the polymerase chain reaction (PCR). The E2 gene was amplified in three overlapping fragments and PCR products sequenced directly. Chromosome abnormalities were identified by interphase cytogenetics using chromosome specific probes for chromosomes 1, 3, 11, 17, 18, and X. Results—E2 gene disruption was present in significantly more invasive carcinomas (eight of 14) than CIN 3 lesions (one of 11) (p = 0.03). No association was found between E2 disruption and the presence of a numerical chromosome abnormality. The E2 gene from the non-disrupted isolates was sequenced and wild-type (n = 5) and variant (n = 11) sequences identified. Variant sequences belonged to European and African classes and contained from one to 15 amino acid substitutions. Although numerical chromosome abnormalities were significantly more frequent in invasive squamous cell carcinoma than CIN 3 (p = 0.04), there was no significant relation between the presence of sequence variation and either histological diagnosis or chromosome abnormality. Conclusions—These data do not support the hypothesis that E2 gene disruption or variation is important in the induction of chromosome imbalance in these lesions. However, there is a relation between E2 gene disruption and the presence of invasive disease.
- Published
- 2000
39. Prediction of the interacting surfaces in a trimolecular complex formed between the major dust mite allergen Der p1, a mouse monoclonal anti-Der p 1 antibody and its anti-idiotype
- Author
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Furtado, P. B., Furmonaviciene, R., McElveen, J., Clark, M. R., Sewell, H. F., and Shakib, F.
- Subjects
Idiotype ,Paper ,Antigen-Antibody Complex ,DNA, Complementary ,medicine.drug_class ,Molecular Sequence Data ,Complementarity determining region ,Biology ,Monoclonal antibody ,Polymerase Chain Reaction ,Epitope ,Pathology and Forensic Medicine ,Antigen ,medicine ,Animals ,Humans ,Antigens, Dermatophagoides ,Framework region ,Glycoproteins ,Mites ,Base Sequence ,Antibodies, Monoclonal ,Allergens ,Immunoglobulin E ,Molecular biology ,Complementarity Determining Regions ,Antibodies, Anti-Idiotypic ,biology.protein ,Antibody - Abstract
Background—Two mouse monoclonal antibodies (mAbs) have been described recently; namely, mAb 2C7 (IgG2bκ), which is directed against the major house dust mite allergen Der p 1, and mAb 2G10 (IgG1κ), which is an anti-idiotypic antibody raised against mAb 2C7. The anti-idiotype mAb 2G10 does not block the binding of mAb 2C7 to Der p 1, which means that mAb 2C7 can simultaneously bind to Der p 1 and to mAb 2G10, thereby generating a trimolecular complex consisting of antigen–idiotype–anti-idiotype. Aims—To sequence and model the V region of the anti-idiotypic antibody mAb 2G10 to enable the prediction of the interacting surfaces in the trimolecular complex consisting of Der p 1–mAb 2C7–mAb 2G10. Methods—DNA sequencing of mAb 2G10 was carried out and the Swiss Model and Swiss PDB-Viewer programs were used to build a three dimensional model of the trimolecular complex. Results—Complementarity of shape and charge was revealed when comparing the protrusion of the previously determined Der p 1 epitope (Leu147–Gln160) with the cavity formed by the complementarity determining regions (CDRs) of mAb 2C7. Such complementarity was also observed between the mAb 2C7 epitope predicted to be recognised by mAb 2G10 (residues Lys19 from framework region 1 (FRW1) and Ser74–Gln81 from FRW3) and residues from the CDRs of mAb 2G10 (a negatively charged patch flanked by the residues Asp55H/Glu58H and Glu27L/Glu27cL). As expected, the location of the mAb 2C7 epitope recognised by mAb 2G10 does not appear to interfere with the binding of Der p 1 to mAb 2C7. Conclusion—Although the results obtained represent only an approximation, they nevertheless provide a rare insight into how an antigen (Der p 1) might bind to its antibody (mAb 2C7) while in complex with an anti-idiotype (mAb 2G10).
- Published
- 2000
- Full Text
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40. Cloning of a new endoglucanase gene from Bacillus sp. BP-23 and characterisation of the enzyme. Performance in paper manufacture from cereal straw
- Author
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Teresa Vidal, Ana Blanco, Antonio L. Torres, F.I.J. Pastor, Josefina Martínez, and Pilar Diaz
- Subjects
DNA, Bacterial ,Paper ,Molecular Sequence Data ,Bacillus ,Cellulase ,medicine.disease_cause ,Applied Microbiology and Biotechnology ,Substrate Specificity ,Gene product ,medicine ,Escherichia coli ,Amino Acid Sequence ,Cloning, Molecular ,Cellulose ,Phosphoenolpyruvate Sugar Phosphotransferase System ,Peptide sequence ,Triticum ,Electrophoresis, Agar Gel ,Bacillaceae ,biology ,Base Sequence ,Sequence Homology, Amino Acid ,Nucleic acid sequence ,General Medicine ,Sequence Analysis, DNA ,biology.organism_classification ,Bacillales ,Open reading frame ,Biochemistry ,Carboxymethylcellulose Sodium ,biology.protein ,Electrophoresis, Polyacrylamide Gel ,Isoelectric Focusing ,Sequence Alignment ,Biotechnology - Abstract
The gene ce1A, encoding an endoglucanase from the strain Bacillus sp. BP-23, was cloned and expressed in Escherichia coli. The nucleotide sequence of a 1867-bp DNA fragment containing the ce1A gene was determined, revealing an open reading frame of 1200 nucleotides that encodes a protein of 44,803 Da. The deduced amino acid sequence of the encoded enzyme shows high homology to those of enzymes belonging to subtype 4 of the family-A cellulases. The ce1A gene product synthesized in E. coli showed activity on carboxymethylcellulose and lichenan but no activity was found on Avicel. Activity was enhanced in the presence of 10 mM Mg2+ and Ca2+ and showed its maximum at 40 degrees C and pH 4.0. Study of the performance of Ce1A on paper manufacture from agricultural fibres showed that treatment with the enzyme improved the properties of the pulp and the quality of paper. Ce1A treatment enhanced the physical properties (stretch and tensile index) of paper from wheat straw, while dewatering properties were slightly diminished. Electron-microscope analysis showed that the surface of straw fibres was modified by Ce1A.
- Published
- 1998
41. Isolation and molecular characterization of high-performance cellobiose-fermenting spontaneous mutants of ethanologenic Escherichia coli KO11 containing the Klebsiella oxytoca casAB operon
- Author
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Xiaokuang Lai, Sean W. York, Mohammed Moniruzzaman, and Lonnie O. Ingram
- Subjects
DNA, Bacterial ,Paper ,Cellobiose ,Operon ,Molecular Sequence Data ,Gene Expression ,Cellulase ,Ethanol fermentation ,medicine.disease_cause ,Applied Microbiology and Biotechnology ,Zymomonas mobilis ,Microbiology ,chemistry.chemical_compound ,Klebsiella ,medicine ,Escherichia coli ,DNA Primers ,Ecology ,biology ,Base Sequence ,Ethanol ,Klebsiella oxytoca ,biology.organism_classification ,chemistry ,Biochemistry ,Genes, Bacterial ,Fermentation ,Mutation ,biology.protein ,bacteria ,Pyruvate decarboxylase ,Food Science ,Biotechnology ,Research Article - Abstract
Escherichia coli KO11 was previously constructed to produce ethanol from acid hydrolysates of hemicellulose (pentoses and hexoses) by the chromosomal integration of Zymomonas mobilis genes encoding pyruvate decarboxylase (pdc) and alcohol dehydrogenase (adhB). Klebsiella oxytoca P2 was constructed in an analogous fashion for the simultaneous saccharification and fermentation of cellulose and contains PTS enzymes for cellobiose. In this study, KO11 was further engineered for the fermentation of cellulose by adding the K. oxytoca casAB genes encoding Enzyme IIcellobiose and phospho-beta-glucosidase. Although the two K. oxytoca genes were well expressed in cloning hosts such as DH5 alpha, both were expressed poorly in E. coli KO11, a derivative of E. coli B. Spontaneous mutants which exhibited more than 15-fold-higher specific activities for cellobiose metabolism were isolated. The mutations of these mutants resided in the plasmid rather than the host. Three mutants were characterized by sequence analysis. All contained similar internal deletions which eliminated the casAB promoter and operator regions and placed the lacZ Shine-Dalgarno region immediately upstream from the casA Shine-Dalgarno region. KO11 harboring mutant plasmids (pLOI1908, pLOI1909, or pLOI1910) rapidly fermented cellobiose to ethanol, and the yield was more than 90% of the theoretical yield. Two of these strains were used with commercial cellulase to ferment mixed-waste office paper to ethanol.
- Published
- 1997
42. Structural basis of the properties of an industrially relevant thermophilic xylanase
- Author
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G W, Harris, R W, Pickersgill, I, Connerton, P, Debeire, J P, Touzel, C, Breton, and S, Pérez
- Subjects
Models, Molecular ,Paper ,Base Sequence ,Molecular Sequence Data ,Temperature ,Bacillus ,Hydrogen-Ion Concentration ,Crystallography, X-Ray ,Molecular Weight ,Xylan Endo-1,3-beta-Xylosidase ,Structure-Activity Relationship ,Xylosidases ,Chemical Industry ,Enzyme Stability ,Amino Acid Sequence ,Cloning, Molecular ,Crystallization - Abstract
A thermophilic xylanase from Bacillus strain D3 suitable for use as a bleach booster in the paper pulping industry has been identified and characterized. The enzyme is suited to the high temperature and alkaline conditions needed for using xylanases in the pulp industry. The xylanase is stable at 60 degrees C and relatively stable at high temperatures, with a temperature optimum of 75 degrees C. The pH optimum is 6, but the enzyme is active over a broad pH range. The xylanase has been cloned and sequenced, and the crystal structure has been determined. The structure of Bacillus D3 xylanase reveals an unusual feature of surface aromatic residues, which form clusters or "sticky patches" between pairs of molecules. These "sticky patches" on the surface of the enzyme are responsible for the tendency of the protein to aggregate at high concentrations in the absence of reagents such as ethylene glycol. The formation of dimers and higher order polymers via these hydrophobic contacts may also contribute to the thermostability of this xylanase.
- Published
- 1997
43. Photoelectrochemical lab-on-paper device equipped with a porous Au-paper electrode and fluidic delay-switch for sensitive detection of DNA hybridization
- Author
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Jiadong Huang, Nianqiang Li, Panpan Wang, Mei Yan, Shenguang Ge, Lei Ge, Jinghua Yu, and Yanhu Wang
- Subjects
Paper ,Working electrode ,Materials science ,Microfluidics ,Biomedical Engineering ,DNA, Single-Stranded ,Bioengineering ,Nanotechnology ,Sulfides ,Electric Capacitance ,Biochemistry ,Cadmium Compounds ,Electrochemistry ,Fluidics ,Electrodes ,Photocurrent ,Base Sequence ,Amplifier ,Detector ,Nucleic Acid Hybridization ,DNA ,General Chemistry ,Microfluidic Analytical Techniques ,Photochemical Processes ,Quaternary Ammonium Compounds ,Electrode ,Gold ,Polyethylenes ,Porosity ,Biosensor - Abstract
The sequence-specific detection of DNA hybridization has attracted considerable interest in numerous fields. Although traditional DNA biosensors have been widely explored due to their high sensitivity, it is still challenging to develop a low-cost, portable, disposable, fast, and easy-to-use DNA detection method for public use at home or in the field. To address these challenges, herein, we report a novel microfluidic photoelectrochemical (PEC) paper-based analytical platform, integrated with an internal chemiluminescent light source, a novel paper supercapacitor (PS) amplifier, and a terminal digital multi-meter (DMM) detector, for sensitive DNA detection using a graphene-modified porous Au-paper electrode as the working electrode to obtain enhanced PEC responses. The quantification mechanism of this strategy is based on the charging of this PS, which was constructed on a paper-based analytical platform through a simple "drawing and soaking" method, by the generated photocurrent. After a fixed period, the PS was automatically shorted under the control of a novel built-in fluidic delay-switch to output an instantaneously amplified current, which could be sensitively detected by the DMM. At optimal conditions, this paper-based analytical platform can detect DNA at concentrations at femtomolar level. This approach also shows excellent specificity toward single nucleotide mismatches.
- Published
- 2013
44. Detection of cystic fibrosis delta F508 mutation by anti-double-stranded DNA antibody
- Author
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S M, Hopfer, G S, Makowski, E L, Davis, and J, Aslanzadeh
- Subjects
Paper ,Base Sequence ,Cystic Fibrosis ,Genotype ,Molecular Sequence Data ,DNA ,Polymerase Chain Reaction ,Immunoenzyme Techniques ,Antibodies, Antinuclear ,Mutation ,Humans ,Electrophoresis, Polyacrylamide Gel ,DNA Probes ,Filtration - Abstract
This study evaluated an enzyme immunoassay (EIA) as a screening tool for detection of the most common mutation (delta F508) in cystic fibrosis (CF). Guthrie card bloodspots were extracted to remove whole blood polymerase chain reaction (PCR) inhibitors. The washed filter paper was directly amplified under standard (98 bp amplicons) or modified PCR conditions (491 bp amplicons) for the delta F508 mutation. Monoclonal anti-double stranded deoxyribonucleic acid antibody was used to detect competent hybrid formation between PCR products and normal (N) and mutant (M) cDNA (deoxyribonucleic acid) probes coated to microtiter plate wells. Under standard conditions, mean relative color production (N/M) via an enzyme-linked horseradish peroxidase secondary antibody was 8.8, 0.6 and 0.04 for individuals normal, heterozygous and homozygous for the CF delta F508 mutation, respectively (n = 27). Comparison of EIA results to those obtained by tris-borate-EDTA/8 percent polyacrylamide gel electrophoresis (TBE-PAGE) yielded excellent correlation (100 percent) for all three genotypes (n = 27). In comparison to TBE-PAGE, the EIA was 5 to 10 fold more sensitive when serially diluted PCR samples were evaluated. Larger PCR products (491 bp amplicons) for the CF delta F508 mutation obtained under modified conditions were not resolved by TBE-PAGE. The EIA, however, demonstrated equal sensitivity to the 98 bp and 491 bp amplicons. Performance time for TBE-PAGE analysis was substantially shorter (25 percent) than the EIA (3.5 to 4 h and 4.5 to 5 h, respectively) when small batches of samples (n = 5) were analyzed. The TBE-PAGE was not, however, convenient for screening large numbers of PCR-amplified samples (n15).
- Published
- 1995
45. Stabilization of nucleic acids in whole blood: an alternative to Guthrie cards
- Author
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R, Ramanujan, M, Anhalt, P, Blair, and B, Burdick
- Subjects
Paper ,Base Sequence ,Molecular Sequence Data ,Carbohydrates ,DNA ,Polymerase Chain Reaction ,Globins ,Drug Stability ,Blood Preservation ,Animals ,Humans ,Glass ,RNA, Messenger ,Rabbits ,Filtration - Published
- 1993
46. Rapid colony hybridization on Whatman 541 paper using oligonucleotide probes
- Author
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G V, Paddock
- Subjects
Paper ,Base Sequence ,Molecular Sequence Data ,DNA, Recombinant ,Escherichia coli ,Autoradiography ,Nucleic Acid Hybridization ,Oligonucleotide Probes ,Phosphorus Radioisotopes ,Gene Library ,Plasmids - Published
- 1993
47. A novel polymerase chain reaction method for detection of human immunodeficiency virus in dried blood spots on filter paper
- Author
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J Yourno and J Conroy
- Subjects
Microbiology (medical) ,Paper ,Molecular Sequence Data ,HIV Infections ,Biology ,Polymerase Chain Reaction ,Virus ,law.invention ,chemistry.chemical_compound ,law ,Humans ,Child ,Polymerase chain reaction ,Blood Specimen Collection ,Filter paper ,Base Sequence ,Infant, Newborn ,HIV ,Infant ,Provirus ,Reference Standards ,Virology ,Genes, gag ,chemistry ,Evaluation Studies as Topic ,Child, Preschool ,DNA, Viral ,Human Immunodeficiency Virus DNA ,Primer (molecular biology) ,Nested polymerase chain reaction ,DNA ,Research Article - Abstract
A method for detection of proviral human immunodeficiency virus DNA in dried blood spots on filter paper by direct polymerase chain reaction (PCR) has been developed. To develop the method, a standard system was used which was prepared from cells each containing a single integrated provirus and titrated with normal donor blood. This rapid procedure provides virtually quantitative yields of nuclear DNA and exploits most of the standard methodology described for blood specimens. A nested PCR using SK38-SK39 gag as the internal primer pair was also designed; this PCR detected a single copy of provirus per filter at near theoretical frequency with SK19 probe. The utility of the procedure was demonstrated with clinical specimens. Blood spot filters from human immunodeficiency virus-infected and uninfected individuals were readily and unequivocally discriminated. The method is designed for ultimate use with large (1.5-ml) sample preparation tubes that are compatible as PCR tubes with thermal cyclers. This will permit convenient, direct single-tube PCR of dried blood specimens on filters. It should be adaptable to analysis of dried blood spots for a variety of infectious or genetic diseases.
- Published
- 1992
48. Detection of DNA using bioactive paper strips
- Author
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Yaqin Xu, Robert Pelton, M. Monsur Ali, Carlos D. M. Filipe, Yingfu Li, and Sergio D. Aguirre
- Subjects
Paper ,DNA nanoball sequencing ,Base Sequence ,Chemistry ,Hybridization probe ,Metals and Alloys ,Nucleic Acid Hybridization ,DNA ,General Chemistry ,Molecular biology ,Catalysis ,Surfaces, Coatings and Films ,Electronic, Optical and Magnetic Materials ,chemistry.chemical_compound ,Nucleic acid thermodynamics ,Rolling circle replication ,Materials Chemistry ,Ceramics and Composites ,A-DNA ,Primer (molecular biology) ,Ligation - Abstract
Paper strips containing DNA-conjugated microgels (MG) are used to achieve sensitive DNA detection in three steps: target DNA promoted ligation of a DNA primer to the MG-bound DNA, rolling circle amplification (RCA) between the primer and a circle DNA, and hybridization of the RCA products and a fluorescent DNA probe.
- Published
- 2009
49. Biochemical identification of cutaneous leishmanias by analysis of kinetoplast DNA. II. Sequence homologies in Leishmania kDNA
- Author
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Douglas C. Barker and David E. Arnot
- Subjects
Male ,Paper ,Buoyant density ,DNA, Mitochondrial ,DNA sequencing ,chemistry.chemical_compound ,Species Specificity ,parasitic diseases ,Methods ,Humans ,A-DNA ,Leishmaniasis ,Molecular Biology ,Southern blot ,Sequence (medicine) ,Electrophoresis, Agar Gel ,Leishmania ,Genetics ,Base Sequence ,biology ,Nucleic Acid Hybridization ,DNA Restriction Enzymes ,Diazonium Compounds ,biology.organism_classification ,Molecular biology ,chemistry ,Kinetoplast ,Female ,Parasitology ,DNA - Abstract
Kinetoplast DNA (kDNA) has been isolated from the human cutaneous Leishmania isolates, L. tropica major, L. aethiopica and an unknown Kenyan isolate, Leishmania SP48. DNA sequence relationships among these isolates have been studied by restriction enzyme digestion and two phase hybridisation to Southern blots of kDNA covalently coupled to diazobenzyloxymethyl (DBM) paper. The results of this analysis confirm that rapid kDNA sequence evolution is occurring in the Old World leishmanias although some sequence conservation in defined regions of the mini-circle sequence is present. These results emphasise the danger of constructing a rigid Leishmania classification on buoyant density data alone. The covalent binding of kDNA electrophoretic separations to DBM paper permits the construction of a DNA sequence "library' which can be used in the classification and diagnosis of unknown Leishmania isolates.
- Published
- 1981
50. Analysis of Expression of Yeast Enolase 1 Gene Containing a Longer Pyrimidine-Rich Region Located between the TATA Box and Transcription Start Site
- Author
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Hiroshi Uemura, Hirofumi Fujisawa, Yoshifumi Jigami, Hideaki Tanaka, Satoshi Nakasato, and Nobumasa Toshimitsu
- Subjects
Paper ,TATA box ,Saccharomyces cerevisiae ,Biochemistry ,Transformation, Genetic ,Transcription (biology) ,Gene expression ,Escherichia coli ,Consensus sequence ,RNA, Messenger ,Promoter Regions, Genetic ,Molecular Biology ,Gene ,Messenger RNA ,Base Sequence ,biology ,Collodion ,Promoter ,General Medicine ,beta-Galactosidase ,biology.organism_classification ,Molecular biology ,Isoenzymes ,Pyrimidines ,Gene Expression Regulation ,Lac Operon ,Phosphopyruvate Hydratase ,Electrophoresis, Polyacrylamide Gel ,Transcription Factors - Abstract
Yeast ENO1 promoter was prepared by a chemical synthetic method. Two variant promoters containing a pyrimidine-rich region (CT block), located between the TATA box and the transcription start site, either 32 or 34 base pairs (bp) longer than the native ENO1 promoter were isolated during the chemical synthesis. Gene expression of variant promoters was compared with that of the native promoter by measuring the amount of mRNA and the activity of beta-galactosidase by constructing ENO1-lacZ gene fusions. No significant differences were observed between the native and variant promoters in transcription levels. The start site of transcription was mapped on CAAG, a consensus sequence of transcription start site of yeast glycolytic genes. The results suggest a longer CT block in ENO1 promoter may not affect the expression of the yeast ENO1 gene. In addition, the level of ENO1 gene expression was found to be higher in stationary phase cells than in log phase cells.
- Published
- 1986
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