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Sample records for automated dna assembly

  1. Automated Robotic Liquid Handling Assembly of Modular DNA Devices.

    Science.gov (United States)

    Ortiz, Luis; Pavan, Marilene; McCarthy, Lloyd; Timmons, Joshua; Densmore, Douglas M

    2017-12-01

    Recent advances in modular DNA assembly techniques have enabled synthetic biologists to test significantly more of the available "design space" represented by "devices" created as combinations of individual genetic components. However, manual assembly of such large numbers of devices is time-intensive, error-prone, and costly. The increasing sophistication and scale of synthetic biology research necessitates an efficient, reproducible way to accommodate large-scale, complex, and high throughput device construction. Here, a DNA assembly protocol using the Type-IIS restriction endonuclease based Modular Cloning (MoClo) technique is automated on two liquid-handling robotic platforms. Automated liquid-handling robots require careful, often times tedious optimization of pipetting parameters for liquids of different viscosities (e.g. enzymes, DNA, water, buffers), as well as explicit programming to ensure correct aspiration and dispensing of DNA parts and reagents. This makes manual script writing for complex assemblies just as problematic as manual DNA assembly, and necessitates a software tool that can automate script generation. To this end, we have developed a web-based software tool, http://mocloassembly.com, for generating combinatorial DNA device libraries from basic DNA parts uploaded as Genbank files. We provide access to the tool, and an export file from our liquid handler software which includes optimized liquid classes, labware parameters, and deck layout. All DNA parts used are available through Addgene, and their digital maps can be accessed via the Boston University BDC ICE Registry. Together, these elements provide a foundation for other organizations to automate modular cloning experiments and similar protocols. The automated DNA assembly workflow presented here enables the repeatable, automated, high-throughput production of DNA devices, and reduces the risk of human error arising from repetitive manual pipetting. Sequencing data show the automated DNA

  2. Automated Assembly Center (AAC)

    Science.gov (United States)

    Stauffer, Robert J.

    1993-01-01

    The objectives of this project are as follows: to integrate advanced assembly and assembly support technology under a comprehensive architecture; to implement automated assembly technologies in the production of high-visibility DOD weapon systems; and to document the improved cost, quality, and lead time. This will enhance the production of DOD weapon systems by utilizing the latest commercially available technologies combined into a flexible system that will be able to readily incorporate new technologies as they emerge. Automated assembly encompasses the following areas: product data, process planning, information management policies and framework, three schema architecture, open systems communications, intelligent robots, flexible multi-ability end effectors, knowledge-based/expert systems, intelligent workstations, intelligent sensor systems, and PDES/PDDI data standards.

  3. ex vivo DNA assembly

    Directory of Open Access Journals (Sweden)

    Adam B Fisher

    2013-10-01

    Full Text Available Even with decreasing DNA synthesis costs there remains a need for inexpensive, rapid and reliable methods for assembling synthetic DNA into larger constructs or combinatorial libraries. Advances in cloning techniques have resulted in powerful in vitro and in vivo assembly of DNA. However, monetary and time costs have limited these approaches. Here, we report an ex vivo DNA assembly method that uses cellular lysates derived from a commonly used laboratory strain of Escherichia coli for joining double-stranded DNA with short end homologies embedded within inexpensive primers. This method concurrently shortens the time and decreases costs associated with current DNA assembly methods.

  4. A Demonstration of Automated DNA Sequencing.

    Science.gov (United States)

    Latourelle, Sandra; Seidel-Rogol, Bonnie

    1998-01-01

    Details a simulation that employs a paper-and-pencil model to demonstrate the principles behind automated DNA sequencing. Discusses the advantages of automated sequencing as well as the chemistry of automated DNA sequencing. (DDR)

  5. Automated ensemble assembly and validation of microbial genomes

    Science.gov (United States)

    2014-01-01

    Background The continued democratization of DNA sequencing has sparked a new wave of development of genome assembly and assembly validation methods. As individual research labs, rather than centralized centers, begin to sequence the majority of new genomes, it is important to establish best practices for genome assembly. However, recent evaluations such as GAGE and the Assemblathon have concluded that there is no single best approach to genome assembly. Instead, it is preferable to generate multiple assemblies and validate them to determine which is most useful for the desired analysis; this is a labor-intensive process that is often impossible or unfeasible. Results To encourage best practices supported by the community, we present iMetAMOS, an automated ensemble assembly pipeline; iMetAMOS encapsulates the process of running, validating, and selecting a single assembly from multiple assemblies. iMetAMOS packages several leading open-source tools into a single binary that automates parameter selection and execution of multiple assemblers, scores the resulting assemblies based on multiple validation metrics, and annotates the assemblies for genes and contaminants. We demonstrate the utility of the ensemble process on 225 previously unassembled Mycobacterium tuberculosis genomes as well as a Rhodobacter sphaeroides benchmark dataset. On these real data, iMetAMOS reliably produces validated assemblies and identifies potential contamination without user intervention. In addition, intelligent parameter selection produces assemblies of R. sphaeroides comparable to or exceeding the quality of those from the GAGE-B evaluation, affecting the relative ranking of some assemblers. Conclusions Ensemble assembly with iMetAMOS provides users with multiple, validated assemblies for each genome. Although computationally limited to small or mid-sized genomes, this approach is the most effective and reproducible means for generating high-quality assemblies and enables users to

  6. ReseqChip: Automated integration of multiple local context probe data from the MitoChip array in mitochondrial DNA sequence assembly

    Directory of Open Access Journals (Sweden)

    Spang Rainer

    2009-12-01

    Full Text Available Abstract Background The Affymetrix MitoChip v2.0 is an oligonucleotide tiling array for the resequencing of the human mitochondrial (mt genome. For each of 16,569 nucleotide positions of the mt genome it holds two sets of four 25-mer probes each that match the heavy and the light strand of a reference mt genome and vary only at their central position to interrogate all four possible alleles. In addition, the MitoChip v2.0 carries alternative local context probes to account for known mtDNA variants. These probes have been neglected in most studies due to the lack of software for their automated analysis. Results We provide ReseqChip, a free software that automates the process of resequencing mtDNA using multiple local context probes on the MitoChip v2.0. ReseqChip significantly improves base call rate and sequence accuracy. ReseqChip is available at http://code.open-bio.org/svnweb/index.cgi/bioperl/browse/bioperl-live/trunk/Bio/Microarray/Tools/. Conclusions ReseqChip allows for the automated consolidation of base calls from alternative local mt genome context probes. It thereby improves the accuracy of resequencing, while reducing the number of non-called bases.

  7. DNA-guided nanoparticle assemblies

    Science.gov (United States)

    Gang, Oleg; Nykypanchuk, Dmytro; Maye, Mathew; van der Lelie, Daniel

    2013-07-16

    In some embodiments, DNA-capped nanoparticles are used to define a degree of crystalline order in assemblies thereof. In some embodiments, thermodynamically reversible and stable body-centered cubic (bcc) structures, with particles occupying <.about.10% of the unit cell, are formed. Designs and pathways amenable to the crystallization of particle assemblies are identified. In some embodiments, a plasmonic crystal is provided. In some aspects, a method for controlling the properties of particle assemblages is provided. In some embodiments a catalyst is formed from nanoparticles linked by nucleic acid sequences and forming an open crystal structure with catalytically active agents attached to the crystal on its surface or in interstices.

  8. Automated extraction of DNA from clothing

    DEFF Research Database (Denmark)

    Stangegaard, Michael; Hjort, Benjamin Benn; Nøhr Hansen, Thomas

    2011-01-01

    Presence of PCR inhibitors in extracted DNA may interfere with the subsequent quantification and short tandem repeat (STR) reactions used in forensic genetic DNA typing. We have compared three automated DNA extraction methods based on magnetic beads with a manual method with the aim of reducing...... the amount of PCR inhibitors in the DNA extracts and increasing the proportion of reportable DNA profiles....

  9. Automated DNA extraction from pollen in honey.

    Science.gov (United States)

    Guertler, Patrick; Eicheldinger, Adelina; Muschler, Paul; Goerlich, Ottmar; Busch, Ulrich

    2014-04-15

    In recent years, honey has become subject of DNA analysis due to potential risks evoked by microorganisms, allergens or genetically modified organisms. However, so far, only a few DNA extraction procedures are available, mostly time-consuming and laborious. Therefore, we developed an automated DNA extraction method from pollen in honey based on a CTAB buffer-based DNA extraction using the Maxwell 16 instrument and the Maxwell 16 FFS Nucleic Acid Extraction System, Custom-Kit. We altered several components and extraction parameters and compared the optimised method with a manual CTAB buffer-based DNA isolation method. The automated DNA extraction was faster and resulted in higher DNA yield and sufficient DNA purity. Real-time PCR results obtained after automated DNA extraction are comparable to results after manual DNA extraction. No PCR inhibition was observed. The applicability of this method was further successfully confirmed by analysis of different routine honey samples. Copyright © 2013 Elsevier Ltd. All rights reserved.

  10. Regulating DNA Self-assembly by DNA-Surface Interactions.

    Science.gov (United States)

    Liu, Longfei; Li, Yulin; Wang, Yong; Zheng, Jianwei; Mao, Chengde

    2017-12-14

    DNA self-assembly provides a powerful approach for preparation of nanostructures. It is often studied in bulk solution and involves only DNA-DNA interactions. When confined to surfaces, DNA-surface interactions become an additional, important factor to DNA self-assembly. However, the way in which DNA-surface interactions influence DNA self-assembly is not well studied. In this study, we showed that weak DNA-DNA interactions could be stabilized by DNA-surface interactions to allow large DNA nanostructures to form. In addition, the assembly can be conducted isothermally at room temperature in as little as 5 seconds. © 2017 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim.

  11. DNA Assembly in 3D Printed Fluidics.

    Directory of Open Access Journals (Sweden)

    William G Patrick

    Full Text Available The process of connecting genetic parts-DNA assembly-is a foundational technology for synthetic biology. Microfluidics present an attractive solution for minimizing use of costly reagents, enabling multiplexed reactions, and automating protocols by integrating multiple protocol steps. However, microfluidics fabrication and operation can be expensive and requires expertise, limiting access to the technology. With advances in commodity digital fabrication tools, it is now possible to directly print fluidic devices and supporting hardware. 3D printed micro- and millifluidic devices are inexpensive, easy to make and quick to produce. We demonstrate Golden Gate DNA assembly in 3D-printed fluidics with reaction volumes as small as 490 nL, channel widths as fine as 220 microns, and per unit part costs ranging from $0.61 to $5.71. A 3D-printed syringe pump with an accompanying programmable software interface was designed and fabricated to operate the devices. Quick turnaround and inexpensive materials allowed for rapid exploration of device parameters, demonstrating a manufacturing paradigm for designing and fabricating hardware for synthetic biology.

  12. "First generation" automated DNA sequencing technology.

    Science.gov (United States)

    Slatko, Barton E; Kieleczawa, Jan; Ju, Jingyue; Gardner, Andrew F; Hendrickson, Cynthia L; Ausubel, Frederick M

    2011-10-01

    Beginning in the 1980s, automation of DNA sequencing has greatly increased throughput, reduced costs, and enabled large projects to be completed more easily. The development of automation technology paralleled the development of other aspects of DNA sequencing: better enzymes and chemistry, separation and imaging technology, sequencing protocols, robotics, and computational advancements (including base-calling algorithms with quality scores, database developments, and sequence analysis programs). Despite the emergence of high-throughput sequencing platforms, automated Sanger sequencing technology remains useful for many applications. This unit provides background and a description of the "First-Generation" automated DNA sequencing technology. It also includes protocols for using the current Applied Biosystems (ABI) automated DNA sequencing machines. © 2011 by John Wiley & Sons, Inc.

  13. Mobius Assembly: A versatile Golden-Gate framework towards universal DNA assembly.

    Directory of Open Access Journals (Sweden)

    Andreas I Andreou

    Full Text Available Synthetic biology builds upon the foundation of engineering principles, prompting innovation and improvement in biotechnology via a design-build-test-learn cycle. A community-wide standard in DNA assembly would enable bio-molecular engineering at the levels of predictivity and universality in design and construction that are comparable to other engineering fields. Golden Gate Assembly technology, with its robust capability to unidirectionally assemble numerous DNA fragments in a one-tube reaction, has the potential to deliver a universal standard framework for DNA assembly. While current Golden Gate Assembly frameworks (e.g. MoClo and Golden Braid render either high cloning capacity or vector toolkit simplicity, the technology can be made more versatile-simple, streamlined, and cost/labor-efficient, without compromising capacity. Here we report the development of a new Golden Gate Assembly framework named Mobius Assembly, which combines vector toolkit simplicity with high cloning capacity. It is based on a two-level, hierarchical approach and utilizes a low-frequency cutter to reduce domestication requirements. Mobius Assembly embraces the standard overhang designs designated by MoClo, Golden Braid, and Phytobricks and is largely compatible with already available Golden Gate part libraries. In addition, dropout cassettes encoding chromogenic proteins were implemented for cost-free visible cloning screening that color-code different cloning levels. As proofs of concept, we have successfully assembled up to 16 transcriptional units of various pigmentation genes in both operon and multigene arrangements. Taken together, Mobius Assembly delivers enhanced versatility and efficiency in DNA assembly, facilitating improved standardization and automation.

  14. Quantifying quality in DNA self-assembly

    Science.gov (United States)

    Wagenbauer, Klaus F.; Wachauf, Christian H.; Dietz, Hendrik

    2014-01-01

    Molecular self-assembly with DNA is an attractive route for building nanoscale devices. The development of sophisticated and precise objects with this technique requires detailed experimental feedback on the structure and composition of assembled objects. Here we report a sensitive assay for the quality of assembly. The method relies on measuring the content of unpaired DNA bases in self-assembled DNA objects using a fluorescent de-Bruijn probe for three-base ‘codons’, which enables a comparison with the designed content of unpaired DNA. We use the assay to measure the quality of assembly of several multilayer DNA origami objects and illustrate the use of the assay for the rational refinement of assembly protocols. Our data suggests that large and complex objects like multilayer DNA origami can be made with high strand integration quality up to 99%. Beyond DNA nanotechnology, we speculate that the ability to discriminate unpaired from paired nucleic acids in the same macromolecule may also be useful for analysing cellular nucleic acids. PMID:24751596

  15. Automated assembly of micro mechanical parts in a Microfactory setup

    DEFF Research Database (Denmark)

    Eriksson, Torbjörn Gerhard; Hansen, Hans Nørgaard; Gegeckaite, Asta

    2006-01-01

    Many micro products in use today are manufactured using semi-automatic assembly. Handling, assembly and transport of the parts are especially labour intense processes. Automation of these processes holds a large potential, especially if flexible, modular microfactories can be developed. This paper...... focuses on the issues that have to be taken into consideration in order to go from a semi-automatic production into an automated microfactory. The application in this study is a switch consisting of 7 parts. The development of a microfactory setup to take care of the automated assembly of the switch...

  16. DNA-templated assembly of nanoscale architectures

    Science.gov (United States)

    Stanca, Sarmiza Elena; Ongaro, Andrea; Eritja, Ramon; Fitzmaurice, Donald

    2005-09-01

    The assembly and detailed structural characterization of a Y-shaped DNA template incorporating a central biotin moiety is reported. Also reported is the use of this template to assemble a model protein-functionalized three-electrode architecture. Of particular significance is the finding that a biotin-modified nanoparticle will recognize and selectively bind the central biotin moiety, once functionalized by the protein streptavidin. Potential applications of the above and related DNA templates in the emerging field of nanoelectronics are considered.

  17. DNA controlled assembly of soft nanoparticles

    DEFF Research Database (Denmark)

    Vogel, Stefan; Simonsen, Adam Cohen; Jakobsen, Ulla

    2008-01-01

    DNA-encoding of solid nanoparticles requires surfacechemistry, which is often tedious and not generally applicable. In the present study non-covalently attached DNA are used to assemble soft nanoparticles (liposomes) in solution. This process displays remarkably sharp thermal transitions from ass...

  18. Terminating DNA Tile Assembly with Nanostructured Caps.

    Science.gov (United States)

    Agrawal, Deepak K; Jiang, Ruoyu; Reinhart, Seth; Mohammed, Abdul M; Jorgenson, Tyler D; Schulman, Rebecca

    2017-10-24

    Precise control over the nucleation, growth, and termination of self-assembly processes is a fundamental tool for controlling product yield and assembly dynamics. Mechanisms for altering these processes programmatically could allow the use of simple components to self-assemble complex final products or to design processes allowing for dynamic assembly or reconfiguration. Here we use DNA tile self-assembly to develop general design principles for building complexes that can bind to a growing biomolecular assembly and terminate its growth by systematically characterizing how different DNA origami nanostructures interact with the growing ends of DNA tile nanotubes. We find that nanostructures that present binding interfaces for all of the binding sites on a growing facet can bind selectively to growing ends and stop growth when these interfaces are presented on either a rigid or floppy scaffold. In contrast, nucleation of nanotubes requires the presentation of binding sites in an arrangement that matches the shape of the structure's facet. As a result, it is possible to build nanostructures that can terminate the growth of existing nanotubes but cannot nucleate a new structure. The resulting design principles for constructing structures that direct nucleation and termination of the growth of one-dimensional nanostructures can also serve as a starting point for programmatically directing two- and three-dimensional crystallization processes using nanostructure design.

  19. Automating System Assembly of Aerospace Systems

    Science.gov (United States)

    Manolios, Panagiotis

    2008-01-01

    One of the major challenges in modern aerospace designs is the integration and assembly of independently developed components. We have formalized this as the system assembly problem: from a sea of available components, which should be selected and how should they be connected, integrated, and assembled so that the overall system requirements are satisfied in a certifiable way? We present a powerful framework for automatically solving the system assembly problem directly from system requirements by using formal verification technology. We also present a case study where we applied our work to large-scale industrial examples from the Boeing Dreamliner.

  20. Solid-phase cloning for high-throughput assembly of single and multiple DNA parts

    DEFF Research Database (Denmark)

    Lundqvist, Magnus; Edfors, Fredrik; Sivertsson, Åsa

    2015-01-01

    present a robust automated protocol for restriction enzyme based SPC and its performance for the cloning of >60 000 unique human gene fragments into expression vectors. In addition, we report on SPC-based single-strand assembly for applications where exact control of the sequence between fragments...... is needed or where multiple inserts are to be assembled. In this approach, the solid support allows for head-to-tail assembly of DNA fragments based on hybridization and polymerase fill-in. The usefulness of head-to-tail SPC was demonstrated by assembly of >150 constructs with up to four DNA parts...

  1. DNA-nanostructure-assembly by sequential spotting

    Directory of Open Access Journals (Sweden)

    Breitenstein Michael

    2011-11-01

    Full Text Available Abstract Background The ability to create nanostructures with biomolecules is one of the key elements in nanobiotechnology. One of the problems is the expensive and mostly custom made equipment which is needed for their development. We intended to reduce material costs and aimed at miniaturization of the necessary tools that are essential for nanofabrication. Thus we combined the capabilities of molecular ink lithography with DNA-self-assembling capabilities to arrange DNA in an independent array which allows addressing molecules in nanoscale dimensions. Results For the construction of DNA based nanostructures a method is presented that allows an arrangement of DNA strands in such a way that they can form a grid that only depends on the spotted pattern of the anchor molecules. An atomic force microscope (AFM has been used for molecular ink lithography to generate small spots. The sequential spotting process allows the immobilization of several different functional biomolecules with a single AFM-tip. This grid which delivers specific addresses for the prepared DNA-strand serves as a two-dimensional anchor to arrange the sequence according to the pattern. Once the DNA-nanoarray has been formed, it can be functionalized by PNA (peptide nucleic acid to incorporate advanced structures. Conclusions The production of DNA-nanoarrays is a promising task for nanobiotechnology. The described method allows convenient and low cost preparation of nanoarrays. PNA can be used for complex functionalization purposes as well as a structural element.

  2. DNA origami as a nanoscale template for protein assembly

    International Nuclear Information System (INIS)

    Kuzyk, Anton; Laitinen, Kimmo T; Toermae, Paeivi

    2009-01-01

    We describe two general approaches to the utilization of DNA origami structures for the assembly of materials. In one approach, DNA origami is used as a prefabricated template for subsequent assembly of materials. In the other, materials are assembled simultaneously with the DNA origami, i.e. the DNA origami technique is used to drive the assembly of materials. Fabrication of complex protein structures is demonstrated by these two approaches. The latter approach has the potential to be extended to the assembly of multiple materials with single attachment chemistry.

  3. Enhanced throughput for infrared automated DNA sequencing

    Science.gov (United States)

    Middendorf, Lyle R.; Gartside, Bill O.; Humphrey, Pat G.; Roemer, Stephen C.; Sorensen, David R.; Steffens, David L.; Sutter, Scott L.

    1995-04-01

    Several enhancements have been developed and applied to infrared automated DNA sequencing resulting in significantly higher throughput. A 41 cm sequencing gel (31 cm well- to-read distance) combines high resolution of DNA sequencing fragments with optimized run times yielding two runs per day of 500 bases per sample. A 66 cm sequencing gel (56 cm well-to-read distance) produces sequence read lengths of up to 1000 bases for ds and ss templates using either T7 polymerase or cycle-sequencing protocols. Using a multichannel syringe to load 64 lanes allows 16 samples (compatible with 96-well format) to be visualized for each run. The 41 cm gel configuration allows 16,000 bases per day (16 samples X 500 bases/sample X 2 ten hour runs/day) to be sequenced with the advantages of infrared technology. Enhancements to internal labeling techniques using an infrared-labeled dATP molecule (Boehringer Mannheim GmbH, Penzberg, Germany; Sequenase (U.S. Biochemical) have also been made. The inclusion of glycerol in the sequencing reactions yields greatly improved results for some primer and template combinations. The inclusion of (alpha) -Thio-dNTP's in the labeling reaction increases signal intensity two- to three-fold.

  4. Engineering of automated assembly of beam-shaping optics

    Science.gov (United States)

    Haag, Sebastian; Sinhoff, Volker; Müller, Tobias; Brecher, Christian

    2014-03-01

    Beam-shaping is essential for any kind of laser application. Assembly technologies for beam-shaping subassemblies are subject to intense research and development activities and their technical feasibility has been proven in recent years while economic viability requires more efficient engineering tools for process planning and production ramp up of complex assembly tasks for micro-optical systems. The work presented in this paper aims for significant reduction of process development and production ramp up times for the automated assembly of micro-optical subassemblies for beam-collimation and beam-tilting. The approach proposed bridges the gap between the product development phase and the realization of automation control through integration of established software tools such as optics simulation and CAD modeling as well as through introduction of novel software tools and methods to efficiently describe active alignment strategies. The focus of the paper is put on the methodological approach regarding the engineering of assembly processes for beam-shaping micro-optics and the formal representation of assembly objectives similar to representation in mechanical assemblies. Main topic of the paper is the engineering methodology for active alignment processes based on the classification of optical functions for beam-shaping optics and corresponding standardized measurement setups including adaptable alignment algorithms. The concepts are applied to industrial use-cases: (1) integrated collimation module for fast- and slow-axis and (2) beam-tilting subassembly consisting of a fast-axis collimator and micro-lens array. The paper concludes with an overview of current limitations as well as an outlook on the next development steps considering adhesive bonding processes.

  5. Software-Supported USER Cloning Strategies for Site-Directed Mutagenesis and DNA Assembly

    DEFF Research Database (Denmark)

    Genee, Hans Jasper; Bonde, Mads Tvillinggaard; Bagger, Frederik Otzen

    2015-01-01

    USER cloning is a fast and versatile method for engineering of plasmid DNA. We have developed a user friendly Web server tool that automates the design of optimal PCR primers for several distinct USER cloning-based applications. Our Web server, named AMUSER (Automated DNA Modifications with USER...... cloning), facilitates DNA assembly and introduction of virtually any type of site-directed mutagenesis by designing optimal PCR primers for the desired genetic changes. To demonstrate the utility, we designed primers for a simultaneous two-position site-directed mutagenesis of green fluorescent protein...... (GFP) to yellow fluorescent protein (YFP), which in a single step reaction resulted in a 94% cloning efficiency. AMUSER also supports degenerate nucleotide primers, single insert combinatorial assembly, and flexible parameters for PCR amplification. AMUSER is freely available online at ....

  6. Automated assembly of a tetrahedral truss structure using machine vision

    Science.gov (United States)

    Doggett, William R.

    1992-01-01

    The Automated Structures Assembly Laboratory is a unique facility at NASA Langley Research Center used to investigate the robotic assembly of truss structures. Two special-purpose end-effectors have been used to assemble 102 truss members and 12 panels into an 8-meter diameter structure. One end-effector is dedicated to truss member insertion, while a second end-effector is used to install panels. Until recently, the robot motions required to construct the structure were developed iteratively using the facility hardware. Recent work at Langley has resulted in a compact machine vision system capable of providing position information relative to targets on the structure. Use of the vision system to guide the robot from an approach point 10 to 18 inches from the structure, offsetting model inaccuracies, permits robot motion based on calculated points as a first step toward use of preplanned paths from an automated path planner. This paper presents recent work at Langley highlighting the application of the machine vision system during truss member insertion.

  7. Automated assembly in the construction of silicon microstrip detector modules

    CERN Document Server

    Eckert, S; Meinhardt, J; Runge, K; Benes, J

    2002-01-01

    The paper concerns silicon microstrip trackers for future experiments at the Large Hadron Collider (LHC). It describes a system for the automated assembly of the trackers. The aim is uniform quality and a mechanical precision of better than 5 mu m. It has been implemented based on an industrial gantry robot. The gantry is equipped with a complex vacuum system which dispenses glue, and places the mechanical parts and the ASICS and the four silicon sensors with the required precision. The modules are double sided and 18 cm * 6 cm in dimension. (5 refs).

  8. Functional-to-form mapping for assembly design automation

    Science.gov (United States)

    Xu, Z. G.; Liu, W. M.; Shen, W. D.; Yang, D. Y.; Liu, T. T.

    2017-11-01

    Assembly-level function-to-form mapping is the most effective procedure towards design automation. The research work mainly includes: the assembly-level function definitions, product network model and the two-step mapping mechanisms. The function-to-form mapping is divided into two steps, i.e. mapping of function-to-behavior, called the first-step mapping, and the second-step mapping, i.e. mapping of behavior-to-structure. After the first step mapping, the three dimensional transmission chain (or 3D sketch) is studied, and the feasible design computing tools are developed. The mapping procedure is relatively easy to be implemented interactively, but, it is quite difficult to finish it automatically. So manual, semi-automatic, automatic and interactive modification of the mapping model are studied. A mechanical hand F-F mapping process is illustrated to verify the design methodologies.

  9. Design of a miniaturized solid state laser for automated assembly

    Science.gov (United States)

    Funck, Max C.; Dolkemeyer, Jan; Morasch, Valentin; Loosen, Peter

    2010-05-01

    A miniaturized solid state laser for marking applications has been developed featuring novel assembly strategies to reduce size, cost and assembly effort. Design and setup have been laid out with future automation of the assembly in mind. Using a high precision robot the optical components composing the laser system are directly placed on a planar substrate providing accurate positioning and alignment within a few microns. No adjustable mounts for mirrors and lenses are necessary, greatly simplifying the setup. Consisting of either a ND:YAG or a Nd:YVO4 crystal pumped with a fiber coupled diode laser, a q-switch for pulse generation and a beam expander the entire assembly is confined in a 100ml space and delivers 4 W of continuous output power at 1.064 μm with an efficiency greater than 40%. Pulse lengths of 10-20 ns and repetition rates of up to 150 kHz have been obtained with an acousto-optic modulator. In addition, a custom designed electro-optic modulator with integrated high voltage switch has been realized. A supply unit for the entire system, including scanner and water cooling, is integrated in a 19" industrial chassis and can be operated via a graphical user interface on a standard personal computer.

  10. Circuits and programmable self-assembling DNA structures

    OpenAIRE

    Carbone, Alessandra; Seeman, Nadrian C.

    2002-01-01

    Self-assembly is beginning to be seen as a practical vehicle for computation. We investigate how basic ideas on tiling can be applied to the assembly and evaluation of circuits. We suggest that these procedures can be realized on the molecular scale through the medium of self-assembled DNA tiles. One layer of self-assembled DNA tiles will be used as the program or circuit that leads to the computation of a particular Boolean expression. This layer templates the assembly of tiles, and their as...

  11. Development of a machine vision system for automated structural assembly

    Science.gov (United States)

    Sydow, P. Daniel; Cooper, Eric G.

    1992-01-01

    Research is being conducted at the LaRC to develop a telerobotic assembly system designed to construct large space truss structures. This research program was initiated within the past several years, and a ground-based test-bed was developed to evaluate and expand the state of the art. Test-bed operations currently use predetermined ('taught') points for truss structural assembly. Total dependence on the use of taught points for joint receptacle capture and strut installation is neither robust nor reliable enough for space operations. Therefore, a machine vision sensor guidance system is being developed to locate and guide the robot to a passive target mounted on the truss joint receptacle. The vision system hardware includes a miniature video camera, passive targets mounted on the joint receptacles, target illumination hardware, and an image processing system. Discrimination of the target from background clutter is accomplished through standard digital processing techniques. Once the target is identified, a pose estimation algorithm is invoked to determine the location, in three-dimensional space, of the target relative to the robots end-effector. Preliminary test results of the vision system in the Automated Structural Assembly Laboratory with a range of lighting and background conditions indicate that it is fully capable of successfully identifying joint receptacle targets throughout the required operational range. Controlled optical bench test results indicate that the system can also provide the pose estimation accuracy to define the target position.

  12. Directed self-assembly of DNA tiles into complex nanocages.

    Science.gov (United States)

    Tian, Cheng; Li, Xiang; Liu, Zhiyu; Jiang, Wen; Wang, Guansong; Mao, Chengde

    2014-07-28

    Tile-based self-assembly is a powerful method in DNA nanotechnology and has produced a wide range of well-defined nanostructures. But the resulting structures are relatively simple. Increasing the structural complexity and the scope of the accessible structures is an outstanding challenge in molecular self-assembly. A strategy to partially address this problem by introducing flexibility into assembling DNA tiles and employing directing agents to control the self-assembly process is presented. To demonstrate this strategy, a range of DNA nanocages have been rationally designed and constructed. Many of them can not be assembled otherwise. All of the resulting structures have been thoroughly characterized by gel electrophoresis and cryogenic electron microscopy. This strategy greatly expands the scope of accessible DNA nanostructures and would facilitate technological applications such as nanoguest encapsulation, drug delivery, and nanoparticle organization. © 2014 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

  13. Phase behavior and selectivity of DNA-linked nanoparticle assemblies

    NARCIS (Netherlands)

    Lukatsky, D.B.; Frenkel, D.

    2004-01-01

    We propose a model that can account for the experimentally observed phase behavior of DNA-nanoparticle assemblies [R. Jin et al., J. Am. Chem. Soc. 125, 1643 (2003); T. A. Taton et al., Science 289, 1757 (2000)]. The binding of DNA-coated nanoparticles by dissolved DNA linkers can be described by

  14. Assembly of DNA Architectures in a Non-Aqueous Solution

    Science.gov (United States)

    2012-08-31

    templated semiconductor nanocrystal growth for controlled DNA packing and gene delivery. ACS Nano 2012, 6, 689–695. 21. Zang, D.Y.; Grote, J. DNA...189–212. 23. Séverac, F.; Alphonse, P.; Estève, A.; Bancaud, A.; Rossi, C. High-energy Al/ Cuo nanocomposites obtained by DNA-directed assembly. Adv

  15. pH-Controlled Assembly of DNA Tiles.

    Science.gov (United States)

    Amodio, Alessia; Adedeji, Abimbola Feyisara; Castronovo, Matteo; Franco, Elisa; Ricci, Francesco

    2016-10-05

    We demonstrate a strategy to trigger and finely control the assembly of supramolecular DNA nanostructures with pH. Control is achieved via a rationally designed strand displacement circuit that responds to pH and activates a downstream DNA tile self-assembly process. We observe that the DNA structures form under neutral/basic conditions, while the self-assembly process is suppressed under acidic conditions. The strategy presented here demonstrates a modular approach toward building systems capable of processing biochemical inputs and finely controlling the assembly of DNA-based nanostructures under isothermal conditions. In particular, the presented architecture is relevant for the development of complex DNA devices able to sense and respond to molecular markers associated with abnormal metabolism.

  16. DNA-Controlled Assembly of Soft Nanoparticles

    DEFF Research Database (Denmark)

    Vogel, Stefan

    2015-01-01

    This book covers the emerging topic of DNA nanotechnology and DNA supramolecular chemistry in its broader sense. By taking DNA out of its biological role, this biomolecule has become a very versatile building block in materials chemistry, supramolecular chemistry and bio-nanotechnology. Many nove......-covalent systems, DNA origami, DNA based switches, DNA machines, and alternative structures and templates. This broad coverage is very appealing since it combines both the synthesis of modified DNA as well as designer concepts to successfully plan and make DNA nanostructures....

  17. Physical principles for DNA tile self-assembly.

    Science.gov (United States)

    Evans, Constantine G; Winfree, Erik

    2017-06-19

    DNA tiles provide a promising technique for assembling structures with nanoscale resolution through self-assembly by basic interactions rather than top-down assembly of individual structures. Tile systems can be programmed to grow based on logical rules, allowing for a small number of tile types to assemble large, complex assemblies that can retain nanoscale resolution. Such algorithmic systems can even assemble different structures using the same tiles, based on inputs that seed the growth. While programming and theoretical analysis of tile self-assembly often makes use of abstract logical models of growth, experimentally implemented systems are governed by nanoscale physical processes that can lead to very different behavior, more accurately modeled by taking into account the thermodynamics and kinetics of tile attachment and detachment in solution. This review discusses the relationships between more abstract and more physically realistic tile assembly models. A central concern is how consideration of model differences enables the design of tile systems that robustly exhibit the desired abstract behavior in realistic physical models and in experimental implementations. Conversely, we identify situations where self-assembly in abstract models can not be well-approximated by physically realistic models, putting constraints on physical relevance of the abstract models. To facilitate the discussion, we introduce a unified model of tile self-assembly that clarifies the relationships between several well-studied models in the literature. Throughout, we highlight open questions regarding the physical principles for DNA tile self-assembly.

  18. Modified Classical Graph Algorithms for the DNA Fragment Assembly Problem

    Directory of Open Access Journals (Sweden)

    Guillermo M. Mallén-Fullerton

    2015-09-01

    Full Text Available DNA fragment assembly represents an important challenge to the development of efficient and practical algorithms due to the large number of elements to be assembled. In this study, we present some graph theoretical linear time algorithms to solve the problem. To achieve linear time complexity, a heap with constant time operations was developed, for the special case where the edge weights are integers and do not depend on the problem size. The experiments presented show that modified classical graph theoretical algorithms can solve the DNA fragment assembly problem efficiently.

  19. DNA-Based Self-Assembly of Fluorescent Nanodiamonds.

    Science.gov (United States)

    Zhang, Tao; Neumann, Andre; Lindlau, Jessica; Wu, Yuzhou; Pramanik, Goutam; Naydenov, Boris; Jelezko, Fedor; Schüder, Florian; Huber, Sebastian; Huber, Marinus; Stehr, Florian; Högele, Alexander; Weil, Tanja; Liedl, Tim

    2015-08-12

    As a step toward deterministic and scalable assembly of ordered spin arrays we here demonstrate a bottom-up approach to position fluorescent nanodiamonds (NDs) with nanometer precision on DNA origami structures. We have realized a reliable and broadly applicable surface modification strategy that results in DNA-functionalized and perfectly dispersed NDs that were then self-assembled in predefined geometries. With optical studies we show that the fluorescence properties of the nitrogen-vacancy color centers in NDs are preserved during surface modification and DNA assembly. As this method allows the nanoscale arrangement of fluorescent NDs together with other optically active components in complex geometries, applications based on self-assembled spin lattices or plasmon-enhanced spin sensors as well as improved fluorescent labeling for bioimaging could be envisioned.

  20. Directed Formation of DNA Nanoarrays through Orthogonal Self-Assembly

    Directory of Open Access Journals (Sweden)

    Eugen Stulz

    2011-06-01

    Full Text Available We describe the synthesis of terpyridine modified DNA strands which selectively form DNA nanotubes through orthogonal hydrogen bonding and metal complexation interactions. The short DNA strands are designed to self-assemble into long duplexes through a sticky-end approach. Addition of weakly binding metals such as Zn(II and Ni(II induces the formation of tubular arrays consisting of DNA bundles which are 50-200 nm wide and 2-50 nm high. TEM shows additional long distance ordering of the terpy-DNA complexes into fibers.

  1. Gigadalton-scale shape-programmable DNA assemblies

    Science.gov (United States)

    Wagenbauer, Klaus F.; Sigl, Christian; Dietz, Hendrik

    2017-12-01

    Natural biomolecular assemblies such as molecular motors, enzymes, viruses and subcellular structures often form by self-limiting hierarchical oligomerization of multiple subunits. Large structures can also assemble efficiently from a few components by combining hierarchical assembly and symmetry, a strategy exemplified by viral capsids. De novo protein design and RNA and DNA nanotechnology aim to mimic these capabilities, but the bottom-up construction of artificial structures with the dimensions and complexity of viruses and other subcellular components remains challenging. Here we show that natural assembly principles can be combined with the methods of DNA origami to produce gigadalton-scale structures with controlled sizes. DNA sequence information is used to encode the shapes of individual DNA origami building blocks, and the geometry and details of the interactions between these building blocks then control their copy numbers, positions and orientations within higher-order assemblies. We illustrate this strategy by creating planar rings of up to 350 nanometres in diameter and with atomic masses of up to 330 megadaltons, micrometre-long, thick tubes commensurate in size to some bacilli, and three-dimensional polyhedral assemblies with sizes of up to 1.2 gigadaltons and 450 nanometres in diameter. We achieve efficient assembly, with yields of up to 90 per cent, by using building blocks with validated structure and sufficient rigidity, and an accurate design with interaction motifs that ensure that hierarchical assembly is self-limiting and able to proceed in equilibrium to allow for error correction. We expect that our method, which enables the self-assembly of structures with sizes approaching that of viruses and cellular organelles, can readily be used to create a range of other complex structures with well defined sizes, by exploiting the modularity and high degree of addressability of the DNA origami building blocks used.

  2. Integrating DNA strand-displacement circuitry with DNA tile self-assembly

    Science.gov (United States)

    Zhang, David Yu; Hariadi, Rizal F.; Choi, Harry M.T.; Winfree, Erik

    2013-01-01

    DNA nanotechnology has emerged as a reliable and programmable way of controlling matter at the nanoscale through the specificity of Watson–Crick base pairing, allowing both complex self-assembled structures with nanometer precision and complex reaction networks implementing digital and analog behaviors. Here we show how two well-developed frameworks, DNA tile self-assembly and DNA strand-displacement circuits, can be systematically integrated to provide programmable kinetic control of self-assembly. We demonstrate the triggered and catalytic isothermal self-assembly of DNA nanotubes over 10 μm long from precursor DNA double-crossover tiles activated by an upstream DNA catalyst network. Integrating more sophisticated control circuits and tile systems could enable precise spatial and temporal organization of dynamic molecular structures. PMID:23756381

  3. Assembly of DNA Architectures in a Non-Aqueous Solution

    Directory of Open Access Journals (Sweden)

    Thomas J. Proctor

    2012-08-01

    Full Text Available In the present work, the procedures for the creation of self-assembled DNA nanostructures in aqueous and non-aqueous media are described. DNA-Surfactant complex formation renders the DNA soluble in organic solvents offering an exciting way to bridge the transition of DNA origami materials electronics applications. The DNA retains its structural features, and these unique geometries provide an interesting candidate for future electronics and nanofabrication applications with potential for new properties. The DNA architectures were first assembled under aqueous conditions, and then characterized in solution (using circular dichroism (CD spectroscopy and on the surface (using atomic force microscopy (AFM. Following aqueous assembly, the DNA nanostructures were transitioned to a non-aqueous environment, where butanol was chosen for optical compatibility and thermal properties. The retention of DNA hierarchical structure and thermal stability in non-aqueous conditions were confirmed via CD spectroscopy. The formation and characterization of these higher order DNA-surfactant complexes is described in this paper.

  4. An automated annotation tool for genomic DNA sequences using ...

    Indian Academy of Sciences (India)

    Genomic sequence data are often available well before the annotated sequence is published. We present a method for analysis of genomic DNA to identify coding sequences using the GeneScan algorithm and characterize these resultant sequences by BLAST. The routines are used to develop a system for automated ...

  5. An automated annotation tool for genomic DNA sequences using ...

    Indian Academy of Sciences (India)

    Unknown

    Introduction. DNA sequencing has evolved from a complicated labo- ratory process to an automated technique using high- throughput sequencers with fluorescent-dye-based chemistry. This technological advance coupled with the replacement of the traditional mapping and sequencing of clones in series to an integrated ...

  6. An automated annotation tool for genomic DNA sequences using

    Indian Academy of Sciences (India)

    Genomic sequence data are often available well before the annotated sequence is published. We present a method for analysis of genomic DNA to identify coding sequences using the GeneScan algorithm and characterize these resultant sequences by BLAST. The routines are used to develop a system for automated ...

  7. Process development for automated solar cell and module production. Task 4. Automated array assembly. Annual report

    Energy Technology Data Exchange (ETDEWEB)

    Witham, C.R.

    1979-06-12

    MBA has been working on the automated array assembly task of the Low-Cost Solar Array project. A baseline sequence for the manufacture of solar cell modules is specified. Starting with silicon wafers, the process goes through damage etching, texture etching, junction formation, plasma edge etch, aluminum back surface field formation, and screen printed metallization to produce finished solar cells which are then series connected on a ribbon and bonded into a finished glass, PVB, tedlar module. A number of steps required additional developmental effort to verify technical and economic feasibility. These steps include texture etching, plasma edge etch, aluminum back surface field formation, array layup and interconnect, and module edge sealing and framing.

  8. Phase Transition and Optical Properties of DNA-Gold Nanoparticle Assemblies

    OpenAIRE

    Sun, Young; Harris, Nolan C.; Kiang, Ching-Hwa

    2007-01-01

    We review recent work on DNA-linked gold nanoparticle assemblies. The synthesis, properties, and phase behavior of such DNA-gold nanoparticle assemblies are described. These nanoparticle assemblies have strong optical extinction in the ultraviolet and visible light regions; hence, the technique is used to study the kinetics and phase transitions of DNA-gold nanoparticle assemblies. The melting transition of DNA-gold nanoparticle assemblies shows unusual trends compared to those of free DNA. T...

  9. Programmable DNA scaffolds for spatially-ordered protein assembly

    Science.gov (United States)

    Chandrasekaran, Arun Richard

    2016-02-01

    Ever since the notion of using DNA as a material was realized, it has been employed in the construction of complex structures that facilitate the assembly of nanoparticles or macromolecules with nanometer-scale precision. Specifically, tiles fashioned from DNA strands and DNA origami sheets have been shown to be suitable as scaffolds for immobilizing proteins with excellent control over their spatial positioning. Supramolecular assembly of proteins into periodic arrays in one or more dimensions is one of the most challenging aspects in the design of scaffolds for biomolecular investigations and macromolecular crystallization. This review provides a brief overview of how various biomolecular interactions with high degree of specificity such as streptavidin-biotin, antigen-antibody, and aptamer-protein interactions have been used to fabricate linear and multidimensional assemblies of structurally intact and functional proteins. The use of DNA-binding proteins as adaptors, polyamide recognition on DNA scaffolds and oligonucleotide linkers for protein assembly are also discussed.Ever since the notion of using DNA as a material was realized, it has been employed in the construction of complex structures that facilitate the assembly of nanoparticles or macromolecules with nanometer-scale precision. Specifically, tiles fashioned from DNA strands and DNA origami sheets have been shown to be suitable as scaffolds for immobilizing proteins with excellent control over their spatial positioning. Supramolecular assembly of proteins into periodic arrays in one or more dimensions is one of the most challenging aspects in the design of scaffolds for biomolecular investigations and macromolecular crystallization. This review provides a brief overview of how various biomolecular interactions with high degree of specificity such as streptavidin-biotin, antigen-antibody, and aptamer-protein interactions have been used to fabricate linear and multidimensional assemblies of structurally

  10. The Automated Assembly Team contributions to the APRIMED Agile Manufacturing Project

    Energy Technology Data Exchange (ETDEWEB)

    Jones, R.E.; Ames, A.L.; Calton, T.L. [and others

    1995-06-01

    The Automated Assembly Team of the APRIMED Project (abbreviated as A{prime}) consists of two parts: the Archimedes Project, which is an ongoing project developing automated assembly technology, and the A{prime} Robot Team. Archimedes is a second generation assembly planning system that both provides a general high-level assembly sequencing capability and, for a smaller class of products, facilitates automatic programming of a robotic workcell to assemble them. The A{prime} robot team designed, developed, and implemented a flexible robot workcell which served as the automated factory of the A{prime} project. In this document we briefly describe the role of automated assembly planning in agile manufacturing, and specifically describe the contributions of the Archimedes project and the A{prime} robot team to the A{prime} project. We introduce the concepts of the Archimedes automated assembly planning project, and discuss the enhancements to Archimedes which were developed in response to the needs of the A{prime} project. We also present the work of the A{prime} robot team in designing and developing the A{prime} robot workcell, including all tooling and programming to support assembly of the A{prime} discriminator devices. Finally, we discuss the process changes which these technologies have enabled in the A{prime} project.

  11. The Automated Assembly Team contributions to the APRIMED Agile Manufacturing Project

    International Nuclear Information System (INIS)

    Jones, R.E.; Ames, A.L.; Calton, T.L.

    1995-06-01

    The Automated Assembly Team of the APRIMED Project (abbreviated as A') consists of two parts: the Archimedes Project, which is an ongoing project developing automated assembly technology, and the A' Robot Team. Archimedes is a second generation assembly planning system that both provides a general high-level assembly sequencing capability and, for a smaller class of products, facilitates automatic programming of a robotic workcell to assemble them. The A' robot team designed, developed, and implemented a flexible robot workcell which served as the automated factory of the A' project. In this document we briefly describe the role of automated assembly planning in agile manufacturing, and specifically describe the contributions of the Archimedes project and the A' robot team to the A' project. We introduce the concepts of the Archimedes automated assembly planning project, and discuss the enhancements to Archimedes which were developed in response to the needs of the A' project. We also present the work of the A' robot team in designing and developing the A' robot workcell, including all tooling and programming to support assembly of the A' discriminator devices. Finally, we discuss the process changes which these technologies have enabled in the A' project

  12. Process development for automated solar-cell and module production. Task 4. Automated array assembly. Quarterly report No. 3

    Energy Technology Data Exchange (ETDEWEB)

    Hagerty, J. J.; Gifford, M.

    1981-04-15

    The Automated Lamination Station is mechanically complete and is currently undergoing final wiring. The high current driver and isolator boards have been completed and installed, and the main interface board is under construction. The automated vacuum chamber has had a minor redesign to increase stiffness and improve the cover open/close mechanism. Design of the Final Assembly Station has been completed and construction is underway.

  13. Hierarchically assembled DNA origami tubules with reconfigurable chirality

    International Nuclear Information System (INIS)

    Chen, Haorong; Cha, Tae-Gon; Pan, Jing; Choi, Jong Hyun

    2013-01-01

    The dynamic reconfiguration of a hierarchically assembled tubular structure is demonstrated using the DNA origami technique. Short cylindrical DNA origami monomers are synthesized and linked into elongated tubules, which can then be disassembled via toehold-mediated strand displacement. The disassembled subunits are subsequently linked into tubules of a different chirality. The reconfiguration is performed with the subunits carrying dumbbell hairpin DNA oligonucleotides or gold nanoparticles (AuNPs). The reconfiguration of higher order origami structures presented here is useful for constructing dynamic nanostructures that exceed the size limit of single DNA origami and may facilitate the study of molecular or particle interactions by tuning their relative distance and organization. (paper)

  14. AI tools for use in assembly automation and some examples of recent applications

    OpenAIRE

    Sanders, David; Gegov, Alexander

    2013-01-01

    Purpose – This paper aims to review seven artificial intelligence tools that are useful in assembly automation: knowledge‐based systems, fuzzy logic, automatic knowledge acquisition, neural networks, genetic algorithms, case‐based reasoning and ambient‐intelligence. Design/methodology/approach – Each artificial intelligence tool is outlined, together with some examples of their use in assembly automation. Findings – Artificial intelligence has produced a number of useful and powerful tools. T...

  15. Engineering of DNA-mediated assemblies for biosensing applications

    Science.gov (United States)

    Xu, Phyllis F.

    Traditionally known as the genetic code, in recent years DNA has been engineered in new ways and applied toward novel applications in materials science and bio-nanotechnology. The nanometer features and highly specific base-pairing of DNA has enabled its use to build complex 2- and 3D nano-architectures and nano-structures. Specific DNA scaffolds include branched DNA junctions, 2D and 3D DNA "tiles" and "bricks", and DNA hydrogels that can be used for applications across various fields of materials science. Another unique feature of DNA is its ability to bind other materials through molecular recognition which has made the use of DNA aptamer or DNA-protein conjugates highly useful for applications in biosensing and identification. The main focus of this thesis is to demonstrate the use of DNA as an invaluable tool for engineering specific nanostructures for biosensing. The first chapter introduces the basics of DNA and current research in various DNA structures and assemblies and their applications. The second and third chapters describe the self-assembly of two structurally different nanomaterials using DNA, carbon nanotubes and gold nanoparticles. In the second chapter, the successful large-scale alignment of carbon nanotubes on a surface is shown. The study goes into the factors that affect the quality of alignment, including salt concentration, length of DNA, and annealing. In the third chapter, we show how DNA can be used to engineer discrete gold nanocrystal assemblies-"nanodumbbells"- that can adopt different structural conformations through DNA interactions. By incorporating DNA aptamers in the nanoparticle structure, these nanodumbbells can be used to sense particular analytes in solution. Chapter 4 continues the study of the nanodumbbell structures and their potential use as surface-enhanced Raman spectroscopy (SERS) biosensors. A large measurable difference in Raman signal was experimentally obtained from the two distinct nanodumbbell conformations. This

  16. Sequential self-assembly of DNA functionalized droplets.

    Science.gov (United States)

    Zhang, Yin; McMullen, Angus; Pontani, Lea-Laetitia; He, Xiaojin; Sha, Ruojie; Seeman, Nadrian C; Brujic, Jasna; Chaikin, Paul M

    2017-06-16

    Complex structures and devices, both natural and manmade, are often constructed sequentially. From crystallization to embryogenesis, a nucleus or seed is formed and built upon. Sequential assembly allows for initiation, signaling, and logical programming, which are necessary for making enclosed, hierarchical structures. Although biology relies on such schemes, they have not been available in materials science. Here, we demonstrate programmed sequential self-assembly of DNA functionalized emulsions. The droplets are initially inert because the grafted DNA strands are pre-hybridized in pairs. Active strands on initiator droplets then displace one of the paired strands and thus release its complement, which in turn activates the next droplet in the sequence, akin to living polymerization. Our strategy provides time and logic control during the self-assembly process, and offers a new perspective on the synthesis of materials.Natural complex systems are often constructed by sequential assembly but this is not readily available for synthetic systems. Here, the authors program the sequential self-assembly of DNA functionalized emulsions by altering the DNA grafted strands.

  17. Automated Array Assembly. Phase 2. Annual technical progress report, 1978

    Energy Technology Data Exchange (ETDEWEB)

    Carbajal, B G

    1979-02-01

    The Automated Array Assembly Task, Phase 2 of the Low Cost Silicon Solar Array (LSSA) Project is a process development task. This contract includes solar cell module process development activities in the areas of Surface Preparation. Plasma Processing, Diffusion, Cell Processing and Module Fabrication. In addition, a High Efficiency Cell Development Activity is included. The overall goal is to advance solar cell module process technology to meet the 1986 goal of a production capacity of 500 megawatts per year at a cost of less than $500 per kilowatt. This contract will focus on the process element developments stated above and will propose an overall module process. During 1978, process step development was carried out on texture etching including the evolution of a conceptual process model for the texturing process; plasma etching; and diffusion studies tat focused on doped polymer diffusion sources. Cell processing was carried out to test process steps and a simplified diode solar cell process was developed. Cell processing was also run to fabricate square cells to populate sample minimodules. Module fabrication featured the demonstration of a porcelainized steel-glass structure that should exceed the 20 year life goal of the LSA program. In a related set of studies, high efficiency cell development was carried out on the Texas Instruments developed Tandem Junction Cell (TJC) and a modification of the TJC called the Front Surface Field cell. These cells feature planar backslide contacts with no metallization of the frontside. Cell efficiencies in excess of 16% at AM1 have been attained with only modest fill factors. Photo generated current densities as high as 44 mA/cm/sup 2/ at AM0 have been attained. A transistor-like model has been proposed that fits the cell performance and provides a guideline for future improvements in cell performance.

  18. Technologies for the Fast Set-Up of Automated Assembly Processes

    DEFF Research Database (Denmark)

    Krüger, Norbert; Ude, Ales; Petersen, Henrik Gordon

    2014-01-01

    In this article, we describe technologies facilitating the set-up of automated assembly solutions which have been developed in the context of the IntellAct project (2011–2014). Tedious procedures are currently still required to establish such robot solutions. This hinders especially the automation...

  19. Collective helicity switching of a DNA-coat assembly

    Science.gov (United States)

    Kim, Yongju; Li, Huichang; He, Ying; Chen, Xi; Ma, Xiaoteng; Lee, Myongsoo

    2017-07-01

    Hierarchical assemblies of biomolecular subunits can carry out versatile tasks at the cellular level with remarkable spatial and temporal precision. As an example, the collective motion and mutual cooperation between complex protein machines mediate essential functions for life, such as replication, synthesis, degradation, repair and transport. Nucleic acid molecules are far less dynamic than proteins and need to bind to specific proteins to form hierarchical structures. The simplest example of these nucleic acid-based structures is provided by a rod-shaped tobacco mosaic virus, which consists of genetic material surrounded by coat proteins. Inspired by the complexity and hierarchical assembly of viruses, a great deal of effort has been devoted to design similarly constructed artificial viruses. However, such a wrapping approach makes nucleic acid dynamics insensitive to environmental changes. This limitation generally restricts, for example, the amplification of the conformational dynamics between the right-handed B form to the left-handed Z form of double-stranded deoxyribonucleic acid (DNA). Here we report a virus-like hierarchical assembly in which the native DNA and a synthetic coat undergo repeated collective helicity switching triggered by pH change under physiological conditions. We also show that this collective helicity inversion occurs during translocation of the DNA-coat assembly into intracellular compartments. Translating DNA conformational dynamics into a higher level of hierarchical dynamics may provide an approach to create DNA-based nanomachines.

  20. DNA-Promoted Auto-Assembly of Gold Nanoparticles: Effect of the DNA Sequence on the Stability of the Assemblies

    Directory of Open Access Journals (Sweden)

    Matthieu Doyen

    2013-07-01

    Full Text Available The use of deoxyribonucleic acid (DNA oligonucleotides has proven to be a powerful and versatile strategy to assemble nanomaterials into two (2D and three-dimensional (3D superlattices. With the aim of contributing to the elucidation of the factors that affect the stability of this type of superlattices, the assembly of gold nanoparticles grafted with different DNA oligonucleotides was characterized by UV-Vis absorption spectroscopy as a function of temperature. After establishing an appropriate methodology the effect of (i the length of the grafted oligonucleotides; (ii the length of their complementary parts and also of (iii the simultaneous grafting of different oligonucleotides was investigated. Our results indicate that the electrostatic repulsion between the particles and the cooperativity of the assembly process play crucial roles in the stability of the assemblies while the grafting density of the oligonucleotide strands seems to have little influence.

  1. Development of a machine vision guidance system for automated assembly of space structures

    Science.gov (United States)

    Cooper, Eric G.; Sydow, P. Daniel

    1992-01-01

    The topics are presented in viewgraph form and include: automated structural assembly robot vision; machine vision requirements; vision targets and hardware; reflective efficiency; target identification; pose estimation algorithms; triangle constraints; truss node with joint receptacle targets; end-effector mounted camera and light assembly; vision system results from optical bench tests; and future work.

  2. Electron Microscopic Visualization of Protein Assemblies on Flattened DNA Origami.

    Science.gov (United States)

    Mallik, Leena; Dhakal, Soma; Nichols, Joseph; Mahoney, Jacob; Dosey, Anne M; Jiang, Shuoxing; Sunahara, Roger K; Skiniotis, Georgios; Walter, Nils G

    2015-07-28

    DNA provides an ideal substrate for the engineering of versatile nanostructures due to its reliable Watson-Crick base pairing and well-characterized conformation. One of the most promising applications of DNA nanostructures arises from the site-directed spatial arrangement with nanometer precision of guest components such as proteins, metal nanoparticles, and small molecules. Two-dimensional DNA origami architectures, in particular, offer a simple design, high yield of assembly, and large surface area for use as a nanoplatform. However, such single-layer DNA origami were recently found to be structurally polymorphous due to their high flexibility, leading to the development of conformationally restrained multilayered origami that lack some of the advantages of the single-layer designs. Here we monitored single-layer DNA origami by transmission electron microscopy (EM) and discovered that their conformational heterogeneity is dramatically reduced in the presence of a low concentration of dimethyl sulfoxide, allowing for an efficient flattening onto the carbon support of an EM grid. We further demonstrated that streptavidin and a biotinylated target protein (cocaine esterase, CocE) can be captured at predesignated sites on these flattened origami while maintaining their functional integrity. Our demonstration that protein assemblies can be constructed with high spatial precision (within ∼2 nm of their predicted position on the platforms) by using strategically flattened single-layer origami paves the way for exploiting well-defined guest molecule assemblies for biochemistry and nanotechnology applications.

  3. Self-assembly of DNA rings from scaffold-free DNA tiles.

    Science.gov (United States)

    Yang, Yang; Zhao, Zhao; Zhang, Fei; Nangreave, Jeanette; Liu, Yan; Yan, Hao

    2013-04-10

    We report a scaffold-free approach in which four- and six-helix DNA bundle units, assembled from a small number of single stranded DNA oligonucleotides precisely arranged in networks of contiguous and semicrossover strands, are connected into DNA nano rings. Nearly uniform structures with well-defined diameters of 53 ± 7, 81 ± 9, 85 ± 8, and 166 ± 13 nm were achieved by introducing uniform, in-plane curvature to the repeating units. We demonstrate that precise higher order assemblies can be achieved by fine tuning the particular features of the individual building blocks.

  4. Automation of a single-DNA molecule stretching device

    DEFF Research Database (Denmark)

    Sørensen, Kristian Tølbøl; Lopacinska, Joanna M.; Tommerup, Niels

    2015-01-01

    We automate the manipulation of genomic-length DNA in a nanofluidic device based on real-time analysis of fluorescence images. In our protocol, individual molecules are picked from a microchannel and stretched with pN forces using pressure driven flows. The millimeter-long DNA fragments free...... flowing in micro- and nanofluidics emit low fluorescence and change shape, thus challenging the image analysis for machine vision. We demonstrate a set of image processing steps that increase the intrinsically low signal-to-noise ratio associated with single-molecule fluorescence microscopy. Furthermore......, we demonstrate how to estimate the length of molecules by continuous real-time image stitching and how to increase the effective resolution of a pressure controller by pulse width modulation. The sequence of image-processing steps addresses the challenges of genomic-length DNA visualization; however...

  5. DNA biosensor by self-assembly of carbon nanotubes and DNA to detect riboflavin

    Energy Technology Data Exchange (ETDEWEB)

    Li Jing [College of Chemistry and Chemical Engineering. Chongqing University, ChongQing, 400044 (China); Zhang Yunhuai, E-mail: xp2031@163.com [College of Chemistry and Chemical Engineering. Chongqing University, ChongQing, 400044 (China); Yang Tongyi [School of Life Science. NanJing University, Nanjing, 210093 (China); Zhang Huai [Liming Research Institute of Chemical Industry, LuoYang, 471001 (China); Yang Yixuan [State Key Laboratory of Chemical Resource Engineering. Beijing University of Chemical Technology, Beijing 100029 (China); Xiao Peng [College of Mathematics and Physics, Chongqing University, Chongqing 400044 (China)

    2009-10-15

    The fabrication of biosensors via self-assembly of single-walled carbon nanotubes (SWNTs) and DNA on a platinum electrode was presented in this paper. The carboxylic SWNTs were assembled on an amine-modified platinum electrode surface and followed by the assembly of NH{sub 2}-DNA with the carboxyl-amine coupling. The decorated surface was characterized by Field Emission Electron Microscopy (FEG-SEM) and electrochemical experiments, which showed that the reaction of DNA-SWNTs biosensor was quasi-reversible. The mechanism of DNA and riboflavin (VB{sub 2}) was studied by cyclic voltammetry and UV-Vis spectroscopy. The fabricated SWNTs-reinforced biosensor exhibits high sensitivity and low detection limit for the tested VB{sub 2} compared to the reported methods.

  6. An integer programming approach to DNA sequence assembly.

    Science.gov (United States)

    Chang, Youngjung; Sahinidis, Nikolaos V

    2011-08-10

    De novo sequence assembly is a ubiquitous combinatorial problem in all DNA sequencing technologies. In the presence of errors in the experimental data, the assembly problem is computationally challenging, and its solution may not lead to a unique reconstruct. The enumeration of all alternative solutions is important in drawing a reliable conclusion on the target sequence, and is often overlooked in the heuristic approaches that are currently available. In this paper, we develop an integer programming formulation and global optimization solution strategy to solve the sequence assembly problem with errors in the data. We also propose an efficient technique to identify all alternative reconstructs. When applied to examples of sequencing-by-hybridization, our approach dramatically increases the length of DNA sequences that can be handled with global optimality certificate to over 10,000, which is more than 10 times longer than previously reported. For some problem instances, alternative solutions exhibited a wide range of different ability in reproducing the target DNA sequence. Therefore, it is important to utilize the methodology proposed in this paper in order to obtain all alternative solutions to reliably infer the true reconstruct. These alternative solutions can be used to refine the obtained results and guide the design of further experiments to correctly reconstruct the target DNA sequence. Copyright © 2011 Elsevier Ltd. All rights reserved.

  7. Cellular Uptake of Tile-Assembled DNA Nanotubes.

    Science.gov (United States)

    Kocabey, Samet; Meinl, Hanna; MacPherson, Iain S; Cassinelli, Valentina; Manetto, Antonio; Rothenfusser, Simon; Liedl, Tim; Lichtenegger, Felix S

    2014-12-30

    DNA-based nanostructures have received great attention as molecular vehicles for cellular delivery of biomolecules and cancer drugs. Here, we report on the cellular uptake of tubule-like DNA tile-assembled nanostructures 27 nm in length and 8 nm in diameter that carry siRNA molecules, folic acid and fluorescent dyes. In our observations, the DNA structures are delivered to the endosome and do not reach the cytosol of the GFP -expressing HeLa cells that were used in the experiments. Consistent with this observation, no elevated silencing of the GFP gene could be detected. Furthermore, the presence of up to six molecules of folic acid on the carrier surface did not alter the uptake behavior and gene silencing. We further observed several challenges that have to be considered when performing in vitro and in vivo experiments with DNA structures: (i) DNA tile tubes consisting of 42 nt-long oligonucleotides and carrying single- or double-stranded extensions degrade within one hour in cell medium at 37 °C, while the same tubes without extensions are stable for up to eight hours. The degradation is caused mainly by the low concentration of divalent ions in the media. The lifetime in cell medium can be increased drastically by employing DNA tiles that are 84 nt long. (ii) Dyes may get cleaved from the oligonucleotides and then accumulate inside the cell close to the mitochondria, which can lead to misinterpretation of data generated by flow cytometry and fluorescence microscopy. (iii) Single-stranded DNA carrying fluorescent dyes are internalized at similar levels as the DNA tile-assembled tubes used here.

  8. Arduino-based automation of a DNA extraction system.

    Science.gov (United States)

    Kim, Kyung-Won; Lee, Mi-So; Ryu, Mun-Ho; Kim, Jong-Won

    2015-01-01

    There have been many studies to detect infectious diseases with the molecular genetic method. This study presents an automation process for a DNA extraction system based on microfluidics and magnetic bead, which is part of a portable molecular genetic test system. This DNA extraction system consists of a cartridge with chambers, syringes, four linear stepper actuators, and a rotary stepper actuator. The actuators provide a sequence of steps in the DNA extraction process, such as transporting, mixing, and washing for the gene specimen, magnetic bead, and reagent solutions. The proposed automation system consists of a PC-based host application and an Arduino-based controller. The host application compiles a G code sequence file and interfaces with the controller to execute the compiled sequence. The controller executes stepper motor axis motion, time delay, and input-output manipulation. It drives the stepper motor with an open library, which provides a smooth linear acceleration profile. The controller also provides a homing sequence to establish the motor's reference position, and hard limit checking to prevent any over-travelling. The proposed system was implemented and its functionality was investigated, especially regarding positioning accuracy and velocity profile.

  9. Electrostatic assembly of Cu2O nanoparticles on DNA templates

    International Nuclear Information System (INIS)

    Wang Li; Wei Gang; Qi Bin; Zhou Hualan; Liu Zhiguo; Song Yonghai; Yang Xiurong; Li Zhuang

    2006-01-01

    In this paper, a method for highly ordered assembly of cuprous oxide (Cu 2 O) nanoparticles (NPs) by DNA templates was reported. Cetyltrimethylammonium bromide (CTAB)-capped Cu 2 O NPs were adsorbed onto well-aligned λ-DNA chains to form necklace-like one-dimensional (1D) nanostructures. UV-vis, atomic force microscopy (AFM) and X-ray photoelectron spectroscopy (XPS) were used to characterize the nanostructure. The Cu 2 O nanostructures fabricated with the method are both highly ordered and quite straight

  10. Effect of water-DNA interactions on elastic properties of DNA self-assembled monolayers.

    Science.gov (United States)

    Domínguez, Carmen M; Ramos, Daniel; Mendieta-Moreno, Jesús I; Fierro, José L G; Mendieta, Jesús; Tamayo, Javier; Calleja, Montserrat

    2017-04-03

    DNA-water interactions have revealed as very important actor in DNA mechanics, from the molecular to the macroscopic scale. Given the particularly useful properties of DNA molecules to engineer novel materials through self-assembly and by bridging organic and inorganic materials, the interest in understanding DNA elasticity has crossed the boundaries of life science to reach also materials science and engineering. Here we show that thin films of DNA constructed through the self-assembly of sulfur tethered ssDNA strands demonstrate a Young's modulus tuning range of about 10 GPa by simply varying the environment relative humidity from 0% up to 70%. We observe that the highest tuning range occurs for ssDNA grafting densities of about 3.5 × 10 13 molecules/cm 2 , where the distance between the molecules maximizes the water mediated interactions between the strands. Upon hybridization with the complementary strand, the DNA self-assembled monolayers significantly soften by one order of magnitude and their Young's modulus dependency on the hydration state drastically decreases. The experimental observations are in agreement with molecular dynamics simulations.

  11. Low Cost Automated Module Assembly for 180 GHz Devices Project

    Data.gov (United States)

    National Aeronautics and Space Administration — Despite the obvious advantages of millimeter wave technology, a major barrier to expanded use is high assembly costs due to: need for specialized equipments; need...

  12. Process development for automated solar cell and module production. Task 4: automated array assembly

    Energy Technology Data Exchange (ETDEWEB)

    Hagerty, J.J.

    1980-06-30

    The scope of work under this contract involves specifying a process sequence which can be used in conjunction with automated equipment for the mass production of solar cell modules for terrestrial use. This process sequence is then critically analyzed from a technical and economic standpoint to determine the technological readiness of each process step for implementation. The process steps are ranked according to the degree of development effort required and according to their significance to the overall process. Under this contract the steps receiving analysis were: back contact metallization, automated cell array layup/interconnect, and module edge sealing. For automated layup/interconnect both hard automation and programmable automation (using an industrial robot) were studied. The programmable automation system was then selected for actual hardware development. Economic analysis using the SAMICS system has been performed during these studies to assure that development efforts have been directed towards the ultimate goal of price reduction. Details are given. (WHK)

  13. Automated Eukaryotic Gene Structure Annotation Using EVidenceModeler and the Program to Assemble Spliced Alignments

    Energy Technology Data Exchange (ETDEWEB)

    Haas, B J; Salzberg, S L; Zhu, W; Pertea, M; Allen, J E; Orvis, J; White, O; Buell, C R; Wortman, J R

    2007-12-10

    EVidenceModeler (EVM) is presented as an automated eukaryotic gene structure annotation tool that reports eukaryotic gene structures as a weighted consensus of all available evidence. EVM, when combined with the Program to Assemble Spliced Alignments (PASA), yields a comprehensive, configurable annotation system that predicts protein-coding genes and alternatively spliced isoforms. Our experiments on both rice and human genome sequences demonstrate that EVM produces automated gene structure annotation approaching the quality of manual curation.

  14. Surface-assisted DNA self-assembly: An enzyme-free strategy towards formation of branched DNA lattice.

    Science.gov (United States)

    Bhanjadeo, Madhabi M; Nayak, Ashok K; Subudhi, Umakanta

    2017-04-01

    DNA based self-assembled nanostructures and DNA origami has proven useful for organizing nanomaterials with firm precision. However, for advanced applications like nanoelectronics and photonics, large-scale organization of self-assembled branched DNA (bDNA) into periodic lattices is desired. In this communication for the first time we report a facile method of self-assembly of Y-shaped bDNA nanostructures on the cationic surface of Aluminum (Al) foil to prepare periodic two dimensional (2D) bDNA lattice. Particularly those Y-shaped bDNA structures having smaller overhangs and unable to self-assemble in solution, they are easily assembled on the surface of Al foil in the absence of ligase. Field emission scanning electron microscopy (FESEM) analysis shows homogenous distribution of two-dimensional bDNA lattices across the Al foil. When the assembled bDNA structures were recovered from the Al foil and electrophoresed in nPAGE only higher order polymeric bDNA structures were observed without a trace of monomeric structures which confirms the stability and high yield of the bDNA lattices. Therefore, this enzyme-free economic and efficient strategy for developing bDNA lattices can be utilized in assembling various nanomaterials for functional molecular components towards development of DNA based self-assembled nanodevices. Copyright © 2017 Elsevier Inc. All rights reserved.

  15. Automated assembly of Gallium Arsenide and 50-micron thick silicon solar cell modules

    Science.gov (United States)

    Mesch, H. G.

    1984-01-01

    The TRW automated solar array assembly equipment was used for the module assembly of 300 GaAs solar cells and 300 50 micron thick silicon solar cells (2 x 4 cm in size). These cells were interconnected with silver plated Invar tabs by means of welding. The GaAs cells were bonded to Kapton graphite aluminum honeycomb graphite substrates and the thin silicon cells were bonded to 0.002 inch thick single layer Kapton substrates. The GaAs solar cell module assembly resulted in a yield of 86% and the thin cell assembly produced a yield of 46% due to intermittent sticking of weld electrodes during the front cell contact welding operation. (Previously assembled thin cell solar modules produced an overall assembly yield of greater than 80%).

  16. Nanopore DNA Sequencing and Genome Assembly on the International Space Station.

    Science.gov (United States)

    Castro-Wallace, Sarah L; Chiu, Charles Y; John, Kristen K; Stahl, Sarah E; Rubins, Kathleen H; McIntyre, Alexa B R; Dworkin, Jason P; Lupisella, Mark L; Smith, David J; Botkin, Douglas J; Stephenson, Timothy A; Juul, Sissel; Turner, Daniel J; Izquierdo, Fernando; Federman, Scot; Stryke, Doug; Somasekar, Sneha; Alexander, Noah; Yu, Guixia; Mason, Christopher E; Burton, Aaron S

    2017-12-21

    We evaluated the performance of the MinION DNA sequencer in-flight on the International Space Station (ISS), and benchmarked its performance off-Earth against the MinION, Illumina MiSeq, and PacBio RS II sequencing platforms in terrestrial laboratories. Samples contained equimolar mixtures of genomic DNA from lambda bacteriophage, Escherichia coli (strain K12, MG1655) and Mus musculus (female BALB/c mouse). Nine sequencing runs were performed aboard the ISS over a 6-month period, yielding a total of 276,882 reads with no apparent decrease in performance over time. From sequence data collected aboard the ISS, we constructed directed assemblies of the ~4.6 Mb E. coli genome, ~48.5 kb lambda genome, and a representative M. musculus sequence (the ~16.3 kb mitochondrial genome), at 100%, 100%, and 96.7% consensus pairwise identity, respectively; de novo assembly of the E. coli genome from raw reads yielded a single contig comprising 99.9% of the genome at 98.6% consensus pairwise identity. Simulated real-time analyses of in-flight sequence data using an automated bioinformatic pipeline and laptop-based genomic assembly demonstrated the feasibility of sequencing analysis and microbial identification aboard the ISS. These findings illustrate the potential for sequencing applications including disease diagnosis, environmental monitoring, and elucidating the molecular basis for how organisms respond to spaceflight.

  17. Assembly of a biocompatible triazole-linked gene by one-pot click-DNA ligation

    Science.gov (United States)

    Kukwikila, Mikiembo; Gale, Nittaya; El-Sagheer, Afaf H.; Brown, Tom; Tavassoli, Ali

    2017-11-01

    The chemical synthesis of oligonucleotides and their enzyme-mediated assembly into genes and genomes has significantly advanced multiple scientific disciplines. However, these approaches are not without their shortcomings; enzymatic amplification and ligation of oligonucleotides into genes and genomes makes automation challenging, and site-specific incorporation of epigenetic information and/or modified bases into large constructs is not feasible. Here we present a fully chemical one-pot method for the assembly of oligonucleotides into a gene by click-DNA ligation. We synthesize the 335 base-pair gene that encodes the green fluorescent protein iLOV from ten functionalized oligonucleotides that contain 5ʹ-azide and 3ʹ-alkyne units. The resulting click-linked iLOV gene contains eight triazoles at the sites of chemical ligation, and yet is fully biocompatible; it is replicated by DNA polymerases in vitro and encodes a functional iLOV protein in Escherichia coli. We demonstrate the power and potential of our one-pot gene-assembly method by preparing an epigenetically modified variant of the iLOV gene.

  18. Automated hybridization/imaging device for fluorescent multiplex DNA sequencing

    Science.gov (United States)

    Weiss, Robert B.; Kimball, Alvin W.; Gesteland, Raymond F.; Ferguson, F. Mark; Dunn, Diane M.; Di Sera, Leonard J.; Cherry, Joshua L.

    1995-01-01

    A method is disclosed for automated multiplex sequencing of DNA with an integrated automated imaging hybridization chamber system. This system comprises an hybridization chamber device for mounting a membrane containing size-fractionated multiplex sequencing reaction products, apparatus for fluid delivery to the chamber device, imaging apparatus for light delivery to the membrane and image recording of fluorescence emanating from the membrane while in the chamber device, and programmable controller apparatus for controlling operation of the system. The multiplex reaction products are hybridized with a probe, then an enzyme (such as alkaline phosphatase) is bound to a binding moiety on the probe, and a fluorogenic substrate (such as a benzothiazole derivative) is introduced into the chamber device by the fluid delivery apparatus. The enzyme converts the fluorogenic substrate into a fluorescent product which, when illuminated in the chamber device with a beam of light from the imaging apparatus, excites fluorescence of the fluorescent product to produce a pattern of hybridization. The pattern of hybridization is imaged by a CCD camera component of the imaging apparatus to obtain a series of digital signals. These signals are converted by the controller apparatus into a string of nucleotides corresponding to the nucleotide sequence an automated sequence reader. The method and apparatus are also applicable to other membrane-based applications such as colony and plaque hybridization and Southern, Northern, and Western blots.

  19. DNA origami-based nanoribbons: assembly, length distribution, and twist

    International Nuclear Information System (INIS)

    Jungmann, Ralf; Scheible, Max; Kuzyk, Anton; Pardatscher, Guenther; Simmel, Friedrich C; Castro, Carlos E

    2011-01-01

    A variety of polymerization methods for the assembly of elongated nanoribbons from rectangular DNA origami structures are investigated. The most efficient method utilizes single-stranded DNA oligonucleotides to bridge an intermolecular scaffold seam between origami monomers. This approach allows the fabrication of origami ribbons with lengths of several micrometers, which can be used for long-range ordered arrangement of proteins. It is quantitatively shown that the length distribution of origami ribbons obtained with this technique follows the theoretical prediction for a simple linear polymerization reaction. The design of flat single layer origami structures with constant crossover spacing inevitably results in local underwinding of the DNA helix, which leads to a global twist of the origami structures that also translates to the nanoribbons.

  20. DNA origami-based nanoribbons: assembly, length distribution, and twist

    Energy Technology Data Exchange (ETDEWEB)

    Jungmann, Ralf; Scheible, Max; Kuzyk, Anton; Pardatscher, Guenther; Simmel, Friedrich C [Lehrstuhl fuer Bioelektronik, Physik-Department and ZNN/WSI, Technische Universitaet Muenchen, Am Coulombwall 4a, 85748 Garching (Germany); Castro, Carlos E, E-mail: simmel@ph.tum.de [Labor fuer Biomolekulare Nanotechnologie, Physik-Department and ZNN/WSI, Technische Universitaet Muenchen, Am Coulombwall 4a, 85748 Garching (Germany)

    2011-07-08

    A variety of polymerization methods for the assembly of elongated nanoribbons from rectangular DNA origami structures are investigated. The most efficient method utilizes single-stranded DNA oligonucleotides to bridge an intermolecular scaffold seam between origami monomers. This approach allows the fabrication of origami ribbons with lengths of several micrometers, which can be used for long-range ordered arrangement of proteins. It is quantitatively shown that the length distribution of origami ribbons obtained with this technique follows the theoretical prediction for a simple linear polymerization reaction. The design of flat single layer origami structures with constant crossover spacing inevitably results in local underwinding of the DNA helix, which leads to a global twist of the origami structures that also translates to the nanoribbons.

  1. Topics in programmable automation. [for materials handling, inspection, and assembly

    Science.gov (United States)

    Rosen, C. A.

    1975-01-01

    Topics explored in the development of integrated programmable automation systems include: numerically controlled and computer controlled machining; machine intelligence and the emulation of human-like capabilities; large scale semiconductor integration technology applications; and sensor technology for asynchronous local computation without burdening the executive minicomputer which controls the whole system. The role and development of training aids, and the potential application of these aids to augmented teleoperator systems are discussed.

  2. Design and Assembly of DNA Nano-Objects and 2D DNA Origami Arrays

    Science.gov (United States)

    Liu, Wenyan

    DNA, which plays a central role in biology as the carrier of genetic information, is also an excellent candidate for structural nanotechnology. Researches have proven that a variety of complicated DNA assemblies, such as objects, 2D & 3D crystals, and nanomechanical devices, can be fabricated through the combination of robust branched DNA motifs and sticky ends. This dissertation focuses on the design and construction of DNA nano--objects and 2D DNA origami arrays. In this dissertation, we first describe the formation of a triangular species that has four strands per edge, held together by PX interactions. We demonstrate by nondenaturing gel electrophoresis and by atomic force microscopy (AFM) that we can combine a partial triangle with other strands to form a robust four--stranded molecule. By combining them with a novel three--domain molecule, we also demonstrate by AFM that these triangles can be self--assembled into a linear array. Second, we demonstrate our attempts to design and self--assemble 2D DNA origami arrays using several different strategies. Specifically, we introduce the self--assembly of 2D DNA origami lattices using a symmetric cross--like design. This design strategy resulted in a well--ordered woven latticework array with edge dimensions of 2--3 mum. This size is likely to be large enough to connect bottom-up methods of patterning with top--down approaches. Third, we illustrate the design and construction of DNA nano--objects for exploring the substrate preferences of topoisomerase (topo) II. We designed and fabricated four double rhombus--like DNA molecules, each of which contains a different conformation of crossover in the middle, as possible substrates to establish the structural preferences for topo II. We characterized the formation of each substrate molecule by gel electrophoresis. Finally, we study the effect of M13 DNA knotting on the formation of the DNA origami tiles. We demonstrate by atomic force microscopy (AFM) that knotted M13

  3. Module and System Design in Flexible Automated Assembly

    NARCIS (Netherlands)

    Vos, J.A.W.M.

    2001-01-01

    There is an ongoing trend of increasing variety of products and a decreasing time-to-market of new products. These trends require flexibility in the production and extensive control of the production processes. This is especially true for the assembly: the joining of parts to form a product. These

  4. DNA Trojan Horses: Self-Assembled Floxuridine-Containing DNA Polyhedra for Cancer Therapy.

    Science.gov (United States)

    Mou, Quanbing; Ma, Yuan; Pan, Gaifang; Xue, Bai; Yan, Deyue; Zhang, Chuan; Zhu, Xinyuan

    2017-10-02

    Based on their structural similarity to natural nucleobases, nucleoside analogue therapeutics were integrated into DNA strands through conventional solid-phase synthesis. By elaborately designing their sequences, floxuridine-integrated DNA strands were synthesized and self-assembled into well-defined DNA polyhedra with definite drug-loading ratios as well as tunable size and morphology. As a novel drug delivery system, these drug-containing DNA polyhedra could ideally mimic the Trojan Horse to deliver chemotherapeutics into tumor cells and fight against cancer. Both in vitro and in vivo results demonstrate that the DNA Trojan horse with buckyball architecture exhibits superior anticancer capability over the free drug and other formulations. With precise control over the drug-loading ratio and structure of the nanocarriers, the DNA Trojan horse may play an important role in anticancer treatment and exhibit great potential in translational nanomedicine. © 2017 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim.

  5. Isothermal hybridization kinetics of DNA assembly of two-dimensional DNA origami.

    Science.gov (United States)

    Song, Jie; Zhang, Zhao; Zhang, Shuai; Liu, Lei; Li, Qiang; Xie, Erqing; Gothelf, Kurt Vesterager; Besenbacher, Flemming; Dong, Mingdong

    2013-09-09

    The Watson-Crick base-pairing with specificity and predictability makes DNA molecules suitable for building versatile nanoscale structures and devices, and the DNA origami method enables researchers to incorporate more complexities into DNA-based devices. Thermally controlled atomic force microscopy in combination with nanomechanical spectroscopy with forces controlled in the pico Newton (pN) range as a novel technique is introduced to directly investigate the kinetics of multistrand DNA hybridization events on DNA origami nanopores under defined isothermal conditions. For the synthesis of DNA nanostructures under isothermal conditions at 60 °C, a higher hybridization rate, fewer defects, and a higher stability are achieved compared to room-temperature studies. By quantifying the assembly times for filling pores in origami structures at several constant temperatures, the fill factors show a consistent exponential increase over time. Furthermore, the local hybridization rate can be accelerated by adding a higher concentration of the staples. The new insight gained on the kinetics of staple-scaffold hybridization on the synthesis of two dimensional DNA origami structures may open up new routes and ideas for designing DNA assembly systems with increased potential for their application. Copyright © 2013 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

  6. Supramolecular Hydrogels Based on DNA Self-Assembly.

    Science.gov (United States)

    Shao, Yu; Jia, Haoyang; Cao, Tianyang; Liu, Dongsheng

    2017-04-18

    Extracellular matrix (ECM) provides essential supports three dimensionally to the cells in living organs, including mechanical support and signal, nutrition, oxygen, and waste transportation. Thus, using hydrogels to mimic its function has attracted much attention in recent years, especially in tissue engineering, cell biology, and drug screening. However, a hydrogel system that can merit all parameters of the natural ECM is still a challenge. In the past decade, deoxyribonucleic acid (DNA) has arisen as an outstanding building material for the hydrogels, as it has unique properties compared to most synthetic or natural polymers, such as sequence designability, precise recognition, structural rigidity, and minimal toxicity. By simple attachment to polymers as a side chain, DNA has been widely used as cross-links in hydrogel preparation. The formed secondary structures could confer on the hydrogel designable responsiveness, such as response to temperature, pH, metal ions, proteins, DNA, RNA, and small signal molecules like ATP. Moreover, single or multiple DNA restriction enzyme sites could be incorporated into the hydrogels by sequence design and greatly expand the latitude of their responses. Compared with most supramolecular hydrogels, these DNA cross-linked hydrogels could be relatively strong and easily adjustable via sequence variation, but it is noteworthy that these hydrogels still have excellent thixotropic properties and could be easily injected through a needle. In addition, the quick formation of duplex has also enabled the multilayer three-dimensional injection printing of living cells with the hydrogel as matrix. When the matrix is built purely by DNA assembly structures, the hydrogel inherits all the previously described characteristics; however, the long persistence length of DNA structures excluded the small size meshes of the network and made the hydrogel permeable to nutrition for cell proliferation. This unique property greatly expands the cell

  7. JPLEX: Java Simplex Implementation with Branch-and-Bound Search for Automated Test Assembly

    Science.gov (United States)

    Park, Ryoungsun; Kim, Jiseon; Dodd, Barbara G.; Chung, Hyewon

    2011-01-01

    JPLEX, short for Java simPLEX, is an automated test assembly (ATA) program. It is a mixed integer linear programming (MILP) solver written in Java. It reads in a configuration file, solves the minimization problem, and produces an output file for postprocessing. It implements the simplex algorithm to create a fully relaxed solution and…

  8. Automated Test Assembly for Cognitive Diagnosis Models Using a Genetic Algorithm

    Science.gov (United States)

    Finkelman, Matthew; Kim, Wonsuk; Roussos, Louis A.

    2009-01-01

    Much recent psychometric literature has focused on cognitive diagnosis models (CDMs), a promising class of instruments used to measure the strengths and weaknesses of examinees. This article introduces a genetic algorithm to perform automated test assembly alongside CDMs. The algorithm is flexible in that it can be applied whether the goal is to…

  9. A Case Study of the Likes and Dislikes of DNA and RNA in Self-Assembly.

    Science.gov (United States)

    Zuo, Hua; Wu, Siyu; Li, Mo; Li, Yulin; Jiang, Wen; Mao, Chengde

    2015-12-07

    Programmed self-assembly of nucleic acids (DNA and RNA) is an active research area as it promises a general approach for nanoconstruction. Whereas DNA self-assembly has been extensively studied, RNA self-assembly lags much behind. One strategy to boost RNA self-assembly is to adapt the methods of DNA self-assembly for RNA self-assembly because of the chemical and structural similarities of DNA and RNA. However, these two types of molecules are still significantly different. To enable the rational design of RNA self-assembly, a thorough examination of their likes and dislikes in programmed self-assembly is needed. The current work begins to address this task. It was found that similar, two-stranded motifs of RNA and DNA lead to similar, but clearly different nanostructures. © 2015 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

  10. DNA-PK dependent targeting of DNA-ends to a protein complex assembled on matrix attachment region DNA sequences

    International Nuclear Information System (INIS)

    Mauldin, S.K.; Getts, R.C.; Perez, M.L.; DiRienzo, S.; Stamato, T.D.

    2003-01-01

    Full text: We find that nuclear protein extracts from mammalian cells contain an activity that allows DNA ends to associate with circular pUC18 plasmid DNA. This activity requires the catalytic subunit of DNA-PK (DNA-PKcs) and Ku since it was not observed in mutants lacking Ku or DNA-PKcs but was observed when purified Ku/DNA-PKcs was added to these mutant extracts. Competition experiments between pUC18 and pUC18 plasmids containing various nuclear matrix attachment region (MAR) sequences suggest that DNA ends preferentially associate with plasmids containing MAR DNA sequences. At a 1:5 mass ratio of MAR to pUC18, approximately equal amounts of DNA end binding to the two plasmids were observed, while at a 1:1 ratio no pUC18 end-binding was observed. Calculation of relative binding activities indicates that DNA-end binding activities to MAR sequences was 7 to 21 fold higher than pUC18. Western analysis of proteins bound to pUC18 and MAR plasmids indicates that XRCC4, DNA ligase IV, scaffold attachment factor A, topoisomerase II, and poly(ADP-ribose) polymerase preferentially associate with the MAR plasmid in the absence or presence of DNA ends. In contrast, Ku and DNA-PKcs were found on the MAR plasmid only in the presence of DNA ends. After electroporation of a 32P-labeled DNA probe into human cells and cell fractionation, 87% of the total intercellular radioactivity remained in nuclei after a 0.5M NaCl extraction suggesting the probe was strongly bound in the nucleus. The above observations raise the possibility that DNA-PK targets DNA-ends to a repair and/or DNA damage signaling complex which is assembled on MAR sites in the nucleus

  11. Protein Self-Assembly and Protein-Induced DNA Morphologies

    Science.gov (United States)

    Mawhinney, Matthew T.

    The ability of biomolecules to associate into various structural configurations has a substantial impact on human physiology. The synthesis of protein polypeptide chains using the information encoded by DNA is mediated through the use of regulatory proteins, known as transcription factors. Some transcription factors perform function by inducing local curvature in deoxyribonucleic acid (DNA) strands, the mechanisms of which are not entirely known. An important architectural protein, eleven zinc finger CTCF (11 ZF CTCF) is involved in genome organization and hypothesized to mediate DNA loop formation. Direct evidence for these CTCF-induced DNA loops has yet to be observed. In this thesis, the effect of 11 ZF CTCF on DNA morphology is examined using atomic force microscopy, a powerful technique for visualizing biomolecules with nanometer resolution. The presence of CTCF is revealed to induce a variety of morphologies deviating from the relaxed state of control DNA samples, including compact circular complexes, meshes, and networks. Images reveal quasi-circular DNA/CTCF complexes consistent with a single DNA molecule twice wrapped around the protein. The structures of DNA and proteins are highly important for operations in the cell. Structural irregularities may lead to a variety of issues, including more than twenty human pathologies resulting from aberrant protein misfolding into amyloid aggregates of elongated fibrils. Insulin deficiency and resistance characterizing type 2 diabetes often requires administration of insulin. Injectable and inhalable delivery methods have been documented to result in the deposition of amyloid fibrils. Oligomers, soluble multiprotein assemblies, are believed to play an important role in this process. Insulin aggregation under physiological conditions is not well understood and oligomers have not yet been fully characterized. In this thesis, in vitro insulin aggregation at acidic and neutral pH is explored using a variety of techniques

  12. High molecular weight DNA assembly in vivo for synthetic biology applications.

    Science.gov (United States)

    Juhas, Mario; Ajioka, James W

    2017-05-01

    DNA assembly is the key technology of the emerging interdisciplinary field of synthetic biology. While the assembly of smaller DNA fragments is usually performed in vitro, high molecular weight DNA molecules are assembled in vivo via homologous recombination in the host cell. Escherichia coli, Bacillus subtilis and Saccharomyces cerevisiae are the main hosts used for DNA assembly in vivo. Progress in DNA assembly over the last few years has paved the way for the construction of whole genomes. This review provides an update on recent synthetic biology advances with particular emphasis on high molecular weight DNA assembly in vivo in E. coli, B. subtilis and S. cerevisiae. Special attention is paid to the assembly of whole genomes, such as those of the first synthetic cell, synthetic yeast and minimal genomes.

  13. Assembly of presynaptic filaments. Factors affecting the assembly of RecA protein onto single-stranded DNA

    DEFF Research Database (Denmark)

    Thresher, RJ; Christiansen, Gunna; Griffith, JD

    1988-01-01

    We have previously shown that the assembly of RecA protein onto single-stranded DNA (ssDNA) facilitated by SSB protein occurs in three steps: (1) rapid binding of SSB protein to the ssDNA; (2) nucleation of RecA protein onto this template; and (3) co-operative polymerization of additional Rec...... assembled onto ssDNA at net rates that varied from 250 to 900 RecA protein monomers per minute, with the rate inversely related to the concentration of SSB protein. Combined sucrose sedimentation and electron microscope analysis established that SSB protein was displaced from the ssDNA during RecA protein...

  14. The Problem of Automation of Solar Concentrator Assembly and Adjustment

    Directory of Open Access Journals (Sweden)

    Ernst Kussul

    2011-09-01

    Full Text Available Recently we have proposed a low cost solar concentrator based on a large number of small flat mirrors that approximates parabolic surface needed for solar light concentration. In this work we describe an improvement of the concentrator support frame assembly. The improvement is connected with the design of the gauge/guages for support frame adjustment. The gauge has the parabolic edge and rotates in the central tube. Special nuts are moved up to the contact with parabolic edge of the gauge

  15. Low-Cost, High-Throughput Sequencing of DNA Assemblies Using a Highly Multiplexed Nextera Process.

    Science.gov (United States)

    Shapland, Elaine B; Holmes, Victor; Reeves, Christopher D; Sorokin, Elena; Durot, Maxime; Platt, Darren; Allen, Christopher; Dean, Jed; Serber, Zach; Newman, Jack; Chandran, Sunil

    2015-07-17

    In recent years, next-generation sequencing (NGS) technology has greatly reduced the cost of sequencing whole genomes, whereas the cost of sequence verification of plasmids via Sanger sequencing has remained high. Consequently, industrial-scale strain engineers either limit the number of designs or take short cuts in quality control. Here, we show that over 4000 plasmids can be completely sequenced in one Illumina MiSeq run for less than $3 each (15× coverage), which is a 20-fold reduction over using Sanger sequencing (2× coverage). We reduced the volume of the Nextera tagmentation reaction by 100-fold and developed an automated workflow to prepare thousands of samples for sequencing. We also developed software to track the samples and associated sequence data and to rapidly identify correctly assembled constructs having the fewest defects. As DNA synthesis and assembly become a centralized commodity, this NGS quality control (QC) process will be essential to groups operating high-throughput pipelines for DNA construction.

  16. Revealing Structural Transformations during Crystallization of DNA-Nanoparticle Assemblies

    Science.gov (United States)

    Zhang, Yugang; Lu, Fang; van der Lelie, Daniel; Gang, Oleg

    2013-03-01

    Nanoparticle assembly via sequence-specific DNA recognition emerges as a powerful strategy for the fabrication of nanoparticle (NP)-based crystalline materials. Generally, a delicate thermal annealing is essential for the crystallization of NPs from kinetically trapped disordered states. Due to the complex coupling between interactions, entropic and chain effects in these systems, the crystallization pathway remains an intricate and open question. Herein, we present an experimental study of the crystallization process for DNA-directed nanoparticle assembly systems using synchrotron-based small angle x-ray scattering (SAXS). We demonstrated the effects of two crystallization-dominant factors, namely, temperature and volume fraction, on the structural transformation and order development. By combining a single component and binary systems we uncovered the evolution of global and local particle arrangements, such as correlation length, compositional disorder and coordination number, during the phase transformation. Research was supported by the U.S. Department of Energy, Office of Basic Energy Sciences, under Contract No. DE-AC02-98CH10886.

  17. Shared robotic system: automated pipette calibration and pipette tip filter assembly

    International Nuclear Information System (INIS)

    Addison, J.H. Jr.; Dyches, G.M.

    1985-01-01

    At the Savannah River Laboratory a Zymate Laboratory Automation System has been developed to perform two completely independent tasks within one work cell. One operation is the precise calibration of pipettes; the other is the assembly of a filter in a pipette tip. Since neither task requires full robot time, the shared system is an economical means of robotizing both processes. These are tedious, repetitive, time consuming tasks; and human operators fail to yield constant results. Automation insures a repeatable process which increases product quality

  18. Assembly, Structure and Properties of DNA Programmable Nanoclusters

    Science.gov (United States)

    Chi, Cheng

    Finite size nanoclusters can be viewed as a nanoscale analogue of molecules. Just as molecules, synthesized from atoms, give access to new properties, clusters composed of nanoparticles modulate of their functional properties of nanoparticles. In contrast to synthetic chemistry which is a mature field, the creation of nanoscale clusters with well-defined architectures is a new and challenging area of research. My work explores how to assemble model systems of nanoclusters using DNA-programmable interparticle linkages. The simplest clusters of two particles, dimers, allow one to investigate fundamental effects in these systems. Such clusters serve as a versatile platform to understand DNA-mediated interactions, especially in the non-trivial regime where the nanoparticle and DNA chains are comparable in size. I systematically studied a few fundamental questions as follows: Firstly, we examined the structure of nanoparticle dimers in detail by a combination of X-ray scattering experiments and molecular simulations. We found that, for a given DNA length, the interparticle separation within the dimer is controlled primarily by the number of linking DNA. We summarized our findings in a simple model that captures the interplay of the number of DNA bridges, their length, the particle's curvature and the excluded volume effects. We demonstrated the applicability of the model to our results, without any free parameters. As a consequence, the increase of dimer separation with increasing temperature can be understood as a result of changing the number of connecting DNA. Later, we investigated the self-assembly process of DNA-functionalized particles in the presence of various lengths of the DNA linkage strands using 3 different pathways. We observed a high yield of dimer formation when significantly long linkers were applied. Small Angle X-ray Scattering revealed two configurations of the small clusters by different pathways. In one case, the interparticle distance increases

  19. Automation and integration of multiplexed on-line sample preparation with capillary electrophoresis for DNA sequencing

    Energy Technology Data Exchange (ETDEWEB)

    Tan, H.

    1999-03-31

    The purpose of this research is to develop a multiplexed sample processing system in conjunction with multiplexed capillary electrophoresis for high-throughput DNA sequencing. The concept from DNA template to called bases was first demonstrated with a manually operated single capillary system. Later, an automated microfluidic system with 8 channels based on the same principle was successfully constructed. The instrument automatically processes 8 templates through reaction, purification, denaturation, pre-concentration, injection, separation and detection in a parallel fashion. A multiplexed freeze/thaw switching principle and a distribution network were implemented to manage flow direction and sample transportation. Dye-labeled terminator cycle-sequencing reactions are performed in an 8-capillary array in a hot air thermal cycler. Subsequently, the sequencing ladders are directly loaded into a corresponding size-exclusion chromatographic column operated at {approximately} 60 C for purification. On-line denaturation and stacking injection for capillary electrophoresis is simultaneously accomplished at a cross assembly set at {approximately} 70 C. Not only the separation capillary array but also the reaction capillary array and purification columns can be regenerated after every run. DNA sequencing data from this system allow base calling up to 460 bases with accuracy of 98%.

  20. Single-step rapid assembly of DNA origami nanostructures for addressable nanoscale bioreactors

    DEFF Research Database (Denmark)

    Fu, Yanming; Zeng, Dongdong; Chao, Jie

    2013-01-01

    Self-assembled DNA origami nanostructures have shown great promise for bottom-up construction of complex objects with nanoscale addressability. Here we show that DNA origami-based 1D nanoribbons and nanotubes are one-pot assembled with controllable sizes and nanoscale addressability with high speed...... (within only 10-20 min), exhibiting extraordinarily high cooperativity that is often observed in assembly of natural molecular machines in cells (e.g. ribosome). By exploiting the high specificity of DNA-based self-assembly, we can precisely anchor proteins on these DNA origami nanostructures with sub-10...

  1. Framework to Implement Collaborative Robots in Manual Assembly: A Lean Automation Approach

    DEFF Research Database (Denmark)

    Malik, Ali Ahmad; Bilberg, Arne

    The recent proliferation of smart manufacturing technologies has emerged the concept of hybrid automation for assembly systems utilizing the best of humans and robots in a combination. Based on the ability to work alongside human-workers the next generation of industrial robots (or robotics 2.......0) are referred to as collaborative robots or cobots. This paper presents a systematic framework based on Roozenburg’s engineering design cycle for the deployment of cobots in existing assembly cells for enhanced productivity. A model for defining evaluation-parameters for a cobot are presented. The effectiveness...

  2. A Capping Step During Automated Glycan Assembly Enables Access to Complex Glycans in High Yield.

    Science.gov (United States)

    Yu, Yang; Kononov, Andrew; Delbianco, Martina; Seeberger, Peter H

    2018-04-20

    The products of multi-step automated solid phase syntheses are purified after release from the resin. Capping of unreacted nucleophiles is commonplace in automated oligonucleotide synthesis to minimize accumulation of deletion sequences. To date, capping was not used routinely during automated glycan assembly (AGA) since previous capping protocols suffered from long reaction times and conditions incompatible with some protective groups. Here, a method using methanesulfonic acid and acetic anhydride for the fast and quantitative capping of hydroxyl groups that failed to be glycosylated is reported. Commonly used protective groups in AGA are stable under these capping conditions. The introduction of a capping step into the coupling cycle drastically improved overall yields by decreasing side-products and simplifying purification, while reducing building block consumption. To illustrate the method, the biologically important tetrasaccharide Lc4, as well as a 50-mer polymannoside were prepared. © 2018 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim.

  3. Automated assembly of fast-axis collimation (FAC) lenses for diode laser bar modules

    Science.gov (United States)

    Miesner, Jörn; Timmermann, Andre; Meinschien, Jens; Neumann, Bernhard; Wright, Steve; Tekin, Tolga; Schröder, Henning; Westphalen, Thomas; Frischkorn, Felix

    2009-02-01

    Laser diodes and diode laser bars are key components in high power semiconductor lasers and solid state laser systems. During manufacture, the assembly of the fast axis collimation (FAC) lens is a crucial step. The goal of our activities is to design an automated assembly system for high volume production. In this paper the results of an intermediate milestone will be reported: a demonstration system was designed, realized and tested to prove the feasibility of all of the system components and process features. The demonstration system consists of a high precision handling system, metrology for process feedback, a powerful digital image processing system and tooling for glue dispensing, UV curing and laser operation. The system components as well as their interaction with each other were tested in an experimental system in order to glean design knowledge for the fully automated assembly system. The adjustment of the FAC lens is performed by a series of predefined steps monitored by two cameras concurrently imaging the far field and the near field intensity distributions. Feedback from these cameras processed by a powerful and efficient image processing algorithm control a five axis precision motion system to optimize the fast axis collimation of the laser beam. Automated cementing of the FAC to the diode bar completes the process. The presentation will show the system concept, the algorithm of the adjustment as well as experimental results. A critical discussion of the results will close the talk.

  4. An Automated Sample Preparation System for Large-Scale DNA Sequencing

    Science.gov (United States)

    Marziali, Andre; Willis, Thomas D.; Federspiel, Nancy A.; Davis, Ronald W.

    1999-01-01

    Recent advances in DNA sequencing technologies, both in the form of high lane-density gels and automated capillary systems, will lead to an increased requirement for sample preparation systems that operate at low cost and high throughput. As part of the development of a fully automated sequencing system, we have developed an automated subsystem capable of producing 10,000 sequence-ready ssDNA templates per day from libraries of M13 plaques at a cost of $0.29 per sample. This Front End has been in high throughput operation since June, 1997 and has produced > 400,000 high-quality DNA templates. PMID:10330125

  5. Self-Assembly of Complex DNA Tessellations by Using Low-Symmetry Multi-arm DNA Tiles.

    Science.gov (United States)

    Zhang, Fei; Jiang, Shuoxing; Li, Wei; Hunt, Ashley; Liu, Yan; Yan, Hao

    2016-07-25

    Modular DNA tile-based self-assembly is a versatile way to engineer basic tessellation patterns on the nanometer scale, but it remains challenging to achieve high levels of structural complexity. We introduce a set of general design principles to create intricate DNA tessellations by employing multi-arm DNA motifs with low symmetry. We achieved two novel Archimedean tiling patterns, (4.8.8) and (3.6.3.6), and one pattern with higher-order structures beyond the complexity observed in Archimedean tiling. Our success in assembling complicated DNA tessellations demonstrates the broad design space of DNA structural motifs, enriching the toolbox of DNA tile-based self-assembly and expanding the complexity boundaries of DNA tile-based tessellation. © 2016 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

  6. OligArch: A software tool to allow artificially expanded genetic information systems (AEGIS to guide the autonomous self-assembly of long DNA constructs from multiple DNA single strands

    Directory of Open Access Journals (Sweden)

    Kevin M. Bradley

    2014-08-01

    Full Text Available Synthetic biologists wishing to self-assemble large DNA (L-DNA constructs from small DNA fragments made by automated synthesis need fragments that hybridize predictably. Such predictability is difficult to obtain with nucleotides built from just the four standard nucleotides. Natural DNA's peculiar combination of strong and weak G:C and A:T pairs, the context-dependence of the strengths of those pairs, unimolecular strand folding that competes with desired interstrand hybridization, and non-Watson–Crick interactions available to standard DNA, all contribute to this unpredictability. In principle, adding extra nucleotides to the genetic alphabet can improve the predictability and reliability of autonomous DNA self-assembly, simply by increasing the information density of oligonucleotide sequences. These extra nucleotides are now available as parts of artificially expanded genetic information systems (AEGIS, and tools are now available to generate entirely standard DNA from AEGIS DNA during PCR amplification. Here, we describe the OligArch (for "oligonucleotide architecting" software, an application that permits synthetic biologists to engineer optimally self-assembling DNA constructs from both six- and eight-letter AEGIS alphabets. This software has been used to design oligonucleotides that self-assemble to form complete genes from 20 or more single-stranded synthetic oligonucleotides. OligArch is therefore a key element of a scalable and integrated infrastructure for the rapid and designed engineering of biology.

  7. DNA Self-Assembly and Computation Studied with a Coarse-grained Dynamic Bonded Model

    DEFF Research Database (Denmark)

    Svaneborg, Carsten; Fellermann, Harold; Rasmussen, Steen

    2012-01-01

    We utilize a coarse-grained directional dynamic bonding DNA model [C. Svaneborg, Comp. Phys. Comm. (In Press DOI:10.1016/j.cpc.2012.03.005)] to study DNA self-assembly and DNA computation. In our DNA model, a single nucleotide is represented by a single interaction site, and complementary sites can...

  8. Logical NAND and NOR Operations Using Algorithmic Self-assembly of DNA Molecules

    Science.gov (United States)

    Wang, Yanfeng; Cui, Guangzhao; Zhang, Xuncai; Zheng, Yan

    DNA self-assembly is the most advanced and versatile system that has been experimentally demonstrated for programmable construction of patterned systems on the molecular scale. It has been demonstrated that the simple binary arithmetic and logical operations can be computed by the process of self assembly of DNA tiles. Here we report a one-dimensional algorithmic self-assembly of DNA triple-crossover molecules that can be used to execute five steps of a logical NAND and NOR operations on a string of binary bits. To achieve this, abstract tiles were translated into DNA tiles based on triple-crossover motifs. Serving as input for the computation, long single stranded DNA molecules were used to nucleate growth of tiles into algorithmic crystals. Our method shows that engineered DNA self-assembly can be treated as a bottom-up design techniques, and can be capable of designing DNA computer organization and architecture.

  9. Advanced engineering design capabilities applied for developing a technological device for automated assembly

    Directory of Open Access Journals (Sweden)

    Pista Ionut Madalin

    2017-01-01

    Full Text Available The theoretical approach and the related case study presented in the paper are part of a larger research regarding the optimization of assembled products and assembly processes by enhancing the effectiveness and efficiency of design activities, including by embedment of DfX/DFMA principles. First of all, a conceptual model for the theoretical approach is proposed and discussed. The case study refers to the engineering design of an indexing mounting table for automated assembly of an electrical plug inlet. Some advanced principles and theories are discussed in relation to the technical solution design. Advanced engineering design capabilities provided by Solid Edge have been used for solving the 3D modelling task and for the other detailed design activities. Finally, some related conclusions are included, together with some directions of further researches.

  10. Toolchain concept for the automated assembly of micro-optical systems

    Science.gov (United States)

    Haag, Sebastian; Müller, Tobias; Brecher, Christian

    2014-05-01

    In micro-optical assembly, the mastering of the steps of passive and active alignment, bonding, and part feeding as well as their interdependencies are crucial to the success of an automation solution. Process development is therefore complex and time consuming. Separation of assembly process planning and assembly execution decouples both phases so that production and process development can take place in parallel and even in spatially separated stations. The work presented in this paper refines the concept of flexible assembly systems by separating the phases of assembly process planning and assembly execution by providing a dedicated process development platform on the one hand and by providing automatisms regarding the transfer from the development platform into industrial production on the other. For this purpose, two key concepts are being developed by the research carried out at Fraunhofer IPT. The paper introduces the overall approach and formalisms as well as a form of notation based on part lists, product features and key characteristics and it shows industrial use cases the approach has been applied to. Key characteristics are constraints on spatial relations and they are expressed in terms of optical functions or geometric constraints which need to be fulfilled. In the paper, special attention is paid to the illustration of the end-user perspective.

  11. Modelling DNA origami self-assembly at the domain level

    Energy Technology Data Exchange (ETDEWEB)

    Dannenberg, Frits; Kwiatkowska, Marta [Department of Computer Science, University of Oxford, Wolfson Building, Parks Road, Oxford OX1 3QD (United Kingdom); Dunn, Katherine E. [Department of Physics, University of Oxford, Clarendon Laboratory, Parks Road, Oxford OX1 3PU (United Kingdom); Department of Electronics, University of York, York YO10 5DD (United Kingdom); Bath, Jonathan; Turberfield, Andrew J. [Department of Physics, University of Oxford, Clarendon Laboratory, Parks Road, Oxford OX1 3PU (United Kingdom); Ouldridge, Thomas E. [Department of Physics, University of Oxford, Rudolf Peierls Centre for Theoretical Physics, 1 Keble Road, Oxford OX1 3NP (United Kingdom); Department of Mathematics, Imperial College, 180 Queen’s Gate, London SW7 2AZ (United Kingdom)

    2015-10-28

    We present a modelling framework, and basic model parameterization, for the study of DNA origami folding at the level of DNA domains. Our approach is explicitly kinetic and does not assume a specific folding pathway. The binding of each staple is associated with a free-energy change that depends on staple sequence, the possibility of coaxial stacking with neighbouring domains, and the entropic cost of constraining the scaffold by inserting staple crossovers. A rigorous thermodynamic model is difficult to implement as a result of the complex, multiply connected geometry of the scaffold: we present a solution to this problem for planar origami. Coaxial stacking of helices and entropic terms, particularly when loop closure exponents are taken to be larger than those for ideal chains, introduce interactions between staples. These cooperative interactions lead to the prediction of sharp assembly transitions with notable hysteresis that are consistent with experimental observations. We show that the model reproduces the experimentally observed consequences of reducing staple concentration, accelerated cooling, and absent staples. We also present a simpler methodology that gives consistent results and can be used to study a wider range of systems including non-planar origami.

  12. Modelling DNA origami self-assembly at the domain level

    International Nuclear Information System (INIS)

    Dannenberg, Frits; Kwiatkowska, Marta; Dunn, Katherine E.; Bath, Jonathan; Turberfield, Andrew J.; Ouldridge, Thomas E.

    2015-01-01

    We present a modelling framework, and basic model parameterization, for the study of DNA origami folding at the level of DNA domains. Our approach is explicitly kinetic and does not assume a specific folding pathway. The binding of each staple is associated with a free-energy change that depends on staple sequence, the possibility of coaxial stacking with neighbouring domains, and the entropic cost of constraining the scaffold by inserting staple crossovers. A rigorous thermodynamic model is difficult to implement as a result of the complex, multiply connected geometry of the scaffold: we present a solution to this problem for planar origami. Coaxial stacking of helices and entropic terms, particularly when loop closure exponents are taken to be larger than those for ideal chains, introduce interactions between staples. These cooperative interactions lead to the prediction of sharp assembly transitions with notable hysteresis that are consistent with experimental observations. We show that the model reproduces the experimentally observed consequences of reducing staple concentration, accelerated cooling, and absent staples. We also present a simpler methodology that gives consistent results and can be used to study a wider range of systems including non-planar origami

  13. DNA templates silver clusters with magic sizes and colors for multi-cluster fluorescent assemblies

    Science.gov (United States)

    Copp, Stacy

    2015-03-01

    The natural inclusion of information in DNA, a vital part of life's rich complexity, can also be exploited to create diverse structures with multiple scales of complexity. Now emerging in novel photonic applications, DNA-stabilized silver clusters (AgN-DNA) are compelling examples of multi-scale DNA-directed assembly: individual fluorescent clusters, each templated by specific DNA base motifs, can then be arranged together in DNA-mediated multi-cluster assemblies with nanoscale precision. We discuss how DNA imbues AgN-DNA with unique features. Our optical data on pure AgN-DNA show that DNA base-cationic silver ligands impose rod-like shapes for neutral silver clusters, whose length primarily determines fluorescence color. This shape anisotropy leads to the aspherical AgN-DNA magic number cluster sizes and ``magic color'' groupings. We exploit DNA's sequence properties to extract multi-base motifs that select certain magic cluster sizes, using machine learning algorithms applied to large data sets. With these base motifs, we design DNA scaffolds to arrange multiple atomically precise AgN together in nanoscale proximity. We demonstrate that clusters are stable when held at separations below 10 nm, both in bicolor, dual cluster DNA clamp assemblies and in one-dimensional assemblies of atomically precise clusters arrayed on DNA nanotubes. Supported by NSF-CHE-1213895 and NSF-DMR-1309410. SMC acknowledges NSF-DGE-1144085, a NSF GRFP.

  14. Hierarchical Self Assembly of Patterns from the Robinson Tilings: DNA Tile Design in an Enhanced Tile Assembly Model

    Science.gov (United States)

    Padilla, Jennifer E.; Liu, Wenyan; Seeman, Nadrian C.

    2012-01-01

    We introduce a hierarchical self assembly algorithm that produces the quasiperiodic patterns found in the Robinson tilings and suggest a practical implementation of this algorithm using DNA origami tiles. We modify the abstract Tile Assembly Model, (aTAM), to include active signaling and glue activation in response to signals to coordinate the hierarchical assembly of Robinson patterns of arbitrary size from a small set of tiles according to the tile substitution algorithm that generates them. Enabling coordinated hierarchical assembly in the aTAM makes possible the efficient encoding of the recursive process of tile substitution. PMID:23226722

  15. Hierarchical Self Assembly of Patterns from the Robinson Tilings: DNA Tile Design in an Enhanced Tile Assembly Model.

    Science.gov (United States)

    Padilla, Jennifer E; Liu, Wenyan; Seeman, Nadrian C

    2012-06-01

    We introduce a hierarchical self assembly algorithm that produces the quasiperiodic patterns found in the Robinson tilings and suggest a practical implementation of this algorithm using DNA origami tiles. We modify the abstract Tile Assembly Model, (aTAM), to include active signaling and glue activation in response to signals to coordinate the hierarchical assembly of Robinson patterns of arbitrary size from a small set of tiles according to the tile substitution algorithm that generates them. Enabling coordinated hierarchical assembly in the aTAM makes possible the efficient encoding of the recursive process of tile substitution.

  16. Evaluation of Four Automated Protocols for Extraction of DNA from FTA Cards

    DEFF Research Database (Denmark)

    Stangegaard, Michael; Børsting, Claus; Ferrero-Miliani, Laura

    2013-01-01

    Extraction of DNA using magnetic bead-based techniques on automated DNA extraction instruments provides a fast, reliable, and reproducible method for DNA extraction from various matrices. Here, we have compared the yield and quality of DNA extracted from FTA cards using four automated extraction...... protocols on three different instruments. The extraction processes were repeated up to six times with the same pieces of FTA cards. The sample material on the FTA cards was either blood or buccal cells. With the QIAamp DNA Investigator and QIAsymphony DNA Investigator kits, it was possible to extract DNA...... from the FTA cards in all six rounds of extractions in sufficient amount and quality to obtain complete short tandem repeat (STR) profiles on a QIAcube and a QIAsymphony SP. With the PrepFiler Express kit, almost all the extractable DNA was extracted in the first two rounds of extractions. Furthermore...

  17. A new building block for DNA network formation by self-assembly and polymerase chain reaction.

    Science.gov (United States)

    Bußkamp, Holger; Keller, Sascha; Robotta, Marta; Drescher, Malte; Marx, Andreas

    2014-01-01

    The predictability of DNA self-assembly is exploited in many nanotechnological approaches. Inspired by naturally existing self-assembled DNA architectures, branched DNA has been developed that allows self-assembly to predesigned architectures with dimensions on the nanometer scale. DNA is an attractive material for generation of nanostructures due to a plethora of enzymes which modify DNA with high accuracy, providing a toolbox for many different manipulations to construct nanometer scaled objects. We present a straightforward synthesis of a rigid DNA branching building block successfully used for the generation of DNA networks by self-assembly and network formation by enzymatic DNA synthesis. The Y-shaped 3-armed DNA construct, bearing 3 primer strands is accepted by Taq DNA polymerase. The enzyme uses each arm as primer strand and incorporates the branched construct into large assemblies during PCR. The networks were investigated by agarose gel electrophoresis, atomic force microscopy, dynamic light scattering, and electron paramagnetic resonance spectroscopy. The findings indicate that rather rigid DNA networks were formed. This presents a new bottom-up approach for DNA material formation and might find applications like in the generation of functional hydrogels.

  18. A new building block for DNA network formation by self-assembly and polymerase chain reaction

    Directory of Open Access Journals (Sweden)

    Holger Bußkamp

    2014-05-01

    Full Text Available The predictability of DNA self-assembly is exploited in many nanotechnological approaches. Inspired by naturally existing self-assembled DNA architectures, branched DNA has been developed that allows self-assembly to predesigned architectures with dimensions on the nanometer scale. DNA is an attractive material for generation of nanostructures due to a plethora of enzymes which modify DNA with high accuracy, providing a toolbox for many different manipulations to construct nanometer scaled objects. We present a straightforward synthesis of a rigid DNA branching building block successfully used for the generation of DNA networks by self-assembly and network formation by enzymatic DNA synthesis. The Y-shaped 3-armed DNA construct, bearing 3 primer strands is accepted by Taq DNA polymerase. The enzyme uses each arm as primer strand and incorporates the branched construct into large assemblies during PCR. The networks were investigated by agarose gel electrophoresis, atomic force microscopy, dynamic light scattering, and electron paramagnetic resonance spectroscopy. The findings indicate that rather rigid DNA networks were formed. This presents a new bottom-up approach for DNA material formation and might find applications like in the generation of functional hydrogels.

  19. FOLDNA, a Web Server for Self-Assembled DNA Nanostructure Autoscaffolds and Autostaples

    Directory of Open Access Journals (Sweden)

    Chensheng Zhou

    2012-01-01

    Full Text Available DNA self-assembly is a nanotechnology that folds DNA into desired shapes. Self-assembled DNA nanostructures, also known as origami, are increasingly valuable in nanomaterial and biosensing applications. Two ways to use DNA nanostructures in medicine are to form nanoarrays, and to work as vehicles in drug delivery. The DNA nanostructures perform well as a biomaterial in these areas because they have spatially addressable and size controllable properties. However, manually designing complementary DNA sequences for self-assembly is a technically demanding and time consuming task, which makes it advantageous for computers to do this job instead. We have developed a web server, FOLDNA, which can automatically design 2D self-assembled DNA nanostructures according to custom pictures and scaffold sequences provided by the users. It is the first web server to provide an entirely automatic design of self-assembled DNA nanostructure, and it takes merely a second to generate comprehensive information for molecular experiments including: scaffold DNA pathways, staple DNA directions, and staple DNA sequences. This program could save as much as several hours in the designing step for each DNA nanostructure. We randomly selected some shapes and corresponding outputs from our server and validated its performance in molecular experiments.

  20. Process development for automated solar cell and module production. Task 4. Automated array assembly. Quarterly report No. 1

    Energy Technology Data Exchange (ETDEWEB)

    Hagerty, J. J.

    1980-10-15

    Work has been divided into five phases. The first phase is to modify existing hardware and controlling computer software to: (1) improve cell-to-cell placement accuracy, (2) improve the solder joint while reducing the amount of solder and flux smear on the cell's surface, and (3) reduce the system cycle time to 10 seconds. The second phase involves expanding the existing system's capabilities to be able to reject broken cells and make post-solder electrical tests. Phase 3 involves developing new hardware to allow for the automated encapsulation of solar modules. This involves three discrete pieces of hardware: (1) a vacuum platen end effector for the robot which allows it to pick up the 1' x 4' array of 35 inter-connected cells. With this, it can also pick up the cover glass and completed module, (2) a lamination preparation station which cuts the various encapsulation components from roll storage and positions them for encapsulation, and (3) an automated encapsulation chamber which interfaces with the above two and applies the heat and vacuum to cure the encapsulants. Phase 4 involves the final assembly of the encapsulated array into a framed, edge-sealed module completed for installation. For this we are using MBA's Glass Reinforced Concrete (GRC) in panels such as those developed by MBA for JPL under contract No. 955281. The GRC panel plays the multiple role of edge frame, substrate and mounting structure. An automated method of applying the edge seal will also be developed. The final phase (5) is the fabrication of six 1' x 4' electrically active solar modules using the above developed equipment. Progress is reported. (WHK)

  1. Multi-scale coarse-graining for the study of assembly pathways in DNA-brick self-assembly

    Science.gov (United States)

    Fonseca, Pedro; Romano, Flavio; Schreck, John S.; Ouldridge, Thomas E.; Doye, Jonathan P. K.; Louis, Ard A.

    2018-04-01

    Inspired by recent successes using single-stranded DNA tiles to produce complex structures, we develop a two-step coarse-graining approach that uses detailed thermodynamic calculations with oxDNA, a nucleotide-based model of DNA, to parametrize a coarser kinetic model that can reach the time and length scales needed to study the assembly mechanisms of these structures. We test the model by performing a detailed study of the assembly pathways for a two-dimensional target structure made up of 334 unique strands each of which are 42 nucleotides long. Without adjustable parameters, the model reproduces a critical temperature for the formation of the assembly that is close to the temperature at which assembly first occurs in experiments. Furthermore, the model allows us to investigate in detail the nucleation barriers and the distribution of critical nucleus shapes for the assembly of a single target structure. The assembly intermediates are compact and highly connected (although not maximally so), and classical nucleation theory provides a good fit to the height and shape of the nucleation barrier at temperatures close to where assembly first occurs.

  2. Robust Automated Test Assembly for Testlet-Based Tests: An Illustration with Analytical Reasoning Items

    Directory of Open Access Journals (Sweden)

    Bernard P. Veldkamp

    2017-12-01

    Full Text Available In many high-stakes testing programs, testlets are used to increase efficiency. Since responses to items belonging to the same testlet not only depend on the latent ability but also on correct reading, understanding, and interpretation of the stimulus, the assumption of local independence does not hold. Testlet response theory (TRT models have been developed to deal with this dependency. For both logit and probit testlet models, a random testlet effect is added to the standard logit and probit item response theory (IRT models. Even though this testlet effect might make the IRT models more realistic, application of these models in practice leads to new questions, for example, in automated test assembly (ATA. In many test assembly models, goals have been formulated for the amount of information the test should provide about the candidates. The amount of Fisher Information is often maximized or it has to meet a prespecified target. Since TRT models have a random testlet effect, Fisher Information contains a random effect as well. The question arises as to how this random effect in ATA should be dealt with. A method based on robust optimization techniques for dealing with uncertainty in test assembly due to random testlet effects is presented. The method is applied in the context of a high-stakes testing program, and the impact of this robust test assembly method is studied. Results are discussed, advantages of the use of robust test assembly are mentioned, and recommendations about the use of the new method are given.

  3. Automated solar cell assembly teamed process research. Semiannual subcontract report, December 6, 1993--June 30, 1994

    Energy Technology Data Exchange (ETDEWEB)

    Nowlan, M. [Spire Corp., Bedford, MA (United States)

    1995-01-01

    This is the second Semiannual Technical Progress Report for the program titled `Automated Solar Cell Assembly Teamed Process Research` funded under National Renewable Energy Laboratory (NREL) subcontract No. ZAG-3-11219-01. This report describes the work done on Phase II of the program in the period from December 6, 1993 to June 30, 1994. Spire`s objective in this program is to develop high throughput (5 MW/yr) automated processes for interconnecting thin (200 {mu}m) silicon solar cells. High yield will be achieved with these fragile cells through the development of low mechanical stress and low thermal stress processes. For example, a machine vision system is being developed for cell alignment without mechanically contacting the cell edges, while a new soldering process is being developed to solder metal interconnect ribbons simultaneously to a cells` front and back contacts, eliminating one of the two heating steps normally used for soldering each cell.

  4. Size-controllable DNA nanoribbons assembled from three types of reusable brick single-strand DNA tiles.

    Science.gov (United States)

    Shi, Xiaolong; Chen, Congzhou; Li, Xin; Song, Tao; Chen, Zhihua; Zhang, Zheng; Wang, Yanfeng

    2015-11-21

    Precise control of nanostructure is a significant goal shared by supramolecular chemistry, nanotechnology and materials science. In DNA nanotechnology, methods of constructing desired DNA nanostructures using programmable DNA strands have been studied extensively and have become a promising branch of research, but developing universal and low-cost (in the sense of using fewer types of DNA strands) methods remains a challenge. In this work, we propose a novel approach to assemble size-controllable DNA nanoribbons with three types of reusable brick SSTs (single-stranded DNA tiles), where the control of ribbon size is achieved by regulating the concentration ratio between manipulative strands and packed single-stranded DNA tiles. In our method, three types of brick SSTs are sufficient in assembling DNA nanoribbons of different sizes, which is much less than the number of types of unique tile-programmable assembling strategy, thus achieving a universal and low-cost method. The assembled DNA nanoribbons are observed and analyzed by atomic force microscopy (AFM). Experimental observations strongly suggest the feasibility and reliability of our method.

  5. Electrostatic assembly of protein lysozyme on DNA visualized by atomic force microscopy

    International Nuclear Information System (INIS)

    Yang Tao; Wei Gang; Li Zhuang

    2007-01-01

    In the present work, atomic force microscopy (AFM) has been used to study the assembly of protein lysozyme on DNA molecule. Based on the electrostatic interaction, the positively charged lysozyme can easily bind onto the negatively charged DNA molecule surface. The protein molecules appear as globular objects on the DNA scaffold, which are distinguishable in the AFM images. At the same time, lysozyme molecules can be assembled onto DNA as dense or sporadic pattern by varying the protein concentration. This work may provide fundamental aspects for building protein nanostructures and studying of DNA-protein interaction

  6. APPROACH TO AUTOMATION OF LENS COMPONENTS CENTERING FOR ASSEMBLING OF DIFFERENT DESIGN OBJECTIVES

    Directory of Open Access Journals (Sweden)

    A. Y. Pimenov

    2016-01-01

    Full Text Available Subject of Research.Existing adjustment methods of lens objectives in two mechanical designs have been analyzed, the main ways of centering error compensation have been considered. The algorithm of automated search of optical elements position has been developed based on the existing methods of centering for compensation of residual decentering. The methods of joint application of this algorithm and centering machine “Trioptics Opticentric” have been suggested. Method. Centering errors of the objective lens components are measured on the centering machine. Aberrations of optical system are calculated by using the received data of centering errors entered to the objective mathematical model. For the calculation of aberration compensation, iteration algorithm of searching of optical elements optimal position is used. The image quality is analyzed for each obtained realization of the optical system. The result of program performance is data matrix about optical components location such that compensation of residual aberrations occurs. Based on the obtained information the system assembling is carried out with the use of centering machine “Trioptics Opticentric”. Main Results. Computer modeling of centering errors for 100 lens apochromatic objectives of drop-in mountings lens design has been carried out. The coma of object axial point image has been chosen as quality estimation criterion. The developed algorithm has been applied for the worst realization of the optical system. In this case the value of integral coma of object axial point image has been decreased by two orders and reduced practically to zero. It is noted that the modeling of adjustment process via using the algorithm of automated search enables to predict the possibility of objective assembly with required quality parameters. Practical Relevance. The received results can be applied on the lines of automated assembly of modern lens objectives.

  7. Plasmonic photonic crystals realized through DNA-programmable assembly.

    Science.gov (United States)

    Park, Daniel J; Zhang, Chuan; Ku, Jessie C; Zhou, Yu; Schatz, George C; Mirkin, Chad A

    2015-01-27

    Three-dimensional dielectric photonic crystals have well-established enhanced light-matter interactions via high Q factors. Their plasmonic counterparts based on arrays of nanoparticles, however, have not been experimentally well explored owing to a lack of available synthetic routes for preparing them. However, such structures should facilitate these interactions based on the small mode volumes associated with plasmonic polarization. Herein we report strong light-plasmon interactions within 3D plasmonic photonic crystals that have lattice constants and nanoparticle diameters that can be independently controlled in the deep subwavelength size regime by using a DNA-programmable assembly technique. The strong coupling within such crystals is probed with backscattering spectra, and the mode splitting (0.10 and 0.24 eV) is defined based on dispersion diagrams. Numerical simulations predict that the crystal photonic modes (Fabry-Perot modes) can be enhanced by coating the crystals with a silver layer, achieving moderate Q factors (∼10(2)) over the visible and near-infrared spectrum.

  8. Proteomics reveals dynamic assembly of repair complexes during bypass of DNA cross-links

    DEFF Research Database (Denmark)

    Räschle, Markus; Smeenk, Godelieve; Hansen, Rebecca K

    2015-01-01

    a technique called chromatin mass spectrometry (CHROMASS) to study protein recruitment dynamics during perturbed DNA replication in Xenopus egg extracts. Using CHROMASS, we systematically monitored protein assembly and disassembly on ICL-containing chromatin. Among numerous prospective DNA repair factors, we...... to damaged DNA in vertebrate cells....

  9. Unique nucleotide sequence-guided assembly of repetitive DNA parts for synthetic biology applications

    Energy Technology Data Exchange (ETDEWEB)

    Torella, JP; Lienert, F; Boehm, CR; Chen, JH; Way, JC; Silver, PA

    2014-08-07

    Recombination-based DNA construction methods, such as Gibson assembly, have made it possible to easily and simultaneously assemble multiple DNA parts, and they hold promise for the development and optimization of metabolic pathways and functional genetic circuits. Over time, however, these pathways and circuits have become more complex, and the increasing need for standardization and insulation of genetic parts has resulted in sequence redundancies-for example, repeated terminator and insulator sequences-that complicate recombination-based assembly. We and others have recently developed DNA assembly methods, which we refer to collectively as unique nucleotide sequence (UNS)-guided assembly, in which individual DNA parts are flanked with UNSs to facilitate the ordered, recombination-based assembly of repetitive sequences. Here we present a detailed protocol for UNS-guided assembly that enables researchers to convert multiple DNA parts into sequenced, correctly assembled constructs, or into high-quality combinatorial libraries in only 2-3 d. If the DNA parts must be generated from scratch, an additional 2-5 d are necessary. This protocol requires no specialized equipment and can easily be implemented by a student with experience in basic cloning techniques.

  10. Automated assembly of species metabolomes through data submission into a public repository.

    Science.gov (United States)

    Salek, Reza M; Conesa, Pablo; Cochrane, Keeva; Haug, Kenneth; Williams, Mark; Kale, Namrata; Moreno, Pablo; Jayaseelan, Kalai Vanii; Macias, Jose Ramon; Nainala, Venkata Chandrasekhar; Hall, Robert D; Reed, Laura K; Viant, Mark R; O'Donovan, Claire; Steinbeck, Christoph

    2017-08-01

    Following similar global efforts to exchange genomic and other biomedical data, global databases in metabolomics have now been established. MetaboLights, the first general purpose, publically available, cross-species, cross-application database in metabolomics, has become the fastest growing data repository at the European Bioinformatics Institute in terms of data volume. Here we present the automated assembly of species metabolomes in MetaboLights, a crucial reference for chemical biology, which is growing through user submissions. © The Authors 2017. Published by Oxford University Press.

  11. Automated Glycan Assembly of Complex Oligosaccharides Related to Blood Group Determinants.

    Science.gov (United States)

    Hahm, Heung Sik; Liang, Chien-Fu; Lai, Chian-Hui; Fair, Richard J; Schuhmacher, Frank; Seeberger, Peter H

    2016-07-15

    Lactotetraosyl (Lc4) and neo-lactotetraosyl (nLc4) are backbones that are common to many glycans. Using automated glycan assembly, these common core structures were constructed and elaborated to access synthetically challenging glycans of biological relevance. The incorporation of α-fucoses is demonstrated for H-type I and II; α(1,3)-galactose epitopes were prepared, and the pentasaccharide HNK-1 required incorporation of a 3-O-sulfate. In addition to preparing the target structures, essential insights were gained regarding the relationships of glycosylating agents and nucleophiles as well as the linker stability.

  12. Assembly and melting of DNA nanotubes from single-sequence tiles

    International Nuclear Information System (INIS)

    Sobey, T L; Renner, S; Simmel, F C

    2009-01-01

    DNA melting and renaturation studies are an extremely valuable tool to study the kinetics and thermodynamics of duplex dissociation and reassociation reactions. These are important not only in a biological or biotechnological context, but also for DNA nanotechnology which aims at the construction of molecular materials by DNA self-assembly. We here study experimentally the formation and melting of a DNA nanotube structure, which is composed of many copies of an oligonucleotide containing several palindromic sequences. This is done using temperature-controlled UV absorption measurements correlated with atomic force microscopy, fluorescence microscopy and transmission electron microscopy techniques. In the melting studies, important factors such as DNA strand concentration, hierarchy of assembly and annealing protocol are investigated. Assembly and melting of the nanotubes are shown to proceed via different pathways. Whereas assembly occurs in several hierarchical steps related to the formation of tiles, lattices and tubes, melting of DNA nanotubes appears to occur in a single step. This is proposed to relate to fundamental differences between closed, three-dimensional tube-like structures and open, two-dimensional lattices. DNA melting studies can lead to a better understanding of the many factors that affect the assembly process which will be essential for the assembly of increasingly complex DNA nanostructures.

  13. Noncanonical self-assembly of multifunctional DNA nanoflowers for biomedical applications.

    Science.gov (United States)

    Zhu, Guizhi; Hu, Rong; Zhao, Zilong; Chen, Zhuo; Zhang, Xiaobing; Tan, Weihong

    2013-11-06

    DNA nanotechnology has been extensively explored to assemble various functional nanostructures for versatile applications. Mediated by Watson-Crick base-pairing, these DNA nanostructures have been conventionally assembled through hybridization of many short DNA building blocks. Here we report the noncanonical self-assembly of multifunctional DNA nanostructures, termed as nanoflowers (NFs), and the versatile biomedical applications. These NFs were assembled from long DNA building blocks generated via rolling circle replication (RCR) of a designer template. NF assembly was driven by liquid crystallization and dense packaging of building blocks, without relying on Watson-Crick base-pairing between DNA strands, thereby avoiding the otherwise conventional complicated DNA sequence design. NF sizes were readily tunable in a wide range, by simply adjusting such parameters as assembly time and template sequences. NFs were exceptionally resistant to nuclease degradation, denaturation, or dissociation at extremely low concentration, presumably resulting from the dense DNA packaging in NFs. The exceptional biostability is critical for biomedical applications. By rational design, NFs can be readily incorporated with myriad functional moieties. All these properties make NFs promising for versatile applications. As a proof-of-principle demonstration, in this study, NFs were integrated with aptamers, bioimaging agents, and drug loading sites, and the resultant multifunctional NFs were demonstrated for selective cancer cell recognition, bioimaging, and targeted anticancer drug delivery.

  14. Monitoring the hydration of DNA self-assembled monolayers using an extensional nanomechanical resonator

    DEFF Research Database (Denmark)

    Cagliani, Alberto; Kosaka, Priscila; Tamayo, Javier

    2012-01-01

    We have fabricated an ultrasensitive nanomechanical resonator based on the extensional vibration mode to weigh the adsorbed water on self-assembled monolayers of DNA as a function of the relative humidity. The water adsorption isotherms provide the number of adsorbed water molecules per nucleotide...... for monolayers of single stranded (ss) DNA and after hybridization with the complementary DNA strand. Our results differ from previous data obtained with bulk samples, showing the genuine behavior of these self-assembled monolayers. The hybridization cannot be inferred from the water adsorption isotherms due...... on the interaction between water and self-assembled monolayers of nucleic acids....

  15. The Assembly of DNA Amphiphiles at Liquid Crystal-Aqueous Interface

    Directory of Open Access Journals (Sweden)

    Jingsheng Zhou

    2016-12-01

    Full Text Available In this article, we synthesized a type of DNA amphiphiles (called DNA-lipids and systematically studied its assembly behavior at the liquid crystal (LC—aqueous interface. It turned out that the pure DNA-lipids at various concentrations cannot trigger the optical transition of liquid crystals from planar anchoring to homeotropic anchoring at the liquid crystal—aqueous interface. The co-assembly of DNA-lipid and l-dilauroyl phosphatidylcholine (l-DLPC indicated that the DLPC assembled all over the LC-aqueous interface, and DNA-lipids prefer to couple with LC in certain areas, particularly in polarized and fluorescent image, forming micron sized net-like structures. The addition of DNA complementary to DNA-lipids forming double stranded DNA-lipids caused de-assembly of DNA-lipids from LC-aqueous interface, resulting in the disappearance of net-like structures, which can be visualized through polarized microscope. The optical changes combined with DNA unique designable property and specific interaction with wide range of target molecules, the DNA-lipids decorated LC-aqueous interface would provide a new platform for biological sensing and diagnosis.

  16. Applications of DNA self-assembled structures in nanoelectronics and plasmonics

    OpenAIRE

    Shen, Boxuan

    2018-01-01

    In this thesis, the potential applications of DNA self-assembled structures were explored in both nanoelectronics and plasmonics. The works can be divided into two parts: electrical characterization of unmodified multilayered DNA origami and DNA-gold-nanoparticle conjugates after they were trapped between gold nanoelectrodes by dielectrophoresis, and the development of a novel fabrication method using DNA origami as a template for smooth, high resolution metallic nanostructures as ...

  17. Programmable DNA tile self-assembly using a hierarchical sub-tile strategy.

    Science.gov (United States)

    Shi, Xiaolong; Lu, Wei; Wang, Zhiyu; Pan, Linqiang; Cui, Guangzhao; Xu, Jin; LaBean, Thomas H

    2014-02-21

    DNA tile based self-assembly provides a bottom-up approach to construct desired nanostructures. DNA tiles have been directly constructed from ssDNA and readily self-assembled into 2D lattices and 3D superstructures. However, for more complex lattice designs including algorithmic assemblies requiring larger tile sets, a more modular approach could prove useful. This paper reports a new DNA 'sub-tile' strategy to easily create whole families of programmable tiles. Here, we demonstrate the stability and flexibility of our sub-tile structures by constructing 3-, 4- and 6-arm DNA tiles that are subsequently assembled into 2D lattices and 3D nanotubes according to a hierarchical design. Assembly of sub-tiles, tiles, and superstructures was analyzed using polyacrylamide gel electrophoresis and atomic force microscopy. DNA tile self-assembly methods provide a bottom-up approach to create desired nanostructures; the sub-tile strategy adds a useful new layer to this technique. Complex units can be made from simple parts. The sub-tile approach enables the rapid redesign and prototyping of complex DNA tile sets and tiles with asymmetric designs.

  18. Programmed self-assembly of DNA/RNA for biomedical applications

    Science.gov (United States)

    Wang, Pengfei

    Three self-assembly strategies were utilized for assembly of novel functional DNA/RNA nanostructures. RNA-DNA hybrid origami method was developed to fabricate nano-objects (ribbon, rectangle, and triangle) with precisely controlled geometry. Unlike conventional DNA origami which use long DNA single strand as scaffold, a long RNA single strand was used instead, which was folded by short DNA single strands (staples) into prescribed objects through sequence specific hybridization between RNA and DNA. Single stranded tiles (SST) and RNA-DNA hybrid origami were utilized to fabricate a variety of barcode-like nanostructures with unique patterns by expanding a plain rectangle via introducing spacers (10-bp dsDNA segment) between parallel duplexes. Finally, complex 2D array and 3D polyhedrons with multiple patterns within one structure were assembled from simple DNA motifs. Two demonstrations of biomedical applications of DNA nanotechnology were presented. Firstly, lambda-DNA was used as template to direct the fabrication of multi-component magnetic nanoparticle chains. Nuclear magnetic relaxation (NMR) characterization showed superb magnetic relaxativity of the nanoparticle chains which have large potential to be utilized as MRI contrast agents. Secondly, DNA nanotechnology was introduced into the conformational study of a routinely used catalytic DNAzyme, the RNA-cleaving 10-23 DNAzyme. The relative angle between two flanking duplexes of the catalytic core was determined (94.8°), which shall be able to provide a clue to further understanding of the cleaving mechanism of this DNAzyme from a conformational perspective.

  19. An automated spin-assisted approach for molecular layer-by-layer assembly of crosslinked polymer thin films.

    Science.gov (United States)

    Chan, Edwin P; Lee, Jung-Hyun; Chung, Jun Young; Stafford, Christopher M

    2012-11-01

    We present the design of an automated spin-coater that facilitates fabrication of polymer films based on molecular layer-by-layer (mLbL) assembly. Specifically, we demonstrate the synthesis of ultrathin crosslinked fully-aromatic polyamide (PA) films that are chemically identical to polymer membranes used in water desalination applications as measured by X-ray photoelectron spectroscopy. X-ray reflectivity measurements indicate that the automated mLbL assembly creates films with a constant film growth rate and minimal roughness compared with the traditional interfacial polymerization of PA. This automated spin-coater improves the scalability and sample-to-sample consistency by reducing human involvement in the mLbL assembly.

  20. An automated spin-assisted approach for molecular layer-by-layer assembly of crosslinked polymer thin films

    Energy Technology Data Exchange (ETDEWEB)

    Chan, Edwin P.; Chung, Jun Young; Stafford, Christopher M. [Polymers Division, National Institute of Standards and Technology, Gaithersburg, Maryland 20899 (United States); Lee, Jung-Hyun [Center for Materials Architecturing, Korea Institute of Science and Technology, Seoul (Korea, Republic of)

    2012-11-15

    We present the design of an automated spin-coater that facilitates fabrication of polymer films based on molecular layer-by-layer (mLbL) assembly. Specifically, we demonstrate the synthesis of ultrathin crosslinked fully-aromatic polyamide (PA) films that are chemically identical to polymer membranes used in water desalination applications as measured by X-ray photoelectron spectroscopy. X-ray reflectivity measurements indicate that the automated mLbL assembly creates films with a constant film growth rate and minimal roughness compared with the traditional interfacial polymerization of PA. This automated spin-coater improves the scalability and sample-to-sample consistency by reducing human involvement in the mLbL assembly.

  1. Katome: de novo DNA assembler implemented in rust

    Science.gov (United States)

    Neumann, Łukasz; Nowak, Robert M.; Kuśmirek, Wiktor

    2017-08-01

    Katome is a new de novo sequence assembler written in the Rust programming language, designed with respect to future parallelization of the algorithms, run time and memory usage optimization. The application uses new algorithms for the correct assembly of repetitive sequences. Performance and quality tests were performed on various data, comparing the new application to `dnaasm', `ABySS' and `Velvet' genome assemblers. Quality tests indicate that the new assembler creates more contigs than well-established solutions, but the contigs have better quality with regard to mismatches per 100kbp and indels per 100kbp. Additionally, benchmarks indicate that the Rust-based implementation outperforms `dnaasm', `ABySS' and `Velvet' assemblers, written in C++, in terms of assembly time. Lower memory usage in comparison to `dnaasm' is observed.

  2. Automated genomic DNA purification options in agricultural applications using MagneSil paramagnetic particles

    Science.gov (United States)

    Bitner, Rex M.; Koller, Susan C.

    2002-06-01

    The automated high throughput purification of genomic DNA form plant materials can be performed using MagneSil paramagnetic particles on the Beckman-Coulter FX, BioMek 2000, and the Tecan Genesis robot. Similar automated methods are available for DNA purifications from animal blood. These methods eliminate organic extractions, lengthy incubations and cumbersome filter plates. The DNA is suitable for applications such as PCR and RAPD analysis. Methods are described for processing traditionally difficult samples such as those containing large amounts of polyphenolics or oils, while still maintaining a high level of DNA purity. The robotic protocols have ben optimized for agricultural applications such as marker assisted breeding, seed-quality testing, and SNP discovery and scoring. In addition to high yield purification of DNA from plant samples or animal blood, the use of Promega's DNA-IQ purification system is also described. This method allows for the purification of a narrow range of DNA regardless of the amount of additional DNA that is present in the initial sample. This simultaneous Isolation and Quantification of DNA allows the DNA to be used directly in applications such as PCR, SNP analysis, and RAPD, without the need for separate quantitation of the DNA.

  3. Automated DNA extraction from genetically modified maize using aminosilane-modified bacterial magnetic particles.

    Science.gov (United States)

    Ota, Hiroyuki; Lim, Tae-Kyu; Tanaka, Tsuyoshi; Yoshino, Tomoko; Harada, Manabu; Matsunaga, Tadashi

    2006-09-18

    A novel, automated system, PNE-1080, equipped with eight automated pestle units and a spectrophotometer was developed for genomic DNA extraction from maize using aminosilane-modified bacterial magnetic particles (BMPs). The use of aminosilane-modified BMPs allowed highly accurate DNA recovery. The (A(260)-A(320)):(A(280)-A(320)) ratio of the extracted DNA was 1.9+/-0.1. The DNA quality was sufficiently pure for PCR analysis. The PNE-1080 offered rapid assay completion (30 min) with high accuracy. Furthermore, the results of real-time PCR confirmed that our proposed method permitted the accurate determination of genetically modified DNA composition and correlated well with results obtained by conventional cetyltrimethylammonium bromide (CTAB)-based methods.

  4. Assembly of presynaptic filaments. Factors affecting the assembly of RecA protein onto single-stranded DNA

    DEFF Research Database (Denmark)

    Thresher, RJ; Christiansen, Gunna; Griffith, JD

    1988-01-01

    We have previously shown that the assembly of RecA protein onto single-stranded DNA (ssDNA) facilitated by SSB protein occurs in three steps: (1) rapid binding of SSB protein to the ssDNA; (2) nucleation of RecA protein onto this template; and (3) co-operative polymerization of additional Rec......M in the presence of 12 mM-Mg2+), and relatively low concentrations of SSB protein (1 monomer per 18 nucleotides). Assembly was depressed threefold when SSB protein was added to one monomer per nine nucleotides. These effects appeared to be exerted at the nucleation step. Following nucleation, RecA protein...... assembled onto ssDNA at net rates that varied from 250 to 900 RecA protein monomers per minute, with the rate inversely related to the concentration of SSB protein. Combined sucrose sedimentation and electron microscope analysis established that SSB protein was displaced from the ssDNA during RecA protein...

  5. Mic60/Mitofilin determines MICOS assembly essential for mitochondrial dynamics and mtDNA nucleoid organization

    OpenAIRE

    Li, H; Ruan, Y; Zhang, K; Jian, F; Hu, C; Miao, L; Gong, L; Sun, L; Zhang, X; Chen, S; Chen, H; Liu, D; Song, Z

    2015-01-01

    The MICOS complex (mitochondrial contact site and cristae organizing system) is essential for mitochondrial inner membrane organization and mitochondrial membrane contacts, however, the molecular regulation of MICOS assembly and the physiological functions of MICOS in mammals remain obscure. Here, we report that Mic60/Mitofilin has a critical role in the MICOS assembly, which determines the mitochondrial morphology and mitochondrial DNA (mtDNA) organization. The downregulati...

  6. Fractal assembly of micrometre-scale DNA origami arrays with arbitrary patterns

    Science.gov (United States)

    Tikhomirov, Grigory; Petersen, Philip; Qian, Lulu

    2017-12-01

    Self-assembled DNA nanostructures enable nanometre-precise patterning that can be used to create programmable molecular machines and arrays of functional materials. DNA origami is particularly versatile in this context because each DNA strand in the origami nanostructure occupies a unique position and can serve as a uniquely addressable pixel. However, the scale of such structures has been limited to about 0.05 square micrometres, hindering applications that demand a larger layout and integration with more conventional patterning methods. Hierarchical multistage assembly of simple sets of tiles can in principle overcome this limitation, but so far has not been sufficiently robust to enable successful implementation of larger structures using DNA origami tiles. Here we show that by using simple local assembly rules that are modified and applied recursively throughout a hierarchical, multistage assembly process, a small and constant set of unique DNA strands can be used to create DNA origami arrays of increasing size and with arbitrary patterns. We illustrate this method, which we term ‘fractal assembly’, by producing DNA origami arrays with sizes of up to 0.5 square micrometres and with up to 8,704 pixels, allowing us to render images such as the Mona Lisa and a rooster. We find that self-assembly of the tiles into arrays is unaffected by changes in surface patterns on the tiles, and that the yield of the fractal assembly process corresponds to about 0.95m - 1 for arrays containing m tiles. When used in conjunction with a software tool that we developed that converts an arbitrary pattern into DNA sequences and experimental protocols, our assembly method is readily accessible and will facilitate the construction of sophisticated materials and devices with sizes similar to that of a bacterium using DNA nanostructures.

  7. Dynamic protein assemblies in homologous recombination with single DNA molecules

    NARCIS (Netherlands)

    van der Heijden, A.H.

    2007-01-01

    What happens when your DNA breaks? This thesis describes experimental work on the single-molecule level focusing on the interaction between DNA and DNA-repair proteins, in particular bacterial RecA and human Rad51, involved in homologous recombination. Homologous recombination and its central event

  8. Molecular Behavior of DNA Origami in Higher-Order Self-Assembly

    Energy Technology Data Exchange (ETDEWEB)

    Li, Zhe [Arizona State Univ., Tempe, AZ (United States); Liu, Minghui [Arizona State Univ., Tempe, AZ (United States); Lei, Wang [Arizona State Univ., Tempe, AZ (United States); Shandong Univ., Jinan (China); Nangreave, Jeanette [Arizona State Univ., Tempe, AZ (United States); Yan, Hao [Arizona State Univ., Tempe, AZ (United States); Liu, Yan [Arizona State Univ., Tempe, AZ (United States)

    2010-09-08

    DNA-based self-assembly is a unique method for achieving higher-order molecular architectures made possible by the fact that DNA is a programmable information-coding polymer. In the past decade, two main types of DNA nanostructures have been developed: branch-shaped DNA tiles with small dimensions (commonly up to ~20 nm) and DNA origami tiles with larger dimensions (up to ~100 nm). Here we aimed to determine the important factors involved in the assembly of DNA origami superstructures. We constructed a new series of rectangular-shaped DNA origami tiles in which parallel DNA helices are arranged in a zigzag pattern when viewed along the DNA helical axis, a design conceived in order to relax an intrinsic global twist found in the original planar, rectangular origami tiles. Self-associating zigzag tiles were found to form linear arrays in both diagonal directions, while planar tiles showed significant growth in only one direction. Although the series of zigzag tiles were designed to promote two-dimensional array formation, one-dimensional linear arrays and tubular structures were observed instead. We discovered that the dimensional aspect ratio of the origami unit tiles and intertile connection design play important roles in determining the final products, as revealed by atomic force microscopy imaging. This study provides insight into the formation of higher-order structures from self-assembling DNA origami tiles, revealing their unique behavior in comparison with conventional DNA tiles having smaller dimensions.

  9. Electrochemical DNA sensor by the assembly of graphene and DNA-conjugated gold nanoparticles with silver enhancement strategy.

    Science.gov (United States)

    Lin, Lei; Liu, Yang; Tang, Longhua; Li, Jinghong

    2011-11-21

    Sensitive and selective detection of DNA is in urgent need due to its important role in human bodies. Many disorders, such as Alzheimer's disease and various cancers, are closely related with DNA damage. In this work, a novel electrochemical DNA biosensor was constructed on a DNA-assembling graphene platform which provided a robust, simple and biocompatible platform with large surface area for DNA immobilization. The as-designed DNA sensor was fabricated by directly assembling captured ssDNA on a graphene-modified electrode through the π-π stacking interaction between graphene and ssDNA bases. Then, the target DNA sequence and oligonucleotide probes-labeled AuNPs were able to hybridize in a sandwich assay format, following the AuNPs-catalyzed silver deposition. The deposited silver was further detected by differential pulse voltammetry. Owing to the high DNA loading ability of graphene and the distinct signal amplification by AuNPs-catalyzed silver staining, the resulting biosensor exhibited a good analytical performance with a wide detection linear range from 200 pM to 500 nM, and a low detection limit of 72 pM. Additionally, the biosensor was proved to be able to discriminate the complementary sequence from the single-base mismatch sequence. The simple biosensor is promising in developing electronic, on-chip assays in clinical diagnosis, environmental control, and drug discovery.

  10. AFEAP cloning: a precise and efficient method for large DNA sequence assembly.

    Science.gov (United States)

    Zeng, Fanli; Zang, Jinping; Zhang, Suhua; Hao, Zhimin; Dong, Jingao; Lin, Yibin

    2017-11-14

    Recent development of DNA assembly technologies has spurred myriad advances in synthetic biology, but new tools are always required for complicated scenarios. Here, we have developed an alternative DNA assembly method named AFEAP cloning (Assembly of Fragment Ends After PCR), which allows scarless, modular, and reliable construction of biological pathways and circuits from basic genetic parts. The AFEAP method requires two-round of PCRs followed by ligation of the sticky ends of DNA fragments. The first PCR yields linear DNA fragments and is followed by a second asymmetric (one primer) PCR and subsequent annealing that inserts overlapping overhangs at both sides of each DNA fragment. The overlapping overhangs of the neighboring DNA fragments annealed and the nick was sealed by T4 DNA ligase, followed by bacterial transformation to yield the desired plasmids. We characterized the capability and limitations of new developed AFEAP cloning and demonstrated its application to assemble DNA with varying scenarios. Under the optimized conditions, AFEAP cloning allows assembly of an 8 kb plasmid from 1-13 fragments with high accuracy (between 80 and 100%), and 8.0, 11.6, 19.6, 28, and 35.6 kb plasmids from five fragments at 91.67, 91.67, 88.33, 86.33, and 81.67% fidelity, respectively. AFEAP cloning also is capable to construct bacterial artificial chromosome (BAC, 200 kb) with a fidelity of 46.7%. AFEAP cloning provides a powerful, efficient, seamless, and sequence-independent DNA assembly tool for multiple fragments up to 13 and large DNA up to 200 kb that expands synthetic biologist's toolbox.

  11. Evaluation of four automated protocols for extraction of DNA from FTA cards.

    Science.gov (United States)

    Stangegaard, Michael; Børsting, Claus; Ferrero-Miliani, Laura; Frank-Hansen, Rune; Poulsen, Lena; Hansen, Anders J; Morling, Niels

    2013-10-01

    Extraction of DNA using magnetic bead-based techniques on automated DNA extraction instruments provides a fast, reliable, and reproducible method for DNA extraction from various matrices. Here, we have compared the yield and quality of DNA extracted from FTA cards using four automated extraction protocols on three different instruments. The extraction processes were repeated up to six times with the same pieces of FTA cards. The sample material on the FTA cards was either blood or buccal cells. With the QIAamp DNA Investigator and QIAsymphony DNA Investigator kits, it was possible to extract DNA from the FTA cards in all six rounds of extractions in sufficient amount and quality to obtain complete short tandem repeat (STR) profiles on a QIAcube and a QIAsymphony SP. With the PrepFiler Express kit, almost all the extractable DNA was extracted in the first two rounds of extractions. Furthermore, we demonstrated that it was possible to successfully extract sufficient DNA for STR profiling from previously processed FTA card pieces that had been stored at 4 °C for up to 1 year. This showed that rare or precious FTA card samples may be saved for future analyses even though some DNA was already extracted from the FTA cards.

  12. Highly efficient automated extraction of DNA from old and contemporary skeletal remains.

    Science.gov (United States)

    Zupanič Pajnič, Irena; Debska, Magdalena; Gornjak Pogorelc, Barbara; Vodopivec Mohorčič, Katja; Balažic, Jože; Zupanc, Tomaž; Štefanič, Borut; Geršak, Ksenija

    2016-01-01

    We optimised the automated extraction of DNA from old and contemporary skeletal remains using the AutoMate Express system and the PrepFiler BTA kit. 24 Contemporary and 25 old skeletal remains from WWII were analysed. For each skeleton, extraction using only 0.05 g of powder was performed according to the manufacturer's recommendations (no demineralisation - ND method). Since only 32% of full profiles were obtained from aged and 58% from contemporary casework skeletons, the extraction protocol was modified to acquire higher quality DNA and genomic DNA was obtained after full demineralisation (FD method). The nuclear DNA of the samples was quantified using the Investigator Quantiplex kit and STR typing was performed using the NGM kit to evaluate the performance of tested extraction methods. In the aged DNA samples, 64% of full profiles were obtained using the FD method. For the contemporary skeletal remains the performance of the ND method was closer to the FD method compared to the old skeletons, giving 58% of full profiles with the ND method and 71% of full profiles using the FD method. The extraction of DNA from only 0.05 g of bone or tooth powder using the AutoMate Express has proven highly successful in the recovery of DNA from old and contemporary skeletons, especially with the modified FD method. We believe that the results obtained will contribute to the possibilities of using automated devices for extracting DNA from skeletal remains, which would shorten the procedures for obtaining high-quality DNA from skeletons in forensic laboratories. Copyright © 2015 Elsevier Ltd and Faculty of Forensic and Legal Medicine. All rights reserved.

  13. Automated serial extraction of DNA and RNA from biobanked tissue specimens.

    Science.gov (United States)

    Mathot, Lucy; Wallin, Monica; Sjöblom, Tobias

    2013-08-19

    With increasing biobanking of biological samples, methods for large scale extraction of nucleic acids are in demand. The lack of such techniques designed for extraction from tissues results in a bottleneck in downstream genetic analyses, particularly in the field of cancer research. We have developed an automated procedure for tissue homogenization and extraction of DNA and RNA into separate fractions from the same frozen tissue specimen. A purpose developed magnetic bead based technology to serially extract both DNA and RNA from tissues was automated on a Tecan Freedom Evo robotic workstation. 864 fresh-frozen human normal and tumor tissue samples from breast and colon were serially extracted in batches of 96 samples. Yields and quality of DNA and RNA were determined. The DNA was evaluated in several downstream analyses, and the stability of RNA was determined after 9 months of storage. The extracted DNA performed consistently well in processes including PCR-based STR analysis, HaloPlex selection and deep sequencing on an Illumina platform, and gene copy number analysis using microarrays. The RNA has performed well in RT-PCR analyses and maintains integrity upon storage. The technology described here enables the processing of many tissue samples simultaneously with a high quality product and a time and cost reduction for the user. This reduces the sample preparation bottleneck in cancer research. The open automation format also enables integration with upstream and downstream devices for automated sample quantitation or storage.

  14. Surface and bulk dissolution properties, and selectivity of DNA-linked nanoparticle assemblies

    NARCIS (Netherlands)

    Lukatsky, D.B.; Frenkel, D.

    2005-01-01

    Using a simple mean-field model, we analyze the surface and bulk dissolution properties of DNA-linked nanoparticle assemblies. We find that the dissolution temperature and the sharpness of the dissolution profiles increase with the grafting density of the single-stranded DNA "probes" on the surface

  15. Searching for avidity by chemical ligation of combinatorially self-assembled DNA-encoded ligand libraries.

    Science.gov (United States)

    Matysiak, Stefan; Hellmuth, Klaus; El-Sagheer, Afaf H; Shivalingam, Arun; Ariyurek, Yavuz; de Jong, Marco; Hollestelle, Martine J; Out, Ruud; Brown, Tom

    2017-12-19

    DNA encoded ligands are self-assembled into bivalent complexes and chemically ligated to link their identities. To demonstrate their potential as a combinatorial screening platform for avidity interactions, the optimal bivalent aptamer design (examplar ligands) for human alpha-thrombin is determined in a single round of selection and the DNA scaffold replaced with minimal impact on the final design.

  16. Accurate DNA assembly and genome engineering with optimized uracil excision cloning

    DEFF Research Database (Denmark)

    Cavaleiro, Mafalda; Kim, Se Hyeuk; Seppala, Susanna

    2015-01-01

    Simple and reliable DNA editing by uracil excision (a.k.a. USER cloning) has been described by several research groups, but the optimal design of cohesive DNA ends for multigene assembly remains elusive. Here, we use two model constructs based on expression of gfp and a four-gene pathway that pro...

  17. Automated array assembly. Quarterly report No. 1, February--March 1976

    Energy Technology Data Exchange (ETDEWEB)

    Williams, B.F.

    1976-03-01

    We have begun our study for the Automated Array Assembly Task by simultaneously evaluating present manufacturing techniques using expenses based on experience and studying basic cost factors for each step to evaluate expenses from a first-principles point of view. We are developing a formal cost accounting procedure which will be used throughout the study for cost comparisons. The first test of this procedure is a comparison of its predicted costs for array module manufacturing with costs from a study we have now completed which is based on experience factors. In this completed study, which is described in this report, we estimate a manufacturing cost for array modules of $10/W, based on present-day manufacturing techniques, expenses, and materials costs. Our analytical system to provide the input data for the cost analysis is also described. The analysis of different input sheet forms and quality has begun.

  18. DNA origami: a quantum leap for self-assembly of complex structures

    DEFF Research Database (Denmark)

    Tørring, Thomas; Voigt, Niels Vinther; Nangreave, Jeanette

    2011-01-01

    The spatially controlled positioning of functional materials by self-assembly is one of the fundamental visions of nanotechnology. Major steps towards this goal have been achieved using DNA as a programmable building block. This tutorial review will focus on one of the most promising methods: DNA...... origami. The basic design principles, organization of a variety of functional materials and recent implementation of DNA robotics are discussed together with future challenges and opportunities....

  19. Automated protein-DNA interaction screening of Drosophila regulatory elements.

    Science.gov (United States)

    Hens, Korneel; Feuz, Jean-Daniel; Isakova, Alina; Iagovitina, Antonina; Massouras, Andreas; Bryois, Julien; Callaerts, Patrick; Celniker, Susan E; Deplancke, Bart

    2011-10-30

    Drosophila melanogaster has one of the best characterized metazoan genomes in terms of functionally annotated regulatory elements. To explore how these elements contribute to gene regulation, we need convenient tools to identify the proteins that bind to them. Here we describe the development and validation of a high-throughput yeast one-hybrid platform, which enables screening of DNA elements versus an array of full-length, sequence-verified clones containing over 85% of predicted Drosophila transcription factors. Using six well-characterized regulatory elements, we identified 33 transcription factor-DNA interactions of which 27 were previously unidentified. To simultaneously validate these interactions and locate the binding sites of involved transcription factors, we implemented a powerful microfluidics-based approach that enabled us to retrieve DNA-occupancy data for each transcription factor throughout the respective target DNA elements. Finally, we biologically validated several interactions and identified two new regulators of sine oculis gene expression and hence eye development.

  20. Comparison of Integer Programming (IP) Solvers for Automated Test Assembly (ATA). Research Report. ETS RR-15-05

    Science.gov (United States)

    Donoghue, John R.

    2015-01-01

    At the heart of van der Linden's approach to automated test assembly (ATA) is a linear programming/integer programming (LP/IP) problem. A variety of IP solvers are available, ranging in cost from free to hundreds of thousands of dollars. In this paper, I compare several approaches to solving the underlying IP problem. These approaches range from…

  1. Automated extraction of DNA from biological stains on fabric from crime cases. A comparison of a manual and three automated methods

    DEFF Research Database (Denmark)

    Stangegaard, Michael; Hjort, Benjamin B; Hansen, Thomas N

    2013-01-01

    The presence of PCR inhibitors in extracted DNA may interfere with the subsequent quantification and short tandem repeat (STR) reactions used in forensic genetic DNA typing. DNA extraction from fabric for forensic genetic purposes may be challenging due to the occasional presence of PCR inhibitors...... that may be co-extracted with the DNA. Using 120 forensic trace evidence samples consisting of various types of fabric, we compared three automated DNA extraction methods based on magnetic beads (PrepFiler Express Forensic DNA Extraction Kit on an AutoMate Express, QIAsyphony DNA Investigator kit either...... with the sample pre-treatment recommended by Qiagen or an in-house optimized sample pre-treatment on a QIAsymphony SP) and one manual method (Chelex) with the aim of reducing the amount of PCR inhibitors in the DNA extracts and increasing the proportion of reportable STR-profiles. A total of 480 samples were...

  2. Automated glycan assembly of a S. pneumoniae serotype 3 CPS antigen

    Directory of Open Access Journals (Sweden)

    Markus W. Weishaupt

    2016-07-01

    Full Text Available Vaccines against S. pneumoniae, one of the most prevalent bacterial infections causing severe disease, rely on isolated capsular polysaccharide (CPS that are conjugated to proteins. Such isolates contain a heterogeneous oligosaccharide mixture of different chain lengths and frame shifts. Access to defined synthetic S. pneumoniae CPS structures is desirable. Known syntheses of S. pneumoniae serotype 3 CPS rely on a time-consuming and low-yielding late-stage oxidation step, or use disaccharide building blocks which limits variability. Herein, we report the first iterative automated glycan assembly (AGA of a conjugation-ready S. pneumoniae serotype 3 CPS trisaccharide. This oligosaccharide was assembled using a novel glucuronic acid building block to circumvent the need for a late-stage oxidation. The introduction of a washing step with the activator prior to each glycosylation cycle greatly increased the yields by neutralizing any residual base from deprotection steps in the synthetic cycle. This process improvement is applicable to AGA of many other oligosaccharides.

  3. Rnnotator: an automated de novo transcriptome assembly pipeline from stranded RNA-Seq reads

    Energy Technology Data Exchange (ETDEWEB)

    Martin, Jeffrey; Bruno, Vincent M.; Fang, Zhide; Meng, Xiandong; Blow, Matthew; Zhang, Tao; Sherlock, Gavin; Snyder, Michael; Wang, Zhong

    2010-11-19

    Background: Comprehensive annotation and quantification of transcriptomes are outstanding problems in functional genomics. While high throughput mRNA sequencing (RNA-Seq) has emerged as a powerful tool for addressing these problems, its success is dependent upon the availability and quality of reference genome sequences, thus limiting the organisms to which it can be applied. Results: Here, we describe Rnnotator, an automated software pipeline that generates transcript models by de novo assembly of RNA-Seq data without the need for a reference genome. We have applied the Rnnotator assembly pipeline to two yeast transcriptomes and compared the results to the reference gene catalogs of these organisms. The contigs produced by Rnnotator are highly accurate (95percent) and reconstruct full-length genes for the majority of the existing gene models (54.3percent). Furthermore, our analyses revealed many novel transcribed regions that are absent from well annotated genomes, suggesting Rnnotator serves as a complementary approach to analysis based on a reference genome for comprehensive transcriptomics. Conclusions: These results demonstrate that the Rnnotator pipeline is able to reconstruct full-length transcripts in the absence of a complete reference genome.

  4. Self-Assembly of 3D DNA Crystals Containing a Torsionally Stressed Component.

    Science.gov (United States)

    Hernandez, Carina; Birktoft, Jens J; Ohayon, Yoel P; Chandrasekaran, Arun Richard; Abdallah, Hatem; Sha, Ruojie; Stojanoff, Vivian; Mao, Chengde; Seeman, Nadrian C

    2017-11-16

    There is an increasing appreciation for structural diversity of DNA that is of interest to both DNA nanotechnology and basic biology. Here, we have explored how DNA responds to torsional stress by building on a previously reported two-turn DNA tensegrity triangle and demonstrating that we could introduce an extra nucleotide pair (np) into the original sequence without affecting assembly and crystallization. The extra np imposes a significant torsional stress, which is accommodated by global changes throughout the B-DNA duplex and the DNA lattice. The work reveals a near-atomic structure of naked DNA under a torsional stress of approximately 14%, and thus provides an example of DNA distortions that occur without a requirement for either an external energy source or the free energy available from protein or drug binding. Copyright © 2017 Elsevier Ltd. All rights reserved.

  5. Controlling Self-Assembly Kinetics of DNA-Functionalized Liposomes Using Toehold Exchange Mechanism.

    Science.gov (United States)

    Parolini, Lucia; Kotar, Jurij; Di Michele, Lorenzo; Mognetti, Bortolo M

    2016-02-23

    The selectivity of Watson-Crick base pairing has allowed the design of DNA-based functional materials bearing an unprecedented level of accuracy. Examples include DNA origami, made of tiles assembling into arbitrarily complex shapes, and DNA coated particles featuring rich phase behaviors. Frequently, the realization of conceptual DNA-nanotechnology designs has been hampered by the lack of strategies for effectively controlling relaxations. In this article, we address the problem of kinetic control on DNA-mediated interactions between Brownian objects. We design a kinetic pathway based on toehold-exchange mechanisms that enables rearrangement of DNA bonds without the need for thermal denaturation, and test it on suspensions of DNA-functionalized liposomes, demonstrating tunability of aggregation rates over more than 1 order of magnitude. While the possibility to design complex phase behaviors using DNA as a glue is already well recognized, our results demonstrate control also over the kinetics of such systems.

  6. Adsorption behavior of DNA-wrapped carbon nanotubes on self-assembled monolayer surfaces.

    Science.gov (United States)

    Zangmeister, Rebecca A; Maslar, James E; Opdahl, Aric; Tarlov, Michael J

    2007-05-22

    We have examined the adsorption of DNA-wrapped single-walled carbon nanotubes (DNA-SWNTs) on hydrophobic, hydrophilic, and charged surfaces of alkylthiol self-assembled monolayers (SAMs) on gold. Our goal is to understand how DNA-SWNTs interact with surfaces of varying chemical functionality. These samples were characterized using reflection absorption FTIR (RAIRS), X-ray photoelectron spectroscopy (XPS), and Raman spectroscopy. We have found that DNA-SWNTs preferentially adsorb to positively charged amine-terminated SAMs and to bare gold surfaces versus hydrophobic methyl-terminated or negatively charged carboxylic acid-terminated SAMs. Examination of the adsorption on gold of single-strand DNA (ssDNA) of the same sequence used to wrap the SWNTs suggests that the DNA wrapping plays a role in the adsorption behavior of DNA-SWNTs.

  7. Automated extraction of DNA and PCR setup using a Tecan Freedom EVO® liquid handler

    DEFF Research Database (Denmark)

    Frøslev, Tobias Guldberg; Hansen, Anders Johannes; Stangegaard, Michael

    2009-01-01

    We have implemented and validated automated protocols for DNA extraction and PCR setup using a Tecan Freedom EVO® liquid handler mounted with the TeMagS magnetic separation device. The methods were validated for accredited, forensic genetic work according to ISO 17025 using the Qiagen Mag...

  8. Self-assembling protein arrays on DNA chips by auto-labeling fusion proteins with a single DNA address

    NARCIS (Netherlands)

    Jongsma, M.A.; Litjens, R.H.G.M.

    2006-01-01

    The high-throughput deposition of recombinant proteins on chips, beads or biosensor devices would be greatly facilitated by the implementation of self-assembly concepts. DNA-directed immobilization via conjugation of proteins to an oligonucleotide would be preeminently suited for this purpose. Here,

  9. DNA self-assembly-driven positioning of molecular components on nanopatterned surfaces

    Science.gov (United States)

    Szymonik, M.; Davies, A. G.; Wälti, C.

    2016-09-01

    We present a method for the specific, spatially targeted attachment of DNA molecules to lithographically patterned gold surfaces—demonstrated by bridging DNA strands across nanogap electrode structures. An alkanethiol self-assembled monolayer was employed as a molecular resist, which could be selectively removed via electrochemical desorption, allowing the binding of thiolated DNA anchoring oligonucleotides to each electrode. After introducing a bridging DNA molecule with single-stranded ends complementary to the electrode-tethered anchoring oligonucleotides, the positioning of the DNA molecule across the electrode gap, driven by self-assembly, occurred autonomously. This demonstrates control of molecule positioning with resolution limited only by the underlying patterned structure, does not require any alignment, is carried out entirely under biologically compatible conditions, and is scalable.

  10. Coupling mechanisms between nucleosome assembly and the cellular response to DNA damage

    International Nuclear Information System (INIS)

    Lautrette, Aurelie

    2006-01-01

    Cells are continuously exposed to genotoxic stresses that induce a variety of DNA lesions. To protect their genome, cells have specific pathways that orchestrate the detection, signaling and repair of DNA damages. This work is dedicated to the characterization of such pathways that couple the DNA damage response to the assembly of chromatin, a complex that protects and regulates DNA accessibility. We have focused our study on two multifunctional proteins: Rad53, a central checkpoint kinase in the cellular response to DNA damage and Asf1, a histone chaperone involved in chromatin assembly. We have characterized in vitro the binding mode of Asf1 with Rad53 and Asfl with histones. This study is associated with the functional analysis of the role of these interactions in vivo in yeast cells. (author) [fr

  11. An automated annotation tool for genomic DNA sequences using ...

    Indian Academy of Sciences (India)

    Unknown

    , New Delhi 110 067, India. Abstract ... analysis of genomic DNA to identify coding sequences using the GeneScan algorithm and characterize these resultant sequences by .... genes for the TCA cycle, while in mitochondria only a subset of the ...

  12. Mic60/Mitofilin determines MICOS assembly essential for mitochondrial dynamics and mtDNA nucleoid organization.

    Science.gov (United States)

    Li, H; Ruan, Y; Zhang, K; Jian, F; Hu, C; Miao, L; Gong, L; Sun, L; Zhang, X; Chen, S; Chen, H; Liu, D; Song, Z

    2016-03-01

    The MICOS complex (mitochondrial contact site and cristae organizing system) is essential for mitochondrial inner membrane organization and mitochondrial membrane contacts, however, the molecular regulation of MICOS assembly and the physiological functions of MICOS in mammals remain obscure. Here, we report that Mic60/Mitofilin has a critical role in the MICOS assembly, which determines the mitochondrial morphology and mitochondrial DNA (mtDNA) organization. The downregulation of Mic60/Mitofilin or Mic19/CHCHD3 results in instability of other MICOS components, disassembly of MICOS complex and disorganized mitochondrial cristae. We show that there exists direct interaction between Mic60/Mitofilin and Mic19/CHCHD3, which is crucial for their stabilization in mammals. Importantly, we identified that the mitochondrial i-AAA protease Yme1L regulates Mic60/Mitofilin homeostasis. Impaired MICOS assembly causes the formation of 'giant mitochondria' because of dysregulated mitochondrial fusion and fission. Also, mtDNA nucleoids are disorganized and clustered in these giant mitochondria in which mtDNA transcription is attenuated because of remarkable downregulation of some key mtDNA nucleoid-associated proteins. Together, these findings demonstrate that Mic60/Mitofilin homeostasis regulated by Yme1L is central to the MICOS assembly, which is required for maintenance of mitochondrial morphology and organization of mtDNA nucleoids.

  13. Automated Device for Asynchronous Extraction of RNA, DNA, or Protein Biomarkers from Surrogate Patient Samples.

    Science.gov (United States)

    Bitting, Anna L; Bordelon, Hali; Baglia, Mark L; Davis, Keersten M; Creecy, Amy E; Short, Philip A; Albert, Laura E; Karhade, Aditya V; Wright, David W; Haselton, Frederick R; Adams, Nicholas M

    2016-12-01

    Many biomarker-based diagnostic methods are inhibited by nontarget molecules in patient samples, necessitating biomarker extraction before detection. We have developed a simple device that purifies RNA, DNA, or protein biomarkers from complex biological samples without robotics or fluid pumping. The device design is based on functionalized magnetic beads, which capture biomarkers and remove background biomolecules by magnetically transferring the beads through processing solutions arrayed within small-diameter tubing. The process was automated by wrapping the tubing around a disc-like cassette and rotating it past a magnet using a programmable motor. This device recovered biomarkers at ~80% of the operator-dependent extraction method published previously. The device was validated by extracting biomarkers from a panel of surrogate patient samples containing clinically relevant concentrations of (1) influenza A RNA in nasal swabs, (2) Escherichia coli DNA in urine, (3) Mycobacterium tuberculosis DNA in sputum, and (4) Plasmodium falciparum protein and DNA in blood. The device successfully extracted each biomarker type from samples representing low levels of clinically relevant infectivity (i.e., 7.3 copies/µL of influenza A RNA, 405 copies/µL of E. coli DNA, 0.22 copies/µL of TB DNA, 167 copies/µL of malaria parasite DNA, and 2.7 pM of malaria parasite protein). © 2015 Society for Laboratory Automation and Screening.

  14. Self-assembly of fully addressable DNA nanostructures from double crossover tiles.

    Science.gov (United States)

    Wang, Wen; Lin, Tong; Zhang, Suoyu; Bai, Tanxi; Mi, Yongli; Wei, Bryan

    2016-09-19

    DNA origami and single-stranded tile (SST) are two proven approaches to self-assemble finite-size complex DNA nanostructures. The construction elements appeared in structures from these two methods can also be found in multi-stranded DNA tiles such as double crossover tiles. Here we report the design and observation of four types of finite-size lattices with four different double crossover tiles, respectively, which, we believe, in terms of both complexity and robustness, will be rival to DNA origami and SST structures. © The Author(s) 2016. Published by Oxford University Press on behalf of Nucleic Acids Research.

  15. DNA self-assembly on graphene surface studied by SERS mapping

    DEFF Research Database (Denmark)

    Botti, Sabina; Rufoloni, Alessandro; Laurenzi, Susanna

    2016-01-01

    The self-assembly of double-stranded DNA (dsDNA) segments on two variations of graphene surfaces having nano-platelets with different lateral sizes and thicknesses was investigated using surface enhanced Raman spectroscopy (SERS) and electrical impedance spectroscopy (EIS) techniques. Due...... nano-platelets. Results from the EIS analysis supported the SERS findings and confirmed that SERS mapping is a reliable method for a rapid monitoring of the procedures used to interface DNA with graphene surfaces. The present study, linking DNA anchoring morphology to the conductive properties of nano...

  16. Gold-nanoparticle-mediated jigsaw-puzzle-like assembly of supersized plasmonic DNA origami.

    Science.gov (United States)

    Yao, Guangbao; Li, Jiang; Chao, Jie; Pei, Hao; Liu, Huajie; Zhao, Yun; Shi, Jiye; Huang, Qing; Wang, Lianhui; Huang, Wei; Fan, Chunhai

    2015-03-02

    DNA origami has rapidly emerged as a powerful and programmable method to construct functional nanostructures. However, the size limitation of approximately 100 nm in classic DNA origami hampers its plasmonic applications. Herein, we report a jigsaw-puzzle-like assembly strategy mediated by gold nanoparticles (AuNPs) to break the size limitation of DNA origami. We demonstrated that oligonucleotide-functionalized AuNPs function as universal joint units for the one-pot assembly of parent DNA origami of triangular shape to form sub-microscale super-origami nanostructures. AuNPs anchored at predefined positions of the super-origami exhibited strong interparticle plasmonic coupling. This AuNP-mediated strategy offers new opportunities to drive macroscopic self-assembly and to fabricate well-defined nanophotonic materials and devices. © 2015 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

  17. Optimizing DNA assembly based on statistical language modelling.

    Science.gov (United States)

    Fang, Gang; Zhang, Shemin; Dong, Yafei

    2017-12-15

    By successively assembling genetic parts such as BioBrick according to grammatical models, complex genetic constructs composed of dozens of functional blocks can be built. However, usually every category of genetic parts includes a few or many parts. With increasing quantity of genetic parts, the process of assembling more than a few sets of these parts can be expensive, time consuming and error prone. At the last step of assembling it is somewhat difficult to decide which part should be selected. Based on statistical language model, which is a probability distribution P(s) over strings S that attempts to reflect how frequently a string S occurs as a sentence, the most commonly used parts will be selected. Then, a dynamic programming algorithm was designed to figure out the solution of maximum probability. The algorithm optimizes the results of a genetic design based on a grammatical model and finds an optimal solution. In this way, redundant operations can be reduced and the time and cost required for conducting biological experiments can be minimized. © The Author(s) 2017. Published by Oxford University Press on behalf of Nucleic Acids Research.

  18. Stepwise Assembly and Characterization of DNA Linked Two-Color Quantum Dot Clusters.

    Science.gov (United States)

    Coopersmith, Kaitlin; Han, Hyunjoo; Maye, Mathew M

    2015-07-14

    The DNA-mediated self-assembly of multicolor quantum dot (QD) clusters via a stepwise approach is described. The CdSe/ZnS QDs were synthesized and functionalized with an amphiphilic copolymer, followed by ssDNA conjugation. At each functionalization step, the QDs were purified via gradient ultracentrifugation, which was found to remove excess polymer and QD aggregates, allowing for improved conjugation yields and assembly reactivity. The QDs were then assembled and disassembled in a stepwise manner at a ssDNA functionalized magnetic colloid, which provided a convenient way to remove unreacted QDs and ssDNA impurities. After assembly/disassembly, the clusters' optical characteristics were studied by fluorescence spectroscopy and the assembly morphology and stoichiometry was imaged via electron microscopy. The results indicate that a significant amount of QD-to-QD energy transfer occurred in the clusters, which was studied as a function of increasing acceptor-to-donor ratios, resulting in increased QD acceptor emission intensities compared to controls.

  19. Understanding the Elementary Steps in DNA Tile-Based Self-Assembly.

    Science.gov (United States)

    Jiang, Shuoxing; Hong, Fan; Hu, Huiyu; Yan, Hao; Liu, Yan

    2017-09-26

    Although many models have been developed to guide the design and implementation of DNA tile-based self-assembly systems with increasing complexity, the fundamental assumptions of the models have not been thoroughly tested. To expand the quantitative understanding of DNA tile-based self-assembly and to test the fundamental assumptions of self-assembly models, we investigated DNA tile attachment to preformed "multi-tile" arrays in real time and obtained the thermodynamic and kinetic parameters of single tile attachment in various sticky end association scenarios. With more sticky ends, tile attachment becomes more thermostable with an approximately linear decrease in the free energy change (more negative). The total binding free energy of sticky ends is partially compromised by a sequence-independent energy penalty when tile attachment forms a constrained configuration: "loop". The minimal loop is a 2 × 2 tetramer (Loop4). The energy penalty of loops of 4, 6, and 8 tiles was analyzed with the independent loop model assuming no interloop tension, which is generalizable to arbitrary tile configurations. More sticky ends also contribute to a faster on-rate under isothermal conditions when nucleation is the rate-limiting step. Incorrect sticky end contributes to neither the thermostability nor the kinetics. The thermodynamic and kinetic parameters of DNA tile attachment elucidated here will contribute to the future improvement and optimization of tile assembly modeling, precise control of experimental conditions, and structural design for error-free self-assembly.

  20. Evaluation of an automated protocol for efficient and reliable DNA extraction of dietary samples.

    Science.gov (United States)

    Wallinger, Corinna; Staudacher, Karin; Sint, Daniela; Thalinger, Bettina; Oehm, Johannes; Juen, Anita; Traugott, Michael

    2017-08-01

    Molecular techniques have become an important tool to empirically assess feeding interactions. The increased usage of next-generation sequencing approaches has stressed the need of fast DNA extraction that does not compromise DNA quality. Dietary samples here pose a particular challenge, as these demand high-quality DNA extraction procedures for obtaining the minute quantities of short-fragmented food DNA. Automatic high-throughput procedures significantly decrease time and costs and allow for standardization of extracting total DNA. However, these approaches have not yet been evaluated for dietary samples. We tested the efficiency of an automatic DNA extraction platform and a traditional CTAB protocol, employing a variety of dietary samples including invertebrate whole-body extracts as well as invertebrate and vertebrate gut content samples and feces. Extraction efficacy was quantified using the proportions of successful PCR amplifications of both total and prey DNA, and cost was estimated in terms of time and material expense. For extraction of total DNA, the automated platform performed better for both invertebrate and vertebrate samples. This was also true for prey detection in vertebrate samples. For the dietary analysis in invertebrates, there is still room for improvement when using the high-throughput system for optimal DNA yields. Overall, the automated DNA extraction system turned out as a promising alternative to labor-intensive, low-throughput manual extraction methods such as CTAB. It is opening up the opportunity for an extensive use of this cost-efficient and innovative methodology at low contamination risk also in trophic ecology.

  1. Designing and Implementing Algorithmic DNA Assembly Pipelines for Multi-Gene Systems.

    Science.gov (United States)

    Hsu, Szu-Yi; Smanski, Michael J

    2018-01-01

    Advances in DNA synthesis and assembly technology allow for the high-throughput fabrication of hundreds to thousands of multi-part genetic constructs in a short time. This allows for rapid hypothesis-testing and genetic optimization in multi-gene biological systems. Here, we discuss key considerations to design and implement an algorithmic DNA assembly pipeline that provides the freedom to change nearly any design variable in a multi-gene system. In addition to considerations for pipeline design, we describe protocols for three useful molecular biology techniques in plasmid construction.

  2. Parallel Assisted Assembly of Multilayer DNA and Protein Nanoparticle Structures Using a CMOS Electronic Array

    Science.gov (United States)

    Heller, Michael J.; Dehlinger, Dietrich A.; Sullivan, Benjamin D.

    2006-09-01

    A CMOS electronic microarray device was used to carry out the rapid parallel assembly of functionalized nanoparticles into multilayer structures. Electronic microarrays produce reconfigurable DC electric fields that allow DNA, proteins as well as charged molecules to be rapidly transported from the bulk solution and addressed to specifically activated sites on the array surface. Such a device was used to carry out the assisted self-assembly DNA, biotin and streptavidin derivatized fluorescent nanoparticles into multilayer structures. Nanoparticle addressing could be carried out in about 15 seconds, and forty depositions of nanoparticles were completed in less than one hour. The final multilayered 3D nanostructures were verified by scanning electron microscopy.

  3. GenePublisher: automated analysis of DNA microarray data

    DEFF Research Database (Denmark)

    Knudsen, Steen; Workman, Christopher; Sicheritz-Ponten, T.

    2003-01-01

    , statistical analysis and visualization of the data. The results are run against databases of signal transduction pathways, metabolic pathways and promoter sequences in order to extract more information. The results of the entire analysis are summarized in report form and returned to the user.......GenePublisher, a system for automatic analysis of data from DNA microarray experiments, has been implemented with a web interface at http://www.cbs.dtu.dk/services/GenePublisher. Raw data are uploaded to the server together with aspecification of the data. The server performs normalization...

  4. RADOM, an efficient in vivo method for assembling designed DNA fragments up to 10 kb long in Saccharomyces cerevisiae.

    Science.gov (United States)

    Lin, Qiuhui; Jia, Bin; Mitchell, Leslie A; Luo, Jingchuan; Yang, Kun; Zeller, Karen I; Zhang, Wenqian; Xu, Zhuwei; Stracquadanio, Giovanni; Bader, Joel S; Boeke, Jef D; Yuan, Ying-Jin

    2015-03-20

    We describe rapid assembly of DNA overlapping multifragments (RADOM), an improved assembly method via homologous recombination in Saccharomyces cerevisiae, which combines assembly in yeasto with blue/white screening in Escherichia coli. We show that RADOM can successfully assemble ∼3 and ∼10 kb DNA fragments that are highly similar to the yeast genome rapidly and accurately. This method was tested in the Build-A-Genome course by undergraduate students, where 125 ∼3 kb "minichunks" from the synthetic yeast genome project Sc2.0 were assembled. Here, 122 out of 125 minichunks achieved insertions with correct sizes, and 102 minichunks were sequenced verified. As this method reduces the time-consuming and labor-intensive efforts of yeast assembly by improving the screening efficiency for correct assemblies, it may find routine applications in the construction of DNA fragments, especially in hierarchical assembly projects.

  5. Recruitment, assembly, and molecular architecture of the SpoIIIE DNA pump revealed by superresolution microscopy.

    Directory of Open Access Journals (Sweden)

    Jean-Bernard Fiche

    Full Text Available ATP-fuelled molecular motors are responsible for rapid and specific transfer of double-stranded DNA during several fundamental processes, such as cell division, sporulation, bacterial conjugation, and viral DNA transport. A dramatic example of intercompartmental DNA transfer occurs during sporulation in Bacillus subtilis, in which two-thirds of a chromosome is transported across a division septum by the SpoIIIE ATPase. Here, we use photo-activated localization microscopy, structured illumination microscopy, and fluorescence fluctuation microscopy to investigate the mechanism of recruitment and assembly of the SpoIIIE pump and the molecular architecture of the DNA translocation complex. We find that SpoIIIE assembles into ∼45 nm complexes that are recruited to nascent sites of septation, and are subsequently escorted by the constriction machinery to the center of sporulation and division septa. SpoIIIE complexes contain 47±20 SpoIIIE molecules, a majority of which are assembled into hexamers. Finally, we show that directional DNA translocation leads to the establishment of a compartment-specific, asymmetric complex that exports DNA. Our data are inconsistent with the notion that SpoIIIE forms paired DNA conducting channels across fused membranes. Rather, our results support a model in which DNA translocation occurs through an aqueous DNA-conducting pore that could be structurally maintained by the divisional machinery, with SpoIIIE acting as a checkpoint preventing membrane fusion until completion of chromosome segregation. Our findings and proposed mechanism, and our unique combination of innovating methodologies, are relevant to the understanding of bacterial cell division, and may illuminate the mechanisms of other complex machineries involved in DNA conjugation and protein transport across membranes.

  6. Evidence of impurities in thiolated single-stranded DNA oligomers and their effect on DNA self-assembly on gold.

    Science.gov (United States)

    Lee, Chi-Ying; Canavan, Heather E; Gamble, Lara J; Castner, David G

    2005-05-24

    The diversity of techniques used in the synthesis, treatment, and purification of the single-stranded DNA oligomers containing a thiol anchor group (SH-ssDNA) has led to a significant variation in the purity of commercially available SH-ssDNA. In this work, we use X-ray photoelectron spectroscopy (XPS) and time-of-flight secondary ion mass spectrometry (ToF-SIMS) to study how the impurities present in commercially synthesized SH-ssDNA oligomers affected the structure of the resulting DNA films on Au. XPS results indicate that two of the purchased SH-ssDNA oligomers contain excess carbon and sulfur. The molecular fragmentation patterns obtained with ToF-SIMS were used to determine the identity of several contaminants in the DNA films, including poly(dimethylsiloxane) (PDMS), lipid molecules, and sulfur-containing molecules. In particular, the ToF-SIMS results determined that the excess sulfur detected by XPS was due to the presence of dithiothreitol, a reductant often used to cleave disulfide precursors. Furthermore, we found that the SH-ssDNA self-assembly process is affected by the presence of these contaminants. When relatively pure SH-ssDNA is used to prepare the DNA films, the P, N, O, and C atomic percentages were observed by XPS to increase over a 24-h time period. In contrast, surfaces prepared using SH-ssDNA containing higher levels of contaminants did not follow this trend. XPS result indicates that, after the initial SH-ssDNA adsorption, the remaining material incorporated into these films was due to contamination.

  7. Building superlattices from individual nanoparticles via template-confined DNA-mediated assembly

    Science.gov (United States)

    Lin, Qing-Yuan; Mason, Jarad A.; Li, Zhongyang; Zhou, Wenjie; O’Brien, Matthew N.; Brown, Keith A.; Jones, Matthew R.; Butun, Serkan; Lee, Byeongdu; Dravid, Vinayak P.; Aydin, Koray; Mirkin, Chad A.

    2018-02-01

    A polymer pore template can control the order of assembly of nanoparticles into well-defined stacks and create superlattices. Lin et al. used DNA strands on gold nanoparticles to control interparticle distance. The DNA strands contained modified adenines with more rigid ribose groups that formed stronger base pairs. The height of the stacks of three different types of gold nanoparticle could be changed with different solvents, which in turn changed their optical response.

  8. Simple cloning and DNA assembly in Escherichia coli by prolonged overlap extension PCR.

    Science.gov (United States)

    You, Chun; Zhang, Y-H Percival

    2014-01-01

    We developed a simple method (Simple Cloning) for subcloning one, two, or three DNA fragments into any location of a targeted vector without the need for restriction enzyme, ligase, exonuclease, or recombinase. This cloning technology can be applied to a few common Escherichia coli hosts (e.g., BL21(DE3), DH5α, JM109, TOP10). The protocol includes three steps: (a) linear DNA fragments (i.e., the insert DNA and the vector backbone) with two overlap ends were generated by regular high-fidelity PCR, (b) the DNA multimers were generated based on these equimolar DNA templates by using prolonged overlap extension PCR (POE-PCR) without primers added, and (c) the POE-PCR product was transformed to E. coli strains directly. Because positive colony efficiencies are very high, it is not necessary to identify desired clones by using colony PCR. Simple Cloning provides a new cloning and DNA assembly method with great simplicity and flexibility.

  9. Dynamic protein assembly by programmable DNA strand displacement

    Science.gov (United States)

    Chen, Rebecca P.; Blackstock, Daniel; Sun, Qing; Chen, Wilfred

    2018-03-01

    Inspired by the remarkable ability of natural protein switches to sense and respond to a wide range of environmental queues, here we report a strategy to engineer synthetic protein switches by using DNA strand displacement to dynamically organize proteins with highly diverse and complex logic gate architectures. We show that DNA strand displacement can be used to dynamically control the spatial proximity and the corresponding fluorescence resonance energy transfer between two fluorescent proteins. Performing Boolean logic operations enabled the explicit control of protein proximity using multi-input, reversible and amplification architectures. We further demonstrate the power of this technology beyond sensing by achieving dynamic control of an enzyme cascade. Finally, we establish the utility of the approach as a synthetic computing platform that drives the dynamic reconstitution of a split enzyme for targeted prodrug activation based on the sensing of cancer-specific miRNAs.

  10. Automated array assembly. Phase II. Final report, October 1977-December 1979

    Energy Technology Data Exchange (ETDEWEB)

    D' Aiello, R. V.

    1980-10-01

    The philosophy of this project was to establish an experimental process line starting with 3-in.-diameter silicon wafers and consisting of junction formation using POCl/sub 3/ gaseous diffusion, screen-printed thick-film metallization, reflow solder interconnect, and double-glass lamination panel assembly. This experimental production line produced a sufficient number of solar cells to demonstrate the technological readiness of each of those process steps. Variations (of each process) were made to set limits on the usable range of each process step and to determine the interaction with adjoining steps. Inspections, measurements, and tests were included to determine the output requirement characteristics of each step, obtain statistical variations, and evaluate the performance of the solar cells and panels. A description of this work, which was conducted from October 1977 through December 1978, is given. This was followed by an 18-month study in which three manufacturing sequences synthesized from the previous work and from studies conducted by other participants in the LSA program were exercised. The objectives were to assess the compatibility between process steps for each sequence, to generate sufficient data for comparative SAMICS cost analysis, and to make recommendations of the suitability of one or more of these sequences for the large-scale automated production of solar cells within the cost goal of $0.70/pW. The detailed experimental results of this study are described, followed by SAMICS cost analysis, recommendations, and conclusions.

  11. Assembly of positioner of automated two-dimensional scan coupled to X-ray fluorescence spectrometry

    International Nuclear Information System (INIS)

    Silva, Leonardo Santiago Melgaço

    2011-01-01

    This work describes the design and assembling of a prototype automated positioner two-dimensional scanning coupled to X-ray fluorescence spectrometry. The work aims to achieve a portable and easy to use, device of broad utility in the analysis of samples by X-ray fluorescence area of expertise and research. The two-dimensional scanning of the positioner is by means of two stepper motors controlled by a microcontroller PIC 16F877A, encoder and optical sensors. The user interacts with the XY table through an interface program for the Windows operating system, which communicates with the microcontroller through the serial port. The system of Fluorescence Spectroscopy incorporated into the positioner consists of a system commercially available system from the company AMPTEK, where the primary source of excitation of the sample was a source of 241 Am of 59.5 KeV emissions. Resolution and accuracy of tests were performed in the XY scanning process and reproducibility of the same kit with the fluorescence spectrometry X-ray. Qualitative tests by X-ray fluorescence spectrometry in samples were performed to demonstrate the applicability and versatility of the project. It follows that the prototype illustrates a possible adequately to portable device for X-ray spectrometry of two-dimensional. (author)

  12. Structurally Ordered Nanowire Formation from Co-Assembly of DNA Origami and Collagen-Mimetic Peptides

    Energy Technology Data Exchange (ETDEWEB)

    Jiang, Tao [Department; Meyer, Travis A. [Wallace; Modlin, Charles [Department; Zuo, Xiaobing [X-ray; Conticello, Vincent P. [Department; Ke, Yonggang [Wallace

    2017-10-02

    We describe the co-assembly of two different building units: collagen-mimetic peptides and DNA origami. Two peptides CP++ and sCP(++) are designed with a sequence comprising a central block (Pro-Hyp-Gly) and two positively charged domains (Pro-Arg-Gly) at both N- and C-termini. Co-assembly of peptides and DNA origami two-layer (TL) nanosheets affords the formation of one-dimensional nanowires with repeating periodicity of similar to 10 nm. Structural analyses suggest a face-to-face stacking of DNA nanosheets with peptides aligned perpendicularly to the sheet surfaces. We demonstrate the potential of selective peptide-DNA association between face-to-face and edge-to-edge packing by tailoring the size of DNA nanostructures. This study presents an attractive strategy to create hybrid biomolecular assemblies from peptide and DNA-based building blocks that takes advantage of the intrinsic chemical and physical properties of the respective components to encode structural and, potentially, functional complexity within readily accessible biomimetic materials.

  13. Norgal: extraction and de novo assembly of mitochondrial DNA from whole-genome sequencing data.

    Science.gov (United States)

    Al-Nakeeb, Kosai; Petersen, Thomas Nordahl; Sicheritz-Pontén, Thomas

    2017-11-21

    Whole-genome sequencing (WGS) projects provide short read nucleotide sequences from nuclear and possibly organelle DNA depending on the source of origin. Mitochondrial DNA is present in animals and fungi, while plants contain DNA from both mitochondria and chloroplasts. Current techniques for separating organelle reads from nuclear reads in WGS data require full reference or partial seed sequences for assembling. Norgal (de Novo ORGAneLle extractor) avoids this requirement by identifying a high frequency subset of k-mers that are predominantly of mitochondrial origin and performing a de novo assembly on a subset of reads that contains these k-mers. The method was applied to WGS data from a panda, brown algae seaweed, butterfly and filamentous fungus. We were able to extract full circular mitochondrial genomes and obtained sequence identities to the reference sequences in the range from 98.5 to 99.5%. We also assembled the chloroplasts of grape vines and cucumbers using Norgal together with seed-based de novo assemblers. Norgal is a pipeline that can extract and assemble full or partial mitochondrial and chloroplast genomes from WGS short reads without prior knowledge. The program is available at: https://bitbucket.org/kosaidtu/norgal .

  14. Toward reliable algorithmic self-assembly of DNA tiles: a fixed-width cellular automaton pattern.

    Science.gov (United States)

    Fujibayashi, Kenichi; Hariadi, Rizal; Park, Sung Ha; Winfree, Erik; Murata, Satoshi

    2008-07-01

    Bottom-up fabrication of nanoscale structures relies on chemical processes to direct self-assembly. The complexity, precision, and yield achievable by a one-pot reaction are limited by our ability to encode assembly instructions into the molecules themselves. Nucleic acids provide a platform for investigating these issues, as molecular structure and intramolecular interactions can encode growth rules. Here, we use DNA tiles and DNA origami to grow crystals containing a cellular automaton pattern. In a one-pot annealing reaction, 250 DNA strands first assemble into a set of 10 free tile types and a seed structure, then the free tiles grow algorithmically from the seed according to the automaton rules. In our experiments, crystals grew to approximately 300 nm long, containing approximately 300 tiles with an initial assembly error rate of approximately 1.4% per tile. This work provides evidence that programmable molecular self-assembly may be sufficient to create a wide range of complex objects in one-pot reactions.

  15. One-pot DNA construction for synthetic biology: the Modular Overlap-Directed Assembly with Linkers (MODAL) strategy

    Science.gov (United States)

    Casini, Arturo; MacDonald, James T.; Jonghe, Joachim De; Christodoulou, Georgia; Freemont, Paul S.; Baldwin, Geoff S.; Ellis, Tom

    2014-01-01

    Overlap-directed DNA assembly methods allow multiple DNA parts to be assembled together in one reaction. These methods, which rely on sequence homology between the ends of DNA parts, have become widely adopted in synthetic biology, despite being incompatible with a key principle of engineering: modularity. To answer this, we present MODAL: a Modular Overlap-Directed Assembly with Linkers strategy that brings modularity to overlap-directed methods, allowing assembly of an initial set of DNA parts into a variety of arrangements in one-pot reactions. MODAL is accompanied by a custom software tool that designs overlap linkers to guide assembly, allowing parts to be assembled in any specified order and orientation. The in silico design of synthetic orthogonal overlapping junctions allows for much greater efficiency in DNA assembly for a variety of different methods compared with using non-designed sequence. In tests with three different assembly technologies, the MODAL strategy gives assembly of both yeast and bacterial plasmids, composed of up to five DNA parts in the kilobase range with efficiencies of between 75 and 100%. It also seamlessly allows mutagenesis to be performed on any specified DNA parts during the process, allowing the one-step creation of construct libraries valuable for synthetic biology applications. PMID:24153110

  16. Self-assembly of complex two-dimensional shapes from single-stranded DNA tiles.

    Science.gov (United States)

    Wei, Bryan; Vhudzijena, Michelle K; Robaszewski, Joanna; Yin, Peng

    2015-05-08

    Current methods in DNA nano-architecture have successfully engineered a variety of 2D and 3D structures using principles of self-assembly. In this article, we describe detailed protocols on how to fabricate sophisticated 2D shapes through the self-assembly of uniquely addressable single-stranded DNA tiles which act as molecular pixels on a molecular canvas. Each single-stranded tile (SST) is a 42-nucleotide DNA strand composed of four concatenated modular domains which bind to four neighbors during self-assembly. The molecular canvas is a rectangle structure self-assembled from SSTs. A prescribed complex 2D shape is formed by selecting the constituent molecular pixels (SSTs) from a 310-pixel molecular canvas and then subjecting the corresponding strands to one-pot annealing. Due to the modular nature of the SST approach we demonstrate the scalability, versatility and robustness of this method. Compared with alternative methods, the SST method enables a wider selection of information polymers and sequences through the use of de novo designed and synthesized short DNA strands.

  17. Engineered Diblock Polypeptides Improve DNA and Gold Solubility during Molecular Assembly

    NARCIS (Netherlands)

    Estrich, Nicole A.; Hernandez-Garcia, Armando; Vries, De Renko; LaBean, Thomas H.

    2017-01-01

    Programmed molecular recognition is being developed for the bionanofabrication of mixed organic/inorganic supramolecular assemblies for applications in electronics, photonics, and medicine. For example, DNA-based nanotechnology seeks to exploit the easily programmed complementary base-pairing of

  18. Semi-automated library preparation for high-throughput DNA sequencing platforms.

    Science.gov (United States)

    Farias-Hesson, Eveline; Erikson, Jonathan; Atkins, Alexander; Shen, Peidong; Davis, Ronald W; Scharfe, Curt; Pourmand, Nader

    2010-01-01

    Next-generation sequencing platforms are powerful technologies, providing gigabases of genetic information in a single run. An important prerequisite for high-throughput DNA sequencing is the development of robust and cost-effective preprocessing protocols for DNA sample library construction. Here we report the development of a semi-automated sample preparation protocol to produce adaptor-ligated fragment libraries. Using a liquid-handling robot in conjunction with Carboxy Terminated Magnetic Beads, we labeled each library sample using a unique 6 bp DNA barcode, which allowed multiplex sample processing and sequencing of 32 libraries in a single run using Applied Biosystems' SOLiD sequencer. We applied our semi-automated pipeline to targeted medical resequencing of nuclear candidate genes in individuals affected by mitochondrial disorders. This novel method is capable of preparing as much as 32 DNA libraries in 2.01 days (8-hour workday) for emulsion PCR/high throughput DNA sequencing, increasing sample preparation production by 8-fold.

  19. Interactions between Cytochrome c and DNA Strands Self-Assembled at Gold Electrode

    Science.gov (United States)

    Lao, Ruojun; Wang, Lihua; Wan, Ying; Zhang, Jiong; Song, Shiping; Zhang, Zhizhou; Fan, Chunhai; He, Lin

    2007-01-01

    In this work, we reported the investigation on the interaction between DNA strands self-assembled at gold electrodes and an electron transfer protein, cytochrome c. We observed that cytochrome c exhibited well-defined electrochemistry in both double-stranded and single-stranded DNA films. This suggested that the electron transfer reaction of cytochrome c arose possibly due to the electron hopping along DNA strands rather than wiring along the double helix. We also compared the heterogeneous electron transfer rate of cytochrome c with that of a ruthenium complex, which further confirmed this mechanism.

  20. Automated serial extraction of DNA and RNA from biobanked tissue specimens

    OpenAIRE

    Mathot, Lucy; Wallin, Monica; Sjöblom, Tobias

    2013-01-01

    Background: With increasing biobanking of biological samples, methods for large scale extraction of nucleic acids are in demand. The lack of such techniques designed for extraction from tissues results in a bottleneck in downstream genetic analyses, particularly in the field of cancer research. We have developed an automated procedure for tissue homogenization and extraction of DNA and RNA into separate fractions from the same frozen tissue specimen. A purpose developed magnetic bead based te...

  1. Automated Solar Cell Assembly Teamed Process Research. Final subcontract report, 6 January 1993--31 October 1995

    Energy Technology Data Exchange (ETDEWEB)

    Nowlan, M. J.; Hogan, S. J.; Breen, W. F.; Murach, J. M.; Sutherland, S. F.; Patterson, J. S.; Darkazalli, G. [Spire Corp., Bedford, MA (US)

    1996-02-01

    This is the Final Technical Report for a program entitled ''Automated Solar Cell Assembly Teamed Process Research,'' funded by the US Department of Energy. This program was part of Phase 3A of the Photovoltaic Manufacturing Technology (PVMaT) project, which addressed the generic needs of the photovoltaic (PV) industry for improved quality, accelerated production scale-up, and substantially reduced manufacturing cost. Crystalline silicon solar cells (Czochralski monocrystalline, cast polycrystalline, and ribbon polycrystalline) are used in the great majority of PV modules produced in the US, accounting for 95% of all shipments in 1994. Spire's goal in this program was to reduce the cost of these modules by developing high throughput (5 MW per year) automated processes for interconnecting solar cells made from standard and thin silicon wafers. Spire achieved this goal by developing a completely new automated processing system, designated the SPI-ASSEMBLER{trademark} 5000, which is now offered as a commercial product to the PV industry. A discussion of the project and of the Assembler is provided.

  2. An optimized routing algorithm for the automated assembly of standard multimode ribbon fibers in a full-mesh optical backplane

    Science.gov (United States)

    Basile, Vito; Guadagno, Gianluca; Ferrario, Maddalena; Fassi, Irene

    2018-03-01

    In this paper a parametric, modular and scalable algorithm allowing a fully automated assembly of a backplane fiber-optic interconnection circuit is presented. This approach guarantees the optimization of the optical fiber routing inside the backplane with respect to specific criteria (i.e. bending power losses), addressing both transmission performance and overall costs issues. Graph theory has been exploited to simplify the complexity of the NxN full-mesh backplane interconnection topology, firstly, into N independent sub-circuits and then, recursively, into a limited number of loops easier to be generated. Afterwards, the proposed algorithm selects a set of geometrical and architectural parameters whose optimization allows to identify the optimal fiber optic routing for each sub-circuit of the backplane. The topological and numerical information provided by the algorithm are then exploited to control a robot which performs the automated assembly of the backplane sub-circuits. The proposed routing algorithm can be extended to any array architecture and number of connections thanks to its modularity and scalability. Finally, the algorithm has been exploited for the automated assembly of an 8x8 optical backplane realized with standard multimode (MM) 12-fiber ribbons.

  3. Complex shapes self-assembled from single-stranded DNA tiles.

    Science.gov (United States)

    Wei, Bryan; Dai, Mingjie; Yin, Peng

    2012-05-30

    Programmed self-assembly of strands of nucleic acid has proved highly effective for creating a wide range of structures with desired shapes. A particularly successful implementation is DNA origami, in which a long scaffold strand is folded by hundreds of short auxiliary strands into a complex shape. Modular strategies are in principle simpler and more versatile and have been used to assemble DNA or RNA tiles into periodic and algorithmic two-dimensional lattices, extended ribbons and tubes, three-dimensional crystals, polyhedra and simple finite two-dimensional shapes. But creating finite yet complex shapes from a large number of uniquely addressable tiles remains challenging. Here we solve this problem with the simplest tile form, a 'single-stranded tile' (SST) that consists of a 42-base strand of DNA composed entirely of concatenated sticky ends and that binds to four local neighbours during self-assembly. Although ribbons and tubes with controlled circumferences have been created using the SST approach, we extend it to assemble complex two-dimensional shapes and tubes from hundreds (in some cases more than one thousand) distinct tiles. Our main design feature is a self-assembled rectangle that serves as a molecular canvas, with each of its constituent SST strands--folded into a 3 nm-by-7 nm tile and attached to four neighbouring tiles--acting as a pixel. A desired shape, drawn on the canvas, is then produced by one-pot annealing of all those strands that correspond to pixels covered by the target shape; the remaining strands are excluded. We implement the strategy with a master strand collection that corresponds to a 310-pixel canvas, and then use appropriate strand subsets to construct 107 distinct and complex two-dimensional shapes, thereby establishing SST assembly as a simple, modular and robust framework for constructing nanostructures with prescribed shapes from short synthetic DNA strands.

  4. Programming Self-Assembly of DNA Origami Honeycomb Two-Dimensional Lattices and Plasmonic Metamaterials.

    Science.gov (United States)

    Wang, Pengfei; Gaitanaros, Stavros; Lee, Seungwoo; Bathe, Mark; Shih, William M; Ke, Yonggang

    2016-06-22

    Scaffolded DNA origami has proven to be a versatile method for generating functional nanostructures with prescribed sub-100 nm shapes. Programming DNA-origami tiles to form large-scale 2D lattices that span hundreds of nanometers to the micrometer scale could provide an enabling platform for diverse applications ranging from metamaterials to surface-based biophysical assays. Toward this end, here we design a family of hexagonal DNA-origami tiles using computer-aided design and demonstrate successful self-assembly of micrometer-scale 2D honeycomb lattices and tubes by controlling their geometric and mechanical properties including their interconnecting strands. Our results offer insight into programmed self-assembly of low-defect supra-molecular DNA-origami 2D lattices and tubes. In addition, we demonstrate that these DNA-origami hexagon tiles and honeycomb lattices are versatile platforms for assembling optical metamaterials via programmable spatial arrangement of gold nanoparticles (AuNPs) into cluster and superlattice geometries.

  5. Crystal structure of Mycobacterium tuberculosis O6-methylguanine-DNA methyltransferase protein clusters assembled on to damaged DNA.

    Science.gov (United States)

    Miggiano, Riccardo; Perugino, Giuseppe; Ciaramella, Maria; Serpe, Mario; Rejman, Dominik; Páv, Ondřej; Pohl, Radek; Garavaglia, Silvia; Lahiri, Samarpita; Rizzi, Menico; Rossi, Franca

    2016-01-15

    Mycobacterium tuberculosis O(6)-methylguanine-DNA methyltransferase (MtOGT) contributes to protect the bacterial GC-rich genome against the pro-mutagenic potential of O(6)-methylated guanine in DNA. Several strains of M. tuberculosis found worldwide encode a point-mutated O(6)-methylguanine-DNA methyltransferase (OGT) variant (MtOGT-R37L), which displays an arginine-to-leucine substitution at position 37 of the poorly functionally characterized N-terminal domain of the protein. Although the impact of this mutation on the MtOGT activity has not yet been proved in vivo, we previously demonstrated that a recombinant MtOGT-R37L variant performs a suboptimal alkylated-DNA repair in vitro, suggesting a direct role for the Arg(37)-bearing region in catalysis. The crystal structure of MtOGT complexed with modified DNA solved in the present study reveals details of the protein-protein and protein-DNA interactions occurring during alkylated-DNA binding, and the protein capability also to host unmodified bases inside the active site, in a fully extrahelical conformation. Our data provide the first experimental picture at the atomic level of a possible mode of assembling three adjacent MtOGT monomers on the same monoalkylated dsDNA molecule, and disclose the conformational flexibility of discrete regions of MtOGT, including the Arg(37)-bearing random coil. This peculiar structural plasticity of MtOGT could be instrumental to proper protein clustering at damaged DNA sites, as well as to protein-DNA complexes disassembling on repair. © 2016 Authors; published by Portland Press Limited.

  6. Self-Assembling Molecular Logic Gates Based on DNA Crossover Tiles.

    Science.gov (United States)

    Campbell, Eleanor A; Peterson, Evan; Kolpashchikov, Dmitry M

    2017-07-05

    DNA-based computational hardware has attracted ever-growing attention due to its potential to be useful in the analysis of complex mixtures of biological markers. Here we report the design of self-assembling logic gates that recognize DNA inputs and assemble into crossover tiles when the output signal is high; the crossover structures disassemble to form separate DNA stands when the output is low. The output signal can be conveniently detected by fluorescence using a molecular beacon probe as a reporter. AND, NOT, and OR logic gates were designed. We demonstrate that the gates can connect to each other to produce other logic functions. © 2017 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim.

  7. Self-assembly of genetically encoded DNA-protein hybrid nanoscale shapes.

    Science.gov (United States)

    Praetorius, Florian; Dietz, Hendrik

    2017-03-24

    We describe an approach to bottom-up fabrication that allows integration of the functional diversity of proteins into designed three-dimensional structural frameworks. A set of custom staple proteins based on transcription activator-like effector proteins folds a double-stranded DNA template into a user-defined shape. Each staple protein is designed to recognize and closely link two distinct double-helical DNA sequences at separate positions on the template. We present design rules for constructing megadalton-scale DNA-protein hybrid shapes; introduce various structural motifs, such as custom curvature, corners, and vertices; and describe principles for creating multilayer DNA-protein objects with enhanced rigidity. We demonstrate self-assembly of our hybrid nanostructures in one-pot mixtures that include the genetic information for the designed proteins, the template DNA, RNA polymerase, ribosomes, and cofactors for transcription and translation. Copyright © 2017, American Association for the Advancement of Science.

  8. Tuning the Cavity Size and Chirality of Self-Assembling 3D DNA Crystals

    Energy Technology Data Exchange (ETDEWEB)

    Simmons, Chad R.; Zhang, Fei; MacCulloch, Tara; Fahmi, Noureddine; Stephanopoulos, Nicholas; Liu, Yan; Seeman, Nadrian C. [Department; Yan, Hao

    2017-08-02

    The foundational goal of structural DNA nanotechnology—the field that uses oligonucleotides as a molecular building block for the programmable self-assembly of nanostructured systems—was to use DNA to construct three-dimensional (3D) lattices for solving macromolecular structures. The programmable nature of DNA makes it an ideal system for rationally constructing self-assembled crystals and immobilizing guest molecules in a repeating 3D array through their specific stereospatial interactions with the scaffold. In this work, we have extended a previously described motif (4 × 5) by expanding the structure to a system that links four double-helical layers; we use a central weaving oligonucleotide containing a sequence of four six-base repeats (4 × 6), forming a matrix of layers that are organized and dictated by a series of Holliday junctions. In addition, we have assembled mirror image crystals (l-DNA) with the identical sequence that are completely resistant to nucleases. Bromine and selenium derivatives were obtained for the l- and d-DNA forms, respectively, allowing phase determination for both forms and solution of the resulting structures to 3.0 and 3.05 Å resolution. Both right- and left-handed forms crystallized in the trigonal space groups with mirror image 3-fold helical screw axes P32 and P31 for each motif, respectively. The structures reveal a highly organized array of discrete and well-defined cavities that are suitable for hosting guest molecules and allow us to dictate a priori the assembly of guest–DNA conjugates with a specified crystalline hand.

  9. Establishing a novel automated magnetic bead-based method for the extraction of DNA from a variety of forensic samples.

    Science.gov (United States)

    Witt, Sebastian; Neumann, Jan; Zierdt, Holger; Gébel, Gabriella; Röscheisen, Christiane

    2012-09-01

    Automated systems have been increasingly utilized for DNA extraction by many forensic laboratories to handle growing numbers of forensic casework samples while minimizing the risk of human errors and assuring high reproducibility. The step towards automation however is not easy: The automated extraction method has to be very versatile to reliably prepare high yields of pure genomic DNA from a broad variety of sample types on different carrier materials. To prevent possible cross-contamination of samples or the loss of DNA, the components of the kit have to be designed in a way that allows for the automated handling of the samples with no manual intervention necessary. DNA extraction using paramagnetic particles coated with a DNA-binding surface is predestined for an automated approach. For this study, we tested different DNA extraction kits using DNA-binding paramagnetic particles with regard to DNA yield and handling by a Freedom EVO(®)150 extraction robot (Tecan) equipped with a Te-MagS magnetic separator. Among others, the extraction kits tested were the ChargeSwitch(®)Forensic DNA Purification Kit (Invitrogen), the PrepFiler™Automated Forensic DNA Extraction Kit (Applied Biosystems) and NucleoMag™96 Trace (Macherey-Nagel). After an extensive test phase, we established a novel magnetic bead extraction method based upon the NucleoMag™ extraction kit (Macherey-Nagel). The new method is readily automatable and produces high yields of DNA from different sample types (blood, saliva, sperm, contact stains) on various substrates (filter paper, swabs, cigarette butts) with no evidence of a loss of magnetic beads or sample cross-contamination. Copyright © 2012 Elsevier Ireland Ltd. All rights reserved.

  10. PNA-Peptide Assembly in a 3D DNA Nanocage at Room Temperature

    Energy Technology Data Exchange (ETDEWEB)

    Flory, Justin D. [Center; Shinde, Sandip [Center; Lin, Su [Center; Liu, Yan [Center; Yan, Hao [Center; Ghirlanda, Giovanna [Center; Fromme, Petra [Center

    2013-04-12

    Proteins and peptides fold into dynamic structures that access a broad functional landscape; however, designing artificial polypeptide systems is still a great challenge. Conversely, DNA engineering is now routinely used to build a wide variety of 2D and 3D nanostructures from hybridization based rules, and their functional diversity can be significantly expanded through site specific incorporation of the appropriate guest molecules. Here we demonstrate a new approach to rationally design 3D nucleic acid–amino acid complexes using peptide nucleic acid (PNA) to assemble peptides inside a 3D DNA nanocage. The PNA-peptides were found to bind to the preassembled DNA nanocage in 5–10 min at room temperature, and assembly could be performed in a stepwise fashion. Biophysical characterization of the DNA-PNA-peptide complex was performed using gel electrophoresis as well as steady state and time-resolved fluorescence spectroscopy. Based on these results we have developed a model for the arrangement of the PNA-peptides inside the DNA nanocage. This work demonstrates a flexible new approach to leverage rationally designed nucleic acid (DNA-PNA) nanoscaffolds to guide polypeptide engineering.

  11. Rapid DNA analysis for automated processing and interpretation of low DNA content samples.

    Science.gov (United States)

    Turingan, Rosemary S; Vasantgadkar, Sameer; Palombo, Luke; Hogan, Catherine; Jiang, Hua; Tan, Eugene; Selden, Richard F

    2016-01-01

    Short tandem repeat (STR) analysis of casework samples with low DNA content include those resulting from the transfer of epithelial cells from the skin to an object (e.g., cells on a water bottle, or brim of a cap), blood spatter stains, and small bone and tissue fragments. Low DNA content (LDC) samples are important in a wide range of settings, including disaster response teams to assist in victim identification and family reunification, military operations to identify friend or foe, criminal forensics to identify suspects and exonerate the innocent, and medical examiner and coroner offices to identify missing persons. Processing LDC samples requires experienced laboratory personnel, isolated workstations, and sophisticated equipment, requires transport time, and involves complex procedures. We present a rapid DNA analysis system designed specifically to generate STR profiles from LDC samples in field-forward settings by non-technical operators. By performing STR in the field, close to the site of collection, rapid DNA analysis has the potential to increase throughput and to provide actionable information in real time. A Low DNA Content BioChipSet (LDC BCS) was developed and manufactured by injection molding. It was designed to function in the fully integrated Accelerated Nuclear DNA Equipment (ANDE) instrument previously designed for analysis of buccal swab and other high DNA content samples (Investigative Genet. 4(1):1-15, 2013). The LDC BCS performs efficient DNA purification followed by microfluidic ultrafiltration of the purified DNA, maximizing the quantity of DNA available for subsequent amplification and electrophoretic separation and detection of amplified fragments. The system demonstrates accuracy, precision, resolution, signal strength, and peak height ratios appropriate for casework analysis. The LDC rapid DNA analysis system is effective for the generation of STR profiles from a wide range of sample types. The technology broadens the range of sample

  12. Lipid-bilayer-assisted two-dimensional self-assembly of DNA origami nanostructures

    Science.gov (United States)

    Endo, Masayuki; Sugiyama, Hiroshi

    2015-01-01

    Self-assembly is a ubiquitous approach to the design and fabrication of novel supermolecular architectures. Here we report a strategy termed ‘lipid-bilayer-assisted self-assembly' that is used to assemble DNA origami nanostructures into two-dimensional lattices. DNA origami structures are electrostatically adsorbed onto a mica-supported zwitterionic lipid bilayer in the presence of divalent cations. We demonstrate that the bilayer-adsorbed origami units are mobile on the surface and self-assembled into large micrometre-sized lattices in their lateral dimensions. Using high-speed atomic force microscopy imaging, a variety of dynamic processes involved in the formation of the lattice, such as fusion, reorganization and defect filling, are successfully visualized. The surface modifiability of the assembled lattice is also demonstrated by in situ decoration with streptavidin molecules. Our approach provides a new strategy for preparing versatile scaffolds for nanofabrication and paves the way for organizing functional nanodevices in a micrometer space. PMID:26310995

  13. Gene assembly via one-pot chemical ligation of DNA promoted by DNA nanostructures

    DEFF Research Database (Denmark)

    Manuguerra, Ilenia; Croce, Stefano; El-Sagheer, Afaf H.

    2018-01-01

    Current gene synthesis methods are driven by enzymatic reactions. Here we report the one-pot synthesis of a chemically-ligated gene from 14 oligonucleotides. The chemical ligation benefits from the highly efficient click chemistry approach templated by DNA nanostructures, and produces modified DNA...

  14. Structure and assembly of the essential RNA ring component of a viral DNA packaging motor.

    Science.gov (United States)

    Ding, Fang; Lu, Changrui; Zhao, Wei; Rajashankar, Kanagalaghatta R; Anderson, Dwight L; Jardine, Paul J; Grimes, Shelley; Ke, Ailong

    2011-05-03

    Prohead RNA (pRNA) is an essential component in the assembly and operation of the powerful bacteriophage 29 DNA packaging motor. The pRNA forms a multimeric ring via intermolecular base-pairing interactions between protomers that serves to guide the assembly of the ring ATPase that drives DNA packaging. Here we report the quaternary structure of this rare multimeric RNA at 3.5 Å resolution, crystallized as tetrameric rings. Strong quaternary interactions and the inherent flexibility helped rationalize how free pRNA is able to adopt multiple oligomerization states in solution. These characteristics also allowed excellent fitting of the crystallographic pRNA protomers into previous prohead/pRNA cryo-EM reconstructions, supporting the presence of a pentameric, but not hexameric, pRNA ring in the context of the DNA packaging motor. The pentameric pRNA ring anchors itself directly to the phage prohead by interacting specifically with the fivefold symmetric capsid structures that surround the head-tail connector portal. From these contacts, five RNA superhelices project from the pRNA ring, where they serve as scaffolds for binding and assembly of the ring ATPase, and possibly mediate communication between motor components. Construction of structure-based designer pRNAs with little sequence similarity to the wild-type pRNA were shown to fully support the packaging of 29 DNA.

  15. DNA-mediated self-assembly of tetrahedral plasmonic clusters for metafluids

    Science.gov (United States)

    Schade, Nicholas; Sun, Li; Lee, You-Jin; Fan, Jonathan; Capasso, Federico; Yi, Gi-Ra; Manoharan, Vinothan

    2014-03-01

    We direct the self-assembly of clusters of gold nanospheres with the goal of creating a bulk, isotropic, optical metafluid. We use spherical gold nanoparticles that are exceptionally smooth, monocrystalline, and monodisperse. These particles exhibit highly reproducible scattering spectra compared with commercially available gold colloids. We label them with DNA sequences and mix them together to self-assemble small clusters. By controlling the particle sizes and the interactions between them, we maximize the yield of tetrahedral clusters, the ideal structures for isotropic metamaterials.

  16. Automated High-Volume Manufacturing of Modular Photovoltaic Panel Assemblies for Space Solar Arrays, Phase I

    Data.gov (United States)

    National Aeronautics and Space Administration — Deployable Space Systems, Inc. (DSS) will focus the proposed SBIR program on the creation and development of an automated robotic manufacturing infrastructure...

  17. Automated High-Volume Manufacturing of Modular Photovoltaic Panel Assemblies for Space Solar Arrays Project

    Data.gov (United States)

    National Aeronautics and Space Administration — Deployable Space Systems, Inc. (DSS) will focus the proposed SBIR program on the creation and development of an automated robotic manufacturing infrastructure...

  18. Development of an Automated Microfluidic System for DNA Collection, Amplification, and Detection of Pathogens

    Energy Technology Data Exchange (ETDEWEB)

    Hagan, Bethany S.; Bruckner-Lea, Cynthia J.

    2002-12-01

    This project was focused on developing and testing automated routines for a microfluidic Pathogen Detection System. The basic pathogen detection routine has three primary components; cell concentration, DNA amplification, and detection. In cell concentration, magnetic beads are held in a flow cell by an electromagnet. Sample liquid is passed through the flow cell and bacterial cells attach to the beads. These beads are then released into a small volume of fluid and delivered to the peltier device for cell lysis and DNA amplification. The cells are lysed during initial heating in the peltier device, and the released DNA is amplified using polymerase chain reaction (PCR) or strand displacement amplification (SDA). Once amplified, the DNA is then delivered to a laser induced fluorescence detection unit in which the sample is detected. These three components create a flexible platform that can be used for pathogen detection in liquid and sediment samples. Future developments of the system will include on-line DNA detection during DNA amplification and improved capture and release methods for the magnetic beads during cell concentration.

  19. DNA-nanoparticle assemblies go organic: Macroscopic polymeric materials with nanosized features

    Directory of Open Access Journals (Sweden)

    Mentovich Elad D

    2012-05-01

    Full Text Available Abstract Background One of the goals in the field of structural DNA nanotechnology is the use of DNA to build up 2- and 3-D nanostructures. The research in this field is motivated by the remarkable structural features of DNA as well as by its unique and reversible recognition properties. Nucleic acids can be used alone as the skeleton of a broad range of periodic nanopatterns and nanoobjects and in addition, DNA can serve as a linker or template to form DNA-hybrid structures with other materials. This approach can be used for the development of new detection strategies as well as nanoelectronic structures and devices. Method Here we present a new method for the generation of unprecedented all-organic conjugated-polymer nanoparticle networks guided by DNA, based on a hierarchical self-assembly process. First, microphase separation of amphiphilic block copolymers induced the formation of spherical nanoobjects. As a second ordering concept, DNA base pairing has been employed for the controlled spatial definition of the conjugated-polymer particles within the bulk material. These networks offer the flexibility and the diversity of soft polymeric materials. Thus, simple chemical methodologies could be applied in order to tune the network's electrical, optical and mechanical properties. Results and conclusions One- two- and three-dimensional networks have been successfully formed. Common to all morphologies is the integrity of the micelles consisting of DNA block copolymer (DBC, which creates an all-organic engineered network.

  20. Assembly and structural analysis of a covalently closed nano-scale DNA cage

    DEFF Research Database (Denmark)

    Andersen, Félicie Faucon; Knudsen, Bjarne; Oliveira, Cristiano Luis Pinto De

    2008-01-01

     The inherent properties of DNA as a stable polymer with unique affinity for partner molecules determined by the specific Watson-Crick base pairing makes it an ideal component in self-assembling structures. This has been exploited for decades in the design of a variety of artificial substrates...... are necessary before complex DNA structures can be routinely designed for the use in basal science and/or biotechnology. Here we present the design, construction and structural analysis of a covalently closed and stable 3D DNA structure with the connectivity of an octahedron, as defined by the double...... be described as a nano-scale DNA cage, Hence, in theory it could hold proteins or other bio-molecules to enable their investigation in certain harmful environments or even allow their organization into higher order structures...

  1. Multiple competition reactions for RPA order the assembly of the DNA polymerase delta holoenzyme.

    Science.gov (United States)

    Yuzhakov, A; Kelman, Z; Hurwitz, J; O'Donnell, M

    1999-11-01

    Processive extension of DNA in eukaryotes requires three factors to coordinate their actions. First, DNA polymerase alpha-primase synthesizes the primed site. Then replication factor C loads a proliferating cell nuclear antigen (PCNA) clamp onto the primer. Following this, DNA polymerase delta assembles with PCNA for processive extension. This report shows that these proteins each bind the primed site tightly and trade places in a highly coordinated fashion such that the primer terminus is never left free of protein. Replication protein A (RPA), the single-stranded DNA-binding protein, forms a common touchpoint for each of these proteins and they compete with one another for it. Thus these protein exchanges are driven by competition-based protein switches in which two proteins vie for contact with RPA.

  2. A new biochromatography model based on DNA origami assembled PPARγ: construction and evaluation.

    Science.gov (United States)

    Zhou, Jie; Meng, Lingchang; Sun, Chong; Chen, Shanshan; Sun, Fang; Luo, Pei; Zhao, Yongxing

    2017-05-01

    As drug targets, receptors have potential to screen drugs. Silica is an attractive support to immobilize receptors; however, the lack of biocompatibility makes it easier for receptors to lose bioactivity, which remains an obstacle to its widespread use. With the advantage of biocompatibility, DNA origami can be used as a biological carrier to improve the biocompatibility of silica and assemble receptors. In this study, a new biochromatography model based on DNA origami was constructed. A large quantity of M13ssDNA was used as a scaffold, leading to significant costs, so M13ssDNA was self-produced from the bacteriophage particles. This approach is demonstrated using the ligand binding domain of gamma isoform peroxisome proliferator-activated receptor (PPARγ-LBD) as a research object. PPARγ-LBD was assembled on DNA origami carrier and then coupled on the surface of silica. The products were packed into the column as stationary phase to construct the biochromatography with the ability to recognize drugs. Affinity and specificity of the biochromatography model were evaluated by HPLC. The final results showed that the biochromatography could recognize rosiglitazone specifically, which further proved that the model could screen chemical compositions interacted with PPARγ. It was the first time to take advantage of DNA origami to assemble PPARγ to construct biochromatography. The new biochromatography model has the advantages of being efficient, convenient, and high-throughput. This method affords a new way to rapidly and conveniently screen active ingredients from complex sample plant extracts and natural product-like libraries.

  3. DNA driven self-assembly of micron-sized rods using DNA-grafted bacteriophage fd virions.

    Science.gov (United States)

    Unwin, R R; Cabanas, R A; Yanagishima, T; Blower, T R; Takahashi, H; Salmond, G P C; Edwardson, J M; Fraden, S; Eiser, E

    2015-03-28

    We have functionalized the sides of fd bacteriophage virions with oligonucleotides to induce DNA hybridization driven self-assembly of high aspect ratio filamentous particles. Potential impacts of this new structure range from an entirely new building block in DNA origami structures, inclusion of virions in DNA nanostructures and nanomachines, to a new means of adding thermotropic control to lyotropic liquid crystal systems. A protocol for producing the virions in bulk is reviewed. Thiolated oligonucleotides are attached to the viral capsid using a heterobifunctional chemical linker. A commonly used system is utilized, where a sticky, single-stranded DNA strand is connected to an inert double-stranded spacer to increase inter-particle connectivity. Solutions of fd virions carrying complementary strands are mixed, annealed, and their aggregation is studied using dynamic light scattering (DLS), fluorescence microscopy, and atomic force microscopy (AFM). Aggregation is clearly observed on cooling, with some degree of local order, and is reversible when temperature is cycled through the DNA hybridization transition.

  4. Defined DNA-mediated assemblies of gene-expressing giant unilamellar vesicles

    DEFF Research Database (Denmark)

    Hadorn, M.; Boenzli, E.; Sørensen, Kristian T.

    2013-01-01

    of giant unilamellar vesicles functionalized with a basic cellular machinery to express green fluorescent protein and specified neighbor-to-neighbor interactions. We show both that the local and programmable DNA pairing rules on the nanoscale are able to direct the microscale vesicles into macroscale soft...... matter assemblies and that the highly sensitive gene-expression machinery remains intact and active during multiple experimental steps. An in silico model recapitulates the experiments performed in vitro and covers additional experimental setups highlighting the parameters that control the DNA...

  5. Primer effect in the detection of mitochondrial DNA point heteroplasmy by automated sequencing.

    Science.gov (United States)

    Calatayud, Marta; Ramos, Amanda; Santos, Cristina; Aluja, Maria Pilar

    2013-06-01

    The correct detection of mitochondrial DNA (mtDNA) heteroplasmy by automated sequencing presents methodological constraints. The main goals of this study are to investigate the effect of sense and distance of primers in heteroplasmy detection and to test if there are differences in the accurate determination of heteroplasmy involving transitions or transversions. A gradient of the heteroplasmy levels was generated for mtDNA positions 9477 (transition G/A) and 15,452 (transversion C/A). Amplification and subsequent sequencing with forward and reverse primers, situated at 550 and 150 bp from the heteroplasmic positions, were performed. Our data provide evidence that there is a significant difference between the use of forward and reverse primers. The forward primer is the primer that seems to give a better approximation to the real proportion of the variants. No significant differences were found concerning the distance at which the sequencing primers were placed neither between the analysis of transitions and transversions. The data collected in this study are a starting point that allows to glimpse the importance of the sequencing primers in the accurate detection of point heteroplasmy, providing additional insight into the overall automated sequencing strategy.

  6. Building superlattices from individual nanoparticles via template-confined DNA-mediated assembly

    Energy Technology Data Exchange (ETDEWEB)

    Lin, Qing-Yuan; Mason, Jarad A.; Li, Zhongyang; Zhou, Wenjie; O’Brien, Matthew N.; Brown, Keith A.; Jones, Matthew R.; Butun, Serkan; Lee, Byeongdu; Dravid, Vinayak P.; Aydin, Koray; Mirkin, Chad A.

    2018-01-18

    DNA programmable assembly has been combined with top-down lithography to construct superlattices of discrete, reconfigurable nanoparticle architectures on a gold surface over large areas. Specifically, individual colloidal plasmonic nanoparticles with different shapes and sizes are assembled with ‘locked” nucleic acids in polymer pores into oriented architectures that feature tunable arrangements and independently controllable distances at both nanometer and micrometer length scales. These structures, which would be difficult to construct via other common assembly methods, provide a platform to systematically study and control light-matter interactions in nanoparticle-based optical materials. The generality and potential of this approach is explored by identifying a broadband absorber with a solvent polarity response that allows dynamic tuning of the wavelength response and amplitude of visible light absorption.

  7. Assembling the Streptococcus thermophilus clustered regularly interspaced short palindromic repeats (CRISPR) array for multiplex DNA targeting.

    Science.gov (United States)

    Guo, Lijun; Xu, Kun; Liu, Zhiyuan; Zhang, Cunfang; Xin, Ying; Zhang, Zhiying

    2015-06-01

    In addition to the advantages of scalable, affordable, and easy to engineer, the clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR-associated protein (Cas) technology is superior for multiplex targeting, which is laborious and inconvenient when achieved by cloning multiple gRNA expressing cassettes. Here, we report a simple CRISPR array assembling method which will facilitate multiplex targeting usage. First, the Streptococcus thermophilus CRISPR3/Cas locus was cloned. Second, different CRISPR arrays were assembled with different crRNA spacers. Transformation assays using different Escherichia coli strains demonstrated efficient plasmid DNA targeting, and we achieved targeting efficiency up to 95% with an assembled CRISPR array with three crRNA spacers. Copyright © 2015 Elsevier Inc. All rights reserved.

  8. Cloning Should Be Simple: Escherichia coli DH5α-Mediated Assembly of Multiple DNA Fragments with Short End Homologies

    Science.gov (United States)

    Richardson, Ruth E.; Suzuki, Yo

    2015-01-01

    Numerous DNA assembly technologies exist for generating plasmids for biological studies. Many procedures require complex in vitro or in vivo assembly reactions followed by plasmid propagation in recombination-impaired Escherichia coli strains such as DH5α, which are optimal for stable amplification of the DNA materials. Here we show that despite its utility as a cloning strain, DH5α retains sufficient recombinase activity to assemble up to six double-stranded DNA fragments ranging in size from 150 bp to at least 7 kb into plasmids in vivo. This process also requires surprisingly small amounts of DNA, potentially obviating the need for upstream assembly processes associated with most common applications of DNA assembly. We demonstrate the application of this process in cloning of various DNA fragments including synthetic genes, preparation of knockout constructs, and incorporation of guide RNA sequences in constructs for clustered regularly interspaced short palindromic repeats (CRISPR) genome editing. This consolidated process for assembly and amplification in a widely available strain of E. coli may enable productivity gain across disciplines involving recombinant DNA work. PMID:26348330

  9. Comparison of QIAsymphony Automated and QIAamp Manual DNA Extraction Systems for Measuring Epstein-Barr Virus DNA Load in Whole Blood Using Real-Time PCR

    OpenAIRE

    Laus, Stella; Kingsley, Lawrence A.; Green, Michael; Wadowsky, Robert M.

    2011-01-01

    Automated and manual extraction systems have been used with real-time PCR for quantification of Epstein-Barr virus [human herpesvirus 4 (HHV-4)] DNA in whole blood, but few studies have evaluated relative performances. In the present study, the automated QIAsymphony and manual QIAamp extraction systems (Qiagen, Valencia, CA) were assessed using paired aliquots derived from clinical whole-blood specimens and an in-house, real-time PCR assay. The detection limits using the QIAsymphony and QIAam...

  10. Complex shapes self–assembled from single–stranded DNA tiles

    Science.gov (United States)

    Wei, Bryan; Dai, Mingjie; Yin, Peng

    2014-01-01

    Programmed self-assembly of strands of nucleic acid has proved highly effective for creating a wide range of structures with desired shapes1–25. A particularly successful implementation is DNA origami, in which a long scaffold strand is folded by hundreds of short auxiliary strands into a complex shape9, 14–16,18–21,25. Modular strategies are in principle simpler and more versatile and have been used to assemble DNA2–5,8,10–13,17,23 or RNA7,22 tiles into periodic3,4,7,22 and algorithmic5 two-dimensional lattices, extended ribbons10,12 and tubes4,12,13, three-dimensional crystals17, polyhedra11 and simple finite two-dimensional shapes7,8. But creating finite yet complex shapes from a large number of uniquely addressable tiles remains challenging. Here we solve this problem with the simplest tile form, a ‘single-stranded tile’ (SST) that consists of a 42-base strand of DNA composed entirely of concatenated sticky ends and that binds to four local neighbours during self-assembly12. Although ribbons and tubes with controlled circumferences12 have been created using the SST approach, we extend it to assemble complex two-dimensional shapes and tubes from hundreds (in some cases more than one thousand) distinct tiles. Our main design feature is a self-assembled rectangle that serves as a molecular canvas, with each of its constituent SST strands—folded into a 3nm-by-7 nm tile and attached to four neighbouring tiles—acting as a pixel. A desired shape, drawn on the canvas, is then produced by one-pot annealing of all those strands that correspond to pixels covered by the target shape; the remaining strands are excluded. We implement the strategy with a master strand collection that corresponds to a 310-pixel canvas, and then use appropriate strand subsets to construct 107 distinct and complex two-dimensional shapes, thereby establishing SST assembly as a simple, modular and robust framework for constructing nanostructures with prescribed shapes from short

  11. Pydna: a simulation and documentation tool for DNA assembly strategies using python.

    Science.gov (United States)

    Pereira, Filipa; Azevedo, Flávio; Carvalho, Ângela; Ribeiro, Gabriela F; Budde, Mark W; Johansson, Björn

    2015-05-02

    Recent advances in synthetic biology have provided tools to efficiently construct complex DNA molecules which are an important part of many molecular biology and biotechnology projects. The planning of such constructs has traditionally been done manually using a DNA sequence editor which becomes error-prone as scale and complexity of the construction increase. A human-readable formal description of cloning and assembly strategies, which also allows for automatic computer simulation and verification, would therefore be a valuable tool. We have developed pydna, an extensible, free and open source Python library for simulating basic molecular biology DNA unit operations such as restriction digestion, ligation, PCR, primer design, Gibson assembly and homologous recombination. A cloning strategy expressed as a pydna script provides a description that is complete, unambiguous and stable. Execution of the script automatically yields the sequence of the final molecule(s) and that of any intermediate constructs. Pydna has been designed to be understandable for biologists with limited programming skills by providing interfaces that are semantically similar to the description of molecular biology unit operations found in literature. Pydna simplifies both the planning and sharing of cloning strategies and is especially useful for complex or combinatorial DNA molecule construction. An important difference compared to existing tools with similar goals is the use of Python instead of a specifically constructed language, providing a simulation environment that is more flexible and extensible by the user.

  12. Automated High-Volume Manufacturing of Modular Photovoltaic Panel Assemblies for Space Solar Arrays Project

    Data.gov (United States)

    National Aeronautics and Space Administration — Deployable Space Systems, Inc. (DSS) will focus the proposed SBIR Phase 2 program on the development and demonstration of an automated robotic manufacturing...

  13. Automated High-Volume Manufacturing of Modular Photovoltaic Panel Assemblies for Space Solar Arrays, Phase II

    Data.gov (United States)

    National Aeronautics and Space Administration — Deployable Space Systems, Inc. (DSS) will focus the proposed SBIR Phase 2 program on the development and demonstration of an automated robotic manufacturing...

  14. Automated extraction of DNA and PCR setup using a Tecan Freedom EVO® liquid handler

    DEFF Research Database (Denmark)

    Frøslev, Tobias Guldberg; Hansen, Anders Johannes; Stangegaard, Michael

    2009-01-01

    We have implemented and validated automated protocols for DNA extraction and PCR setup using a Tecan Freedom EVO® liquid handler mounted with the TeMagS magnetic separation device. The methods were validated for accredited, forensic genetic work according to ISO 17025 using the Qiagen Mag......Attract® DNA Mini M48 kit from fresh, whole blood and blood from deceased. The methods were simplified by returning the DNA extracts to the original tubes reducing the risk of misplacing samples. The original tubes that had contained the samples were washed with 700 µl Milli-Q water prior to the return...... of the DNA extracts. The PCR setup protocols were designed for 96 well microtiter plates. The methods were validated for the kits: AmpFlSTR® Identifiler® and Y-filer® (Applied Biosystems), GenePrint® FFFL and PowerPlex® Y (Promega). Within 3.5 hours, 96 samples were extracted and PCR master mix was added...

  15. A dynamic combinatorial approach for identifying side groups that stabilize DNA-templated supramolecular self-assemblies.

    Science.gov (United States)

    Paolantoni, Delphine; Cantel, Sonia; Dumy, Pascal; Ulrich, Sébastien

    2015-02-06

    DNA-templated self-assembly is an emerging strategy for generating functional supramolecular systems, which requires the identification of potent multi-point binding ligands. In this line, we recently showed that bis-functionalized guanidinium compounds can interact with ssDNA and generate a supramolecular complex through the recognition of the phosphodiester backbone of DNA. In order to probe the importance of secondary interactions and to identify side groups that stabilize these DNA-templated self-assemblies, we report herein the implementation of a dynamic combinatorial approach. We used an in situ fragment assembly process based on reductive amination and tested various side groups, including amino acids. The results reveal that aromatic and cationic side groups participate in secondary supramolecular interactions that stabilize the complexes formed with ssDNA.

  16. A Dynamic Combinatorial Approach for Identifying Side Groups that Stabilize DNA-Templated Supramolecular Self-Assemblies

    Directory of Open Access Journals (Sweden)

    Delphine Paolantoni

    2015-02-01

    Full Text Available DNA-templated self-assembly is an emerging strategy for generating functional supramolecular systems, which requires the identification of potent multi-point binding ligands. In this line, we recently showed that bis-functionalized guanidinium compounds can interact with ssDNA and generate a supramolecular complex through the recognition of the phosphodiester backbone of DNA. In order to probe the importance of secondary interactions and to identify side groups that stabilize these DNA-templated self-assemblies, we report herein the implementation of a dynamic combinatorial approach. We used an in situ fragment assembly process based on reductive amination and tested various side groups, including amino acids. The results reveal that aromatic and cationic side groups participate in secondary supramolecular interactions that stabilize the complexes formed with ssDNA.

  17. Stabilization of Pt nanoparticles by single stranded DNA and the binary assembly of Au and Pt nanoparticles without hybridization

    International Nuclear Information System (INIS)

    Yang, J.; Lee, Jim Yang; Too, Heng-Phon; Chow, Gan-Moog; Gan, Leong M.

    2006-01-01

    The non-specific interaction between single stranded DNA (ssDNA) and 12 nm Pt nanoparticles is investigated in this work. The data show a strong and non-specific interaction between the two which can be exploited for the stabilization of Pt nanoparticles in aqueous solutions. Based on the experimental findings, a non-hybridization based protocol to assemble 17 nm Au and Pt nanoparticles (12 nm cubic and 3.6 nm spherical) by single-stranded DNA was developed. Transmission electron microscopy (TEM) and UV-visible spectroscopy confirmed that Au and Pt nanoparticles could be assembled by the non-specific interaction in an orderly manner. The experimental results also caution against the potential pitfalls in using DNA melting point analysis to infer metal nanoparticle assembly by DNA hybridization

  18. Evaluation of Sample Stability and Automated DNA Extraction for Fetal Sex Determination Using Cell-Free Fetal DNA in Maternal Plasma

    Directory of Open Access Journals (Sweden)

    Elena Ordoñez

    2013-01-01

    Full Text Available Objective. The detection of paternally inherited sequences in maternal plasma, such as the SRY gene for fetal sexing or RHD for fetal blood group genotyping, is becoming part of daily routine in diagnostic laboratories. Due to the low percentage of fetal DNA, it is crucial to ensure sample stability and the efficiency of DNA extraction. We evaluated blood stability at 4°C for at least 24 hours and automated DNA extraction, for fetal sex determination in maternal plasma. Methods. A total of 158 blood samples were collected, using EDTA-K tubes, from women in their 1st trimester of pregnancy. Samples were kept at 4°C for at least 24 hours before processing. An automated DNA extraction was evaluated, and its efficiency was compared with a standard manual procedure. The SRY marker was used to quantify cfDNA by real-time PCR. Results. Although lower cfDNA amounts were obtained by automated DNA extraction (mean 107,35 GE/mL versus 259,43 GE/mL, the SRY sequence was successfully detected in all 108 samples from pregnancies with male fetuses. Conclusion. We successfully evaluated the suitability of standard blood tubes for the collection of maternal blood and assessed samples to be suitable for analysis at least 24 hours later. This would allow shipping to a central reference laboratory almost from anywhere in Europe.

  19. DNA nanotubes assembled from tensegrity triangle tiles with circular DNA scaffolds.

    Science.gov (United States)

    Afshan, Noshin; Ali, Mashooq; Wang, Meng; Baig, Mirza Muhammad Faran Ashraf; Xiao, Shou-Jun

    2017-11-16

    Using small circular DNA molecules of different lengths as scaffolds, we successfully synthesised DNA nanotubes consisting of Mao's DNA tensegrity triangle tiles with four-arm junctions (Holliday junctions) at all vertices. Due to the intrinsic curvature of the triangle tile and the consecutive tile alignment, the 2D arrays are organised in the form of nanotubes. Two sized triangle tiles with equilateral side lengths of 1.5 and 2.5 full helical turns are connected by the sticky ended cohesion of a duplex with a length of 2.5 helical turns respectively, and their parallel lozenge tiling lattices were demonstrated by high resolution AFM images, where the former lozenge unit cell has a lattice constant of 13.6 nm, and the latter has a larger lattice constant of 17.0 nm. Modification of the triangle tile with infinitesimal disturbance on side lengths and insertion of one thymine single stranded loop at every vertex resulted in comparably similar nanotubes.

  20. DNA damage response and spindle assembly checkpoint function throughout the cell cycle to ensure genomic integrity.

    Directory of Open Access Journals (Sweden)

    Katherine S Lawrence

    2015-04-01

    Full Text Available Errors in replication or segregation lead to DNA damage, mutations, and aneuploidies. Consequently, cells monitor these events and delay progression through the cell cycle so repair precedes division. The DNA damage response (DDR, which monitors DNA integrity, and the spindle assembly checkpoint (SAC, which responds to defects in spindle attachment/tension during metaphase of mitosis and meiosis, are critical for preventing genome instability. Here we show that the DDR and SAC function together throughout the cell cycle to ensure genome integrity in C. elegans germ cells. Metaphase defects result in enrichment of SAC and DDR components to chromatin, and both SAC and DDR are required for metaphase delays. During persistent metaphase arrest following establishment of bi-oriented chromosomes, stability of the metaphase plate is compromised in the absence of DDR kinases ATR or CHK1 or SAC components, MAD1/MAD2, suggesting SAC functions in metaphase beyond its interactions with APC activator CDC20. In response to DNA damage, MAD2 and the histone variant CENPA become enriched at the nuclear periphery in a DDR-dependent manner. Further, depletion of either MAD1 or CENPA results in loss of peripherally associated damaged DNA. In contrast to a SAC-insensitive CDC20 mutant, germ cells deficient for SAC or CENPA cannot efficiently repair DNA damage, suggesting that SAC mediates DNA repair through CENPA interactions with the nuclear periphery. We also show that replication perturbations result in relocalization of MAD1/MAD2 in human cells, suggesting that the role of SAC in DNA repair is conserved.

  1. Self-assembling of calcium salt of the new DNA base 5-carboxylcytosine

    Energy Technology Data Exchange (ETDEWEB)

    Irrera, Simona [Department of Chemistry, SAPIENZA University of Rome, Piazzale A. Moro 5, 00185 Rome (Italy); Department of Chemistry, University College London, 20 Grodon Street, WC1H0AJ London (United Kingdom); Ruiz-Hernandez, Sergio E. [School of Chemistry, Cardiff University Main Building, Park Place, CF103AT Cardiff (United Kingdom); Reggente, Melania [Department of Basic and Applied Sciences for Engineering, SAPIENZA University of Rome, Via A. Scarpa 16, 00161 Rome (Italy); Passeri, Daniele, E-mail: daniele.passeri@uniroma1.it [Department of Basic and Applied Sciences for Engineering, SAPIENZA University of Rome, Via A. Scarpa 16, 00161 Rome (Italy); Natali, Marco [Department of Basic and Applied Sciences for Engineering, SAPIENZA University of Rome, Via A. Scarpa 16, 00161 Rome (Italy); Gala, Fabrizio [Department of Basic and Applied Sciences for Engineering, SAPIENZA University of Rome, Via A. Scarpa 16, 00161 Rome (Italy); Department of Medical-Surgical, Techno-Biomedical Sciences and Translational Medicine of SAPIENZA University of Rome, Sant’Andrea Hospital, Rome (Italy); Zollo, Giuseppe [Department of Basic and Applied Sciences for Engineering, SAPIENZA University of Rome, Via A. Scarpa 16, 00161 Rome (Italy); Rossi, Marco [Department of Basic and Applied Sciences for Engineering, SAPIENZA University of Rome, Via A. Scarpa 16, 00161 Rome (Italy); Research Center for Nanotechnology applied to Engineering of SAPIENZA University of Rome (CNIS), Piazzale A. Moro 5, 00185 Rome (Italy); Portalone, Gustavo, E-mail: gustavo.portalone@uniroma1.it [Department of Chemistry, SAPIENZA University of Rome, Piazzale A. Moro 5, 00185 Rome (Italy)

    2017-06-15

    Highlights: • Ca salt of 5-carboxylcytosine has been deposited on HOPG substrate. • Molecules self-assembled in monolayers and filaments. • Height of the features were measured by atomic force microscopy. • Ab-initio calculations confirmed the AFM results. - Abstract: Supramolecular architectures involving DNA bases can have a strong impact in several fields such as nanomedicine and nanodevice manufacturing. To date, in addition to the four canonical nucleobases (adenine, thymine, guanine and cytosine), four other forms of cytosine modified at the 5 position have been identified in DNA. Among these four new cytosine derivatives, 5-carboxylcytosine has been recently discovered in mammalian stem cell DNA, and proposed as the final product of the oxidative epigenetic demethylation pathway on the 5 position of cytosine. In this work, a calcium salt of 5-carboxylcytosine has been synthesized and deposited on graphite surface, where it forms self-assembled features as long range monolayers and up to one micron long filaments. These structures have been analyzed in details combining different theoretical and experimental approaches: X-ray single-crystal diffraction data were used to simulate the molecule-graphite interaction, first using molecular dynamics and then refining the results using density functional theory (DFT); finally, data obtained with DFT were used to rationalize atomic force microscopy (AFM) results.

  2. Optimized assembly and covalent coupling of single-molecule DNA origami nanoarrays.

    Science.gov (United States)

    Gopinath, Ashwin; Rothemund, Paul W K

    2014-12-23

    Artificial DNA nanostructures, such as DNA origami, have great potential as templates for the bottom-up fabrication of both biological and nonbiological nanodevices at a resolution unachievable by conventional top-down approaches. However, because origami are synthesized in solution, origami-templated devices cannot easily be studied or integrated into larger on-chip architectures. Electrostatic self-assembly of origami onto lithographically defined binding sites on Si/SiO2 substrates has been achieved, but conditions for optimal assembly have not been characterized, and the method requires high Mg2+ concentrations at which most devices aggregate. We present a quantitative study of parameters affecting origami placement, reproducibly achieving single-origami binding at 94±4% of sites, with 90% of these origami having an orientation within ±10° of their target orientation. Further, we introduce two techniques for converting electrostatic DNA-surface bonds to covalent bonds, allowing origami arrays to be used under a wide variety of Mg2+-free solution conditions.

  3. DNA-library assembly programmed by on-demand nano-liter droplets from a custom microfluidic chip.

    Science.gov (United States)

    Tangen, Uwe; Minero, Gabriel Antonio S; Sharma, Abhishek; Wagler, Patrick F; Cohen, Rafael; Raz, Ofir; Marx, Tzipy; Ben-Yehezkel, Tuval; McCaskill, John S

    2015-07-01

    Nanoscale synthetic biology can benefit from programmable nanoliter-scale processing of DNA in microfluidic chips if they are interfaced effectively to biochemical arrays such as microwell plates. Whereas active microvalve chips require complex fabrication and operation, we show here how a passive and readily fabricated microchip can be employed for customizable nanoliter scale pipetting and reaction control involving DNA. This recently developed passive microfluidic device, supporting nanoliter scale combinatorial droplet generation and mixing, is here used to generate a DNA test library with one member per droplet exported to addressed locations on microwell plates. Standard DNA assembly techniques, such as Gibson assembly, compatible with isothermal on-chip operation, are employed and checked using off-chip PCR and assembly PCR. The control of output droplet sequences and mixing performance was verified using dyes and fluorescently labeled DNA solutions, both on-chip and in external capillary channels. Gel electrophoresis of products and DNA sequencing were employed to further verify controlled combination and functional enzymatic assembly. The scalability of the results to larger DNA libraries is also addressed by combinatorial input expansion using sequential injection plugs from a multiwell plate. Hence, the paper establishes a proof of principle of the production of functional combinatorial mixtures at the nanoliter scale for one sequence per well DNA libraries.

  4. In vivo assembly of DNA-fragments in the moss, Physcomitrella patens

    DEFF Research Database (Denmark)

    King, Brian Christopher; Vavitsas, Konstantinos; Ikram, Nur Kusaira Binti Khairul

    2016-01-01

    enabled the complete replacement of eukaryotic chromosomes with heterologous DNA. The moss Physcomitrella patens, a non-vascular and spore producing land plant (Bryophyte), has a well-established capacity for homologous recombination. Here, we demonstrate the in vivo assembly of multiple DNA fragments...

  5. The dynamic assembly of distinct RNA polymerase I complexes modulates rDNA transcription.

    Science.gov (United States)

    Torreira, Eva; Louro, Jaime Alegrio; Pazos, Irene; González-Polo, Noelia; Gil-Carton, David; Duran, Ana Garcia; Tosi, Sébastien; Gallego, Oriol; Calvo, Olga; Fernández-Tornero, Carlos

    2017-03-06

    Cell growth requires synthesis of ribosomal RNA by RNA polymerase I (Pol I). Binding of initiation factor Rrn3 activates Pol I, fostering recruitment to ribosomal DNA promoters. This fundamental process must be precisely regulated to satisfy cell needs at any time. We present in vivo evidence that, when growth is arrested by nutrient deprivation, cells induce rapid clearance of Pol I-Rrn3 complexes, followed by the assembly of inactive Pol I homodimers. This dual repressive mechanism reverts upon nutrient addition, thus restoring cell growth. Moreover, Pol I dimers also form after inhibition of either ribosome biogenesis or protein synthesis. Our mutational analysis, based on the electron cryomicroscopy structures of monomeric Pol I alone and in complex with Rrn3, underscores the central role of subunits A43 and A14 in the regulation of differential Pol I complexes assembly and subsequent promoter association.

  6. Structural and thermodynamic analysis of modified nucleosides in self-assembled DNA cross-tiles.

    Science.gov (United States)

    Hakker, Lauren; Marchi, Alexandria N; Harris, Kimberly A; LaBean, Thomas H; Agris, Paul F

    2014-01-01

    DNA Holliday junctions are important natural strand-exchange structures that form during homologous recombination. Immobile four-arm junctions, analogs to Holliday junctions, have been designed to self-assemble into cross-tile structures by maximizing Watson-Crick base pairing and fixed crossover points. The cross-tiles, self-assembled from base pair recognition between designed single-stranded DNAs, form higher order lattice structures through cohesion of self-associating sticky ends. These cross-tiles have 16 unpaired nucleosides in the central loop at the junction of the four duplex stems. The importance of the centralized unpaired nucleosides to the structure's thermodynamic stability and self-assembly is unknown. Cross-tile DNA nanostructures were designed and constructed from nine single-stranded DNAs with four shell strands, four arms, and a central loop containing 16 unpaired bases. The 16 unpaired bases were either 2'-deoxyribothymidines, 2'-O-methylribouridines, or abasic 1',2'-dideoxyribonucleosides. Thermodynamic profiles and structural base-stacking contributions were assessed using UV absorption spectroscopy during thermal denaturation and circular dichroism spectroscopy, respectively, and the resulting structures were observed by atomic force microscopy. There were surprisingly significant changes in the thermodynamic and structural properties of lattice formation as a result of altering only the 16 unpaired, centralized nucleosides. The 16 unpaired 2'-O-methyluridines were stabilizing and produced uniform tubular structures. In contrast, the abasic nucleosides were destabilizing producing a mixture of structures. These results strongly indicate the importance of a small number of centrally located unpaired nucleosides within the structures. Since minor modifications lead to palpable changes in lattice formation, DNA cross-tiles present an easily manipulated structure convenient for applications in biomedical and biosensing devices.

  7. Crystal structure of vaccinia virus uracil-DNA glycosylase reveals dimeric assembly

    Directory of Open Access Journals (Sweden)

    DeLucas Lawrence

    2007-07-01

    Full Text Available Abstract Background Uracil-DNA glycosylases (UDGs catalyze excision of uracil from DNA. Vaccinia virus, which is the prototype of poxviruses, encodes a UDG (vvUDG that is significantly different from the UDGs of other organisms in primary, secondary and tertiary structure and characteristic motifs. It adopted a novel catalysis-independent role in DNA replication that involves interaction with a viral protein, A20, to form the processivity factor. UDG:A20 association is essential for assembling of the processive DNA polymerase complex. The structure of the protein must have provisions for such interactions with A20. This paper provides the first glimpse into the structure of a poxvirus UDG. Results Results of dynamic light scattering experiments and native size exclusion chromatography showed that vvUDG is a dimer in solution. The dimeric assembly is also maintained in two crystal forms. The core of vvUDG is reasonably well conserved but the structure contains one additional β-sheet at each terminus. A glycerol molecule is found in the active site of the enzyme in both crystal forms. Interaction of this glycerol molecule with the protein possibly mimics the enzyme-substrate (uracil interactions. Conclusion The crystal structures reveal several distinctive features of vvUDG. The new structural features may have evolved for adopting novel functions in the replication machinery of poxviruses. The mode of interaction between the subunits in the dimers suggests a possible model for binding to its partner and the nature of the processivity factor in the polymerase complex.

  8. Self-assembled alignment of nanorod by using DNA brush (Conference Presentation)

    Science.gov (United States)

    Ijiro, Kuniharu; Nakamura, Satoshi; Mitomo, Hideyuki; Pike, Andrew; Matsuo, Yasutaka; Niikura, Kenichi

    2016-09-01

    Surface modification with polymer is widely applied to various kinds of applications. Recently, polymer brushes, which is a layer of polymers attached with one end to a surface, have attracted much attention as functionalized surfaces. In particular, ionic polymer brushes provide ultra-low friction or anti-fouling because they act as highly hydrated soft film. Almost ionic polymer brushes have been prepared from synthetic polymers. Few biopolymers have been investigated for polymer brush studies. DNA which is one of ionic biopolymers has unique functions and conformations which synthetic polymers don't have. We found that cationic gold nanorods (30 x 10 nm) were adsorbed to DNA bush (148 bp) prepared on a glass surface in an aqueous solution by observation using extinction spectra. When the cationic charge density of gold nanorods were decreased, nanorods were immobilized perpendicularly to the substrate by binding to DNA elongated. This indicates that self-assembled alignment of gold nanorods can be achieved by using DNA brush. Formed aligned gold nanorods can be used for plasmonic color analysis.

  9. A new vision for the automation systems engineering for Automotive Powertrain Assembly

    OpenAIRE

    Haq, Izhar; Monfared, Radmehr P.; Harrison, R; Lee, Leslie; West, A A

    2010-01-01

    Abstract Pressure on the powertrain sector of automotive industry is mounting as market demand for higher variety and lower cost automation systems increases. To maintain the market competitiveness, design-to-market time for new products should be significantly shorter and considerable cost saving needs to be made during the design and manufacture of production facilities. Virtual construction, test and validation of systems prior to build is now identified as crucial because engin...

  10. Assembling high activity phosphotriesterase composites using hybrid nanoparticle peptide-DNA scaffolded architectures

    Science.gov (United States)

    Breger, Joyce C.; Buckhout-White, Susan; Walper, Scott A.; Oh, Eunkeu; Susumu, Kimihiro; Ancona, Mario G.; Medintz, Igor L.

    2017-06-01

    Nanoparticle (NP) display potentially offers a new way to both stabilize and, in many cases, enhance enzyme activity over that seen for native protein in solution. However, the large, globular and sometimes multimeric nature of many enzymes limits their ability to attach directly to the surface of NPs, especially when the latter are colloidally stabilized with bulky PEGylated ligands. Engineering extended protein linkers into the enzymes to achieve direct attachment through the PEG surface often detrimentally alters the enzymes catalytic ability. Here, we demonstrate an alternate, hybrid biomaterials-based approach to achieving directed enzyme assembly on PEGylated NPs. We self-assemble a unique architecture consisting of a central semiconductor quantum dot (QD) scaffold displaying controlled ratios of extended peptide-DNA linkers which penetrate through the PEG surface to directly couple enzymes to the QD surface. As a test case, we utilize phosphotriesterase (PTE), an enzyme of bio-defense interest due to its ability to hydrolyze organophosphate nerve agents. Moreover, this unique approach still allows PTE to maintain enhanced activity while also suggesting the ability of DNA to enhance enzyme activity in and of itself.

  11. Cholesterol-conjugated supramolecular assemblies of low generations polyamidoamine dendrimers for enhanced EGFP plasmid DNA transfection

    Energy Technology Data Exchange (ETDEWEB)

    Golkar, Nasim; Samani, Soliman Mohammadi; Tamaddon, Ali Mohammad, E-mail: amtamadon@gmail.com [Shiraz University of Medical Sciences, Department of Pharmaceutics, School of Pharmacy (Iran, Islamic Republic of)

    2016-05-15

    Aimed to prepare an enhanced gene delivery system with low cytotoxicity and high transfection efficiency, various cholesterol-conjugated derivates of low generation polyamidoamine (PAMAM) dendrimers were prepared. The conjugates were characterized by TNBS assay, FTIR, and {sup 1}H-NMR spectroscopy. Self-assembly of the dendrimer conjugates (G1-Chol, G2-Chol, and G3-Chol) was investigated by pyrene assay. Following formation of the complexes between enhanced green fluorescence protein plasmid and the dendrimer conjugates at various N (primary amine)/P (phosphate) mole ratios, plasmid condensation, biologic stability, cytotoxicity, and protein expression were investigated. The conjugates self-assembled into micellar dispersions with the critical micelle concentration values (<50 µg/ml) depending on the dendrimer generation and cholesterol/amine mole ratio. Cholesterol conjugation resulted in higher resistance of the condensed plasmid DNA in a competition assay with heparin sulfate. Also, the transfection efficiency was determined higher for the cholesterol conjugates than unmodified dendrimers in HepG2 cells, showing the highest for G2-Chol at 40 % degree of cholesterol modification (G2-Chol{sub 40 %}) among various dendrimer generations. Interestingly, such conjugate showed a complete protection of plasmid against serum nucleases. Our results confirmed that the cholesterol conjugation to PAMAM dendrimers of low generations bearing little cytotoxicity improves their several physicochemical and biological characteristics required for an enhanced delivery of plasmid DNA into cells.

  12. Automated simultaneous assembly of multi-stage testing for the uniform CPA examination

    NARCIS (Netherlands)

    Breithaupt, Krista; Ariel, A.; Veldkamp, Bernard P.

    2004-01-01

    Some solutions used in the assembly of the computerized Uniform Certified Public Accountancy (CPA) licensing examination are offered as practical alternatives for operational programs producing large numbers of forms. The Uniform CPA examination will be offered as an adaptive multi-stage test (MST)

  13. Vision feedback driven automated assembly of photopolymerized structures by parallel optical trapping and manipulation

    DEFF Research Database (Denmark)

    Dam, Jeppe Seidelin; Perch-Nielsen, Ivan Ryberg; Rodrigo, Peter John

    2007-01-01

    We demonstrate how optical trapping and manipulation can be used to assemble microstructures. The microstructures we show being automatically recognized and manipulated are produced using the two-photon polymerization (2PP) technique with submicron resolution. In this work, we show identical shape...

  14. A Problem of Particulate Contamination in an Automated Assembly Machine Successfully Solved by CFD and Simple Experiments

    Directory of Open Access Journals (Sweden)

    Jatuporn Thongsri

    2017-01-01

    Full Text Available Assembly of hard disk drives (HDDs needs to be done in an automated assembly machine (AAM virtually free of particulate contamination that can cause them to malfunction. Fan filter units (FFUs are installed above the AAM to reduce the number of suspended particles in the recirculating air flowing over and around them. At one time, several HDDs were found to be defective. To find out the root cause of this problem, computational fluid dynamics (CFD was used to investigate the airflow over and around the AAM. It was found that the cause of the high particle counts was improper air speed from the FFUs. The optimal FFUs air speed needed to be in the range of 0.35–0.65 m/s in which the airflow would block out nearby airborne particles and purge away particles generated by the AAM effectively which would, in effect, reduce the particle counts down below the threshold level of class 100 clean room. A few available measurement tools at the factory were then used to perform validating measurements against the simulation results, and the validation was positive. This optimal speed range was implemented at the factory after which the level of contamination was reduced to an acceptable level.

  15. Application of X-ray digital radiography to online automated inspection of interior assembly structures of complex products

    Energy Technology Data Exchange (ETDEWEB)

    Han Yueping [National Key Laboratory for Electronic Measurement Technology, North University of China, Taiyuan 030051 (China) and Key Laboratory of Instrumentation Science and Dynamic Measurement (North University of China), Ministry of Education, Taiyuan 030051 (China)], E-mail: yuepinghan@163.com; Han Yan [Key Laboratory of Instrumentation Science and Dynamic Measurement (North University of China), Ministry of Education, Taiyuan 030051 (China); Li Ruihong [National Key Laboratory for Electronic Measurement Technology, North University of China, Taiyuan 030051 (China); Wang Liming [Key Laboratory of Instrumentation Science and Dynamic Measurement (North University of China), Ministry of Education, Taiyuan 030051 (China)

    2009-06-11

    The paper proposes an application of X-ray digital radiography to online automated inspection and recognition of the interior assembly structures of complex products by means of the multiple views techniques. First, a vertical hybrid projection function (VHPF) is proposed as the recognition feature of a two-dimensional image. VHPF combines an integral projection function and a standard deviation function so that it can reflect the mean and the variance of the pixels in the vertical direction in an image. Secondly, by considering the different importance grades of objects inside the product and the independence of these objects along the circumference, the paper presents a hierarchical recognition method and uses a neural network system to speed up the computation process with parallel operations. Thirdly, using the whole-orientation features of one standard swatch and by extracting its maximal system of linear independence as the feature basis, the issue of blind areas for recognition is resolved. Based on this approach, the first domestic X-ray multi-view digital detection system has been developed and applied to the online detection of objects containing complicated assembly structures.

  16. Application of X-ray digital radiography to online automated inspection of interior assembly structures of complex products

    International Nuclear Information System (INIS)

    Han Yueping; Han Yan; Li Ruihong; Wang Liming

    2009-01-01

    The paper proposes an application of X-ray digital radiography to online automated inspection and recognition of the interior assembly structures of complex products by means of the multiple views techniques. First, a vertical hybrid projection function (VHPF) is proposed as the recognition feature of a two-dimensional image. VHPF combines an integral projection function and a standard deviation function so that it can reflect the mean and the variance of the pixels in the vertical direction in an image. Secondly, by considering the different importance grades of objects inside the product and the independence of these objects along the circumference, the paper presents a hierarchical recognition method and uses a neural network system to speed up the computation process with parallel operations. Thirdly, using the whole-orientation features of one standard swatch and by extracting its maximal system of linear independence as the feature basis, the issue of blind areas for recognition is resolved. Based on this approach, the first domestic X-ray multi-view digital detection system has been developed and applied to the online detection of objects containing complicated assembly structures.

  17. Application of X-ray digital radiography to online automated inspection of interior assembly structures of complex products

    Science.gov (United States)

    Han, Yueping; Han, Yan; Li, Ruihong; Wang, Liming

    2009-06-01

    The paper proposes an application of X-ray digital radiography to online automated inspection and recognition of the interior assembly structures of complex products by means of the multiple views techniques. First, a vertical hybrid projection function (VHPF) is proposed as the recognition feature of a two-dimensional image. VHPF combines an integral projection function and a standard deviation function so that it can reflect the mean and the variance of the pixels in the vertical direction in an image. Secondly, by considering the different importance grades of objects inside the product and the independence of these objects along the circumference, the paper presents a hierarchical recognition method and uses a neural network system to speed up the computation process with parallel operations. Thirdly, using the whole-orientation features of one standard swatch and by extracting its maximal system of linear independence as the feature basis, the issue of blind areas for recognition is resolved. Based on this approach, the first domestic X-ray multi-view digital detection system has been developed and applied to the online detection of objects containing complicated assembly structures.

  18. Automated Computational Inference of Multi-protein Assemblies from Biochemical Co-purification Data.

    Science.gov (United States)

    Goebels, Florian; Hu, Lucas; Bader, Gary; Emili, Andrew

    2018-01-01

    Biology has amassed a wealth of information about the function of a multitude of protein-coding genes across species. The challenge now is to understand how all these proteins work together to form a living organism, and a crucial step for gaining this knowledge is a complete description of the molecular "wiring circuits" that underlie cellular processes. In this chapter, we describe a general computational framework for predicting multi-protein assemblies from biochemical co-fractionation data.

  19. Automated estimator parameter selection for an IBM head/disk assembly.

    Science.gov (United States)

    Thein, May-Win L; Rendon, Thomas; Misawa, Eduardo A

    2005-07-01

    This paper presents the application of a discrete adaptive observer (DAO) to an IBM head/disk assembly system. Because of the difficulties in tuning, a genetic algorithm is implemented off-line to obtain optimal observer parameters for the DAO. Simulations show that the genetic algorithm is successful in choosing appropriate observer gains. Furthermore, as a result of these optimal gains, the observer state and parameter estimates converge accurately and quickly.

  20. Assembling and auditing a comprehensive DNA barcode reference library for European marine fishes.

    Science.gov (United States)

    Oliveira, L M; Knebelsberger, T; Landi, M; Soares, P; Raupach, M J; Costa, F O

    2016-12-01

    A large-scale comprehensive reference library of DNA barcodes for European marine fishes was assembled, allowing the evaluation of taxonomic uncertainties and species genetic diversity that were otherwise hidden in geographically restricted studies. A total of 4118 DNA barcodes were assigned to 358 species generating 366 Barcode Index Numbers (BIN). Initial examination revealed as much as 141 BIN discordances (more than one species in each BIN). After implementing an auditing and five-grade (A-E) annotation protocol, the number of discordant species BINs was reduced to 44 (13% grade E), while concordant species BINs amounted to 271 (78% grades A and B) and 14 other had insufficient data (grade D). Fifteen species displayed comparatively high intraspecific divergences ranging from 2·6 to 18·5% (grade C), which is biologically paramount information to be considered in fish species monitoring and stock assessment. On balance, this compilation contributed to the detection of 59 European fish species probably in need of taxonomic clarification or re-evaluation. The generalized implementation of an auditing and annotation protocol for reference libraries of DNA barcodes is recommended. © 2016 The Fisheries Society of the British Isles.

  1. Assembly of Francisella novicida Cpf1 endonuclease in complex with guide RNA and target DNA.

    Science.gov (United States)

    Alcón, Pablo; Montoya, Guillermo; Stella, Stefano

    2017-07-01

    Bacteria and archaea use the CRISPR-Cas system as an adaptive response against infection by foreign nucleic acids. Owing to its remarkable flexibility, this mechanism has been harnessed and adopted as a powerful tool for genome editing. The CRISPR-Cas system includes two classes that are subdivided into six types and 19 subtypes according to conservation of the cas gene and loci organization. Recently, a new protein with endonuclease activity belonging to class 2 type V has been identified. This endonuclease, termed Cpf1, in complex with a single CRISPR RNA (crRNA) is able to recognize and cleave a target DNA preceded by a 5'-TTN-3' protospacer-adjacent motif (PAM) complementary to the RNA guide. To obtain structural insight into the inner workings of Cpf1, the crystallization of an active complex containing the full extent of the crRNA and a 31-nucleotide dsDNA target was attempted. The gene encoding Cpf1 from Francisella novicida was cloned, overexpressed and purified. The crRNA was transcribed and purified in vitro. Finally, the ternary FnCpf1-crRNA-DNA complex was assembled and purified by preparative electrophoresis before crystallization. Crystals belonging to space group C222 1 , with unit-cell parameters a = 85.2, b = 137.6, c = 320.5 Å, were obtained and subjected to preliminary diffraction experiments.

  2. Self-assembling of calcium salt of the new DNA base 5-carboxylcytosine

    Science.gov (United States)

    Irrera, Simona; Ruiz-Hernandez, Sergio E.; Reggente, Melania; Passeri, Daniele; Natali, Marco; Gala, Fabrizio; Zollo, Giuseppe; Rossi, Marco; Portalone, Gustavo

    2017-06-01

    Supramolecular architectures involving DNA bases can have a strong impact in several fields such as nanomedicine and nanodevice manufacturing. To date, in addition to the four canonical nucleobases (adenine, thymine, guanine and cytosine), four other forms of cytosine modified at the 5 position have been identified in DNA. Among these four new cytosine derivatives, 5-carboxylcytosine has been recently discovered in mammalian stem cell DNA, and proposed as the final product of the oxidative epigenetic demethylation pathway on the 5 position of cytosine. In this work, a calcium salt of 5-carboxylcytosine has been synthesized and deposited on graphite surface, where it forms self-assembled features as long range monolayers and up to one micron long filaments. These structures have been analyzed in details combining different theoretical and experimental approaches: X-ray single-crystal diffraction data were used to simulate the molecule-graphite interaction, first using molecular dynamics and then refining the results using density functional theory (DFT); finally, data obtained with DFT were used to rationalize atomic force microscopy (AFM) results.

  3. Evaluation of five automated and one manual method for Toxoplasma and human DNA extraction from artificially spiked amniotic fluid.

    Science.gov (United States)

    Yera, H; Ménégaut, L; Brenier-Pinchart, M-P; Touafek, F; Bastien, P; Dalle, F

    2018-01-31

    Molecular detection of Toxoplasma gondii plays a crucial role in the prenatal and neonatal diagnosis of congenital toxoplasmosis (CT). Sensitivity of this diagnosis is partly related to the efficiency of parasite DNA extraction and amplification. DNA extraction methods with automated platforms have been developed. Therefore, it is essential to evaluate them in combination with adequate PCR amplification assays. In this multisite study, we investigated the suitability of two recent automated procedures for the isolation of Toxoplasma DNA from amniotic fluid (AF) (Magtration system 12GC, PSS and Freedom EVO VacS, Tecan), compared with three other automated procedures (MagNAPure Compact, Roche, BioRobot EZ1, Qiagen and modified NucliSens easyMAG, bioMérieux) and with the manual DNA extraction QIAamp DNA Mini kit (Qiagen). Two Toxoplasma PCR assays targeting the '529-bp' repeat DNA element were used, based upon dual hybridization (FRET) or hydrolysis (TaqMan) probes. A total of 1296 PCRs were performed including 972 Toxoplasma PCRs. We showed variable efficacy (4.2%-100% positive results) among the DNA extraction procedures in isolating up to five T. gondii cells/mL in AF samples. Moreover, for a given DNA extraction method, variable results were obtained among the two Toxoplasma PCR assays for detecting up to five T. gondii cells/mL: when using TaqMan PCR, all the automated systems yielded more than 60% positive results. Nevertheless, when testing the DNA extracts in triplicate, four out of six extraction methods allowed a satisfactory detection of low amounts of T. gondii DNA (≥33% of positive results) independently of the PCR assay used. Despite the influence of the subsequent PCR method used, this study should help microbiologists in the choice of DNA extraction methods for the detection of T. gondii in amniotic fluid. The extraction method should be checked as adequate for the PCR assay used. Copyright © 2018 European Society of Clinical Microbiology and

  4. Automated assembling of single fuel cell units for use in a fuel cell stack

    Science.gov (United States)

    Jalba, C. K.; Muminovic, A.; Barz, C.; Nasui, V.

    2017-05-01

    The manufacturing of PEMFC stacks (POLYMER ELEKTROLYT MEMBRAN Fuel Cell) is nowadays still done by hand. Over hundreds of identical single components have to be placed accurate together for the construction of a fuel cell stack. Beside logistic problems, higher total costs and disadvantages in weight the high number of components produce a higher statistic interference because of faulty erection or material defects and summation of manufacturing tolerances. The saving of costs is about 20 - 25 %. Furthermore, the total weight of the fuel cells will be reduced because of a new sealing technology. Overall a one minute cycle time has to be aimed per cell at the manufacturing of these single components. The change of the existing sealing concept to a bonded sealing is one of the important requisites to get an automated manufacturing of single cell units. One of the important steps for an automated gluing process is the checking of the glue application by using of an image processing system. After bonding the single fuel cell the sealing and electrical function can be checked, so that only functional and high qualitative cells can get into further manufacturing processes.

  5. Analysis of the self-assembly of 4x4 DNA tiles by temperature-dependent FRET spectroscopy.

    Science.gov (United States)

    Saccà, Barbara; Meyer, Rebecca; Niemeyer, Christof M

    2009-12-21

    Correct and efficient self-assembly of oligonucleotides into highly ordered superstructures essentially depends on the structural integrity and thermal stability of DNA motifs such as junctions or tiles that build up the superstructure. To investigate the assembly/disassembly process of DNA tiles, we recently described a microplate-based method employing Förster resonance energy transfer (FRET) spectroscopy, which enables the analysis of DNA superstructure formation in real time and with high throughput. This method allows thermodynamic parameters of the self-assembly process to be extracted, and we here apply it for detailed analysis of the self-assembly of five different 4x4 DNA tile motifs. To specifically investigate whether the FRET probes tethered to the DNA motifs report local thermodynamic stabilities in the immediate proximity of the chromophores, or whether the global stability of the entire motif is monitored, systematic variations of the labeling position within one tile are carried out. Combined with gel electrophoretic, UV spectroscopic, and microcalorimetric analysis, this study reveals that the FRET method mainly reports the thermodynamics of local microenvironment assembly, rather than that of the entire motif. Nonetheless, the thermodynamic data derived from FRET analysis are also influenced by the structural surroundings of the motif, and thus rapid and detailed analysis and identification of potential "weak points" within a superstructure which influence the structural integrity of a given tile design are enabled. Therefore, the microplate FRET method readily provides insights into the assembly process of complex DNA superstructures to verify and complement theoretical design approaches.

  6. The self-assembly of particles with isotropic interactions: Using DNA coated colloids to create designer nanomaterials

    International Nuclear Information System (INIS)

    Thompson, R. B.; Dion, S.; Konigslow, K. von

    2014-01-01

    Self-consistent field theory equations are presented that are suitable for use as a coarse-grained model for DNA coated colloids, polymer-grafted nanoparticles and other systems with approximately isotropic interactions. The equations are generalized for arbitrary numbers of chemically distinct colloids. The advantages and limitations of such a coarse-grained approach for DNA coated colloids are discussed, as are similarities with block copolymer self-assembly. In particular, preliminary results for three species self-assembly are presented that parallel results from a two dimensional ABC triblock copolymer phase. The possibility of incorporating crystallization, dynamics, inverse statistical mechanics and multiscale modelling techniques are discussed

  7. NASA Wrangler: Automated Cloud-Based Data Assembly in the RECOVER Wildfire Decision Support System

    Science.gov (United States)

    Schnase, John; Carroll, Mark; Gill, Roger; Wooten, Margaret; Weber, Keith; Blair, Kindra; May, Jeffrey; Toombs, William

    2017-01-01

    NASA Wrangler is a loosely-coupled, event driven, highly parallel data aggregation service designed to take advantageof the elastic resource capabilities of cloud computing. Wrangler automatically collects Earth observational data, climate model outputs, derived remote sensing data products, and historic biophysical data for pre-, active-, and post-wildfire decision making. It is a core service of the RECOVER decision support system, which is providing rapid-response GIS analytic capabilities to state and local government agencies. Wrangler reduces to minutes the time needed to assemble and deliver crucial wildfire-related data.

  8. Detection of anti-dsDNA antibodies by computer-aided automated immunofluorescence analysis.

    Science.gov (United States)

    Lakos, Gabriella; Gonzalez, Marlene; Flaherty, David; Bentow, Chelsea; Ibarra, Claudia; Stimson, Deborah; Nacario, Lori; Hiemann, Rico; Dervieux, Thierry

    2016-06-01

    NOVA View is a computer aided fluorescence microscope that is used for the automated reading and interpretation of indirect immunofluorescent tests in diagnostic immunology. The objective of the present study was to evaluate the performance of the NOVA View® system for the measurement of anti-dsDNA antibodies using the Crithidia luciliae indirect immunofluorescence test (CLIFT) technology. Analytical performance of NOVA View CLIFT was assessed in repeatability (within run) and reproducibility (between runs and instruments) studies. Two hundred-fifty patient samples (N=200 consecutive samples and N=50 samples from systemic lupus erythematosus patients) were tested to evaluate the agreement between results generated with NOVA View CLIFT, and those obtained with manual microscopic reading of the same slides. Positivity rate in SLE was assessed on the 50 SLE samples. The NOVA View system showed high level of repeatability and reproducibility within runs, between runs, and between instruments. Agreement of NOVA View software interpretation and digital image reading results with manual microscopic reading results was 96.0%, and the same positivity rate was obtained on SLE samples by NOVA View digital image reading as that of manual microscopic reading (36.0% vs. 38.0%, respectively). Results generated by NOVA View CLIFT were equivalent to those obtained by manual microscopic reading on a large routine sample set. NOVA View demonstrated consistency within and between runs, and between instruments. Automation of CLIFT provides reliability and is a suitable alternative for routine clinical laboratories. Copyright © 2016 The Authors. Published by Elsevier B.V. All rights reserved.

  9. Functional interfaces for biomimetic energy harvesting: CNTs-DNA matrix for enzyme assembly.

    Science.gov (United States)

    Hjelm, Rachel M E; Garcia, Kristen E; Babanova, Sofia; Artyushkova, Kateryna; Matanovic, Ivana; Banta, Scott; Atanassov, Plamen

    2016-05-01

    The development of 3D structures exploring the properties of nano-materials and biological molecules has been shown through the years as an effective path forward for the design of advanced bio-nano architectures for enzymatic fuel cells, photo-bio energy harvesting devices, nano-biosensors and bio-actuators and other bio-nano-interfacial architectures. In this study we demonstrate a scaffold design utilizing carbon nanotubes, deoxyribose nucleic acid (DNA) and a specific DNA binding transcription factor that allows for directed immobilization of a single enzyme. Functionalized carbon nanotubes were covalently bonded to a diazonium salt modified gold surface through carbodiimide chemistry creating a brush-type nanotube alignment. The aligned nanotubes created a highly ordered structure with high surface area that allowed for the attachment of a protein assembly through a designed DNA scaffold. The enzyme immobilization was controlled by a zinc finger (ZNF) protein domain that binds to a specific dsDNA sequence. ZNF 268 was genetically fused to the small laccase (SLAC) from Streptomyces coelicolor, an enzyme belonging to the family of multi-copper oxidases, and used to demonstrate the applicability of the developed approach. Analytical techniques such as X-ray photoelectron spectroscopy (XPS), atomic force microscopy (AFM), and enzymatic activity analysis, allowed characterization at each stage of development of the bio-nano architecture. This article is part of a Special Issue entitled Biodesign for Bioenergetics--the design and engineering of electronic transfer cofactors, proteins and protein networks, edited by Ronald L. Koder and J.L. Ross Anderson. Copyright © 2015 Elsevier B.V. All rights reserved.

  10. CasEMBLR: Cas9-Facilitated Multiloci Genomic Integration of in Vivo Assembled DNA Parts in Saccharomyces cerevisiae

    DEFF Research Database (Denmark)

    Jakociunas, Tadas; Rajkumar, Arun Stephen; Zhang, Jie

    2015-01-01

    , we present a method for marker-free multiloci integration of in vivo assembled DNA parts. By the use of CRISPR/Cas9-mediated one-step double-strand breaks at single, double and triple integration sites we report the successful in vivo assembly and chromosomal integration of DNA parts. We call our......Homologous recombination (HR) in Saccharomyces cerevisiae has been harnessed for both plasmid construction and chromosomal integration of foreign DNA. Still, native HR machinery is not efficient enough for complex and marker-free genome engineering required for modern metabolic engineering. Here...... method CasEMBLR and validate its applicability for genome engineering and cell factory development in two ways: (i) introduction of the carotenoid pathway from 15 DNA parts into three targeted loci, and (ii) creation of a tyrosine production strain using ten parts into two loci, simultaneously knocking...

  11. Automated DNA mutation detection using universal conditions direct sequencing: application to ten muscular dystrophy genes

    Directory of Open Access Journals (Sweden)

    Wu Bai-Lin

    2009-10-01

    patients. Methods and assay sequences are reported in this paper. Conclusion This automated process allows laboratories to discover DNA variations in a short time and at low cost.

  12. Automated DNA mutation detection using universal conditions direct sequencing: application to ten muscular dystrophy genes.

    Science.gov (United States)

    Bennett, Richard R; Schneider, Hal E; Estrella, Elicia; Burgess, Stephanie; Cheng, Andrew S; Barrett, Caitlin; Lip, Va; Lai, Poh San; Shen, Yiping; Wu, Bai-Lin; Darras, Basil T; Beggs, Alan H; Kunkel, Louis M

    2009-10-18

    this paper. This automated process allows laboratories to discover DNA variations in a short time and at low cost.

  13. Healing assessment of tile sets for error tolerance in DNA self-assembly.

    Science.gov (United States)

    Hashempour, M; Mashreghian Arani, Z; Lombardi, F

    2008-12-01

    An assessment of the effectiveness of healing for error tolerance in DNA self-assembly tile sets for algorithmic/nano-manufacturing applications is presented. Initially, the conditions for correct binding of a tile to an existing aggregate are analysed using a Markovian approach; based on this analysis, it is proved that correct aggregation (as identified with a so-called ideal tile set) is not always met for the existing tile sets for nano-manufacturing. A metric for assessing tile sets for healing by utilising punctures is proposed. Tile sets are investigated and assessed with respect to features such as error (mismatched tile) movement, punctured area and bond types. Subsequently, it is shown that the proposed metric can comprehensively assess the healing effectiveness of a puncture type for a tile set and its capability to attain error tolerance for the desired pattern. Extensive simulation results are provided.

  14. Mapping Nanoscale Hotspots with Single-Molecule Emitters Assembled into Plasmonic Nanocavities Using DNA Origami

    Science.gov (United States)

    Chikkaraddy, Rohit; Turek, V. A.; Kongsuwan, Nuttawut; Benz, Felix; Carnegie, Cloudy; van de Goor, Tim; de Nijs, Bart; Demetriadou, Angela; Hess, Ortwin; Keyser, Ulrich F.; Baumberg, Jeremy J.

    2018-01-01

    Fabricating nanocavities in which optically-active single quantum emitters are precisely positioned, is crucial for building nanophotonic devices. Here we show that self-assembly based on robust DNA-origami constructs can precisely position single molecules laterally within sub-5nm gaps between plasmonic substrates that support intense optical confinement. By placing single-molecules at the center of a nanocavity, we show modification of the plasmon cavity resonance before and after bleaching the chromophore, and obtain enhancements of $\\geq4\\times10^3$ with high quantum yield ($\\geq50$%). By varying the lateral position of the molecule in the gap, we directly map the spatial profile of the local density of optical states with a resolution of $\\pm1.5$ nm. Our approach introduces a straightforward non-invasive way to measure and quantify confined optical modes on the nanoscale.

  15. Discovery of human inversion polymorphisms by comparative analysis of human and chimpanzee DNA sequence assemblies.

    Directory of Open Access Journals (Sweden)

    Lars Feuk

    2005-10-01

    Full Text Available With a draft genome-sequence assembly for the chimpanzee available, it is now possible to perform genome-wide analyses to identify, at a submicroscopic level, structural rearrangements that have occurred between chimpanzees and humans. The goal of this study was to investigate chromosomal regions that are inverted between the chimpanzee and human genomes. Using the net alignments for the builds of the human and chimpanzee genome assemblies, we identified a total of 1,576 putative regions of inverted orientation, covering more than 154 mega-bases of DNA. The DNA segments are distributed throughout the genome and range from 23 base pairs to 62 mega-bases in length. For the 66 inversions more than 25 kilobases (kb in length, 75% were flanked on one or both sides by (often unrelated segmental duplications. Using PCR and fluorescence in situ hybridization we experimentally validated 23 of 27 (85% semi-randomly chosen regions; the largest novel inversion confirmed was 4.3 mega-bases at human Chromosome 7p14. Gorilla was used as an out-group to assign ancestral status to the variants. All experimentally validated inversion regions were then assayed against a panel of human samples and three of the 23 (13% regions were found to be polymorphic in the human genome. These polymorphic inversions include 730 kb (at 7p22, 13 kb (at 7q11, and 1 kb (at 16q24 fragments with a 5%, 30%, and 48% minor allele frequency, respectively. Our results suggest that inversions are an important source of variation in primate genome evolution. The finding of at least three novel inversion polymorphisms in humans indicates this type of structural variation may be a more common feature of our genome than previously realized.

  16. Cholesterol-conjugated supramolecular assemblies of low generations polyamidoamine dendrimers for enhanced EGFP plasmid DNA transfection

    Science.gov (United States)

    Golkar, Nasim; Samani, Soliman Mohammadi; Tamaddon, Ali Mohammad

    2016-05-01

    Aimed to prepare an enhanced gene delivery system with low cytotoxicity and high transfection efficiency, various cholesterol-conjugated derivates of low generation polyamidoamine (PAMAM) dendrimers were prepared. The conjugates were characterized by TNBS assay, FTIR, and 1H-NMR spectroscopy. Self-assembly of the dendrimer conjugates (G1-Chol, G2-Chol, and G3-Chol) was investigated by pyrene assay. Following formation of the complexes between enhanced green fluorescence protein plasmid and the dendrimer conjugates at various N (primary amine)/P (phosphate) mole ratios, plasmid condensation, biologic stability, cytotoxicity, and protein expression were investigated. The conjugates self-assembled into micellar dispersions with the critical micelle concentration values (transfection efficiency was determined higher for the cholesterol conjugates than unmodified dendrimers in HepG2 cells, showing the highest for G2-Chol at 40 % degree of cholesterol modification (G2-Chol40 %) among various dendrimer generations. Interestingly, such conjugate showed a complete protection of plasmid against serum nucleases. Our results confirmed that the cholesterol conjugation to PAMAM dendrimers of low generations bearing little cytotoxicity improves their several physicochemical and biological characteristics required for an enhanced delivery of plasmid DNA into cells.

  17. A Self-Assembled DNA Origami-Gold Nanorod Complex for Cancer Theranostics.

    Science.gov (United States)

    Jiang, Qiao; Shi, Yuefeng; Zhang, Qian; Li, Na; Zhan, Pengfei; Song, Linlin; Dai, Luru; Tian, Jie; Du, Yang; Cheng, Zhen; Ding, Baoquan

    2015-10-01

    A self-assembled DNA origami (DO)-gold nanorod (GNR) complex, which is a dual-functional nanotheranostics constructed by decorating GNRs onto the surface of DNA origami, is demonstrated. After 24 h incubation of two structured DO-GNR complexes with human MCF7 breast cancer cells, significant enhancement of cell uptake is achieved compared to bare GNRs by two-photon luminescence imaging. Particularly, the triangle shaped DO-GNR complex exhibits optimal cellular accumulation. Compared to GNRs, improved photothermolysis against tumor cells is accomplished for the triangle DO-GNR complex by two-photon laser or NIR laser irradiation. Moreover, the DO-GNR complex exhibits enhanced antitumor efficacy compared with bare GNRs in nude mice bearing breast tumor xenografts. The results demonstrate that the DO-GNR complex can achieve optimal two-photon cell imaging and photothermal effect, suggesting a promising candidate for cancer diagnosis and therapy both in vitro and in vivo. © 2015 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

  18. Hierarchical Assembly of Plasmonic Nanostructures using Virus Capsid Scaffolds on DNA Origami Tiles

    Energy Technology Data Exchange (ETDEWEB)

    Wang, Debin; Capehart, Stacy L.; Pal, Suchetan; Liu, Minghui; Zhang, Lei; Schuck, P. J.; Liu, Yan; Yan, Hao; Francis, Matthew B.; De Yoreo, James J.

    2014-07-07

    Plasmonic nanoarchitectures using biological scaffolds have shown the potential to attain controllable plasmonic fluorescence via precise spatial arrangement of fluorophores and plasmonic antennae. However, previous studies report a predominance of fluorescence quenching for small metal nanoparticles (less than ~60 nm) due to their small scattering cross-sections. In this work, we report the design and performance of fluorescent plasmonic structures composed of fluorophore-modified virus capsids and gold nanoparticles (AuNPs) assembled on DNA origami tiles. The virus capsid creates a scaffold for control over the three dimensional arrangement of the fluorophores, whereas the DNA origami tile provides precise control over the distance between the capsid and the AuNP. Using finite-difference time-domain (FDTD) numerical simulations and multimodal single-particle imaging measurements, we show that the judicial design of these structures places the dye molecules near the hot spot of the AuNP. This effectively increases the fluorescence intensity in the quenching regime of the AuNP, with an enhancement factor that increases with increasing AuNP size. This strategy of using biological scaffolds to control fluorescence paves the way for exploring the parameters that determine plasmonic fluorescence. It may lead to a better understanding of the antenna effects of photon absorption and emission, enabling the construction of multicomponent plasmonic systems.

  19. COBAS AMPLICOR: fully automated RNA and DNA amplification and detection system for routine diagnostic PCR.

    Science.gov (United States)

    DiDomenico, N; Link, H; Knobel, R; Caratsch, T; Weschler, W; Loewy, Z G; Rosenstraus, M

    1996-12-01

    The COBAS AMPLICOR system automates amplification and detection of target nucleic acids, making diagnostic PCR routine for a variety of infectious diseases. The system contains a single thermal cycler with two independently regulated heating/cooling blocks, an incubator, a magnetic particle washer, a pipettor, and a photometer. Amplified products are captured on oligonucleotide-coated paramagnetic microparticles and detected with use of an avidin-horseradish peroxidase (HRP) conjugate. Concentrated solutions of amplicon or HRP were pipetted without detectable carryover. Amplified DNA was detected with an intraassay CV of < 4.5%; the combined intraassay CV for amplification and detection was < 15%. No cross-reactivity was observed when three different target nucleic acids were amplified in a single reaction and detected with three target-specific capture probes. The initial COBAS AMPLICOR menu includes qualitative tests for diagnosing infections with Chlamydia trachomatis, Neisseria gonorrhoeae, Mycobacterium tuberculosis, and hepatitis C virus. All tests include an optional Internal Control to provide assurance that specimens are successfully amplified and detected.

  20. De Novo Assembly of Complete Chloroplast Genomes from Non-model Species Based on a K-mer Frequency-Based Selection of Chloroplast Reads from Total DNA Sequences

    Directory of Open Access Journals (Sweden)

    Shairul Izan

    2017-08-01

    Full Text Available Whole Genome Shotgun (WGS sequences of plant species often contain an abundance of reads that are derived from the chloroplast genome. Up to now these reads have generally been identified and assembled into chloroplast genomes based on homology to chloroplasts from related species. This re-sequencing approach may select against structural differences between the genomes especially in non-model species for which no close relatives have been sequenced before. The alternative approach is to de novo assemble the chloroplast genome from total genomic DNA sequences. In this study, we used k-mer frequency tables to identify and extract the chloroplast reads from the WGS reads and assemble these using a highly integrated and automated custom pipeline. Our strategy includes steps aimed at optimizing assemblies and filling gaps which are left due to coverage variation in the WGS dataset. We have successfully de novo assembled three complete chloroplast genomes from plant species with a range of nuclear genome sizes to demonstrate the universality of our approach: Solanum lycopersicum (0.9 Gb, Aegilops tauschii (4 Gb and Paphiopedilum henryanum (25 Gb. We also highlight the need to optimize the choice of k and the amount of data used. This new and cost-effective method for de novo short read assembly will facilitate the study of complete chloroplast genomes with more accurate analyses and inferences, especially in non-model plant genomes.

  1. Efficient self-assembly of DNA-functionalized fluorophores and gold nanoparticles with DNA functionalized silicon surfaces: the effect of oligomer spacers

    Science.gov (United States)

    Milton, James A.; Patole, Samson; Yin, Huabing; Xiao, Qiang; Brown, Tom; Melvin, Tracy

    2013-01-01

    Although strategies for the immobilization of DNA oligonucleotides onto surfaces for bioanalytical and top-down bio-inspired nanobiofabrication approaches are well developed, the effect of introducing spacer molecules between the surface and the DNA oligonucleotide for the hybridization of nanoparticle–DNA conjugates has not been previously assessed in a quantitative manner. The hybridization efficiency of DNA oligonucleotides end-labelled with gold nanoparticles (1.4 or 10 nm diameter) with DNA sequences conjugated to silicon surfaces via hexaethylene glycol phosphate diester oligomer spacers (0, 1, 2, 6 oligomers) was found to be independent of spacer length. To quantify both the density of DNA strands attached to the surfaces and hybridization with the surface-attached DNA, new methodologies have been developed. Firstly, a simple approach based on fluorescence has been developed for determination of the immobilization density of DNA oligonucleotides. Secondly, an approach using mass spectrometry has been created to establish (i) the mean number of DNA oligonucleotides attached to the gold nanoparticles and (ii) the hybridization density of nanoparticle–oligonucleotide conjugates with the silicon surface–attached complementary sequence. These methods and results will be useful for application with nanosensors, the self-assembly of nanoelectronic devices and the attachment of nanoparticles to biomolecules for single-molecule biophysical studies. PMID:23361467

  2. Design and experimental validation of a generic model for combinatorial assembly of DNA tiles into 1D-structures.

    Science.gov (United States)

    Laisne, Aude; Lesniewska, Eric; Pompon, Denis

    2011-06-01

    Quantitative modeling of the self-assembly of DNA tiles leading either to defined end-products or distribution of biopolymers is of practical importance for biotechnology and synthetic biology. The combinatorial process describing tile assembly was implemented into a generic algorithm allowing quantitative description of the population of significant species accumulating during the reaction course. Experimental formation and characterization by optical and electrophoresis approaches of copolymers resulting from the self-assembly of a limited number of half-complementary tiles were used to define and validate generic rules allowing definition of model parameters. Factors controlling the structure and the dynamic of the oligomer population were evidenced for assemblies leading or not to defined end-products. Primary parameters were experimentally determined using rapid mixing experiments. Adjustment of simulations to experimental profiles allowed definition of generic rules for calculation of secondary parameters that take into account macro- and microenvironment of individual hybridization steps. In the case of copolymers, accurate simulation of experimental profiles was achieved for formation of linear assemblies. Overall length of species and structure of the DNA regions flanking the hybridization sites are critical parameters for which calculation rules were defined. The computational approach quantitatively predicted the parameters affecting time-course and distribution of accumulating products for different experimental designs. The computational and parameter evaluation procedures designed for the assembly of DNA tiles into large 1D-structures are more generally applicable for the construction of non-DNA polymers by extremities-specific recognition of molecular blocks. Copyright © 2011 Elsevier B.V. All rights reserved.

  3. Characterization and Application of DNA-templated Silver Nanoclusters and Polarized Spectroscopy of Self-Assembled Nanostructures

    DEFF Research Database (Denmark)

    Carro-Temboury, Miguel R.

    In this thesis two different systems are investigated envisioning their potential applications: DNA-templated silver nanoclusters (DNA-AgNCs) and ionic self-assembled (ISA) nanostructures based on azo-dyes. Mainly Visible-NIR spectroscopy was used to probe electronic transitions with absorbance...... other applications are possible such as detection of analytes, pH detection or their use as active layer of Organic Light Emitting Diodes (OLEDs). The fluorophores studied here, DNAAgNCs, are few nanometer sized and formed by a few to ca. 20 silver atoms templated by one or two single stranded DNA (ss...... fluorescence decay time as a function of emission wavelength (Average decay time spectra). This proved to be a robust method to characterize the complexity of the system due to the multi-exponential decay of the emitters. In order to study the electroluminescence from the C24-AgNCs in Self-Assembled Monolayer...

  4. De novo assembly of the carrot mitochondrial genome using next generation sequencing of whole genomic DNA provides first evidence of DNA transfer into an angiosperm plastid genome

    Directory of Open Access Journals (Sweden)

    Iorizzo Massimo

    2012-05-01

    Full Text Available Abstract Background Sequence analysis of organelle genomes has revealed important aspects of plant cell evolution. The scope of this study was to develop an approach for de novo assembly of the carrot mitochondrial genome using next generation sequence data from total genomic DNA. Results Sequencing data from a carrot 454 whole genome library were used to develop a de novo assembly of the mitochondrial genome. Development of a new bioinformatic tool allowed visualizing contig connections and elucidation of the de novo assembly. Southern hybridization demonstrated recombination across two large repeats. Genome annotation allowed identification of 44 protein coding genes, three rRNA and 17 tRNA. Identification of the plastid genome sequence allowed organelle genome comparison. Mitochondrial intergenic sequence analysis allowed detection of a fragment of DNA specific to the carrot plastid genome. PCR amplification and sequence analysis across different Apiaceae species revealed consistent conservation of this fragment in the mitochondrial genomes and an insertion in Daucus plastid genomes, giving evidence of a mitochondrial to plastid transfer of DNA. Sequence similarity with a retrotransposon element suggests a possibility that a transposon-like event transferred this sequence into the plastid genome. Conclusions This study confirmed that whole genome sequencing is a practical approach for de novo assembly of higher plant mitochondrial genomes. In addition, a new aspect of intercompartmental genome interaction was reported providing the first evidence for DNA transfer into an angiosperm plastid genome. The approach used here could be used more broadly to sequence and assemble mitochondrial genomes of diverse species. This information will allow us to better understand intercompartmental interactions and cell evolution.

  5. Transcriptator: An Automated Computational Pipeline to Annotate Assembled Reads and Identify Non Coding RNA.

    Directory of Open Access Journals (Sweden)

    Kumar Parijat Tripathi

    Full Text Available RNA-seq is a new tool to measure RNA transcript counts, using high-throughput sequencing at an extraordinary accuracy. It provides quantitative means to explore the transcriptome of an organism of interest. However, interpreting this extremely large data into biological knowledge is a problem, and biologist-friendly tools are lacking. In our lab, we developed Transcriptator, a web application based on a computational Python pipeline with a user-friendly Java interface. This pipeline uses the web services available for BLAST (Basis Local Search Alignment Tool, QuickGO and DAVID (Database for Annotation, Visualization and Integrated Discovery tools. It offers a report on statistical analysis of functional and Gene Ontology (GO annotation's enrichment. It helps users to identify enriched biological themes, particularly GO terms, pathways, domains, gene/proteins features and protein-protein interactions related informations. It clusters the transcripts based on functional annotations and generates a tabular report for functional and gene ontology annotations for each submitted transcript to the web server. The implementation of QuickGo web-services in our pipeline enable the users to carry out GO-Slim analysis, whereas the integration of PORTRAIT (Prediction of transcriptomic non coding RNA (ncRNA by ab initio methods helps to identify the non coding RNAs and their regulatory role in transcriptome. In summary, Transcriptator is a useful software for both NGS and array data. It helps the users to characterize the de-novo assembled reads, obtained from NGS experiments for non-referenced organisms, while it also performs the functional enrichment analysis of differentially expressed transcripts/genes for both RNA-seq and micro-array experiments. It generates easy to read tables and interactive charts for better understanding of the data. The pipeline is modular in nature, and provides an opportunity to add new plugins in the future. Web application is

  6. aTRAM - automated target restricted assembly method: a fast method for assembling loci across divergent taxa from next-generation sequencing data

    OpenAIRE

    Allen, Julie M; Huang, Daisie I; Cronk, Quentin C; Johnson, Kevin P

    2015-01-01

    Background Assembling genes from next-generation sequencing data is not only time consuming but computationally difficult, particularly for taxa without a closely related reference genome. Assembling even a draft genome using de novo approaches can take days, even on a powerful computer, and these assemblies typically require data from a variety of genomic libraries. Here we describe software that will alleviate these issues by rapidly assembling genes from distantly related taxa using a sing...

  7. Optimized Method of Extracting Rice Chloroplast DNA for High-Quality Plastome Resequencing and de Novo Assembly

    Directory of Open Access Journals (Sweden)

    Takeshi Takamatsu

    2018-02-01

    Full Text Available Chloroplasts, which perform photosynthesis, are one of the most important organelles in green plants and algae. Chloroplasts maintain an independent genome that includes important genes encoding their photosynthetic machinery and various housekeeping functions. Owing to its non-recombinant nature, low mutation rates, and uniparental inheritance, the chloroplast genome (plastome can give insights into plant evolution and ecology and in the development of biotechnological and breeding applications. However, efficient methods to obtain high-quality chloroplast DNA (cpDNA are currently not available, impeding powerful sequencing and further functional genomics research. To investigate effects on rice chloroplast genome quality, we compared cpDNA extraction by three extraction protocols: liquid nitrogen coupled with sucrose density gradient centrifugation, high-salt buffer, and Percoll gradient centrifugation. The liquid nitrogen–sucrose gradient method gave a high yield of high-quality cpDNA with reliable purity. The cpDNA isolated by this technique was evaluated, resequenced, and assembled de novo to build a robust framework for genomic and genetic studies. Comparison of this high-purity cpDNA with total DNAs revealed the read coverage of the sequenced regions; next-generation sequencing data showed that the high-quality cpDNA eliminated noise derived from contamination by nuclear and mitochondrial DNA, which frequently occurs in total DNA. The assembly process produced highly accurate, long contigs. We summarize the extent to which this improved method of isolating cpDNA from rice can provide practical progress in overcoming challenges related to chloroplast genomes and in further exploring the development of new sequencing technologies.

  8. Amphiphilic DNA tiles for controlled insertion and 2D assembly on fluid lipid membranes: the effect on mechanical properties.

    Science.gov (United States)

    Dohno, Chikara; Makishi, Shingo; Nakatani, Kazuhiko; Contera, Sonia

    2017-03-02

    Future lipid membrane-associated DNA nanostructures are expected to find applications ranging from synthetic biology to nanomedicine. Here we have designed and synthesized DNA tiles and modified them with amphiphilic covalent moieties. dod-DEG groups, which consist of a hydrophilic diethylene glycol (DEG) and a hydrophobic dodecyl group, are introduced at the phosphate backbone to create amphiphilic DNA strands which are subsequently introduced into one face of the DNA tiles. In this way the tile becomes able to stably bind to lipid membranes by insertion of the hydrophobic groups inside the bilayer core. The functionalized tiles do not aggregate in solution. Our results show that these amphiphilic DNA tiles can bind and assemble into 2D lattices on both gel and fluid lipid bilayers. The binding of the DNA structures to membranes is dependent on the lipid phase of the membrane, the concentration of Mg 2+ cations, the length of the amphiphilic modifications to the DNA as well as on the density of the modifications within the tile. Atomic force microscopy-based force spectroscopy is used to investigate the effect of the inserted DNA tiles on the mechanical properties of the lipid membranes. The results indicate that the insertion of DNA tiles produces an approx. 20% increase of the bilayer breakthrough force.

  9. Self-assembled nanocomplexes of anionic pullulan and polyallylamine for DNA and pH-sensitive intracellular drug delivery

    Energy Technology Data Exchange (ETDEWEB)

    Vora, Lalit [University under Sect. 3 of UGC Act – 1956, Elite Status and Center of Excellence – Govt. of Maharashtra, Center for Novel Drug Delivery Systems, Department of Pharmaceutical Sciences and Technology, Institute of Chemical Technology (India); Tyagi, Monica [Advanced Center for Treatment, Research and Education in Cancer (ACTREC), Tata Memorial Centre, Gupta Lab, Cancer Research Institute (India); Patel, Ketan [University under Sect. 3 of UGC Act – 1956, Elite Status and Center of Excellence – Govt. of Maharashtra, Center for Novel Drug Delivery Systems, Department of Pharmaceutical Sciences and Technology, Institute of Chemical Technology (India); Gupta, Sanjay [Advanced Center for Treatment, Research and Education in Cancer (ACTREC), Tata Memorial Centre, Gupta Lab, Cancer Research Institute (India); Vavia, Pradeep, E-mail: vaviapradeep@yahoo.com [University under Sect. 3 of UGC Act – 1956, Elite Status and Center of Excellence – Govt. of Maharashtra, Center for Novel Drug Delivery Systems, Department of Pharmaceutical Sciences and Technology, Institute of Chemical Technology (India)

    2014-12-15

    The amalgamation of chemotherapy and gene therapy is promising treatment option for cancer. In this study, novel biocompatible self-assembled nanocomplexes (NCs) between carboxylmethylated pullulan t335 (CMP) with polyallylamine (CMP–PAA NCs) were developed for plasmid DNA (pDNA) and pH-sensitive doxorubicin (DOX) delivery. DOX was conjugated to CMP (DOX–CMP) via hydrazone and confirmed by FTIR and {sup 1}H-NMR. In vitro release studies of pH-sensitive DOX–CMP conjugate showed 23 and 85 % release after 48 h at pH 7.4 (physiological pH) and pH 5 (intracellular/tumoral pH), respectively. The CMP–PAA NCs or DOX–CMP–PAA NCs self-assembled into a nanosized (<250 nm) spherical shape as confirmed by DLS and TEM. The hemolysis and cytotoxicity study indicated that the CMP–PAA NCs did not show cytotoxicity in comparison with plain polyallylamine. Gel retardation assay showed complete binding of pDNA with CMP–PAA NCs at 1:2 weight ratio. CMP–PAA NCs/pDNA showed significantly higher transfection in HEK293 cells compared to PAA/pDNA complexes. Confocal imaging demonstrated successful cellular uptake of DOX–CMP–PAA NCs in HEK293 cells. Thus, NCs hold great potential for targeted pDNA and pH-sensitive intratumoral drug delivery.

  10. 75 FR 3253 - Lamb Assembly and Test, LLC, Subsidiary of Mag Industrial Automation Systems, Machesney Park, IL...

    Science.gov (United States)

    2010-01-20

    ... automation equipment and machine tools do not form component parts of aluminum transmissions, cases, parts... automation equipment and machine tools did not contribute to worker separations at the subject facility and...' firm's declining customers. The survey revealed no imports of automation equipment and machine tools by...

  11. Self-Assembled Molecular Hybrids of CoS-DNA for Enhanced Water Oxidation with Low Cobalt Content.

    Science.gov (United States)

    Karthick, Kannimuthu; Anantharaj, Sengeni; Karthik, Pitchiah E; Subramanian, Balasubramanian; Kundu, Subrata

    2017-06-05

    Water oxidation in alkaline medium was efficiently catalyzed by the self-assembled molecular hybrids of CoS-DNA that had 20 times lower Co loading than the commonly used loading. The morphological outcome was directed by varying the molar ratio of metal precursor Co(Ac) 2 and DNA and three different sets of CoS-DNA molecular hybrids, viz. CoS-DNA(0.036), CoS-DNA(0.06), and CoS-DNA(0.084) were prepared. These morphologically distinct hybrids had shown similar electrocatalytic behavior, because of the fact that they all contained the same cobalt content. The CoS-DNA(0.036), CoS-DNA(0.06), and CoS-DNA(0.084) required very low overpotentials of 350, 364, and 373 mV at a current density of 10 mA cm -2 (1 M KOH), respectively. The advantages of lower overpotential, lower Tafel slope (42.7 mV dec -1 ), high Faradaic efficiency (90.28%), high stability and reproducibility after all, with a lower cobalt loading, have certainly shown the worth of these molecular hybrids in large-scale water oxidation. Moreover, since DNA itself a good binder, CoS-DNA molecular hybrids were directly casted on substrate electrodes and used after drying. It also showed minimum intrinsic resistance as DNA is a good ionic and electronic conductor. Besides, the present method may also be extended for the preparation of other active electrocatalysts for water splitting.

  12. Crystal structure of Mycobacterium tuberculosis O-6-methylguanine-DNA methyltransferase protein clusters assembled on to damaged DNA

    Czech Academy of Sciences Publication Activity Database

    Miggiano, R.; Perugino, G.; Ciaramella, M.; Serpe, M.; Rejman, Dominik; Páv, Ondřej; Pohl, Radek; Garavaglia, S.; Lahiri, S.; Rizzi, M.; Rossi, F.

    2016-01-01

    Roč. 473, č. 2 (2016), s. 123-133 ISSN 0264-6021 EU Projects: European Commission(XE) 241587 - SYSTEMTB Institutional support: RVO:61388963 Keywords : DNA repair * DNA-binding protein * Mycobacterium tuberculosis * O-6-methylguanine-DNA methyltransferase * co-operativity * crystal structure Subject RIV: CE - Biochemistry Impact factor: 3.797, year: 2016

  13. Rapid and annealing-free self-assembly of DNA building blocks for 3D hydrogel chaperoned by cationic comb-type copolymers.

    Science.gov (United States)

    Zhang, Zheng; Wu, Yuyang; Yu, Feng; Niu, Chaoqun; Du, Zhi; Chen, Yong; Du, Jie

    2017-10-01

    The construction and self-assembly of DNA building blocks are the foundation of bottom-up development of three-dimensional DNA nanostructures or hydrogels. However, most self-assembly from DNA components is impeded by the mishybridized intermediates or the thermodynamic instability. To enable rapid production of complicated DNA objects with high yields no need for annealing process, herein different DNA building blocks (Y-shaped, L- and L'-shaped units) were assembled in presence of a cationic comb-type copolymer, poly (L-lysine)-graft-dextran (PLL-g-Dex), under physiological conditions. The results demonstrated that PLL-g-Dex not only significantly promoted the self-assembly of DNA blocks with high efficiency, but also stabilized the assembled multi-level structures especially for promoting the complicated 3D DNA hydrogel formation. This study develops a novel strategy for rapid and high-yield production of DNA hydrogel even derived from instable building blocks at relatively low DNA concentrations, which would endow DNA nanotechnology for more practical applications.

  14. A DNA aptamer recognising a malaria protein biomarker can function as part of a DNA origami assembly

    Science.gov (United States)

    Godonoga, Maia; Lin, Ting-Yu; Oshima, Azusa; Sumitomo, Koji; Tang, Marco S. L.; Cheung, Yee-Wai; Kinghorn, Andrew B.; Dirkzwager, Roderick M.; Zhou, Cunshan; Kuzuya, Akinori; Tanner, Julian A.; Heddle, Jonathan G.

    2016-01-01

    DNA aptamers have potential for disease diagnosis and as therapeutics, particularly when interfaced with programmable molecular technology. Here we have combined DNA aptamers specific for the malaria biomarker Plasmodium falciparum lactate dehydrogenase (PfLDH) with a DNA origami scaffold. Twelve aptamers that recognise PfLDH were integrated into a rectangular DNA origami and atomic force microscopy demonstrated that the incorporated aptamers preserve their ability to specifically bind target protein. Captured PfLDH retained enzymatic activity and protein-aptamer binding was observed dynamically using high-speed AFM. This work demonstrates the ability of DNA aptamers to recognise a malaria biomarker whilst being integrated within a supramolecular DNA scaffold, opening new possibilities for malaria diagnostic approaches based on DNA nanotechnology. PMID:26891622

  15. Proximity hybridization-regulated catalytic DNA hairpin assembly for electrochemical immunoassay based on in situ DNA template-synthesized Pd nanoparticles

    Energy Technology Data Exchange (ETDEWEB)

    Zhou, Fuyi [School of Chemistry and Chemical Engineering, Jiangsu Normal University, Xuzhou 221116 (China); Jiangsu Key Laboratory of New Drug Research and Clinical Pharmacy, Department of Pharmaceutical Analysis, School of Pharmacy, Xuzhou Medical College, 221004, Xuzhou (China); Yao, Yao; Luo, Jianjun; Zhang, Xing; Zhang, Yu; Yin, Dengyang [Jiangsu Key Laboratory of New Drug Research and Clinical Pharmacy, Department of Pharmaceutical Analysis, School of Pharmacy, Xuzhou Medical College, 221004, Xuzhou (China); Gao, Fenglei, E-mail: jsxzgfl@sina.com [Jiangsu Key Laboratory of New Drug Research and Clinical Pharmacy, Department of Pharmaceutical Analysis, School of Pharmacy, Xuzhou Medical College, 221004, Xuzhou (China); Wang, Po, E-mail: wangpo@jsnu.edu.cn [School of Chemistry and Chemical Engineering, Jiangsu Normal University, Xuzhou 221116 (China)

    2017-05-29

    Novel hybridization proximity-regulated catalytic DNA hairpin assembly strategy has been proposed for electrochemical immunoassay based on in situ DNA template-synthesized Pd nanoparticles as signal label. The DNA template-synthesized Pd nanoparticles were characterized with atomic force microscopic and X-ray photoelectron spectroscopy. The highly efficient electrocatalysis by DNA template synthesized Pd nanoparticles for NaBH{sub 4} oxidation produced an intense detection signal. The label-free electrochemical method achieved the detection of carcinoembryonic antigen (CEA) with a linear range from 10{sup −15} to 10{sup −11} g mL{sup −1} and a detection limit of 0.43 × 10{sup −15} g mL{sup −1}. Through introducing a supersandwich reaction to increase the DNA length, the electrochemical signal was further amplified, leading to a detection limit of 0.52 × 10{sup −16} g mL{sup −1}. And it rendered satisfactory analytical performance for the determination of CEA in serum samples. Furthermore, it exhibited good reproducibility and stability; meanwhile, it also showed excellent specificity due to the specific recognition of antigen by antibody. Therefore, the DNA template synthesized Pd nanoparticles based signal amplification approach has great potential in clinical applications and is also suitable for quantification of biomarkers at ultralow level. - Graphical abstract: A novel label-free and enzyme-free electrochemical immunoassay based on proximity hybridization-regulated catalytic DNA hairpin assemblies for recycling of the CEA. - Highlights: • A novel enzyme-free electrochemical immunosensor was developed for detection of CEA. • The signal amplification was based on catalytic DNA hairpin assembly and DNA-template-synthesized Pd nanoparticles. • The biosensor could detect CEA down to 0.52 × 10{sup −16} g mL{sup −1} level with a dynamic range spanning 5 orders of magnitude.

  16. Self-Assembled Functional Nanostructure of Plasmid DNA with Ionic Liquid [Bmim][PF₆]: Enhanced Efficiency in Bacterial Gene Transformation.

    Science.gov (United States)

    Soni, Sarvesh K; Sarkar, Sampa; Mirzadeh, Nedaossadat; Selvakannan, P R; Bhargava, Suresh K

    2015-04-28

    The electrostatic interaction between the negatively charged phosphate groups of plasmid DNA and the cationic part of hydrophobic ionic liquid 1-butyl-3-methylimidazolium hexafluorophosphate ([Bmim][PF6]), initiates spontaneous self-assembly to form the functional nanostructures made up of DNA and ionic liquid (IL). These functional nanostructures were demonstrated as promising synthetic nonviral vectors for the efficient bacterial pGFP gene transformation in cells. In particular, the functional nanostructures that were made up of 1 μL of IL ([Bmim][PF6]) and 1 μg of plasmid DNA can increase the transformation efficiency by 300-400% in microbial systems, without showing any toxicity for E. coli DH5α cells. (31)P nuclear magnetic resonance (NMR), Fourier transform infrared (FTIR) spectroscopy, and X-ray photoelectron (XPS) spectroscopic analysis revealed that the electrostatic interaction between negatively charged phosphate oxygen and cationic Bmim(+) tends to initiate the self-assembly process. Thermogravimetric analysis of the DNA-IL functional nanostructures showed that these nanostructures consist of ∼16 wt % ionic liquid, which is considered to provide the stability to the plasmid DNA that eventually enhanced the transformation efficiency.

  17. DNA origami design of 3D nanostructures

    DEFF Research Database (Denmark)

    Andersen, Ebbe Sloth; Nielsen, Morten Muhlig

    2009-01-01

    [8]. We have recently developed a semi-automated DNA origami software package [9] that uses a 2D sequence editor in conjunction with several automated tools to facilitate the design process. Here we extend the use of the program for designing DNA origami structures in 3D and show the application......Structural DNA nanotechnology has been heavily dependent on the development of dedicated software tools for the design of unique helical junctions, to define unique sticky-ends for tile assembly, and for predicting the products of the self-assembly reaction of multiple DNA strands [1-3]. Recently......, several dedicated 3D editors for computer-aided design of DNA structures have been developed [4-7]. However, many of these tools are not efficient for designing DNA origami structures that requires the design of more than 200 unique DNA strands to be folded along a scaffold strand into a defined 3D shape...

  18. Exploring repetitive DNA landscapes using REPCLASS, a tool that automates the classification of transposable elements in eukaryotic genomes.

    Science.gov (United States)

    Feschotte, Cédric; Keswani, Umeshkumar; Ranganathan, Nirmal; Guibotsy, Marcel L; Levine, David

    2009-07-23

    Eukaryotic genomes contain large amount of repetitive DNA, most of which is derived from transposable elements (TEs). Progress has been made to develop computational tools for ab initio identification of repeat families, but there is an urgent need to develop tools to automate the annotation of TEs in genome sequences. Here we introduce REPCLASS, a tool that automates the classification of TE sequences. Using control repeat libraries, we show that the program can classify accurately virtually any known TE types. Combining REPCLASS to ab initio repeat finding in the genomes of Caenorhabditis elegans and Drosophila melanogaster allowed us to recover the contrasting TE landscape characteristic of these species. Unexpectedly, REPCLASS also uncovered several novel TE families in both genomes, augmenting the TE repertoire of these model species. When applied to the genomes of distant Caenorhabditis and Drosophila species, the approach revealed a remarkable conservation of TE composition profile within each genus, despite substantial interspecific covariations in genome size and in the number of TEs and TE families. Lastly, we applied REPCLASS to analyze 10 fungal genomes from a wide taxonomic range, most of which have not been analyzed for TE content previously. The results showed that TE diversity varies widely across the fungi "kingdom" and appears to positively correlate with genome size, in particular for DNA transposons. Together, these data validate REPCLASS as a powerful tool to explore the repetitive DNA landscapes of eukaryotes and to shed light onto the evolutionary forces shaping TE diversity and genome architecture.

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

    DEFF Research Database (Denmark)

    Astakhova, I Kira; Wengel, Jesper

    2013-01-01

    for the first time performed solid-phase copper(I)-catalyzed azide-alkyne cycloaddition (CuAAC) click labeling during the automated phosphoramidite oligonucleotide synthesis followed by postsynthetic click reactions in solution. We demonstrate that our novel strategy is rapid and efficient for the preparation...... Stokes shifts (40-110 nm), quenched fluorescence of single-stranded probes accompanied by up to 7.7-fold light-up effect of emission upon target DNA/RNA binding, remarkable sensitivity to single-nucleotide mismatches, generally high fluorescence brightness values (FB up to 26), and hence low limit...

  20. CasEMBLR: Cas9-Facilitated Multiloci Genomic Integration of in Vivo Assembled DNA Parts in Saccharomyces cerevisiae.

    Science.gov (United States)

    Jakočiūnas, Tadas; Rajkumar, Arun S; Zhang, Jie; Arsovska, Dushica; Rodriguez, Angelica; Jendresen, Christian Bille; Skjødt, Mette L; Nielsen, Alex T; Borodina, Irina; Jensen, Michael K; Keasling, Jay D

    2015-11-20

    Homologous recombination (HR) in Saccharomyces cerevisiae has been harnessed for both plasmid construction and chromosomal integration of foreign DNA. Still, native HR machinery is not efficient enough for complex and marker-free genome engineering required for modern metabolic engineering. Here, we present a method for marker-free multiloci integration of in vivo assembled DNA parts. By the use of CRISPR/Cas9-mediated one-step double-strand breaks at single, double and triple integration sites we report the successful in vivo assembly and chromosomal integration of DNA parts. We call our method CasEMBLR and validate its applicability for genome engineering and cell factory development in two ways: (i) introduction of the carotenoid pathway from 15 DNA parts into three targeted loci, and (ii) creation of a tyrosine production strain using ten parts into two loci, simultaneously knocking out two genes. This method complements and improves the current set of tools available for genome engineering in S. cerevisiae.

  1. Properties of the chromatin assembled on DNA injected into Xenopus oocytes and eggs

    International Nuclear Information System (INIS)

    Gargiulo, G.; Wasserman, W.; Worcel, A.

    1983-01-01

    The onset of DNA synthesis occurs between 10 and 30 minutes after activation of the egg and thus the transition from nuclease-sensitive to nuclease-resistant supercoils may take place on the newly replicated DNA. To test this possibility, the nonradioactive circular 5-kb DNA carrying the Drosophila histone gene repeat and [α -32 P]dCTP were coinjected into fertilized eggs. Such protocol labels both the injected, replicated heterologous DNA and the replicated endogenous, maternal Xenopus DNA. The labeled, presumably replicated, supercoiled DNA is resistant to micrococcal nuclease as expected. The endogenous, high-molecular-weight Xenopus DNA is degraded to 180-bp nucleosomal DNA. Thus, the nuclease resistance is not a general property of chromatin during the cleavage stage of the Xenopus embryo but is a peculiar feature of the injected DNA. 42 references, 5 figures

  2. CometQ: An automated tool for the detection and quantification of DNA damage using comet assay image analysis.

    Science.gov (United States)

    Ganapathy, Sreelatha; Muraleedharan, Aparna; Sathidevi, Puthumangalathu Savithri; Chand, Parkash; Rajkumar, Ravi Philip

    2016-09-01

    DNA damage analysis plays an important role in determining the approaches for treatment and prevention of various diseases like cancer, schizophrenia and other heritable diseases. Comet assay is a sensitive and versatile method for DNA damage analysis. The main objective of this work is to implement a fully automated tool for the detection and quantification of DNA damage by analysing comet assay images. The comet assay image analysis consists of four stages: (1) classifier (2) comet segmentation (3) comet partitioning and (4) comet quantification. Main features of the proposed software are the design and development of four comet segmentation methods, and the automatic routing of the input comet assay image to the most suitable one among these methods depending on the type of the image (silver stained or fluorescent stained) as well as the level of DNA damage (heavily damaged or lightly/moderately damaged). A classifier stage, based on support vector machine (SVM) is designed and implemented at the front end, to categorise the input image into one of the above four groups to ensure proper routing. Comet segmentation is followed by comet partitioning which is implemented using a novel technique coined as modified fuzzy clustering. Comet parameters are calculated in the comet quantification stage and are saved in an excel file. Our dataset consists of 600 silver stained images obtained from 40 Schizophrenia patients with different levels of severity, admitted to a tertiary hospital in South India and 56 fluorescent stained images obtained from different internet sources. The performance of "CometQ", the proposed standalone application for automated analysis of comet assay images, is evaluated by a clinical expert and is also compared with that of a most recent and related software-OpenComet. CometQ gave 90.26% positive predictive value (PPV) and 93.34% sensitivity which are much higher than those of OpenComet, especially in the case of silver stained images. The

  3. Rapid assembly of multiple DNA fragments through direct transformation of PCR products into E. coli and Lactobacillus.

    Science.gov (United States)

    Cao, Pinghua; Wang, Lei; Zhou, Guangxian; Wang, Yaoyue; Chen, Yulin

    2014-11-01

    This article describes a rapid, highly efficient and versatile method for seamlessly assembling multiple DNA fragments into a vector at any desired position. The inserted fragments and vector backbone were amplified by high-fidelity PCR containing 20 bp to 50 bp overlapping regions at 3' and/or 5' termini. These linearised fragments were equimolarly mixed, and then cyclised in a prolonged overlap extension PCR without adding primers. The resulting PCR products were DNA multimers that could be directly transformed into host strains, yielding the desired chimeric plasmid. The proposed method was illustrated by constructing an Escherichia coli co-expression vector. The feasibility of the method in Lactobacillus was further validated by assembling an E. coli-Lactobacillus shuttle vector. Results showed that three to four fragments could be simultaneously and precisely inserted in a vector in only 2-3 days using the proposed method. The acceptable transformation efficiency was determined through the tested host strains; more than 95% of the colonies were positive transformants. Therefore, the proposed method is sufficiently competent for high-efficiency insertion of multiple DNA fragments into a plasmid and has theoretically good application potential for gene cloning and protein expression because it is simple, easy to implement, flexible and yields highly positive clones. Copyright © 2014 Elsevier Inc. All rights reserved.

  4. Enzyme-free amplification for sensitive electrochemical detection of DNA via target-catalyzed hairpin assembly assisted current change.

    Science.gov (United States)

    Qian, Yong; Wang, Chunyan; Gao, Fenglei

    2014-12-01

    An isothermal, enzyme-free and sensitive method for electrochemical detection of DNA is proposed based on target catalyzed hairpin assembly and for signal amplification. Molecular beacon 1 (MB1) contains a ferrocene (Fc) tag, which was immobilized on the gold electrode as recognition probe to hybridize with target DNA. Then, molecular beacon 2 hybridized with the opened MB1, allowing the target to be displaced. The displaced target again triggered the next round of strand exchange reaction resulting in many Fc far away from the GE to achieve signal amplification for sensitive DNA detection. The current signal amplification strategy is relatively simple and inexpensive owing to avoid the use of any kind of enzyme or sophisticated equipment. It can achieve a sensitivity of 42 fM with a wide linear dynamic range from 10(-13) to 10(-9)M and discriminate mismatched DNA from perfect matched target DNA with a high selectivity. The proposed method showed excellent specificity, high sensitivity and low detection limit, and could be applied in analysis of real samples. Copyright © 2014 Elsevier B.V. All rights reserved.

  5. Conserved residues in the delta subunit help the E. coli clamp loader, gamma complex, target primer-template DNA for clamp assembly.

    Science.gov (United States)

    Chen, Siying; Coman, Maria Magdalena; Sakato, Miho; O'Donnell, Michael; Hingorani, Manju M

    2008-06-01

    The Escherichia coli clamp loader, gamma complex (gamma(3)deltadelta'lambdapsi), catalyzes ATP-driven assembly of beta clamps onto primer-template DNA (p/tDNA), enabling processive replication. The mechanism by which gamma complex targets p/tDNA for clamp assembly is not resolved. According to previous studies, charged/polar amino acids inside the clamp loader chamber interact with the double-stranded (ds) portion of p/tDNA. We find that dsDNA, not ssDNA, can trigger a burst of ATP hydrolysis by gamma complex and clamp assembly, but only at far higher concentrations than p/tDNA. Thus, contact between gamma complex and dsDNA is necessary and sufficient, but not optimal, for the reaction, and additional contacts with p/tDNA likely facilitate its selection as the optimal substrate for clamp assembly. We investigated whether a conserved sequence-HRVW(279)QNRR--in delta subunit contributes to such interactions, since Tryptophan-279 specifically cross-links to the primer-template junction. Mutation of delta-W279 weakens gamma complex binding to p/tDNA, hampering its ability to load clamps and promote proccessive DNA replication, and additional mutations in the sequence (delta-R277, delta-R283) worsen the interaction. These data reveal a novel location in the C-terminal domain of the E. coli clamp loader that contributes to DNA binding and helps define p/tDNA as the preferred substrate for the reaction.

  6. Automated property optimization via ab initio O(N) elongation method: Application to (hyper-)polarizability in DNA

    International Nuclear Information System (INIS)

    Orimoto, Yuuichi; Aoki, Yuriko

    2016-01-01

    An automated property optimization method was developed based on the ab initio O(N) elongation (ELG) method and applied to the optimization of nonlinear optical (NLO) properties in DNA as a first test. The ELG method mimics a polymerization reaction on a computer, and the reaction terminal of a starting cluster is attacked by monomers sequentially to elongate the electronic structure of the system by solving in each step a limited space including the terminal (localized molecular orbitals at the terminal) and monomer. The ELG-finite field (ELG-FF) method for calculating (hyper-)polarizabilities was used as the engine program of the optimization method, and it was found to show linear scaling efficiency while maintaining high computational accuracy for a random sequenced DNA model. Furthermore, the self-consistent field convergence was significantly improved by using the ELG-FF method compared with a conventional method, and it can lead to more feasible NLO property values in the FF treatment. The automated optimization method successfully chose an appropriate base pair from four base pairs (A, T, G, and C) for each elongation step according to an evaluation function. From test optimizations for the first order hyper-polarizability (β) in DNA, a substantial difference was observed depending on optimization conditions between “choose-maximum” (choose a base pair giving the maximum β for each step) and “choose-minimum” (choose a base pair giving the minimum β). In contrast, there was an ambiguous difference between these conditions for optimizing the second order hyper-polarizability (γ) because of the small absolute value of γ and the limitation of numerical differential calculations in the FF method. It can be concluded that the ab initio level property optimization method introduced here can be an effective step towards an advanced computer aided material design method as long as the numerical limitation of the FF method is taken into account.

  7. Automated Forward and Reverse Ratcheting of DNA in a Nanopore at Five Angstrom Precision1

    Science.gov (United States)

    Cherf, Gerald M.; Lieberman, Kate R.; Rashid, Hytham; Lam, Christopher E.; Karplus, Kevin; Akeson, Mark

    2012-01-01

    Single-molecule techniques have been developed for commercial DNA sequencing1,2. One emerging strategy uses a nanopore to analyze DNA molecules as they are driven electrophoretically in single file order past a sensor3-5. However, uncontrolled DNA strand electrophoresis through nanopores is too fast for accurate base reads6. A proposed solution would employ processive enzymes to deliver DNA through the pore at a slower average rate7. Here, we describe forward and reverse ratcheting of DNA templates through the α–hemolysin (α-HL) nanopore controlled by wild-type phi29 DNA polymerase (phi29 DNAP). DNA strands were examined in single file order at one nucleotide spatial precision in real time. The registry error probability (either an insertion or deletion during one pass along a template strand) ranged from 10% to 24.5% absent optimization. This general strategy facilitates multiple reads of individual template strands and is transferrable to other nanopore devices for implementation of DNA sequence analysis. PMID:22334048

  8. Label-free DNA biosensor based on resistance change of platinum nanoparticles assemblies.

    Science.gov (United States)

    Skotadis, Evangelos; Voutyras, Konstantinos; Chatzipetrou, Marianneza; Tsekenis, Georgios; Patsiouras, Lampros; Madianos, Leonidas; Chatzandroulis, Stavros; Zergioti, Ioanna; Tsoukalas, Dimitris

    2016-07-15

    A novel nanoparticle based biosensor for the fast and simple detection of DNA hybridization events is presented. The sensor utilizes hybridized DNA's charge transport properties, combining them with metallic nanoparticle networks that act as nano-gapped electrodes. The DNA hybridization events can be detected by a significant reduction in the sensor's resistance due to the conductive bridging offered by hybridized DNA. By modifying the nanoparticle surface coverage, which can be controlled experimentally being a function of deposition time, and the structural properties of the electrodes, an optimized biosensor for the in situ detection of DNA hybridization events is ultimately fabricated. The fabricated biosensor exhibits a wide response range, covering four orders of magnitude, a limit of detection of 1nM and can detect a single base pair mismatch between probe and complementary DNA. Copyright © 2016 Elsevier B.V. All rights reserved.

  9. Mismatch discrimination of lipidated DNA and LNA-probes (LiNAs) in hybridization-controlled liposome assembly

    DEFF Research Database (Denmark)

    Jakobsen, Ulla; Vogel, Stefan

    2016-01-01

    nucleic acids) probe designs, including membrane-anchoring requirements, studies on different probes and target lengths (including overhangs), DNA and RNA targets (including sequences associated with pathogens) for lipidated nucleic acids (LiNAs). Advantages and limitations of the liposome assembly based...... assay in the context of mismatch discrimination and SNP detection are presented. The advantages of membrane-anchored LiNA-probes compared to chemically attached probes on solid nanoparticles (e.g. gold nanoparticles) are described. Key functionalities such as non-covalent attachment of LiNA probes...

  10. Comparison of QIAsymphony automated and QIAamp manual DNA extraction systems for measuring Epstein-Barr virus DNA load in whole blood using real-time PCR.

    Science.gov (United States)

    Laus, Stella; Kingsley, Lawrence A; Green, Michael; Wadowsky, Robert M

    2011-11-01

    Automated and manual extraction systems have been used with real-time PCR for quantification of Epstein-Barr virus [human herpesvirus 4 (HHV-4)] DNA in whole blood, but few studies have evaluated relative performances. In the present study, the automated QIAsymphony and manual QIAamp extraction systems (Qiagen, Valencia, CA) were assessed using paired aliquots derived from clinical whole-blood specimens and an in-house, real-time PCR assay. The detection limits using the QIAsymphony and QIAamp systems were similar (270 and 560 copies/mL, respectively). For samples estimated as having ≥10,000 copies/mL, the intrarun and interrun variations were significantly lower using QIAsymphony (10.0% and 6.8%, respectively), compared with QIAamp (18.6% and 15.2%, respectively); for samples having ≤1000 copies/mL, the two variations ranged from 27.9% to 43.9% and were not significantly different between the two systems. Among 68 paired clinical samples, 48 pairs yielded viral loads ≥1000 copies/mL under both extraction systems. Although the logarithmic linear correlation from these positive samples was high (r(2) = 0.957), the values obtained using QIAsymphony were on average 0.2 log copies/mL higher than those obtained using QIAamp. Thus, the QIAsymphony and QIAamp systems provide similar EBV DNA load values in whole blood. Copyright © 2011 American Society for Investigative Pathology and the Association for Molecular Pathology. Published by Elsevier Inc. All rights reserved.

  11. Automated extraction of DNA from blood and PCR setup using a Tecan Freedom EVO liquid handler for forensic genetic STR typing of reference samples

    DEFF Research Database (Denmark)

    Stangegaard, Michael; Frøslev, Tobias G; Frank-Hansen, Rune

    2011-01-01

    17025 using the Qiagen MagAttract DNA Mini M48 kit (Qiagen GmbH, Hilden, Germany) from fresh whole blood and blood from deceased individuals. The workflow was simplified by returning the DNA extracts to the original tubes minimizing the risk of misplacing samples. The tubes that originally contained...... the samples were washed with MilliQ water before the return of the DNA extracts. The PCR was setup in 96-well microtiter plates. The methods were validated for the kits: AmpFlSTR Identifiler, SGM Plus and Yfiler (Applied Biosystems, Foster City, CA), GenePrint FFFL and PowerPlex Y (Promega, Madison, WI......). The automated protocols allowed for extraction and addition of PCR master mix of 96 samples within 3.5h. In conclusion, we demonstrated that (1) DNA extraction with magnetic beads and (2) PCR setup for accredited, forensic genetic short tandem repeat typing can be implemented on a simple automated liquid...

  12. Restarting and recentering genetic algorithm variations for DNA fragment assembly: The necessity of a multi-strategy approach.

    Science.gov (United States)

    Hughes, James Alexander; Houghten, Sheridan; Ashlock, Daniel

    2016-12-01

    DNA Fragment assembly - an NP-Hard problem - is one of the major steps in of DNA sequencing. Multiple strategies have been used for this problem, including greedy graph-based algorithms, deBruijn graphs, and the overlap-layout-consensus approach. This study focuses on the overlap-layout-consensus approach. Heuristics and computational intelligence methods are combined to exploit their respective benefits. These algorithm combinations were able to produce high quality results surpassing the best results obtained by a number of competitive algorithms specially designed and tuned for this problem on thirteen of sixteen popular benchmarks. This work also reinforces the necessity of using multiple search strategies as it is clearly observed that algorithm performance is dependent on problem instance; without a deeper look into many searches, top solutions could be missed entirely. Copyright © 2016. Published by Elsevier Ireland Ltd.

  13. DNA-hybrid materials : from supramolecular assembly to applications in catalysis and biosensing

    NARCIS (Netherlands)

    Prusty, Deepak Kumar

    2012-01-01

    Het proefschrift van Deepak Prusty heeft nieuwe toepassingen van DNA als onderwerp. DNA heeft het vermogen om heel specifiek andere moleculen te herkennen en is daarom een uitstekende bouwsteen voor het maken van zelf-assemblerende nano structuren. Met behulp van bijvoorbeeld fluoroforen

  14. Self-assembling of large ordered DNA arrays using superhydrophobic patterned surfaces

    International Nuclear Information System (INIS)

    Ciasca, G; Papi, M; Chiarpotto, M; Palmieri, V; De Spirito, M; Businaro, L; Notargiacomo, A; De Ninno, A; Carta, S; Giovine, E; Gerardino, A

    2013-01-01

    In this paper we present a simple and robust method to realize highly ordered arrays of stretched and suspended DNA molecules over the millimeter length scale. To this end we used an ad hoc designed superhydrophobic surface made of high aspect-ratio silicon pillars, where we deposited a droplet containing genomic DNA. A precise positioning of DNA strands was achieved by shaping the silicon pillars so that sharpened features resembling tips were included. Such features allowed us to accurately control the droplet de-wetting dynamics, pinning DNA strands in a well-defined position above pillars. The proposed technique has the potential to positively impact on the development of novel DNA chips for genetic analysis. (paper)

  15. Automated solar cell assembly team process research. Annual subcontract report, 1 January 1993--31 December 1993

    Energy Technology Data Exchange (ETDEWEB)

    Nowlan, M J; Hogan, S J; Darkazalli, G; Breen, W F; Murach, J M; Sutherland, S F; Patterson, J S [Spire Corp., Bedford, MA (United States)

    1994-06-01

    This report describes work done under the Photovoltaic Manufacturing Technology (PVMaT) project, Phase 3A, which addresses problems that are generic to the photovoltaic (PV) industry. Spire`s objective during Phase 3A was to use its light soldering technology and experience to design and fabricate solar cell tabbing and interconnecting equipment to develop new, high-yield, high-throughput, fully automated processes for tabbing and interconnecting thin cells. Areas that were addressed include processing rates, process control, yield, throughput, material utilization efficiency, and increased use of automation. Spire teamed with Solec International, a PV module manufacturer, and the University of Massachusetts at Lowell`s Center for Productivity Enhancement (CPE), automation specialists, who are lower-tier subcontractors. A number of other PV manufacturers, including Siemens Solar, Mobil Solar, Solar Web, and Texas instruments, agreed to evaluate the processes developed under this program.

  16. Target guided synthesis using DNA nano-templates for selectively assembling a G-quadruplex binding c-MYC inhibitor

    Science.gov (United States)

    Panda, Deepanjan; Saha, Puja; Das, Tania; Dash, Jyotirmayee

    2017-07-01

    The development of small molecules is essential to modulate the cellular functions of biological targets in living system. Target Guided Synthesis (TGS) approaches have been used for the identification of potent small molecules for biological targets. We herein demonstrate an innovative example of TGS using DNA nano-templates that promote Huisgen cycloaddition from an array of azide and alkyne fragments. A G-quadruplex and a control duplex DNA nano-template have been prepared by assembling the DNA structures on gold-coated magnetic nanoparticles. The DNA nano-templates facilitate the regioselective formation of 1,4-substituted triazole products, which are easily isolated by magnetic decantation. The G-quadruplex nano-template can be easily recovered and reused for five reaction cycles. The major triazole product, generated by the G-quadruplex inhibits c-MYC expression by directly targeting the c-MYC promoter G-quadruplex. This work highlights that the nano-TGS approach may serve as a valuable strategy to generate target-selective ligands for drug discovery.

  17. Automated seamless DNA co-transformation cloning with direct expression vectors applying positive or negative insert selection

    Directory of Open Access Journals (Sweden)

    Frey Daniel

    2010-08-01

    Full Text Available Abstract Background Molecular DNA cloning is crucial to many experiments and with the trend to higher throughput of modern approaches automated techniques are urgently required. We have established an automated, fast and flexible low-cost expression cloning approach requiring only vector and insert amplification by PCR and co-transformation of the products. Results Our vectors apply positive selection for the insert or negative selection against empty vector molecules and drive strong expression of target proteins in E.coli cells. Variable tags are available both in N-terminal or C-terminal position. A newly developed β-lactamase (ΔW290 selection cassette contains a segment inside the β-lactamase open reading frame encoding a stretch of hydrophilic amino acids that result in a T7 promoter when back-translated. This position of the promoter permits positive selection and attenuated expression of fusion proteins with C-terminal tags. We have tested eight vectors by inserting six target sequences of variable length, provenience and function. The target proteins were cloned, expressed and detected using an automated Tecan Freedom Evo II liquid handling work station. Only two colonies had to be picked to score with 85% correct inserts while 80% of those were positive in expression tests. Conclusions Our results establish co-transformation and positive/negative selection cloning in conjunction with the provided vectors and selection cassettes as an automatable alternative to commercialized high-throughput cloning systems like Gateway® or ligase-independent cloning (LIC .

  18. High-speed automated DNA sequencing utilizing from-the-side laser excitation

    Science.gov (United States)

    Westphall, Michael S.; Brumley, Robert L., Jr.; Buxton, Erin C.; Smith, Lloyd M.

    1995-04-01

    The Human Genome Initiative is an ambitious international effort to map and sequence the three billion bases of DNA encoded in the human genome. If successfully completed, the resultant sequence database will be a tool of unparalleled power for biomedical research. One of the major challenges of this project is in the area of DNA sequencing technology. At this time, virtually all DNA sequencing is based upon the separation of DNA fragments in high resolution polyacrylamide gels. This method, as generally practiced, is one to two orders of magnitude too slow and expensive for the successful completion of the Human Genome projection. One reasonable approach is improved sequencing of DNA fragments is to increase the performance of such gel-based sequencing methods. Decreased sequencing times may be obtained by increasing the magnitude of the electric field employed. This is not possible with conventional sequencing, due to the fact that the additional heat associated with the increased electric field cannot be adequately dissipated. Recent developments in the use of thin gels have addressed this problem. Performing electrophoresis in ultrathin (50 to 100 microns) gels greatly increases the heat transfer efficiency, thus allowing the benefits of larger electric fields to be obtained. An increase in separation speed of about an order of magnitude is readily achieved. Thin gels have successfully been used in capillary and slab formats. A detection system has been designed for use with a multiple fluorophore sequencing strategy in horizontal ultrathin slab gels. The system employs laser through-the-side excitation and a cooled CCD detector; this allows for the parallel detection of up to 24 sets of four fluorescently labeled DNA sequencing reactions during their electrophoretic separation in ultrathin (115 micrometers ) denaturing polyacrylamide gels. Four hundred bases of sequence information is obtained from 100 ng of M13 template DNA in an hour, corresponding to an

  19. The Role of the N-Terminal Domains of Bacterial Initiator DnaA in the Assembly and Regulation of the Bacterial Replication Initiation Complex

    Science.gov (United States)

    Zawilak-Pawlik, Anna; Nowaczyk, Małgorzata; Zakrzewska-Czerwińska, Jolanta

    2017-01-01

    The primary role of the bacterial protein DnaA is to initiate chromosomal replication. The DnaA protein binds to DNA at the origin of chromosomal replication (oriC) and assembles into a filament that unwinds double-stranded DNA. Through interaction with various other proteins, DnaA also controls the frequency and/or timing of chromosomal replication at the initiation step. Escherichia coli DnaA also recruits DnaB helicase, which is present in unwound single-stranded DNA and in turn recruits other protein machinery for replication. Additionally, DnaA regulates the expression of certain genes in E. coli and a few other species. Acting as a multifunctional factor, DnaA is composed of four domains that have distinct, mutually dependent roles. For example, C-terminal domain IV interacts with double-stranded DnaA boxes. Domain III drives ATP-dependent oligomerization, allowing the protein to form a filament that unwinds DNA and subsequently binds to and stabilizes single-stranded DNA in the initial replication bubble; this domain also interacts with multiple proteins that control oligomerization. Domain II constitutes a flexible linker between C-terminal domains III–IV and N-terminal domain I, which mediates intermolecular interactions between DnaA and binds to other proteins that affect DnaA activity and/or formation of the initiation complex. Of these four domains, the role of the N-terminus (domains I–II) in the assembly of the initiation complex is the least understood and appears to be the most species-dependent region of the protein. Thus, in this review, we focus on the function of the N-terminus of DnaA in orisome formation and the regulation of its activity in the initiation complex in different bacteria. PMID:28489024

  20. Detection of bacteria in platelet concentrates: comparison of broad-range real-time 16S rDNA polymerase chain reaction and automated culturing

    NARCIS (Netherlands)

    Mohammadi, Tamimount; Pietersz, Ruby N. I.; Vandenbroucke-Grauls, Christina M. J. E.; Savelkoul, Paul H. M.; Reesink, Henk W.

    2005-01-01

    BACKGROUND: Based on real-time polymerase chain reaction (PCR) technology, a broad-range 16S rDNA assay was validated and its performance was compared to that of an automated culture system to determine its usefulness for rapid routine screening of platelet concentrates (PCs). STUDY DESIGN AND

  1. Cooperative assembly of Co-Smad4 MH1 with R-Smad1/3 MH1 on DNA: a molecular dynamics simulation study.

    Directory of Open Access Journals (Sweden)

    Guihong Wang

    Full Text Available BACKGROUND: Smads, the homologs of Sma and MAD proteins, play a key role in gene expression regulation in the transforming growth factor-β (TGF-β signaling pathway. Recent experimental studies have revealed that Smad4/R-Smad heterodimers bound on DNA are energetically more favorable than homodimeric R-Smad/R-Smad complexes bound on DNA, which indicates that Smad4 might act as binding vehicle to cooperatively assemble with activated R-Smads on DNA in the nucleus. However, the details of interaction mechanism for cooperative recruitment of Smad4 protein to R-Smad proteins on DNA, and allosteric communication between the Smad4-DNA and R-Smad-DNA interfaces via DNA mediating are not yet clear so far. METHODOLOGY: In the present work, we have constructed a series of Smadn+DNA+Smadn (n = 1, 3, 4 models and carried out molecular dynamics simulations, free energy calculations and DNA dynamics analysis for them to study the interaction properties of Smadn (n = 1, 3, 4 with DNA molecule. RESULTS: The results revealed that the binding of Smad4 protein to DNA molecule facilitates energetically the formation of the heteromeric Smad4+DNA+Smad1/3 complex by increasing the affinity of Smad1/3 with DNA molecule. Further investigations through the residue/base motion correlation and DNA dynamics analyses predicted that the binding of Smad4 protein to DNA molecule in the heteromeric Smad4+DNA+Smad1/3 model induces an allosteric communication from the Smad4-DNA interface to Smad1/Smad3-DNA interface via DNA base-pair helical motions, surface conformation changes and new hydrogen bond formations. The present work theoretically explains the mechanism of cooperative recruitment of Smad4 protein to Smad1/3 protein via DNA-mediated indirect readout mode in the nucleus.

  2. Correlation of the UV-induced mutational spectra and the DNA damage distribution of the human HPRT gene: Automating the analysis

    International Nuclear Information System (INIS)

    Kotturi, G.; Erfle, H.; Koop, B.F.; Boer, J.G. de; Glickman, B.W.

    1994-01-01

    Automated DNA sequencers can be readily adapted for various types of sequence-based nucleic acid analysis: more recently it was determined the distribution of UV photoproducts in the E. coli laci gene using techniques developed for automated fluorescence-based analysis. We have been working to improve the automated approach of damage distribution. Our current method is more rigorous. We have new software that integrates the area under the individual peaks, rather than measuring the height of the curve. In addition, we now employ an internal standard. The analysis can also be partially automated. Detection limits for both major types of UV-photoproducts (cyclobutane dimers and pyrimidine (6-4) pyrimidone photoproducts) are reported. The UV-induced damage distribution in the hprt gene is compared to the mutational spectra in human and rodents cells

  3. Assembly and function of DNA double-strand break repair foci in mammalian cells

    DEFF Research Database (Denmark)

    Bekker-Jensen, Simon; Mailand, Niels

    2010-01-01

    DNA double-strand breaks (DSBs) are among the most cytotoxic types of DNA damage, which if left unrepaired can lead to mutations or gross chromosomal aberrations, and promote the onset of diseases associated with genomic instability such as cancer. One of the most discernible hallmarks...... of the cellular response to DSBs is the accumulation and local concentration of a plethora of DNA damage signaling and repair proteins in the vicinity of the lesion, initiated by ATM-mediated phosphorylation of H2AX (¿-H2AX) and culminating in the generation of distinct nuclear compartments, so-called Ionizing...... of such DNA repair foci still remains limited. In this review, we focus on recent discoveries on the mechanisms that govern the formation of IRIF, and discuss the implications of such findings in light of our understanding of the physiological importance of these structures....

  4. Self-Assembly DNA Polyplex Vaccine inside Dissolving Microneedles for High-Potency Intradermal Vaccination

    Science.gov (United States)

    Liao, Jing-Fong; Lee, Jin-Ching; Lin, Chun-Kuang; Wei, Kuo-Chen; Chen, Pin-Yuan; Yang, Hung-Wei

    2017-01-01

    The strong immunogenicity induction is the powerful weapon to prevent the virus infections. This study demonstrated that one-step synthesis of DNA polyplex vaccine in microneedle (MN) patches can induce high immunogenicity through intradermal vaccination and increase the vaccine stability for storage outside the cold chain. More negative charged DNA vaccine was entrapped into the needle region of MNs followed by DNA polyplex formation with branched polyethylenimine (bPEI) pre-coated in the cavities of polydimethylsiloxane (PDMS) molds that can deliver more DNA vaccine to immune-cell rich epidermis with high transfection efficiency. Our data in this study support the safety and immunogenicity of the MN-based vaccine; the MN patch delivery system induced an immune response 3.5-fold as strong as seen with conventional intramuscular administration; the DNA polyplex formulation provided excellent vaccine stability at high temperature (could be stored at 45ºC for at least 4 months); the DNA vaccine is expected to be manufactured at low cost and not generate sharps waste. We think this study is significant to public health because there is a pressing need for an effective vaccination in developing countries. PMID:28819449

  5. Rolling cycle amplification based single-color quantum dots–ruthenium complex assembling dyads for homogeneous and highly selective detection of DNA

    Energy Technology Data Exchange (ETDEWEB)

    Su, Chen; Liu, Yufei; Ye, Tai; Xiang, Xia; Ji, Xinghu; He, Zhike, E-mail: zhkhe@whu.edu.cn

    2015-01-01

    Graphical abstract: A universal, label-free, homogeneous, highly sensitive, and selective fluorescent biosensor for DNA detection is developed by using rolling-circle amplification (RCA) based single-color quantum dots–ruthenium complex (QDs–Ru) assembling dyads. - Highlights: • The single-color QDs–Ru assembling dyads were applied in homogeneous DNA assay. • This biosensor exhibited high selectivity against base mismatched sequences. • This biosensor could be severed as universal platform for the detection of ssDNA. • This sensor could be used to detect the target in human serum samples. • This DNA sensor had a good selectivity under the interference of other dsDNA. - Abstract: In this work, a new, label-free, homogeneous, highly sensitive, and selective fluorescent biosensor for DNA detection is developed by using rolling-circle amplification (RCA) based single-color quantum dots–ruthenium complex (QDs–Ru) assembling dyads. This strategy includes three steps: (1) the target DNA initiates RCA reaction and generates linear RCA products; (2) the complementary DNA hybridizes with the RCA products to form long double-strand DNA (dsDNA); (3) [Ru(phen){sub 2}(dppx)]{sup 2+} (dppx = 7,8-dimethyldipyrido [3,2-a:2′,3′-c] phenanthroline) intercalates into the long dsDNA with strong fluorescence emission. Due to its strong binding propensity with the long dsDNA, [Ru(phen){sub 2}(dppx)]{sup 2+} is removed from the surface of the QDs, resulting in restoring the fluorescence of the QDs, which has been quenched by [Ru(phen){sub 2}(dppx)]{sup 2+} through a photoinduced electron transfer process and is overlaid with the fluorescence of dsDNA bonded Ru(II) polypyridyl complex (Ru-dsDNA). Thus, high fluorescence intensity is observed, and is related to the concentration of target. This sensor exhibits not only high sensitivity for hepatitis B virus (HBV) ssDNA with a low detection limit (0.5 pM), but also excellent selectivity in the complex matrix. Moreover

  6. Norgal: Extraction and de novo assembly of mitochondrial DNA from whole-genome sequencing data

    DEFF Research Database (Denmark)

    Al-Nakeeb, Kosai Ali Ahmed; Petersen, Thomas Nordahl; Sicheritz-Pontén, Thomas

    2017-01-01

    and performing a de novo assembly on a subset of reads that contains these k-mers. The method was applied to WGS data from a panda, brown algae seaweed, butterfly and filamentous fungus. We were able to extract full circular mitochondrial genomes and obtained sequence identities to the reference sequences...

  7. Automated Extraction of Genomic DNA from Medically Important Yeast Species and Filamentous Fungi by Using the MagNA Pure LC System

    OpenAIRE

    Loeffler, Juergen; Schmidt, Kathrin; Hebart, Holger; Schumacher, Ulrike; Einsele, Hermann

    2002-01-01

    A fully automated assay was established for the extraction of DNA from clinically important fungi by using the MagNA Pure LC instrument. The test was evaluated by DNA isolation from 23 species of yeast and filamentous fungi and by extractions (n = 28) of serially diluted Aspergillus fumigatus conidia (105 to 0 CFU/ml). Additionally, DNA from 67 clinical specimens was extracted and compared to the manual protocol. The detection limit of the MagNA Pure LC assay of 10 CFU corresponded to the sen...

  8. Regular Nanoscale Protein Patterns via Directed Adsorption through Self-Assembled DNA Origami Masks.

    Science.gov (United States)

    Ramakrishnan, Saminathan; Subramaniam, Sivaraman; Stewart, A Francis; Grundmeier, Guido; Keller, Adrian

    2016-11-16

    DNA origami has become a widely used method for synthesizing well-defined nanostructures with promising applications in various areas of nanotechnology, biophysics, and medicine. Recently, the possibility to transfer the shape of single DNA origami nanostructures into different materials via molecular lithography approaches has received growing interest due to the great structural control provided by the DNA origami technique. Here, we use ordered monolayers of DNA origami nanostructures with internal cavities on mica surfaces as molecular lithography masks for the fabrication of regular protein patterns over large surface areas. Exposure of the masked sample surface to negatively charged proteins results in the directed adsorption of the proteins onto the exposed surface areas in the holes of the mask. By controlling the buffer and adsorption conditions, the protein coverage of the exposed areas can be varied from single proteins to densely packed monolayers. To demonstrate the versatility of this approach, regular nanopatterns of four different proteins are fabricated: the single-strand annealing proteins Redβ and Sak, the iron-storage protein ferritin, and the blood protein bovine serum albumin (BSA). We furthermore demonstrate the desorption of the DNA origami mask after directed protein adsorption, which may enable the fabrication of hierarchical patterns composed of different protein species. Because selectivity in adsorption is achieved by electrostatic interactions between the proteins and the exposed surface areas, this approach may enable also the large-scale patterning of other charged molecular species or even nanoparticles.

  9. Self-assembled Multifunctional DNA Nanoflowers for the Circumvention of Multidrug Resistance in Targeted Anticancer Drug Delivery.

    Science.gov (United States)

    Mei, Lei; Zhu, Guizhi; Qiu, Liping; Wu, Cuichen; Chen, Huapei; Liang, Hao; Cansiz, Sena; Lv, Yifan; Zhang, Xiaobing; Tan, Weihong

    2015-11-01

    Cancer chemotherapy has been impeded by side effects and multidrug resistance (MDR) partially caused by drug efflux from cancer cells, which call for targeted drug delivery systems additionally able to circumvent MDR. Here we report multifunctional DNA nanoflowers (NFs) for targeted drug delivery to both chemosensitive and MDR cancer cells and circumvent MDR in both leukemia and breast cancer cell models. NFs are self-assembled via liquid crystallization of DNA generated by Rolling Circle Replication, during which NFs are incorporated with aptamers for specific cancer cell recognition, fluorophores for bioimaging, and Doxorubicin (Dox)-binding DNA for drug delivery. NF sizes are tunable (down to ~200 nm in diameter), and the densely packed drug-binding motifs and porous intrastructures endow NFs with high drug loading capacity (71.4%, wt/wt). The Dox-loaded NFs (NF-Dox) are stable at physiological pH, yet drug release is facilitated in acidic or basic conditions. NFs deliver Dox into target chemosensitive and MDR cancer cells, preventing drug efflux and enhancing drug retention in MDR cells. Consequently, NF-Dox induces potent cytotoxicity in both target chemosensitive cells and MDR cells, but not nontarget cells, thus concurrently circumventing MDR and reducing side effects. Overall, these NFs are promising to circumvent MDR in targeted cancer therapy.

  10. Constructing of DNA vectors with controlled nanosize and single dispersion by block copolymer coating gold nanoparticles as template assembly

    Energy Technology Data Exchange (ETDEWEB)

    Li, Junbo, E-mail: Lijunbo@haust.edu.cn [Henan University of Science and Technology, School of Chemical Engineering and Pharmaceutics (China); Wu, Wenlan [Henan University of Science and Technology, School of Medicine (China); Gao, Jiayu; Liang, Ju; Zhou, Huiyun; Liang, Lijuan [Henan University of Science and Technology, School of Chemical Engineering and Pharmaceutics (China)

    2017-03-15

    Synthesized vectors with nanoscale size and stable colloid dispersion are highly desirable for improving gene delivery efficiency. Here, a core-shell template particle was constructed with polyethylene glycol-b-poly1-(3-aminopropyl)-3-(2-methacryloyloxy propylimidazolium bromine) (PEG-b-PAMPImB) coating gold nanoparticles (PEG-b-PAMPImB-@-Au NPs) for loading DNA and delivering in vitro. Data from transmission electron microscopy (TEM) and dynamic light scattering (DLS) suggest that these nanoplexes, by forming an electrostatic complex with DNA at the inner PAMPImB shell, offer steric protection for the outer PEG corona leading to single dispersion and small size. Notably, higher colloid stability and lower cytotoxicity were achieved with these nanoplexes when compared with PAMPImB monolayer-coated gold nanoparticles (Au NPs). Confocal laser scanning microscopy and intracellular trafficking TEM further indicate that the nanoplexes can translocate across the cell membrane and partly enter the nucleus for high efficient expression. Thus, template assembly represents a promising approach to control the size and colloid stability of gene vectors and ensure safety and efficiency of DNA delivery.

  11. DNA bases assembled on the Au(110)/electrolyte interface: A combined experimental and theoretical study

    DEFF Research Database (Denmark)

    Salvatore, Princia; Nazmutdinov, Renat R.; Ulstrup, Jens

    2015-01-01

    , accompanied by a pair of strong voltammetry peaks in the double-layer region in acid solutions. Adsorption of the DNA bases gives featureless voltammograms with lower double-layer capacitance, suggesting that all the bases are chemisorbed on the Au(110) surface. Further investigation of the surface structures...... of the adlayers of the four DNA bases by EC-STM disclosed lifting of the Au(110) reconstruction, specific molecular packing in dense monolayers, and pH dependence of the A and G adsorption. DFT computations based on a cluster model for the Au(110) surface were performed to investigate the adsorption energy...... and geometry of the DNA bases in different adsorbate orientations. The optimized geometry is further used to compute models for STM images which are compared with the recorded STM images. This has provided insight into the physical nature of the adsorption. The specific orientations of A, C, G, and T on Au(110...

  12. Genomic libraries: II. Subcloning, sequencing, and assembling large-insert genomic DNA clones.

    Science.gov (United States)

    Quail, Mike A; Matthews, Lucy; Sims, Sarah; Lloyd, Christine; Beasley, Helen; Baxter, Simon W

    2011-01-01

    Sequencing large insert clones to completion is useful for characterizing specific genomic regions, identifying haplotypes, and closing gaps in whole genome sequencing projects. Despite being a standard technique in molecular laboratories, DNA sequencing using the Sanger method can be highly problematic when complex secondary structures or sequence repeats are encountered in genomic clones. Here, we describe methods to isolate DNA from a large insert clone (fosmid or BAC), subclone the sample, and sequence the region to the highest industry standard. Troubleshooting solutions for sequencing difficult templates are discussed.

  13. Delineation of the role of chromatin assembly and the Rtt101Mms1 E3 ubiquitin ligase in DNA damage checkpoint recovery in budding yeast.

    Directory of Open Access Journals (Sweden)

    Li-Ting Diao

    Full Text Available The DNA damage checkpoint is activated in response to DNA double-strand breaks (DSBs. We had previously shown that chromatin assembly mediated by the histone chaperone Asf1 triggers inactivation of the DNA damage checkpoint in yeast after DSB repair, also called checkpoint recovery. Here we show that chromatin assembly factor 1 (CAF-1 also contributes to chromatin reassembly after DSB repair, explaining its role in checkpoint recovery. Towards understanding how chromatin assembly promotes checkpoint recovery, we find persistent presence of the damage sensors Ddc1 and Ddc2 after DSB repair in asf1 mutants. The genes encoding the E3 ubiquitin ligase complex Rtt101Mms1 are epistatic to ASF1 for survival following induction of a DSB, and Rtt101Mms1 are required for checkpoint recovery after DSB repair but not for chromatin assembly. By contrast, the Mms22 substrate adaptor that is degraded by Rtt101Mms1 is required for DSB repair per se. Deletion of MMS22 blocks loading of Rad51 at the DSB, while deletion of ASF1 or RTT101 leads to persistent Rad51 loading. We propose that checkpoint recovery is promoted by Rtt101Mms1-mediated ubiquitylation of Mms22 in order to halt Mms22-dependent loading of Rad51 onto double-stranded DNA after DSB repair, in concert with the chromatin assembly-mediated displacement of Rad51 and checkpoint sensors from the site of repair.

  14. Use of a DNA film on a self-assembled monolayer for investigating the physical process of DNA damage induced by core electron ionization.

    Science.gov (United States)

    Narita, Ayumi; Fujii, Kentaro; Baba, Yuji; Shimoyama, Iwao

    2016-11-01

    A novel two-layer sample composed of a deoxyribonucleic acid (DNA) film and self-assembled monolayer (SAM) was prepared on an inorganic surface to mimic the processes in which DNA is damaged by soft X-ray irradiation. A mercaptopropyltrimethoxysilane (MPTS) SAM was formed on a sapphire surface, then oligonucleotide (OGN) molecules were adsorbed on the MPTS-SAM. The thicknesses and chemical states of the layers were determined by X-ray photoelectron spectroscopy (XPS) and near-edge X-ray fine structure (NEXAFS) around the phosphorus (P) and sulfur (S) K-edges. To induce the damage to the OGN molecules, the sample was irradiated with synchrotron soft X-rays. The chemical state of the OGN molecules before and after irradiation was examined by NEXAFS around the nitrogen (N) K-edge region. The thickness of the MPTS-OGN layer was approximately 7.7 nm. The S atom of the OGN molecules was located at the bottom of the OGN layer. The peak shape of the N K-edge NEXAFS spectra of the MPTS-OGN layers clearly changed following irradiation. The MPTS-OGN layer formed on the sapphire surface. The chemical states and the structure of the interface were elucidated using synchrotron soft X-rays. The OGN molecules adsorbed on the MPTS films decomposed upon exposure to soft X-ray irradiation.

  15. Automated extraction of genomic DNA from medically important yeast species and filamentous fungi by using the MagNA Pure LC system.

    Science.gov (United States)

    Loeffler, Juergen; Schmidt, Kathrin; Hebart, Holger; Schumacher, Ulrike; Einsele, Hermann

    2002-06-01

    A fully automated assay was established for the extraction of DNA from clinically important fungi by using the MagNA Pure LC instrument. The test was evaluated by DNA isolation from 23 species of yeast and filamentous fungi and by extractions (n = 28) of serially diluted Aspergillus fumigatus conidia (10(5) to 0 CFU/ml). Additionally, DNA from 67 clinical specimens was extracted and compared to the manual protocol. The detection limit of the MagNA Pure LC assay of 10 CFU corresponded to the sensitivity when DNA was extracted manually; in 9 of 28 runs, we could achieve a higher sensitivity of 1 CFU/ml blood, which was found to be significant (p DNA from all fungal species analyzed could be extracted and amplified by real-time PCR. Negative controls from all MagNA Pure isolations remained negative. Sixty-three clinical samples showed identical results by both methods, whereas in 4 of 67 samples, discordant results were obtained. Thus, the MagNA Pure LC technique offers a fast protocol for automated DNA isolation from numerous fungi, revealing high sensitivity and purity.

  16. Design and simulation of integration system between automated material handling system and manufacturing layout in the automotive assembly line

    Science.gov (United States)

    Seha, S.; Zamberi, J.; Fairu, A. J.

    2017-10-01

    Material handling system (MHS) is an important part for the productivity plant and has recognized as an integral part of today’s manufacturing system. Currently, MHS has growth tremendously with its technology and equipment type. Based on the case study observation, the issue involving material handling system contribute to the reduction of production efficiency. This paper aims to propose a new design of integration between material handling and manufacturing layout by investigating the influences of layout and material handling system. A method approach tool using Delmia Quest software is introduced and the simulation result is used to assess the influences of the integration between material handling system and manufacturing layout in the performance of automotive assembly line. The result show, the production of assembly line output increases more than 31% from the current system. The source throughput rate average value went up to 252 units per working hour in model 3 and show the effectiveness of the pick-to-light system as efficient storage equipment. Thus, overall result shows, the application of AGV and the pick-to-light system gave a large significant effect in the automotive assembly line. Moreover, the change of layout also shows a large significant improvement to the performance.

  17. Functionalized Nanostructures: Redox-Active Porphyrin Anchors for Supramolecular DNA Assemblies

    KAUST Repository

    Börjesson, Karl

    2010-09-28

    We have synthesized and studied a supramolecular system comprising a 39-mer DNA with porphyrin-modified thymidine nucleosides anchored to the surface of large unilamellar vesicles (liposomes). Liposome porphyrin binding characteristics, such as orientation, strength, homogeneity, and binding site size, was determined, suggesting that the porphyrin is well suited as a photophysical and redox-active lipid anchor, in comparison to the inert cholesterol anchor commonly used today. Furthermore, the binding characteristics and hybridization capabilities were studied as a function of anchor size and number of anchoring points, properties that are of importance for our future plans to use the addressability of these redox-active nodes in larger DNA-based nanoconstructs. Electron transfer from photoexcited porphyrin to a lipophilic benzoquinone residing in the lipid membrane was characterized by steady-state and time-resolved fluorescence and verified by femtosecond transient absorption. © 2010 American Chemical Society.

  18. Fully Automated Complementary DNA Microarray Segmentation using a Novel Fuzzy-based Algorithm.

    Science.gov (United States)

    Saberkari, Hamidreza; Bahrami, Sheyda; Shamsi, Mousa; Amoshahy, Mohammad Javad; Ghavifekr, Habib Badri; Sedaaghi, Mohammad Hossein

    2015-01-01

    DNA microarray is a powerful approach to study simultaneously, the expression of 1000 of genes in a single experiment. The average value of the fluorescent intensity could be calculated in a microarray experiment. The calculated intensity values are very close in amount to the levels of expression of a particular gene. However, determining the appropriate position of every spot in microarray images is a main challenge, which leads to the accurate classification of normal and abnormal (cancer) cells. In this paper, first a preprocessing approach is performed to eliminate the noise and artifacts available in microarray cells using the nonlinear anisotropic diffusion filtering method. Then, the coordinate center of each spot is positioned utilizing the mathematical morphology operations. Finally, the position of each spot is exactly determined through applying a novel hybrid model based on the principle component analysis and the spatial fuzzy c-means clustering (SFCM) algorithm. Using a Gaussian kernel in SFCM algorithm will lead to improving the quality in complementary DNA microarray segmentation. The performance of the proposed algorithm has been evaluated on the real microarray images, which is available in Stanford Microarray Databases. Results illustrate that the accuracy of microarray cells segmentation in the proposed algorithm reaches to 100% and 98% for noiseless/noisy cells, respectively.

  19. Serine-based gemini surfactants with different spacer linkages: from self-assembly to DNA compaction.

    Science.gov (United States)

    Silva, Sandra G; Oliveira, Isabel S; do Vale, M Luísa C; Marques, Eduardo F

    2014-12-14

    Cationic gemini surfactants have strong potential as compaction agents of nucleic acids for efficient non-viral gene delivery. In this work, we present the aggregation behavior of three novel cationic serine-based gemini surfactants as well as their ability to compact DNA per se and mixed with a helper lipid, 1,2-dioleoyl-sn-glycero-3-phosphoethanolamine (DOPE). All the surfactants have a 12-12-12 configuration, i.e. two main 12-carbon alkyl chains linked to the nitrogen atom of the amino acid residue and a 12 methylene spacer, but they differ in the nature of the spacer linkage: for (12Ser)2N12, an amine bond; for (12Ser)2CON12, an amide bond; and for (12Ser)2COO12, an ester bond. Interestingly, while the amine-based gemini aggregates into micelles, the amide and ester ones spontaneously form vesicles, which denotes a strong influence of the type of linkage on the surfactant packing parameter. The size, ζ-potential and stability of the vesicles have been characterized by light microscopy, cryogenic scanning electron microscopy (cryo-SEM) and dynamic light scattering (DLS). The interaction of the gemini aggregates with DNA at different charge ratios and in the absence and presence of DOPE has been studied by DLS, fluorescence spectroscopy and cryo-SEM. All the compounds are found to efficiently compact DNA (complexation > 90%), but relevant differences are obtained in terms of the size, ζ-potential and stability of the lipoplexes formed. Results are rationalized in terms of headgroup differences and the type of aggregates present prior to DNA condensation.

  20. Mathematical modelling of the automated FADU assay for the quantification of DNA strand breaks and their repair in human peripheral mononuclear blood cells

    International Nuclear Information System (INIS)

    Junk, Michael; Salzwedel, Judy; Sindlinger, Thilo; Bürkle, Alexander; Moreno-Villanueva, Maria

    2014-01-01

    Cells continuously undergo DNA damage from exogenous agents like irradiation or genotoxic chemicals or from endogenous radicals produced by normal cellular metabolic activities. DNA strand breaks are one of the most common genotoxic lesions and they can also arise as intermediates of DNA repair activity. Unrepaired DNA damage can lead to genomic instability, which can massively compromise the health status of organisms. Therefore it is important to measure and quantify DNA damage and its repair. We have previously published an automated method for measuring DNA strand breaks based on fluorimetric detection of alkaline DNA unwinding [1], and here we present a mathematical model of the FADU assay, which enables to an analytic expression for the relation between measured fluorescence and the number of strand breaks. Assessment of the formation and also the repair of DNA strand breaks is a crucial functional parameter to investigate genotoxicity in living cells. A reliable and convenient method to quantify DNA strand breakage is therefore of significant importance for a wide variety of scientific fields, e.g. toxicology, pharmacology, epidemiology and medical sciences

  1. Cost-effective sequencing of full-length cDNA clones powered by a de novo-reference hybrid assembly.

    Directory of Open Access Journals (Sweden)

    Reginaldo M Kuroshu

    Full Text Available BACKGROUND: Sequencing full-length cDNA clones is important to determine gene structures including alternative splice forms, and provides valuable resources for experimental analyses to reveal the biological functions of coded proteins. However, previous approaches for sequencing cDNA clones were expensive or time-consuming, and therefore, a fast and efficient sequencing approach was demanded. METHODOLOGY: We developed a program, MuSICA 2, that assembles millions of short (36-nucleotide reads collected from a single flow cell lane of Illumina Genome Analyzer to shotgun-sequence approximately 800 human full-length cDNA clones. MuSICA 2 performs a hybrid assembly in which an external de novo assembler is run first and the result is then improved by reference alignment of shotgun reads. We compared the MuSICA 2 assembly with 200 pooled full-length cDNA clones finished independently by the conventional primer-walking using Sanger sequencers. The exon-intron structure of the coding sequence was correct for more than 95% of the clones with coding sequence annotation when we excluded cDNA clones insufficiently represented in the shotgun library due to PCR failure (42 out of 200 clones excluded, and the nucleotide-level accuracy of coding sequences of those correct clones was over 99.99%. We also applied MuSICA 2 to full-length cDNA clones from Toxoplasma gondii, to confirm that its ability was competent even for non-human species. CONCLUSIONS: The entire sequencing and shotgun assembly takes less than 1 week and the consumables cost only approximately US$3 per clone, demonstrating a significant advantage over previous approaches.

  2. Cost-effective sequencing of full-length cDNA clones powered by a de novo-reference hybrid assembly.

    Science.gov (United States)

    Kuroshu, Reginaldo M; Watanabe, Junichi; Sugano, Sumio; Morishita, Shinichi; Suzuki, Yutaka; Kasahara, Masahiro

    2010-05-07

    Sequencing full-length cDNA clones is important to determine gene structures including alternative splice forms, and provides valuable resources for experimental analyses to reveal the biological functions of coded proteins. However, previous approaches for sequencing cDNA clones were expensive or time-consuming, and therefore, a fast and efficient sequencing approach was demanded. We developed a program, MuSICA 2, that assembles millions of short (36-nucleotide) reads collected from a single flow cell lane of Illumina Genome Analyzer to shotgun-sequence approximately 800 human full-length cDNA clones. MuSICA 2 performs a hybrid assembly in which an external de novo assembler is run first and the result is then improved by reference alignment of shotgun reads. We compared the MuSICA 2 assembly with 200 pooled full-length cDNA clones finished independently by the conventional primer-walking using Sanger sequencers. The exon-intron structure of the coding sequence was correct for more than 95% of the clones with coding sequence annotation when we excluded cDNA clones insufficiently represented in the shotgun library due to PCR failure (42 out of 200 clones excluded), and the nucleotide-level accuracy of coding sequences of those correct clones was over 99.99%. We also applied MuSICA 2 to full-length cDNA clones from Toxoplasma gondii, to confirm that its ability was competent even for non-human species. The entire sequencing and shotgun assembly takes less than 1 week and the consumables cost only approximately US$3 per clone, demonstrating a significant advantage over previous approaches.

  3. The application of an automated allele concordance analysis system (CompareCalls) to ensure the accuracy of single-source STR DNA profiles.

    Science.gov (United States)

    Ryan, John H; Barrus, Jeffrey K; Budowle, Bruce; Shannon, Cynthia M; Thompson, Victor W; Ward, Brian E

    2004-05-01

    A powerful method for validating a scientific result is to confirm specific results utilizing independent methodologies and processing pathways. Thus, we have designed, developed and validated an automated allele concordance analysis system (CompareCalls, patent pending) that performs comparisons between two independent DNA analysis platforms to ensure the highest accuracy for allele calls. Application of this system in a quality assurance role has shown the potential to eliminate greater than 90% of the STR analysis required of a DNA data analyst. While this system is broadly applicable for use with any two independent STR analysis programs, either prior to or following human data review, we are presenting its application to data generated with the ABI Prism Genotyper software system versus data generated with the SurelockID system. With the automated allele concordance analysis system, the GeneScan DNA fragment data generated from an ABI 377 gel image are analyzed in two independent pathways. In one analysis pathway, the GeneScan data are imported into Genotyper software where STR labels are assigned to the fragment data based upon the criteria of the Kazam 20% macro. The "Kazam" macro provided with the Genotyper program works by labeling all peaks in a category (or locus) and then filtering (or removing) the labels from peaks, such as those in stutter positions, that meet predefined criteria. In the second pathway, the GeneScan data are imported into the SurelockID analysis platform where STR labels and error messages are assigned to the fragment data based upon hard-coded allele calling criteria and quality parameters. The resulting STR allele calls for each analysis platform are then compared, utilizing the automated allele concordance analysis system. Any differences in the STR allele calls between the two systems are flagged in a discordance report for further review by a qualified DNA data analyst. The automated allele concordance analysis system guides the

  4. Prioritizing multiple therapeutic targets in parallel using automated DNA-encoded library screening

    Science.gov (United States)

    Machutta, Carl A.; Kollmann, Christopher S.; Lind, Kenneth E.; Bai, Xiaopeng; Chan, Pan F.; Huang, Jianzhong; Ballell, Lluis; Belyanskaya, Svetlana; Besra, Gurdyal S.; Barros-Aguirre, David; Bates, Robert H.; Centrella, Paolo A.; Chang, Sandy S.; Chai, Jing; Choudhry, Anthony E.; Coffin, Aaron; Davie, Christopher P.; Deng, Hongfeng; Deng, Jianghe; Ding, Yun; Dodson, Jason W.; Fosbenner, David T.; Gao, Enoch N.; Graham, Taylor L.; Graybill, Todd L.; Ingraham, Karen; Johnson, Walter P.; King, Bryan W.; Kwiatkowski, Christopher R.; Lelièvre, Joël; Li, Yue; Liu, Xiaorong; Lu, Quinn; Lehr, Ruth; Mendoza-Losana, Alfonso; Martin, John; McCloskey, Lynn; McCormick, Patti; O'Keefe, Heather P.; O'Keeffe, Thomas; Pao, Christina; Phelps, Christopher B.; Qi, Hongwei; Rafferty, Keith; Scavello, Genaro S.; Steiginga, Matt S.; Sundersingh, Flora S.; Sweitzer, Sharon M.; Szewczuk, Lawrence M.; Taylor, Amy; Toh, May Fern; Wang, Juan; Wang, Minghui; Wilkins, Devan J.; Xia, Bing; Yao, Gang; Zhang, Jean; Zhou, Jingye; Donahue, Christine P.; Messer, Jeffrey A.; Holmes, David; Arico-Muendel, Christopher C.; Pope, Andrew J.; Gross, Jeffrey W.; Evindar, Ghotas

    2017-07-01

    The identification and prioritization of chemically tractable therapeutic targets is a significant challenge in the discovery of new medicines. We have developed a novel method that rapidly screens multiple proteins in parallel using DNA-encoded library technology (ELT). Initial efforts were focused on the efficient discovery of antibacterial leads against 119 targets from Acinetobacter baumannii and Staphylococcus aureus. The success of this effort led to the hypothesis that the relative number of ELT binders alone could be used to assess the ligandability of large sets of proteins. This concept was further explored by screening 42 targets from Mycobacterium tuberculosis. Active chemical series for six targets from our initial effort as well as three chemotypes for DHFR from M. tuberculosis are reported. The findings demonstrate that parallel ELT selections can be used to assess ligandability and highlight opportunities for successful lead and tool discovery.

  5. A fully automated 384 capillary array for DNA sequencer. Final report

    Energy Technology Data Exchange (ETDEWEB)

    Li, Qingbo; Kane, T

    2003-03-20

    Phase I SpectruMedix has successfully developed an automatic 96-capillary array DNA prototype based on the multiplexed capillary electrophoresis system originated from Ames Laboratory-USDOE, Iowa State University. With computer control of all steps involved in a 96-capillary array running cycle, the prototype instrument (the SCE9600) is now capable of sequencing 450 base pairs (bp) per capillary, or 48,000 bp per instrument run within 2 hrs. Phase II of this grant involved the advancement of the core 96 capillary technologies, as well as designing a high density 384 capillary prototype. True commercialization of the 96 capillary instrument involved finalization of the gel matrix, streamlining the instrument hardware, creating a more reliable capillary cartridge, and further advancement of the data processing software. Together these silos of technology create a truly commercializable product (the SCE9610) capable of meeting the operation needs of the sequencing centers.

  6. DNA-Assembled Core-Satellite Upconverting-Metal-Organic Framework Nanoparticle Superstructures for Efficient Photodynamic Therapy.

    Science.gov (United States)

    He, Liangcan; Brasino, Michael; Mao, Chenchen; Cho, Suehyun; Park, Wounjhang; Goodwin, Andrew P; Cha, Jennifer N

    2017-06-01

    DNA-mediated assembly of core-satellite structures composed of Zr(IV)-based porphyrinic metal-organic framework (MOF) and NaYF 4 ,Yb,Er upconverting nanoparticles (UCNPs) for photodynamic therapy (PDT) is reported. MOF NPs generate singlet oxygen ( 1 O 2 ) upon photoirradiation with visible light without the need for additional small molecule, diffusional photosensitizers such as porphyrins. Using DNA as a templating agent, well-defined MOF-UCNP clusters are produced where UCNPs are spatially organized around a centrally located MOF NP. Under NIR irradiation, visible light emitted from the UCNPs is absorbed by the core MOF NP to produce 1 O 2 at significantly greater amounts than what can be produced from simply mixing UCNPs and MOF NPs. The MOF-UCNP core-satellite superstructures also induce strong cell cytotoxicity against cancer cells, which are further enhanced by attaching epidermal growth factor receptor targeting affibodies to the PDT clusters, highlighting their promise as theranostic photodynamic agents. © 2017 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

  7. DNA damage response clamp 9-1-1 promotes assembly of ZMM proteins for formation of crossovers and synaptonemal complex

    Science.gov (United States)

    Shinohara, Miki; Hayashihara, Kayoko; Grubb, Jennifer T.; Bishop, Douglas K.; Shinohara, Akira

    2015-01-01

    Formation of crossovers between homologous chromosomes during meiosis is positively regulated by the ZMM proteins (also known as SIC proteins). DNA damage checkpoint proteins also promote efficient formation of interhomolog crossovers. Here, we examined, in budding yeast, the meiotic role of the heterotrimeric DNA damage response clamp composed of Rad17, Ddc1 and Mec3 (known as ‘9-1-1’ in other organisms) and a component of the clamp loader, Rad24 (known as Rad17 in other organisms). Cytological analysis indicated that the 9-1-1 clamp and its loader are not required for the chromosomal loading of RecA homologs Rad51 or Dmc1, but are necessary for the efficient loading of ZMM proteins. Interestingly, the loading of ZMM proteins onto meiotic chromosomes was independent of the checkpoint kinase Mec1 (the homolog of ATR) as well as Rad51. Furthermore, the ZMM member Zip3 (also known as Cst9) bound to the 9-1-1 complex in a cell-free system. These data suggest that, in addition to promoting interhomolog bias mediated by Rad51–Dmc1, the 9-1-1 clamp promotes crossover formation through a specific role in the assembly of ZMM proteins. Thus, the 9-1-1 complex functions to promote two crucial meiotic recombination processes, the regulation of interhomolog recombination and crossover formation mediated by ZMM. PMID:25736290

  8. A filter paper-based microdevice for low-cost, rapid, and automated DNA extraction and amplification from diverse sample types.

    Science.gov (United States)

    Gan, Wupeng; Zhuang, Bin; Zhang, Pengfei; Han, Junping; Li, Cai-Xia; Liu, Peng

    2014-10-07

    A plastic microfluidic device that integrates a filter disc as a DNA capture phase was successfully developed for low-cost, rapid and automated DNA extraction and PCR amplification from various raw samples. The microdevice was constructed by sandwiching a piece of Fusion 5 filter, as well as a PDMS (polydimethylsiloxane) membrane, between two PMMA (poly(methyl methacrylate)) layers. An automated DNA extraction from 1 μL of human whole blood can be finished on the chip in 7 minutes by sequentially aspirating NaOH, HCl, and water through the filter. The filter disc containing extracted DNA was then taken out directly for PCR. On-chip DNA purification from 0.25-1 μL of human whole blood yielded 8.1-21.8 ng of DNA, higher than those obtained using QIAamp® DNA Micro kits. To realize DNA extraction from raw samples, an additional sample loading chamber containing a filter net with an 80 μm mesh size was designed in front of the extraction chamber to accommodate sample materials. Real-world samples, including whole blood, dried blood stains on Whatman® 903 paper, dried blood stains on FTA™ cards, buccal swabs, saliva, and cigarette butts, can all be processed in the system in 8 minutes. In addition, multiplex amplification of 15 STR (short tandem repeat) loci and Sanger-based DNA sequencing of the 520 bp GJB2 gene were accomplished from the filters that contained extracted DNA from blood. To further prove the feasibility of integrating this extraction method with downstream analyses, "in situ" PCR amplifications were successfully performed in the DNA extraction chamber following DNA purification from blood and blood stains without DNA elution. Using a modified protocol to bond the PDMS and PMMA, our plastic PDMS devices withstood the PCR process without any leakage. This study represents a significant step towards the practical application of on-chip DNA extraction methods, as well as the development of fully integrated genetic analytical systems.

  9. Self-Assembly of Large-Scale Shape-Controlled DNA Nano-Structures

    Science.gov (United States)

    2014-12-16

    have transformative impact. 15. SUBJECT TERMS DNA nanotechnology, nucleic acid structures, patterning, barcoding 16. SECURITY CLASSIFICATION OF...other standard keyboard symbols, 10 emoticons, 9 astrological symbols, 6 Chinese characters and various miscellaneous symbols. F,ach image is 150 nm X...100 \\iM stock in 0.5 x Tris/EDTA buffer (5 niM Tris, pH 7.9, 1 mM I-D’l’A) supplemented with 40 mM MgCl,. For Z-crystals and ZX-crystals, the final

  10. Porcine transcriptome analysis based on 97 non-normalized cDNA libraries and assembly of 1,021,891 expressed sequence tags

    DEFF Research Database (Denmark)

    Gorodkin, Jan; Cirera, Susanna; Hedegaard, Jacob

    2007-01-01

    public databases. The Sino-Danish ESTs were generated from one normalized and 97 non-normalized cDNA libraries representing 35 different tissues and three developmental stages. RESULTS: Using the Distiller package, the ESTs were assembled to roughly 48,000 contigs and 73,000 singletons, of which...

  11. Automated solar cell assembly teamed process research. Semiannual subcontract report, 7 January 1993--30 June 1993

    Energy Technology Data Exchange (ETDEWEB)

    Nowlan, M.J.; Hogan, S.J.; Darkazalli, G.; Breen, W.F.; Murach, J.M.; Sutherland, S.F. [Spire Corp., Bedford, MA (United States)

    1994-02-01

    This report describes work done under Phase 3A of the PVMaT project to address problems that are generic to the photovoltaics (PV) industry. Crystalline silicon solar cells were used in the majority of all terrestrial power modules shipped in 1992. Spire`s analysis in Phase 1 of the PVMaT project indicated that the use of thin ({le}200-{mu}m) silicon cells can substantially reduce module manufacturing costs, provided that processing yields remain as high as they are now for processing standard thickness cells. Because present solar cell tabbing and interconnecting processes have unacceptably high yield losses with such thin cells, the objective of this Phase 3A subcontract is to use Spire`s light soldering technology and experience in designing and fabricating solar cell tabbing and interconnecting equipment to develop high yield throughput, fully automated processes for tabbing and interconnecting thin cells.

  12. Automation of DNA and miRNA co-extraction for miRNA-based identification of human body fluids and tissues.

    Science.gov (United States)

    Kulstein, Galina; Marienfeld, Ralf; Miltner, Erich; Wiegand, Peter

    2016-10-01

    In the last years, microRNA (miRNA) analysis came into focus in the field of forensic genetics. Yet, no standardized and recommendable protocols for co-isolation of miRNA and DNA from forensic relevant samples have been developed so far. Hence, this study evaluated the performance of an automated Maxwell® 16 System-based strategy (Promega) for co-extraction of DNA and miRNA from forensically relevant (blood and saliva) samples compared to (semi-)manual extraction methods. Three procedures were compared on the basis of recovered quantity of DNA and miRNA (as determined by real-time PCR and Bioanalyzer), miRNA profiling (shown by Cq values and extraction efficiency), STR profiles, duration, contamination risk and handling. All in all, the results highlight that the automated co-extraction procedure yielded the highest miRNA and DNA amounts from saliva and blood samples compared to both (semi-)manual protocols. Also, for aged and genuine samples of forensically relevant traces the miRNA and DNA yields were sufficient for subsequent downstream analysis. Furthermore, the strategy allows miRNA extraction only in cases where it is relevant to obtain additional information about the sample type. Besides, this system enables flexible sample throughput and labor-saving sample processing with reduced risk of cross-contamination. © 2016 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

  13. Centrifugal LabTube platform for fully automated DNA purification and LAMP amplification based on an integrated, low-cost heating system.

    Science.gov (United States)

    Hoehl, Melanie M; Weißert, Michael; Dannenberg, Arne; Nesch, Thomas; Paust, Nils; von Stetten, Felix; Zengerle, Roland; Slocum, Alexander H; Steigert, Juergen

    2014-06-01

    This paper introduces a disposable battery-driven heating system for loop-mediated isothermal DNA amplification (LAMP) inside a centrifugally-driven DNA purification platform (LabTube). We demonstrate LabTube-based fully automated DNA purification of as low as 100 cell-equivalents of verotoxin-producing Escherichia coli (VTEC) in water, milk and apple juice in a laboratory centrifuge, followed by integrated and automated LAMP amplification with a reduction of hands-on time from 45 to 1 min. The heating system consists of two parallel SMD thick film resistors and a NTC as heating and temperature sensing elements. They are driven by a 3 V battery and controlled by a microcontroller. The LAMP reagents are stored in the elution chamber and the amplification starts immediately after the eluate is purged into the chamber. The LabTube, including a microcontroller-based heating system, demonstrates contamination-free and automated sample-to-answer nucleic acid testing within a laboratory centrifuge. The heating system can be easily parallelized within one LabTube and it is deployable for a variety of heating and electrical applications.

  14. Final report : LDRD project 79824 carbon nanotube sorting via DNA-directed self-assembly.

    Energy Technology Data Exchange (ETDEWEB)

    Robinson, David B; Leung, Kevin; Rempe, Susan B.; Dossa, Paul D.; Frischknecht, Amalie Lucile; Martin, Marcus Gary

    2007-10-01

    Single-wall carbon nanotubes (SWNTs) have shown great promise in novel applications in molecular electronics, biohazard detection, and composite materials. Commercially synthesized nanotubes exhibit a wide dispersion of geometries and conductivities, and tend to aggregate. Hence the key to using these materials is the ability to solubilize and sort carbon nanotubes according to their geometric/electronic properties. One of the most effective dispersants is single-stranded DNA (ssDNA), but there are many outstanding questions regarding the interaction between nucleic acids and SWNTs. In this work we focus on the interactions of SWNTs with single monomers of nucleic acids, as a first step to answering these outstanding questions. We use atomistic molecular dynamics simulations to calculate the binding energy of six different nucleotide monophosphates (NMPs) to a (6,0) single-wall carbon nanotube in aqueous solution. We find that the binding energies are generally favorable, of the order of a few kcal/mol. The binding energies of the different NMPs were very similar in salt solution, whereas we found a range of binding energies for NMPs in pure water. The binding energies are sensitive to the details of the association of the sodium ions with the phosphate groups and also to the average conformations of the nucleotides. We use electronic structure (Density Functional Theory (DFT) and Moller-Plesset second order perturbation to uncorrelated Hartree Fock theory (MP2)) methods to complement the classical force field study. With judicious choices of DFT exchange correlation functionals, we find that DFT, MP2, and classical force field predictions are in qualitative and even quantitative agreement; all three methods should give reliable and valid predictions. However, in one important case, the interactions between ions and metallic carbon nanotubes--the SWNT polarization-induced affinity for ions, neglected in most classical force field studies, is found to be extremely

  15. Semi-automated high-throughput fluorescent intercalator displacement-based discovery of cytotoxic DNA binding agents from a large compound library.

    Science.gov (United States)

    Glass, Lateca S; Bapat, Aditi; Kelley, Mark R; Georgiadis, Millie M; Long, Eric C

    2010-03-01

    High-throughput fluorescent intercalator displacement (HT-FID) was adapted to the semi-automated screening of a commercial compound library containing 60,000 molecules resulting in the discovery of cytotoxic DNA-targeted agents. Although commercial libraries are routinely screened in drug discovery efforts, the DNA binding potential of the compounds they contain has largely been overlooked. HT-FID led to the rapid identification of a number of compounds for which DNA binding properties were validated through demonstration of concentration-dependent DNA binding and increased thermal melting of A/T- or G/C-rich DNA sequences. Selected compounds were assayed further for cell proliferation inhibition in glioblastoma cells. Seven distinct compounds emerged from this screening procedure that represent structures unknown previously to be capable of targeting DNA leading to cell death. These agents may represent structures worthy of further modification to optimally explore their potential as cytotoxic anti-cancer agents. In addition, the general screening strategy described may find broader impact toward the rapid discovery of DNA targeted agents with biological activity. Copyright 2010 Elsevier Ltd. All rights reserved.

  16. An ImageJ-based algorithm for a semi-automated method for microscopic image enhancement and DNA repair foci counting

    International Nuclear Information System (INIS)

    Klokov, D.; Suppiah, R.

    2015-01-01

    Proper evaluation of the health risks of low-dose ionizing radiation exposure heavily relies on the ability to accurately measure very low levels of DNA damage in cells. One of the most sensitive methods for measuring DNA damage levels is the quantification of DNA repair foci that consist of macromolecular aggregates of DNA repair proteins, such as γH2AX and 53BP1, forming around individual DNA double-strand breaks. They can be quantified using immunofluorescence microscopy and are widely used as markers of DNA double-strand breaks. However this quantification, if performed manually, may be very tedious and prone to inter-individual bias. Low-dose radiation studies are especially sensitive to this potential bias due to a very low magnitude of the effects anticipated. Therefore, we designed and validated an algorithm for the semi-automated processing of microscopic images and quantification of DNA repair foci. The algorithm uses ImageJ, a freely available image analysis software that is customizable to individual cellular properties or experimental conditions. We validated the algorithm using immunolabeled 53BP1 and γH2AX in normal human fibroblast AG01522 cells under both normal and irradiated conditions. This method is easy to learn, can be used by nontrained personnel, and can help avoiding discrepancies in inter-laboratory comparison studies examining the effects of low-dose radiation. (author)

  17. A Novel Supramolecular Assembly Film of Porphyrin Bound DNA: Characterization and Catalytic Behaviors Towards Nitric Oxide

    Directory of Open Access Journals (Sweden)

    Osamu Ikeda

    2005-04-01

    Full Text Available A stable Fe(4-TMPyP-DNA-PADDA (FePyDP film was characterized onpyrolytic graphite electrode (PGE or an indium-tin oxide (ITO electrode through thesupramolecular interaction between water-soluble iron porphyrin (Fe(4-TMPyP and DNAtemplate, where PADDA (poly(acrylamide-co-diallyldimethylammonium chloride isemployed as a co-immobilizing polymer. Cyclic voltammetry of FePyDP film showed a pairof reversible FeIII/FeII redox peaks and an irreversible FeIV/FeIII peak at –0.13 V and 0.89vs. Ag|AgCl in pH 7.4 PBS, respectively. An excellent catalytic reduction of NO wasdisplayed at –0.61 V vs. Ag|AgCl at a FePyDP film modified electrode.Chronoamperometric experiments demonstrated a rapid response to the reduction of NOwith a linear range from 0.1 to 90 μM and a detection limit of 30 nM at a signal-to-noiseratio of 3. On the other hand, it is the first time to apply high-valent iron porphyrin ascatalyst at modified electrode for NO catalytic oxidation at 0.89 vs. Ag|AgCl. The sensorshows a high selectivity of some endogenous electroactive substances in biological systems.The mechanism of response of the sensors to NO is preliminary studied.

  18. DNA biosensor for detection of Salmonella typhi from blood sample of typhoid fever patient using gold electrode modified by self-assembled monolayers of thiols

    Science.gov (United States)

    Suryapratiwi, Windha Novita; Paat, Vlagia Indira; Gaffar, Shabarni; Hartati, Yeni Wahyuni

    2017-05-01

    Electrochemical biosensors are currently being developed in order to handle various clinical problems in diagnosing infectious diseases caused by pathogenic bacteria, or viruses. On this research, voltammetric DNA biosensor using gold electrode modified by thiols with self-assembled monolayers had been developed to detect a certain sequence of Salmonella typhi DNA from blood sample of typhoid fever patient. Thiol groups of cysteamines (Cys) and aldehyde groups from glutaraldehydes (Glu) were used as a link to increase the performance of gold electrode in detecting guanine oxidation signal of hybridized S. typhi DNA and ssDNA probe. Standard calibration method was used to determine analytical parameters from the measurements. The result shown that, the detection of S. typhi DNA from blood sample of typhoid fever patient can be carried out by voltammetry using gold electrode modified by self-assembled monolayers of thiols. A characteristic oxidation potential of guanine using Au/Cys/Gluwas obtained at +0.17 until +0.20 V. Limit of detection and limit of quantification from this measurements were 1.91μg mL-1 and 6.35 μg mL-1. The concentration of complement DNA from sample was 6.96 μg mL-1.

  19. DNA-promoted electrochemical assembly of [Ru(bpy){sub 2}dpp]{sup 3+/2+} on the ITO electrode by introducing copper(II) ion

    Energy Technology Data Exchange (ETDEWEB)

    Yao Su [School of Chemistry and Environment, South China Normal University, Guangzhou 510006 (China); Li Hong [School of Chemistry and Environment, South China Normal University, Guangzhou 510006 (China)], E-mail: lihong@scnu.edu.cn; Guo Qingyu; Xu Zhenghe [School of Chemistry and Environment, South China Normal University, Guangzhou 510006 (China); Ji Liangnian [Department of Chemistry, Zhongshan University, Guangzhou 510275 (China)

    2008-06-30

    The electrochemical assembly of [Ru(bpy){sub 2}dpp]{sup 3+/2+} (where bpy = 2,2'-bipyridine, dpp = 2,3-bis (2-pyridyl) pyrazine) promoted by calf thymus DNA on an ITO electrode based on the introduction of copper(II) ion has been investigated. There exists a diffusion-controlled wave and two prewaves for the complex in the differential pulse voltammetric sweeping process. The formal potential of the high prewave shift ca. 0.530 V negatively compared with that of the diffusion-controlled wave. Dpp ligand with two vacant chelating N sites in the complex can bite Cu{sup 2+} and the resultant heterometallic complex shows a weakened assembly in contrast to that of [Ru(bpy){sub 2}dpp]{sup 3+/2+} alone. Furthermore, double stranded DNA is able to accelerate the assembly of the ruthenium complex and heterometallic complex generated by chelating with Cu{sup 2+} by using the ITO surface, the prompted strength of the latter is far stronger than the former. Their assembled mechanism enhanced by DNA is proposed.

  20. DNA-promoted electrochemical assembly of [Ru(bpy)2dpp]3+/2+ on the ITO electrode by introducing copper(II) ion

    International Nuclear Information System (INIS)

    Yao Su; Li Hong; Guo Qingyu; Xu Zhenghe; Ji Liangnian

    2008-01-01

    The electrochemical assembly of [Ru(bpy) 2 dpp] 3+/2+ (where bpy = 2,2'-bipyridine, dpp = 2,3-bis (2-pyridyl) pyrazine) promoted by calf thymus DNA on an ITO electrode based on the introduction of copper(II) ion has been investigated. There exists a diffusion-controlled wave and two prewaves for the complex in the differential pulse voltammetric sweeping process. The formal potential of the high prewave shift ca. 0.530 V negatively compared with that of the diffusion-controlled wave. Dpp ligand with two vacant chelating N sites in the complex can bite Cu 2+ and the resultant heterometallic complex shows a weakened assembly in contrast to that of [Ru(bpy) 2 dpp] 3+/2+ alone. Furthermore, double stranded DNA is able to accelerate the assembly of the ruthenium complex and heterometallic complex generated by chelating with Cu 2+ by using the ITO surface, the prompted strength of the latter is far stronger than the former. Their assembled mechanism enhanced by DNA is proposed

  1. A Hybrid Parallel Strategy Based on String Graph Theory to Improve De Novo DNA Assembly on the TianHe-2 Supercomputer.

    Science.gov (United States)

    Zhang, Feng; Liao, Xiangke; Peng, Shaoliang; Cui, Yingbo; Wang, Bingqiang; Zhu, Xiaoqian; Liu, Jie

    2016-06-01

    ' The de novo assembly of DNA sequences is increasingly important for biological researches in the genomic era. After more than one decade since the Human Genome Project, some challenges still exist and new solutions are being explored to improve de novo assembly of genomes. String graph assembler (SGA), based on the string graph theory, is a new method/tool developed to address the challenges. In this paper, based on an in-depth analysis of SGA we prove that the SGA-based sequence de novo assembly is an NP-complete problem. According to our analysis, SGA outperforms other similar methods/tools in memory consumption, but costs much more time, of which 60-70 % is spent on the index construction. Upon this analysis, we introduce a hybrid parallel optimization algorithm and implement this algorithm in the TianHe-2's parallel framework. Simulations are performed with different datasets. For data of small size the optimized solution is 3.06 times faster than before, and for data of middle size it's 1.60 times. The results demonstrate an evident performance improvement, with the linear scalability for parallel FM-index construction. This results thus contribute significantly to improving the efficiency of de novo assembly of DNA sequences.

  2. Slotting optimization of automated storage and retrieval system (AS/RS) for efficient delivery of parts in an assembly shop using genetic algorithm: A case Study

    Science.gov (United States)

    Yue, L.; Guan, Z.; He, C.; Luo, D.; Saif, U.

    2017-06-01

    In recent years, the competitive pressure on manufacturing companies shifted them from mass production to mass customization to produce large variety of products. It is a great challenge for companies nowadays to produce customized mixed flow mode of production to meet customized demand on time. Due to large variety of products, the storage system to deliver variety of products to production lines influences on the timely production of variety of products, as investigated from by simulation study of an inefficient storage system of a real Company, in the current research. Therefore, current research proposed a slotting optimization model with mixed model sequence to assemble in consideration of the final flow lines to optimize whole automated storage and retrieval system (AS/RS) and distribution system in the case company. Current research is aimed to minimize vertical height of centre of gravity of AS/RS and total time spent for taking the materials out from the AS/RS simultaneously. Genetic algorithm is adopted to solve the proposed problem and computational result shows significant improvement in stability and efficiency of AS/RS as compared to the existing method used in the case company.

  3. Method to Assemble Genomic DNA Fragments or Genes on Human Artificial Chromosome with Regulated Kinetochore Using a Multi-Integrase System.

    Science.gov (United States)

    Lee, Nicholas C O; Kim, Jung-Hyun; Petrov, Nikolai S; Lee, Hee-Sheung; Masumoto, Hiroshi; Earnshaw, William C; Larionov, Vladimir; Kouprina, Natalay

    2018-01-19

    The production of cells capable of carrying multiple transgenes to Mb-size genomic loci has multiple applications in biomedicine and biotechnology. In order to achieve this goal, three key steps are required: (i) cloning of large genomic segments; (ii) insertion of multiple DNA blocks at a precise location and (iii) the capability to eliminate the assembled region from cells. In this study, we designed the iterative integration system (IIS) that utilizes recombinases Cre, ΦC31 and ΦBT1, and combined it with a human artificial chromosome (HAC) possessing a regulated kinetochore (alphoid tetO -HAC). We have demonstrated that the IIS-alphoid tetO -HAC system is a valuable genetic tool by reassembling a functional gene from multiple segments on the HAC. IIS-alphoid tetO -HAC has several notable advantages over other artificial chromosome-based systems. This includes the potential to assemble an unlimited number of genomic DNA segments; a DNA assembly process that leaves only a small insertion (system that also changes cell color, and counter-selection markers at each DNA insertion step, simplifying selection of correct clones; and presence of an error proofing mechanism to remove cells with misincorporated DNA segments, which improves the integrity of assembly. In addition, the IIS-alphoid tetO -HAC carrying a locus of interest is removable, offering the unique possibility to revert the cell line to its pretransformed state and compare the phenotypes of human cells with and without a functional copy of a gene(s). Thus, IIS-alphoid tetO -HAC allows investigation of complex biomedical pathways, gene(s) regulation, and has the potential to engineer synthetic chromosomes with a predetermined set of genes.

  4. Automated imaging system for single molecules

    Science.gov (United States)

    Schwartz, David Charles; Runnheim, Rodney; Forrest, Daniel

    2012-09-18

    There is provided a high throughput automated single molecule image collection and processing system that requires minimal initial user input. The unique features embodied in the present disclosure allow automated collection and initial processing of optical images of single molecules and their assemblies. Correct focus may be automatically maintained while images are collected. Uneven illumination in fluorescence microscopy is accounted for, and an overall robust imaging operation is provided yielding individual images prepared for further processing in external systems. Embodiments described herein are useful in studies of any macromolecules such as DNA, RNA, peptides and proteins. The automated image collection and processing system and method of same may be implemented and deployed over a computer network, and may be ergonomically optimized to facilitate user interaction.

  5. DNA synthesis and microtubule assembly-related events in fertilized Paracentrotus lividus eggs: reversible inhibition by 10 mM procaine.

    Science.gov (United States)

    Raymond, M N; Foucault, G; Coffe, G; Pudles, J

    1986-04-01

    This report describes the effects of 10 mM procaine on microtubule assembly and on DNA synthesis, as followed by [3H]colchicine binding assays and [3H]thymidine incorporation respectively, in fertilized Paracentrotus lividus eggs. In the absence of microtubule assembly inhibitors, about 25% of the total egg tubulin is submitted to two cycles of polymerization prior to the first cell division, this polymerization process precedes DNA synthesis. If the zygotes are treated with 10 mM procaine in the course of the cell cycle, tubulin polymerization is inhibited or microtubules are disassembled. DNA synthesis is inhibited when procaine treatment is performed 10 min, before the initiation of the S-period. However, when the drug is applied in the course of this synthetic period, the process is normally accomplished, but the next S-period becomes inhibited. Moreover, procaine treatment increases the cytoplasmic pH of the fertilized eggs by about 0.6 to 0.8 pH units. This pH increase precedes microtubule disassembly and inhibition of DNA synthesis. Washing out the drug induces a decrease of the intracellular pH which returns to about the same value as that of the fertilized egg controls. This pH change is then followed by the reinitiation of microtubule assembly, DNA synthesis and cell division. Our results show that the inhibition of both tubulin polymerization and DNA synthesis in fertilized eggs treated with 10 mM procaine, appears to be related to the drug-induced increase in cytoplasmic pH.

  6. Application of a Novel and Automated Branched DNA in Situ Hybridization Method for the Rapid and Sensitive Localization of mRNA Molecules in Plant Tissues

    Directory of Open Access Journals (Sweden)

    Andrew J. Bowling

    2014-04-01

    Full Text Available Premise of the study: A novel branched DNA detection technology, RNAscope in situ hybridization (ISH, originally developed for use on human clinical and animal tissues, was adapted for use in plant tissue in an attempt to overcome some of the limitations associated with traditional ISH assays. Methods and Results: Zea mays leaf tissue was formaldehyde fixed and paraffin embedded (FFPE and then probed with the RNAscope ISH assay for two endogenous genes, phosphoenolpyruvate carboxylase (PEPC and phosphoenolpyruvate carboxykinase (PEPCK. Results from both manual and automated methods showed tissue- and cell-specific mRNA localization patterns expected from these well-studied genes. Conclusions: RNAscope ISH is a sensitive method that generates high-quality, easily interpretable results from FFPE plant tissues. Automation of the RNAscope method on the Ventana Discovery Ultra platform allows significant advantages for repeatability, reduction in variability, and flexibility of workflow processes.

  7. Application of a novel and automated branched DNA in situ hybridization method for the rapid and sensitive localization of mRNA molecules in plant tissues.

    Science.gov (United States)

    Bowling, Andrew J; Pence, Heather E; Church, Jeffrey B

    2014-04-01

    A novel branched DNA detection technology, RNAscope in situ hybridization (ISH), originally developed for use on human clinical and animal tissues, was adapted for use in plant tissue in an attempt to overcome some of the limitations associated with traditional ISH assays. • Zea mays leaf tissue was formaldehyde fixed and paraffin embedded (FFPE) and then probed with the RNAscope ISH assay for two endogenous genes, phosphoenolpyruvate carboxylase (PEPC) and phosphoenolpyruvate carboxykinase (PEPCK). Results from both manual and automated methods showed tissue- and cell-specific mRNA localization patterns expected from these well-studied genes. • RNAscope ISH is a sensitive method that generates high-quality, easily interpretable results from FFPE plant tissues. Automation of the RNAscope method on the Ventana Discovery Ultra platform allows significant advantages for repeatability, reduction in variability, and flexibility of workflow processes.

  8. Toward allotetraploid cotton genome assembly: integration of a high-density molecular genetic linkage map with DNA sequence information

    Science.gov (United States)

    2012-01-01

    Background Cotton is the world’s most important natural textile fiber and a significant oilseed crop. Decoding cotton genomes will provide the ultimate reference and resource for research and utilization of the species. Integration of high-density genetic maps with genomic sequence information will largely accelerate the process of whole-genome assembly in cotton. Results In this paper, we update a high-density interspecific genetic linkage map of allotetraploid cultivated cotton. An additional 1,167 marker loci have been added to our previously published map of 2,247 loci. Three new marker types, InDel (insertion-deletion) and SNP (single nucleotide polymorphism) developed from gene information, and REMAP (retrotransposon-microsatellite amplified polymorphism), were used to increase map density. The updated map consists of 3,414 loci in 26 linkage groups covering 3,667.62 cM with an average inter-locus distance of 1.08 cM. Furthermore, genome-wide sequence analysis was finished using 3,324 informative sequence-based markers and publicly-available Gossypium DNA sequence information. A total of 413,113 EST and 195 BAC sequences were physically anchored and clustered by 3,324 sequence-based markers. Of these, 14,243 ESTs and 188 BACs from different species of Gossypium were clustered and specifically anchored to the high-density genetic map. A total of 2,748 candidate unigenes from 2,111 ESTs clusters and 63 BACs were mined for functional annotation and classification. The 337 ESTs/genes related to fiber quality traits were integrated with 132 previously reported cotton fiber quality quantitative trait loci, which demonstrated the important roles in fiber quality of these genes. Higher-level sequence conservation between different cotton species and between the A- and D-subgenomes in tetraploid cotton was found, indicating a common evolutionary origin for orthologous and paralogous loci in Gossypium. Conclusion This study will serve as a valuable genomic resource

  9. Dysfunction of chromatin assembly Factor 1 induces shortening of telomeres and loss of 45S rDNA in Arabidopsis thaliana

    Czech Academy of Sciences Publication Activity Database

    Mozgová, I.; Mokroš, P.; Fajkus, Jiří

    2010-01-01

    Roč. 22, č. 8 (2010), s. 2768-2780 ISSN 1040-4651 R&D Projects: GA MŠk(CZ) LC06004 Grant - others:GA MŠk(CZ) GD204/08/H054 Institutional research plan: CEZ:AV0Z50040507; CEZ:AV0Z50040702 Keywords : telomere * rDNA * chromatin assembly Subject RIV: BO - Biophysics Impact factor: 9.396, year: 2010

  10. Preparation and phase behaviour of surface-active pharmaceuticals: self-assembly of DNA and surfactants with membranes. Differential adiabatic scanning microcalorimetric study.

    Science.gov (United States)

    Süleymanoğlu, Erhan

    2005-08-01

    Some energetics issues relevant to preparation and surface characterization of zwitterionic phospholipid-DNA self-assemblies, as alternative models of the currently used problematic lipoplexes are presented. Nucleic acid compaction capacities of Mg(2+) and N-alkyl-N,N,N-trimetylammonium ions (C(n)TMA, n=12) were compared, with regard to surface interaction with unilamellar vesicles. Differential adiabatic scanning microcalorimetric measurements of synthetic phosphatidylcholine liposomes and calf thymus DNA and their ternary complexes with Mg(2+) and C(12)TMA, were employed for deduction of the thermodynamic model describing their structural transitions. Small monodisperce and highly stable complexes are established after precompaction of DNA with detergent, followed by addition of liposomes. In contrast, divalent metal cation-mediated aggregation of vesicles either leads to heterogeneous multilamellar DNA-lipid arrangements, or to DNA-induced bilayer destabilization and lipid fusion. Possible dependence of the cellular internalization and gene transfection efficiency on the structure and physicochemical properties of DNA-Mg(2+)-liposomes or DNA-cationic surfactant-liposome systems is emphasized by proposing the structure of their molecular self-organizations with further implications in gene transfer research.

  11. DNA Scaffolds for the Dictated Assembly of Left-/Right-Handed Plasmonic Au NP Helices with Programmed Chiro-Optical Properties.

    Science.gov (United States)

    Cecconello, Alessandro; Kahn, Jason S; Lu, Chun-Hua; Khosravi Khorashad, Larousse; Govorov, Alexander O; Willner, Itamar

    2016-08-10

    Within the broad interest of assembling chiral left- and right-handed helices of plasmonic nanoparticles (NPs), we introduce the DNA-guided organization of left- or right-handed plasmonic Au NPs on DNA scaffolds. The method involves the self-assembly of stacked 12 DNA quasi-rings interlinked by 30 staple-strands. By the functionalization of one group of staple units with programmed tether-nucleic acid strands and additional staple elements with long nucleic acid chains, acting as promoter strands, the promoter-guided assembly of barrels modified with 12 left- or right-handed tethers is achieved. The subsequent hybridization of Au NPs functionalized with single nucleic acid tethers yields left- or right-handed structures of plasmonic NPs. The plasmonic NP structures reveal CD spectra at the plasmon absorbance, and the NPs are imaged by HR-TEM. Using geometrical considerations corresponding to the left- and right-handed helices of the Au NPs, the experimental CD spectra of the plasmonic Au NPs are modeled by theoretical calculations.

  12. Parallel molecular computation of modular-multiplication with two same inputs over finite field GF(2(n)) using self-assembly of DNA tiles.

    Science.gov (United States)

    Li, Yongnan; Xiao, Limin; Ruan, Li

    2014-06-01

    Two major advantages of DNA computing - huge memory capacity and high parallelism - are being explored for large-scale parallel computing, mass data storage and cryptography. Tile assembly model is a highly distributed parallel model of DNA computing. Finite field GF(2(n)) is one of the most commonly used mathematic sets for constructing public-key cryptosystem. It is still an open question that how to implement the basic operations over finite field GF(2(n)) using DNA tiles. This paper proposes how the parallel tile assembly process could be used for computing the modular-square, modular-multiplication with two same inputs, over finite field GF(2(n)). This system could obtain the final result within less steps than another molecular computing system designed in our previous study, because square and reduction are executed simultaneously and the previous system computes reduction after calculating square. Rigorous theoretical proofs are described and specific computing instance is given after defining the basic tiles and the assembly rules. Time complexity of this system is 3n-1 and space complexity is 2n(2). Copyright © 2014 Elsevier Ltd. All rights reserved.

  13. DNA mediated wire-like clusters of self-assembled TiO₂ nanomaterials: supercapacitor and dye sensitized solar cell applications.

    Science.gov (United States)

    Nithiyanantham, U; Ramadoss, Ananthakumar; Ede, Sivasankara Rao; Kundu, Subrata

    2014-07-21

    A new route for the formation of wire-like clusters of TiO₂ nanomaterials self-assembled in DNA scaffold within an hour of reaction time is reported. TiO₂ nanomaterials are synthesized by the reaction of titanium-isopropoxide with ethanol and water in the presence of DNA under continuous stirring and heating at 60 °C. The individual size of the TiO₂ NPs self-assembled in DNA and the diameter of the wires can be tuned by controlling the DNA to Ti-salt molar ratios and other reaction parameters. The eventual diameter of the individual particles varies between 15 ± 5 nm ranges, whereas the length of the nanowires varies in the 2-3 μm range. The synthesized wire-like DNA-TiO₂ nanomaterials are excellent materials for electrochemical supercapacitor and DSSC applications. From the electrochemical supercapacitor experiment, it was found that the TiO₂ nanomaterials showed different specific capacitance (Cs) values for the various nanowires, and the order of Cs values are as follows: wire-like clusters (small size) > wire-like clusters (large size). The highest Cs of 2.69 F g(-1) was observed for TiO₂ having wire-like structure with small sizes. The study of the long term cycling stability of wire-like clusters (small size) electrode were shown to be stable, retaining ca. 80% of the initial specific capacitance, even after 5000 cycles. The potentiality of the DNA-TiO₂ nanomaterials was also tested in photo-voltaic applications and the observed efficiency was found higher in the case of wire-like TiO₂ nanostructures with larger sizes compared to smaller sizes. In future, the described method can be extended for the synthesis of other oxide based materials on DNA scaffold and can be further used in other applications like sensors, Li-ion battery materials or treatment for environmental waste water.

  14. De novo assembly and annotation of the Asian tiger mosquito (Aedes albopictus) repeatome with dnaPipeTE from raw genomic reads and comparative analysis with the yellow fever mosquito (Aedes aegypti).

    OpenAIRE

    Goubert, Clément; Modolo, Laurent; Vieira, Cristina; ValienteMoro, Claire; Mavingui, Patrick; Boulesteix, Matthieu

    2015-01-01

    International audience; Repetitive DNA, including transposable elements (TEs), is found throughout eukaryotic genomes. Annotating and assembling the "repeatome" during genome-wide analysis often poses a challenge. To address this problem, we present dnaPipeTE-a new bioinformatics pipeline that uses a sample of raw genomic reads. It produces precise estimates of repeated DNA content and TE consensus sequences, as well as the relative ages of TE families. We shows that dnaPipeTE performs well u...

  15. An automated flow system incorporating in-line acid dissolution of bismuth metal from a cyclotron irradiated target assembly for use in the isolation of astatine-211

    Energy Technology Data Exchange (ETDEWEB)

    O’Hara, Matthew J.; Krzysko, Anthony J.; Niver, Cynthia M.; Morrison, Samuel S.; Owsley, Stanley L.; Hamlin, Donald K.; Dorman, Eric F.; Scott Wilbur, D.

    2017-04-01

    Astatine-211 (211At) is a promising cyclotron-produced radionuclide being investigated for use in targeted alpha therapy of blood borne and metastatic cancers, as well as treatment of tumor remnants after surgical resections. The isolation of trace quantities of 211At, produced within several grams of a Bi metal cyclotron target, involves a complex, multi-step procedure: (1) Bi metal dissolution in strong HNO3, (2) distillation of the HNO3 to yield Bi salts containing 211At, (3) dissolution of the salts in strong HCl, (4) solvent extraction of 211At from bismuth salts with diisopropyl ether (DIPE), and (5) back-extraction of 211At from DIPE into NaOH, leading to a purified 211At product. Step (1) has been addressed first to begin the process of automating the onerous 211At isolation process. A computer-controlled Bi target dissolution system has been designed. The system performs in-line dissolution of Bi metal from the target assembly using an enclosed target dissolution block, routing the resulting solubilized 211At/Bi mixture to the subsequent process step. The primary parameters involved in Bi metal solubilization (HNO3 concentration and influent flow rate) were optimized prior to evaluation of the system performance on replicate cyclotron irradiated targets. The results indicate that the system performs reproducibly, having nearly quantitative release of 211At from irradiated targets, with cumulative 211At recoveries that follow a sigmoidal function. The predictable nature of the 211At release profile allows the user to tune the system to meet target processing requirements.

  16. Synthesis and studies of polypeptide materials: Self-assembled block copolypeptide amphiphiles, DNA-condensing block copolypeptides and membrane-interactive random copolypeptides

    Science.gov (United States)

    Wyrsta, Michael Dmytro

    A new class of transition metal initiators for the controlled polymerization of alpha-aminoacid-N-carboxyanhydrides (alpha-NCAs), has been developed by Deming et al. This discovery has allowed for the synthesis of well-defined "protein-like" polymers. Using this chemistry we have made distinct block/random copolypeptides for biomedical applications. Drug delivery, gene delivery, and antimicrobial polymers were the focus of our research efforts. The motivation for the synthesis and study of synthetic polypeptide based materials comes from proteins. Natural proteins are able to adopt a staggeringly large amount of uniquely well-defined folded structures. These structures account for the diversity in properties of proteins. As catalysts (enzymes) natural proteins perform some of the most difficult chemistry with ease and precision at ambient pressures and temperatures. They also exhibit incredible structural properties that directly result from formation of complex hierarchical assemblies. Self-assembling block copolymers were synthesized with various compositions and architectures. In general, di- and tri-block amphiphiles were studied for their self-assembling properties. Both spherical and tubular vesicles were found to assemble from di- and tri-block amphiphiles, respectively. In addition to self-assembly, pH responsiveness was engineered into these amphiphiles by the incorporation of basic residues (lysine) into the hydrophobic block. Another form of self-assembly studied was the condensation of DNA using cationic block copolymers. It was found that cationic block copolymers could condense DNA into compact, ordered, water-soluble aggregates on the nanoscale. These aggregates sufficiently protected DNA from nucleases and yet were susceptible to proteases. These studies form the basis of a gene delivery platform. The ease with which NCAs are polymerized renders them completely amenable to parallel synthetic methods. We have employed this technique to discover new

  17. Qualitative detection of Legionella species in bronchoalveolar lavages and induced sputa by automated DNA extraction and real-time polymerase chain reaction.

    Science.gov (United States)

    Raggam, R B; Leitner, E; Mühlbauer, G; Berg, J; Stöcher, M; Grisold, A J; Marth, E; Kessler, H H

    2002-10-01

    Molecular assays for qualitative detection of Legionella spp. in clinical specimens were evaluated. DNA extraction was done either with a fully automated DNA extraction protocol on the MagNA Pure LC System or with manual DNA extraction. Amplification and detection were done by real-time polymerase chain reaction (PCR) on the LightCycler (LC) instrument. Oligonucleotides were derived from the 16S rRNA gene of Legionella spp. The assays included a specially designed DNA fragment as Legionella-specific internal control. For both molecular assays, the detection limit was determined to be 5 CFU per LC PCR run. Sixty-one clinical specimens were tested with the molecular assays. Results were compared to culture. Five samples were found to be positive with the molecular assays. Three of them were positive in culture. No inhibition was found throughout the whole study. In conclusion, the molecular assays described may lead to safe and early diagnosis of Legionnaires' disease. They proved to be suitable for the routine molecular diagnostics laboratory.

  18. Genome Partitioner: A web tool for multi-level partitioning of large-scale DNA constructs for synthetic biology applications.

    Directory of Open Access Journals (Sweden)

    Matthias Christen

    Full Text Available Recent advances in lower-cost DNA synthesis techniques have enabled new innovations in the field of synthetic biology. Still, efficient design and higher-order assembly of genome-scale DNA constructs remains a labor-intensive process. Given the complexity, computer assisted design tools that fragment large DNA sequences into fabricable DNA blocks are needed to pave the way towards streamlined assembly of biological systems. Here, we present the Genome Partitioner software implemented as a web-based interface that permits multi-level partitioning of genome-scale DNA designs. Without the need for specialized computing skills, biologists can submit their DNA designs to a fully automated pipeline that generates the optimal retrosynthetic route for higher-order DNA assembly. To test the algorithm, we partitioned a 783 kb Caulobacter crescentus genome design. We validated the partitioning strategy by assembling a 20 kb test segment encompassing a difficult to synthesize DNA sequence. Successful assembly from 1 kb subblocks into the 20 kb segment highlights the effectiveness of the Genome Partitioner for reducing synthesis costs and timelines for higher-order DNA assembly. The Genome Partitioner is broadly applicable to translate DNA designs into ready to order sequences that can be assembled with standardized protocols, thus offering new opportunities to harness the diversity of microbial genomes for synthetic biology applications. The Genome Partitioner web tool can be accessed at https://christenlab.ethz.ch/GenomePartitioner.

  19. Highly Sensitive and Quality Self-Testable Electrochemiluminescence Assay of DNA Methyltransferase Activity Using Multifunctional Sandwich-Assembled Carbon Nitride Nanosheets.

    Science.gov (United States)

    Chen, Shiyu; Lv, Yanqin; Shen, Yanfei; Ji, Jingjing; Zhou, Qing; Liu, Songqin; Zhang, Yuanjian

    2018-02-28

    DNA methylation catalyzed by methylase plays a key role in many biological activities. However, developing a highly sensitive, simple, and reliable way for evaluation of DNA methyltransferase (MTase) activity is still a challenge. Here, we report a sandwich-assembled electrochemiluminescence (ECL) biosensor using multifunctional carbon nitride nanosheets (CNNS) to evaluate the Dam MTase activity. The CNNS could not only be used as an excellent substrate to conjugate a large amount of hairpin probe DNA to improve the sensitivity but also be utilized as an internal reliability checker and an analyte reporter in the bottom and top layers of the biosensor, respectively. Such a unique sandwich configuration of CNNS well coupled the advantages of ECL luminophor that were generally assembled in the bottom or top layer in a conventional manner. As a result, the biosensor exhibited an ultralow detection limit down to 0.043 U/mL and a linear range between 0.05 and 80 U/mL, superior to the MTase activity assay in most previous reports. We highlighted the great potential of emerging CNNS luminophor in developing highly sensitive and smart quality self-testable ECL sensing systems using a sandwiched configuration for early disease diagnosis, treatment, and management.

  20. Red Fluorescent Proteins for Gene Expression and Protein Localization Studies in Streptococcus pneumoniae and Efficient Transformation with DNA Assembled via the Gibson Assembly Method.

    Science.gov (United States)

    Beilharz, Katrin; van Raaphorst, Renske; Kjos, Morten; Veening, Jan-Willem

    2015-10-01

    During the last decades, a wide range of fluorescent proteins (FPs) have been developed and improved. This has had a great impact on the possibilities in biological imaging and the investigation of cellular processes at the single-cell level. Recently, we have benchmarked a set of green fluorescent proteins (GFPs) and generated a codon-optimized superfolder GFP for efficient use in the important human pathogen Streptococcus pneumoniae and other low-GC Gram-positive bacteria. In the present work, we constructed and compared four red fluorescent proteins (RFPs) in S. pneumoniae. Two orange-red variants, mOrange2 and TagRFP, and two far-red FPs, mKate2 and mCherry, were codon optimized and examined by fluorescence microscopy and plate reader assays. Notably, protein fusions of the RFPs to FtsZ were constructed by direct transformation of linear Gibson assembly (isothermal assembly) products, a method that speeds up the strain construction process significantly. Our data show that mCherry is the fastest-maturing RFP in S. pneumoniae and is best suited for studying gene expression, while mKate2 and TagRFP are more stable and are the preferred choices for protein localization studies. The RFPs described here will be useful for cell biology studies that require multicolor labeling in S. pneumoniae and related organisms. Copyright © 2015, American Society for Microbiology. All Rights Reserved.

  1. Restriction-based Multiple-fragment Assembly Strategy to Avoid Random Mutation during Long cDNA Cloning

    OpenAIRE

    Wang, Shang; Chen, Wen; Zhang, Kai; Jiao, Peng; Mo, Lihua; Yang, Xiaoxu; Hu, Xiang; Zhang, Jian; Wei, Chenxi; Xiang, Shuanglin

    2015-01-01

    Long fragment cloning is a challenge for its difficulty in accurate amplifying and tendency to get unwanted mutation. Here we discuss Restriction-based Multiple-fragment Assembly Strategy's advantages and limitations. In this strategy, rather than PCR amplifying the entire coding sequence (CDS) at one time, we amplified and sequenced smaller fragments which are shorter than 1.5kb spanning the CDS. After that, the sequence-proved fragments were assembled by digestion-ligation cloning to the ta...

  2. Virus-sized self-assembling lamellar complexes between plasmid DNA and cationic micelles promote gene transfer

    Science.gov (United States)

    Pitard, Bruno; Aguerre, Olivier; Airiau, Marc; Lachagès, Anne-Marie; Boukhnikachvili, Tsiala; Byk, Gérardo; Dubertret, Catherine; Herviou, Christian; Scherman, Daniel; Mayaux, Jean-François; Crouzet, Joël

    1997-01-01

    Gene therapy is based on the vectorization of genes to target cells and their subsequent expression. Cationic amphiphile-mediated delivery of plasmid DNA is the nonviral gene transfer method most often used. We examined the supramolecular structure of lipopolyamine/plasmid DNA complexes under various condensing conditions. Plasmid DNA complexation with lipopolyamine micelles whose mean diameter was 5 nm revealed three domains, depending on the lipopolyamine/plasmid DNA ratio. These domains respectively corresponded to negatively, neutrally, and positively charged complexes. Transmission electron microscopy and x-ray scattering experiments on complexes originating from these three domains showed that although their morphology depends on the lipopolyamine/plasmid DNA ratio, their particle structure consists of ordered domains characterized by even spacing of 80 Å, irrespective of the lipid/DNA ratio. The most active lipopolyamine/DNA complexes for gene transfer were positively charged. They were characterized by fully condensed DNA inside spherical particles (diameter: 50 nm) sandwiched between lipid bilayers. These results show that supercoiled plasmid DNA is able to transform lipopolyamine micelles into a supramolecular organization characterized by ordered lamellar domains. PMID:9405626

  3. Auto-assembly of nanometer thick, water soluble layers of plasmid DNA complexed with diamines and basic amino acids on graphite: Greatest DNA protection is obtained with arginine.

    Science.gov (United States)

    Khalil, T T; Boulanouar, O; Heintz, O; Fromm, M

    2017-02-01

    We have investigated the ability of diamines as well as basic amino acids to condense DNA onto highly ordered pyrolytic graphite with minimum damage after re-dissolution in water. Based on a bibliographic survey we briefly summarize DNA binding properties with diamines as compared to basic amino acids. Thus, solutions of DNA complexed with these linkers were drop-cast in order to deposit ultra-thin layers on the surface of HOPG in the absence or presence of Tris buffer. Atomic Force Microscopy analyses showed that, at a fixed ligand-DNA mixing ratio of 16, the mean thickness of the layers can be statistically predicted to lie in the range 0-50nm with a maximum standard deviation ±6nm, using a simple linear law depending on the DNA concentration. The morphology of the layers appears to be ligand-dependent. While the layers containing diamines present holes, those formed in the presence of basic amino acids, except for lysine, are much more compact and dense. X-ray Photoelectron Spectroscopy measurements provide compositional information indicating that, compared to the maximum number of DNA sites to which the ligands may bind, the basic amino acids Arg and His are present in large excess. Conservation of the supercoiled topology of the DNA plasmids was studied after recovery of the complex layers in water. Remarkably, arginine has the best protection capabilities whether Tris was present or not in the initial solution. Copyright © 2016 Elsevier B.V. All rights reserved.

  4. A simplified and versatile system for the simultaneous expression of multiple siRNAs in mammalian cells using Gibson DNA Assembly.

    Science.gov (United States)

    Deng, Fang; Chen, Xiang; Liao, Zhan; Yan, Zhengjian; Wang, Zhongliang; Deng, Youlin; Zhang, Qian; Zhang, Zhonglin; Ye, Jixing; Qiao, Min; Li, Ruifang; Denduluri, Sahitya; Wang, Jing; Wei, Qiang; Li, Melissa; Geng, Nisha; Zhao, Lianggong; Zhou, Guolin; Zhang, Penghui; Luu, Hue H; Haydon, Rex C; Reid, Russell R; Yang, Tian; He, Tong-Chuan

    2014-01-01

    RNA interference (RNAi) denotes sequence-specific mRNA degradation induced by short interfering double-stranded RNA (siRNA) and has become a revolutionary tool for functional annotation of mammalian genes, as well as for development of novel therapeutics. The practical applications of RNAi are usually achieved by expressing short hairpin RNAs (shRNAs) or siRNAs in cells. However, a major technical challenge is to simultaneously express multiple siRNAs to silence one or more genes. We previously developed pSOS system, in which siRNA duplexes are made from oligo templates driven by opposing U6 and H1 promoters. While effective, it is not equipped to express multiple siRNAs in a single vector. Gibson DNA Assembly (GDA) is an in vitro recombination system that has the capacity to assemble multiple overlapping DNA molecules in a single isothermal step. Here, we developed a GDA-based pSOK assembly system for constructing single vectors that express multiple siRNA sites. The assembly fragments were generated by PCR amplifications from the U6-H1 template vector pB2B. GDA assembly specificity was conferred by the overlapping unique siRNA sequences of insert fragments. To prove the technical feasibility, we constructed pSOK vectors that contain four siRNA sites and three siRNA sites targeting human and mouse β-catenin, respectively. The assembly reactions were efficient, and candidate clones were readily identified by PCR screening. Multiple β-catenin siRNAs effectively silenced endogenous β-catenin expression, inhibited Wnt3A-induced β-catenin/Tcf4 reporter activity and expression of Wnt/β-catenin downstream genes. Silencing β-catenin in mesenchymal stem cells inhibited Wnt3A-induced early osteogenic differentiation and significantly diminished synergistic osteogenic activity between BMP9 and Wnt3A in vitro and in vivo. These findings demonstrate that the GDA-based pSOK system has been proven simplistic, effective and versatile for simultaneous expression of multiple

  5. Fluorometric sensing of DNA using curcumin encapsulated in nanoparticle-assembled microcapsules prepared from poly(diallylammonium chloride-co-sulfur dioxide)

    International Nuclear Information System (INIS)

    Patra, D.; Aridi, R.; Bouhadir, K.

    2013-01-01

    We report on the synthesis of microcapsules (MCs) containing self-assembled nanoparticles formed from poly[diallylammonium chloride-co-(sulfur dioxide)] in the presence of citrate and silica sol nanoparticles. The MCs are spherical, and SEM and optical microscopy reveal them to have micrometer size. The fluorescent probe curcumin was encapsulated in the MCs and found to be located in the shell. The fluorescence of curcumin in the MCs is altered depending on their microenvironment. Effects of pH and ammonia on the fluorescence of curcumin in the MCs also were studied. The brightness of the probe in the MCs increases on addition of DNA. The effect was used to determine DNA from fish sperm by fluorometry. The association constant (K) is 4000 mL. g -1 , and the number of binding sites is ∼1.0. (author)

  6. Temperature-Controlled Encapsulation and Release of an Active Enzyme in the Cavity of a Self-Assembled DNA Nanocage

    DEFF Research Database (Denmark)

    Juul, Sissel; Iacovelli, Federico; Falconi, Mattia

    2013-01-01

    ABSTRACT We demonstrate temperature-controlled encapsulation and release of the enzyme horseradish peroxidase using a preassembled and covalently closed three-dimensional DNA cage structure as a controllable encapsulation device. The utilized cage structure was covalently closed and composed of 12...... double-stranded B-DNA helices that constituted the edges of the structure. The double stranded helices were interrupted by short single-stranded thymidine linkers constituting the cage corners except for one, which was composed by four 32 nucleotide long stretches of DNA with a sequence that allowed them...... of the cargo in the central cavity of the cage at 4 C. The entrapped enzyme was catalytically active inside the DNA cage and was able to convert substrate molecules penetrating the apertures in the DNA lattice that surrounded the central cavity of the cage....

  7. A modular cloning system for standardized assembly of multigene constructs.

    Directory of Open Access Journals (Sweden)

    Ernst Weber

    Full Text Available The field of synthetic biology promises to revolutionize biotechnology through the design of organisms with novel phenotypes useful for medicine, agriculture and industry. However, a limiting factor is the ability of current methods to assemble complex DNA molecules encoding multiple genetic elements in various predefined arrangements. We present here a hierarchical modular cloning system that allows the creation at will and with high efficiency of any eukaryotic multigene construct, starting from libraries of defined and validated basic modules containing regulatory and coding sequences. This system is based on the ability of type IIS restriction enzymes to assemble multiple DNA fragments in a defined linear order. We constructed a 33 kb DNA molecule containing 11 transcription units made from 44 individual basic modules in only three successive cloning steps. This modular cloning (MoClo system can be readily automated and will be extremely useful for applications such as gene stacking and metabolic engineering.

  8. Lrp, a major regulatory protein in Escherichia coli, bends DNA and can organize the assembly of a higher-order nucleoprotein structure.

    Science.gov (United States)

    Wang, Q; Calvo, J M

    1993-06-01

    Lrp (Leucine-responsive regulatory protein) is a global regulatory protein that controls the expression of many operons in Escherichia coli. One of those operons, ilvIH, contains six Lrp binding sites located within a several hundred base pair region upstream of the promoter region. Analysis of the binding of Lrp to a set of circularly permuted DNA fragments from this region indicates that Lrp induces DNA bending. The results of DNase I footprinting experiments suggest that Lrp binding to this region facilitates the formation of a higher-order nucleoprotein structure. To define more precisely the degree of bending associated with Lrp binding, one or two binding sites were separately cloned into a pBend vector and analyzed. Lrp induced a bend of approximately 52 degrees upon binding to a single binding site, and the angle of bending is increased to at least 135 degrees when Lrp binds to two adjacent sites. Lrp-induced DNA bending, and a natural sequence-directed bend that exists within ilvIH DNA, may be architectural elements that facilitate the assembly of a nucleoprotein complex.

  9. A microfluidic device for preparing next generation DNA sequencing libraries and for automating other laboratory protocols that require one or more column chromatography steps.

    Science.gov (United States)

    Tan, Swee Jin; Phan, Huan; Gerry, Benjamin Michael; Kuhn, Alexandre; Hong, Lewis Zuocheng; Min Ong, Yao; Poon, Polly Suk Yean; Unger, Marc Alexander; Jones, Robert C; Quake, Stephen R; Burkholder, William F

    2013-01-01

    Library preparation for next-generation DNA sequencing (NGS) remains a key bottleneck in the sequencing process which can be relieved through improved automation and miniaturization. We describe a microfluidic device for automating laboratory protocols that require one or more column chromatography steps and demonstrate its utility for preparing Next Generation sequencing libraries for the Illumina and Ion Torrent platforms. Sixteen different libraries can be generated simultaneously with significantly reduced reagent cost and hands-on time compared to manual library preparation. Using an appropriate column matrix and buffers, size selection can be performed on-chip following end-repair, dA tailing, and linker ligation, so that the libraries eluted from the chip are ready for sequencing. The core architecture of the device ensures uniform, reproducible column packing without user supervision and accommodates multiple routine protocol steps in any sequence, such as reagent mixing and incubation; column packing, loading, washing, elution, and regeneration; capture of eluted material for use as a substrate in a later step of the protocol; and removal of one column matrix so that two or more column matrices with different functional properties can be used in the same protocol. The microfluidic device is mounted on a plastic carrier so that reagents and products can be aliquoted and recovered using standard pipettors and liquid handling robots. The carrier-mounted device is operated using a benchtop controller that seals and operates the device with programmable temperature control, eliminating any requirement for the user to manually attach tubing or connectors. In addition to NGS library preparation, the device and controller are suitable for automating other time-consuming and error-prone laboratory protocols requiring column chromatography steps, such as chromatin immunoprecipitation.

  10. Post-assembly transformations of porphyrin-containing metal-organic framework (MOF) films fabricated via automated layer-by-layer coordination

    KAUST Repository

    So, Monica

    2015-01-01

    Herein, we demonstrate the robustness of layer-by-layer (LbL)-assembled, pillared-paddlewheel-type MOF films toward conversion to new or modified MOFs via solvent-assisted linker exchange (SALE) and post-assembly linker metalation. Further, we show that LbL synthesis can afford MOFs that have proven inaccessible through other de novo strategies.

  11. DNA data

    Data.gov (United States)

    National Oceanic and Atmospheric Administration, Department of Commerce — Raw DNA chromatogram data produced by the ABI 373, 377, 3130 and 3730 automated sequencing machines in ABI format. These are from fish (primarily Sebastes spp.,...

  12. DNA-guided assembly of a five-component enzyme cascade for enhanced conversion of cellulose to gluconic acid and H2O2.

    Science.gov (United States)

    Chen, Qi; Yu, Sooyoun; Myung, Nosang; Chen, Wilfred

    2017-12-10

    Enzymatic fuel cells have received considerable attention because of their potential for direct conversion of abundant raw materials such as cellulose to electricity. The use of multi-enzyme cascades is particularly attractive as they offer the possibility of achieving a series of complex reactions at higher efficiencies. Here we reported the use of a DNA-guided approach to assemble a five-component enzyme cascade for direct conversion of cellulose to gluconic acid and H 2 O 2 . Site-specific co-localization of β-glucosidase and glucose oxidase resulted in over 11-fold improvement in H 2 O 2 production from cellobiose, highlighting the benefit of substrate channeling. Although a more modest 1.5-fold improvement in H 2 O 2 production was observed using a five-enzyme cascade, due to H 2 O 2 inhibition on enzyme activity, these results demonstrated the possibility to enhance the production of gluconic acid and H 2 O 2 directly from cellulose by DNA-guided enzyme assembly. Copyright © 2017 Elsevier B.V. All rights reserved.

  13. De Novo Transcriptome Sequence Assembly from Coconut Leaves and Seeds with a Focus on Factors Involved in RNA-Directed DNA Methylation

    Science.gov (United States)

    Huang, Ya-Yi; Lee, Chueh-Pai; Fu, Jason L.; Chang, Bill Chia-Han; Matzke, Antonius J. M.; Matzke, Marjori

    2014-01-01

    Coconut palm (Cocos nucifera) is a symbol of the tropics and a source of numerous edible and nonedible products of economic value. Despite its nutritional and industrial significance, coconut remains under-represented in public repositories for genomic and transcriptomic data. We report de novo transcript assembly from RNA-seq data and analysis of gene expression in seed tissues (embryo and endosperm) and leaves of a dwarf coconut variety. Assembly of 10 GB sequencing data for each tissue resulted in 58,211 total unigenes in embryo, 61,152 in endosperm, and 33,446 in leaf. Within each unigene pool, 24,857 could be annotated in embryo, 29,731 could be annotated in endosperm, and 26,064 could be annotated in leaf. A KEGG analysis identified 138, 138, and 139 pathways, respectively, in transcriptomes of embryo, endosperm, and leaf tissues. Given the extraordinarily large size of coconut seeds and the importance of small RNA-mediated epigenetic regulation during seed development in model plants, we used homology searches to identify putative homologs of factors required for RNA-directed DNA methylation in coconut. The findings suggest that RNA-directed DNA methylation is important during coconut seed development, particularly in maturing endosperm. This dataset will expand the genomics resources available for coconut and provide a foundation for more detailed analyses that may assist molecular breeding strategies aimed at improving this major tropical crop. PMID:25193496

  14. De novo transcriptome sequence assembly from coconut leaves and seeds with a focus on factors involved in RNA-directed DNA methylation.

    Science.gov (United States)

    Huang, Ya-Yi; Lee, Chueh-Pai; Fu, Jason L; Chang, Bill Chia-Han; Matzke, Antonius J M; Matzke, Marjori

    2014-09-04

    Coconut palm (Cocos nucifera) is a symbol of the tropics and a source of numerous edible and nonedible products of economic value. Despite its nutritional and industrial significance, coconut remains under-represented in public repositories for genomic and transcriptomic data. We report de novo transcript assembly from RNA-seq data and analysis of gene expression in seed tissues (embryo and endosperm) and leaves of a dwarf coconut variety. Assembly of 10 GB sequencing data for each tissue resulted in 58,211 total unigenes in embryo, 61,152 in endosperm, and 33,446 in leaf. Within each unigene pool, 24,857 could be annotated in embryo, 29,731 could be annotated in endosperm, and 26,064 could be annotated in leaf. A KEGG analysis identified 138, 138, and 139 pathways, respectively, in transcriptomes of embryo, endosperm, and leaf tissues. Given the extraordinarily large size of coconut seeds and the importance of small RNA-mediated epigenetic regulation during seed development in model plants, we used homology searches to identify putative homologs of factors required for RNA-directed DNA methylation in coconut. The findings suggest that RNA-directed DNA methylation is important during coconut seed development, particularly in maturing endosperm. This dataset will expand the genomics resources available for coconut and provide a foundation for more detailed analyses that may assist molecular breeding strategies aimed at improving this major tropical crop. Copyright © 2014 Huang et al.

  15. Comparison of Boiling and Robotics Automation Method in DNA Extraction for Metagenomic Sequencing of Human Oral Microbes.

    Directory of Open Access Journals (Sweden)

    Junya Yamagishi

    Full Text Available The rapid improvement of next-generation sequencing performance now enables us to analyze huge sample sets with more than ten thousand specimens. However, DNA extraction can still be a limiting step in such metagenomic approaches. In this study, we analyzed human oral microbes to compare the performance of three DNA extraction methods: PowerSoil (a method widely used in this field, QIAsymphony (a robotics method, and a simple boiling method. Dental plaque was initially collected from three volunteers in the pilot study and then expanded to 12 volunteers in the follow-up study. Bacterial flora was estimated by sequencing the V4 region of 16S rRNA following species-level profiling. Our results indicate that the efficiency of PowerSoil and QIAsymphony was comparable to the boiling method. Therefore, the boiling method may be a promising alternative because of its simplicity, cost effectiveness, and short handling time. Moreover, this method was reliable for estimating bacterial species and could be used in the future to examine the correlation between oral flora and health status. Despite this, differences in the efficiency of DNA extraction for various bacterial species were observed among the three methods. Based on these findings, there is no "gold standard" for DNA extraction. In future, we suggest that the DNA extraction method should be selected on a case-by-case basis considering the aims and specimens of the study.

  16. Comparison of Boiling and Robotics Automation Method in DNA Extraction for Metagenomic Sequencing of Human Oral Microbes.

    Science.gov (United States)

    Yamagishi, Junya; Sato, Yukuto; Shinozaki, Natsuko; Ye, Bin; Tsuboi, Akito; Nagasaki, Masao; Yamashita, Riu

    2016-01-01

    The rapid improvement of next-generation sequencing performance now enables us to analyze huge sample sets with more than ten thousand specimens. However, DNA extraction can still be a limiting step in such metagenomic approaches. In this study, we analyzed human oral microbes to compare the performance of three DNA extraction methods: PowerSoil (a method widely used in this field), QIAsymphony (a robotics method), and a simple boiling method. Dental plaque was initially collected from three volunteers in the pilot study and then expanded to 12 volunteers in the follow-up study. Bacterial flora was estimated by sequencing the V4 region of 16S rRNA following species-level profiling. Our results indicate that the efficiency of PowerSoil and QIAsymphony was comparable to the boiling method. Therefore, the boiling method may be a promising alternative because of its simplicity, cost effectiveness, and short handling time. Moreover, this method was reliable for estimating bacterial species and could be used in the future to examine the correlation between oral flora and health status. Despite this, differences in the efficiency of DNA extraction for various bacterial species were observed among the three methods. Based on these findings, there is no "gold standard" for DNA extraction. In future, we suggest that the DNA extraction method should be selected on a case-by-case basis considering the aims and specimens of the study.

  17. DNA mediated wire-like clusters of self-assembled TiO2 nanomaterials: supercapacitor and dye sensitized solar cell applications

    Science.gov (United States)

    Nithiyanantham, U.; Ramadoss, Ananthakumar; Ede, Sivasankara Rao; Kundu, Subrata

    2014-06-01

    A new route for the formation of wire-like clusters of TiO2 nanomaterials self-assembled in DNA scaffold within an hour of reaction time is reported. TiO2 nanomaterials are synthesized by the reaction of titanium-isopropoxide with ethanol and water in the presence of DNA under continuous stirring and heating at 60 °C. The individual size of the TiO2 NPs self-assembled in DNA and the diameter of the wires can be tuned by controlling the DNA to Ti-salt molar ratios and other reaction parameters. The eventual diameter of the individual particles varies between 15 +/- 5 nm ranges, whereas the length of the nanowires varies in the 2-3 μm range. The synthesized wire-like DNA-TiO2 nanomaterials are excellent materials for electrochemical supercapacitor and DSSC applications. From the electrochemical supercapacitor experiment, it was found that the TiO2 nanomaterials showed different specific capacitance (Cs) values for the various nanowires, and the order of Cs values are as follows: wire-like clusters (small size) > wire-like clusters (large size). The highest Cs of 2.69 F g-1 was observed for TiO2 having wire-like structure with small sizes. The study of the long term cycling stability of wire-like clusters (small size) electrode were shown to be stable, retaining ca. 80% of the initial specific capacitance, even after 5000 cycles. The potentiality of the DNA-TiO2 nanomaterials was also tested in photo-voltaic applications and the observed efficiency was found higher in the case of wire-like TiO2 nanostructures with larger sizes compared to smaller sizes. In future, the described method can be extended for the synthesis of other oxide based materials on DNA scaffold and can be further used in other applications like sensors, Li-ion battery materials or treatment for environmental waste water.A new route for the formation of wire-like clusters of TiO2 nanomaterials self-assembled in DNA scaffold within an hour of reaction time is reported. TiO2 nanomaterials are

  18. Assembly mechanism of the sixty-subunit nanoparticles via interaction of RNA with the reengineered protein connector of phi29 DNA-packaging motor.

    Science.gov (United States)

    Xiao, Feng; Demeler, Borries; Guo, Peixuan

    2010-06-22

    Bacterial virus phi29 genomic DNA is packaged into a procapsid shell with the aid of a motor containing a 12-subunit connector channel and a hexameric pRNA (packaging RNA) ring. The wide end, or the C-terminus, of the cone-shaped connector is embedded within the procapsid shell, whereas the narrow end, or N-terminus, extends outside of the procapsid, providing a binding location for pRNA. Recently, we have reported the mechanism of in vivo assembly of an ellipsoid nanoparticle with seven connectors through an interaction among a peptide tag. Here we report the formation of a similar nanoparticle in vitro via the addition of DNA or RNA oligos to connector proteins. Free connectors guided by one or two copies of oligonucleotides were assembled into a rosette structure containing 60 subunits of reengineered proteins. The number of oligonucleotides within the particle is length-dependent but sequence-independent. Reversible shifting between the 12- and 60-subunit nanoparticles (between individual connectors and rosette structures, respectively) was demonstrated by the alternative addition of oligonucleotides and the treatment of ribonuclease, suggesting a potential application as a switch or regulator in nanobiotechnology. This advancement allows for a simple method to produce multivalent nanoparticles that contain five 12-unit nanoparticles with defined structure and stoichiometry. That is, it will be possible to assemble nanoparticles in vitro with the combination of 60 assortments of ligands, tags, therapeutic drugs, and diagnostic moieties for multivalent delivery or enhancement of signal detection in nanotechnological and nanomedicinal applications.

  19. High throughput detection of Coxiella burnetii by real-time PCR with internal control system and automated DNA preparation

    Directory of Open Access Journals (Sweden)

    Kramme Stefanie

    2008-05-01

    Full Text Available Abstract Background Coxiella burnetii is the causative agent of Q-fever, a widespread zoonosis. Due to its high environmental stability and infectivity it is regarded as a category B biological weapon agent. In domestic animals infection remains either asymptomatic or presents as infertility or abortion. Clinical presentation in humans can range from mild flu-like illness to acute pneumonia and hepatitis. Endocarditis represents the most common form of chronic Q-fever. In humans serology is the gold standard for diagnosis but is inadequate for early case detection. In order to serve as a diagnostic tool in an eventual biological weapon attack or in local epidemics we developed a real-time 5'nuclease based PCR assay with an internal control system. To facilitate high-throughput an automated extraction procedure was evaluated. Results To determine the minimum number of copies that are detectable at 95% chance probit analysis was used. Limit of detection in blood was 2,881 copies/ml [95%CI, 2,188–4,745 copies/ml] with a manual extraction procedure and 4,235 copies/ml [95%CI, 3,143–7,428 copies/ml] with a fully automated extraction procedure, respectively. To demonstrate clinical application a total of 72 specimens of animal origin were compared with respect to manual and automated extraction. A strong correlation between both methods was observed rendering both methods suitable. Testing of 247 follow up specimens of animal origin from a local Q-fever epidemic rendered real-time PCR more sensitive than conventional PCR. Conclusion A sensitive and thoroughly evaluated real-time PCR was established. Its high-throughput mode may show a useful approach to rapidly screen samples in local outbreaks for other organisms relevant for humans or animals. Compared to a conventional PCR assay sensitivity of real-time PCR was higher after testing samples from a local Q-fever outbreak.

  20. Low-dose DNA damage and replication stress responses quantified by optimized automated single-cell image analysis

    DEFF Research Database (Denmark)

    Mistrik, Martin; Oplustilova, Lenka; Lukas, Jiri

    2009-01-01

    Maintenance of genome integrity is essential for homeostasis and survival as impaired DNA damage response (DDR) may predispose to grave pathologies such as neurodegenerative and immunodeficiency syndromes, cancer and premature aging. Therefore, accurate assessment of DNA damage caused...... and demand for trained personnel. Here we present an option how to transform a regular fluorescence microscope and personal computer with common software into a functional alternative to high-throughput screening devices. In two detailed protocols we introduce a new semi-automatic procedure allowing for very...

  1. Automated Temperature and Emission Measure Analysis of Coronal Loops and Active Regions Observed with the Atmospheric Imaging Assembly on the Solar Dynamics Observatory (SDO/AIA)

    Science.gov (United States)

    Aschwanden, Markus J.; Boerner, Paul; Schrijver, Carolus J.; Malanushenko, Anna

    2013-03-01

    We developed numerical codes designed for automated analysis of SDO/AIA image datasets in the six coronal filters, including: i) coalignment test between different wavelengths with measurements of the altitude of the EUV-absorbing chromosphere, ii) self-calibration by empirical correction of instrumental response functions, iii) automated generation of differential emission measure [DEM] distributions with peak-temperature maps [ T p( x, y)] and emission measure maps [ EM p( x, y)] of the full Sun or active region areas, iv) composite DEM distributions [d EM( T)/d T] of active regions or subareas, v) automated detection of coronal loops, and vi) automated background subtraction and thermal analysis of coronal loops, which yields statistics of loop temperatures [ T e], temperature widths [ σ T], emission measures [ EM], electron densities [ n e], and loop widths [ w]. The combination of these numerical codes allows for automated and objective processing of numerous coronal loops. As an example, we present the results of an application to the active region NOAA 11158, observed on 15 February 2011, shortly before it produced the largest (X2.2) flare during the current solar cycle. We detect 570 loop segments at temperatures in the entire range of log( T e)=5.7 - 7.0 K and corroborate previous TRACE and AIA results on their near-isothermality and the validity of the Rosner-Tucker-Vaiana (RTV) law at soft X-ray temperatures ( T≳2 MK) and its failure at lower EUV temperatures.

  2. Low-dose DNA damage and replication stress responses quantified by optimized automated single-cell image analysis

    DEFF Research Database (Denmark)

    Mistrik, Martin; Oplustilova, Lenka; Lukas, Jiri

    2009-01-01

    the advantages and applicability of this technique. Our present data on assessment of low radiation doses, repair kinetics, spontaneous DNA damage in cancer cells, as well as constitutive and replication stress-induced HR events and their dependence on upstream factors within the DDR machinery document...

  3. Semi-automated extraction of microbial DNA from feces for qPCR and phylogenetic microarray analysis

    NARCIS (Netherlands)

    Nylund, L.; Heilig, G.H.J.; Salminen, S.; Vos, de W.M.; Satokari, R.M.

    2010-01-01

    The human gastrointestinal tract (GI-tract) harbors a complex microbial ecosystem, largely composed of so far uncultured species, which can be detected only by using techniques such as PCR and by different hybridization techniques including phylogenetic microarrays. Manual DNA extraction from feces

  4. Using herbarium-derived DNAs to assemble a large-scale DNA barcode library for the vascular plants of Canada.

    Science.gov (United States)

    Kuzmina, Maria L; Braukmann, Thomas W A; Fazekas, Aron J; Graham, Sean W; Dewaard, Stephanie L; Rodrigues, Anuar; Bennett, Bruce A; Dickinson, Timothy A; Saarela, Jeffery M; Catling, Paul M; Newmaster, Steven G; Percy, Diana M; Fenneman, Erin; Lauron-Moreau, Aurélien; Ford, Bruce; Gillespie, Lynn; Subramanyam, Ragupathy; Whitton, Jeannette; Jennings, Linda; Metsger, Deborah; Warne, Connor P; Brown, Allison; Sears, Elizabeth; Dewaard, Jeremy R; Zakharov, Evgeny V; Hebert, Paul D N

    2017-12-01

    Constructing complete, accurate plant DNA barcode reference libraries can be logistically challenging for large-scale floras. Here we demonstrate the promise and challenges of using herbarium collections for building a DNA barcode reference library for the vascular plant flora of Canada. Our study examined 20,816 specimens representing 5076 of 5190 vascular plant species in Canada (98%). For 98% of the specimens, at least one of the DNA barcode regions was recovered from the plastid loci rbcL and matK and from the nuclear ITS2 region. We used beta regression to quantify the effects of age, type of preservation, and taxonomic affiliation (family) on DNA sequence recovery. Specimen age and method of preservation had significant effects on sequence recovery for all markers, but influenced some families more (e.g., Boraginaceae) than others (e.g., Asteraceae). Our DNA barcode library represents an unparalleled resource for metagenomic and ecological genetic research working on temperate and arctic biomes. An observed decline in sequence recovery with specimen age may be associated with poor primer matches, intragenomic variation (for ITS2), or inhibitory secondary compounds in some taxa.

  5. Paramecium tetraurelia chromatin assembly factor-1-like protein PtCAF-1 is involved in RNA-mediated control of DNA elimination.

    Science.gov (United States)

    Ignarski, Michael; Singh, Aditi; Swart, Estienne C; Arambasic, Miroslav; Sandoval, Pamela Y; Nowacki, Mariusz

    2014-10-29

    Genome-wide DNA remodelling in the ciliate Paramecium is ensured by RNA-mediated trans-nuclear crosstalk between the germline and the somatic genomes during sexual development. The rearrangements include elimination of transposable elements, minisatellites and tens of thousands non-coding elements called internally eliminated sequences (IESs). The trans-nuclear genome comparison process employs a distinct class of germline small RNAs (scnRNAs) that are compared against the parental somatic genome to select the germline-specific subset of scnRNAs that subsequently target DNA elimination in the progeny genome. Only a handful of proteins involved in this process have been identified so far and the mechanism of DNA targeting is unknown. Here we describe chromatin assembly factor-1-like protein (PtCAF-1), which we show is required for the survival of sexual progeny and localizes first in the parental and later in the newly developing macronucleus. Gene silencing shows that PtCAF-1 is required for the elimination of transposable elements and a subset of IESs. PTCAF-1 depletion also impairs the selection of germline-specific scnRNAs during development. We identify specific histone modifications appearing during Paramecium development which are strongly reduced in PTCAF-1 depleted cells. Our results demonstrate the importance of PtCAF-1 for the epigenetic trans-nuclear cross-talk mechanism. © The Author(s) 2014. Published by Oxford University Press on behalf of Nucleic Acids Research.

  6. Label-free and ultrasensitive electrochemiluminescence detection of microRNA based on long-range self-assembled DNA nanostructures

    International Nuclear Information System (INIS)

    Liu, Ting; Chen, Xian; Hong, Cheng-Yi; Xu, Xiao-Ping; Yang, Huang-Hao

    2014-01-01

    Electrochemiluminescence (ECL) integrates the advantages of electrochemical detection and chemiluminescent techniques. The method has received particular attention because it is highly sensitive and selective, has a wide linear range but low reagent costs. The use of nanomaterials with their unique physical and chemical properties has led to new kinds of biosensors that exhibit high sensitivity and stability. Compared to other nanomaterials, DNA nanostructures are more biocompatible, more hydrophilic, and thus less prone to nonspecific adsorption onto the electrode surface. We describe here a label-free and ultrasensitive ECL biosensor for detecting a cancer-associated microRNA at a femtomolar level. We have designed two auxiliary probes that cause the formation of a long-range self-assembly in the form of a μm-long 1-dimensional DNA concatamer. These can be used as carriers for signal amplification. The intercalation of the ECL probe Ru(phen) 3 2+ into the grooves of the concatamers leads to a substantial increase in ECL intensity. This amplified sensor shows high selectivity for discriminating complementary target and other mismatched RNAs. The biosensor enables the quantification of the expression of microRNA-21 in MCF-7 cells. It also displays very low limits of detection and provides an alternative approach for the detection of RNA or DNA detection in diagnostics and gene analysis. (author)

  7. Restriction-based Multiple-fragment Assembly Strategy to Avoid Random Mutation during Long cDNA Cloning.

    Science.gov (United States)

    Wang, Shang; Chen, Wen; Zhang, Kai; Jiao, Peng; Mo, Lihua; Yang, Xiaoxu; Hu, Xiang; Zhang, Jian; Wei, Chenxi; Xiang, Shuanglin

    2015-01-01

    Long fragment cloning is a challenge for its difficulty in accurate amplifying and tendency to get unwanted mutation. Here we discuss Restriction-based Multiple-fragment Assembly Strategy's advantages and limitations. In this strategy, rather than PCR amplifying the entire coding sequence (CDS) at one time, we amplified and sequenced smaller fragments which are shorter than 1.5kb spanning the CDS. After that, the sequence-proved fragments were assembled by digestion-ligation cloning to the target vector. We test its universality in our script programmed in Python. Our data shows that, among the entire human and mouse CDS, at least 70% of long CDS cloning will benefit from this strategy.

  8. Advances in inspection automation

    Science.gov (United States)

    Weber, Walter H.; Mair, H. Douglas; Jansen, Dion; Lombardi, Luciano

    2013-01-01

    This new session at QNDE reflects the growing interest in inspection automation. Our paper describes a newly developed platform that makes the complex NDE automation possible without the need for software programmers. Inspection tasks that are tedious, error-prone or impossible for humans to perform can now be automated using a form of drag and drop visual scripting. Our work attempts to rectify the problem that NDE is not keeping pace with the rest of factory automation. Outside of NDE, robots routinely and autonomously machine parts, assemble components, weld structures and report progress to corporate databases. By contrast, components arriving in the NDT department typically require manual part handling, calibrations and analysis. The automation examples in this paper cover the development of robotic thickness gauging and the use of adaptive contour following on the NRU reactor inspection at Chalk River.

  9. An automated microplate-based method for monitoring DNA strand breaks in plasmids and bacterial artificial chromosomes.

    Science.gov (United States)

    Rock, Cassandra; Shamlou, Parviz Ayazi; Levy, M Susana

    2003-06-01

    A method is described for high-throughput monitoring of DNA backbone integrity in plasmids and artificial chromosomes in solution. The method is based on the denaturation properties of double-stranded DNA in alkaline conditions and uses PicoGreen fluorochrome to monitor denaturation. In the present method, fluorescence enhancement of PicoGreen at pH 12.4 is normalised by its value at pH 8 to give a ratio that is proportional to the average backbone integrity of the DNA molecules in the sample. A good regression fit (r2 > 0.98) was obtained when results derived from the present method and those derived from agarose gel electrophoresis were compared. Spiking experiments indicated that the method is sensitive enough to detect a proportion of 6% (v/v) molecules with an average of less than two breaks per molecule. Under manual operation, validation parameters such as inter-assay and intra-assay variation gave values of electrophoresis of sheared samples were in agreement with those obtained using the microplate-based method.

  10. Self-assembled carbon nanotube-DNA hybrids at the nanoscale : Morphological and conductive properties probed by atomic force microscopy

    NARCIS (Netherlands)

    Santonicola, M.; Laurenzi, Susanna; Schön, Peter M.

    2014-01-01

    Our research is focused on the engineering of novel, highly sensitive and miniaturized hybrid materials from carbon nanotubes (CNTs) and DNA molecules for applications in biosensors and medical devices. These hybrid sensors allow for a high degree of miniaturization, a key factor in the design of

  11. Automated Test-Form Generation

    Science.gov (United States)

    van der Linden, Wim J.; Diao, Qi

    2011-01-01

    In automated test assembly (ATA), the methodology of mixed-integer programming is used to select test items from an item bank to meet the specifications for a desired test form and optimize its measurement accuracy. The same methodology can be used to automate the formatting of the set of selected items into the actual test form. Three different…

  12. Anchoring of self-assembled plasmid DNA/ anti-DNA antibody/cationic lipid micelles on bisphosphonate-modified stent for cardiovascular gene delivery

    Directory of Open Access Journals (Sweden)

    Ma G

    2013-03-01

    Full Text Available Guilei Ma,1,# Yong Wang,1,# Ilia Fishbein,2 Mei Yu,1 Linhua Zhang,1 Ivan S Alferiev,2 Jing Yang,1 Cunxian Song,1 Robert J Levy2 1Institute of Biomedical Engineering, Chinese Academy of Medical Sciences and Peking Union Medical College, Tianjin, People's Republic of China; 2Children's Hospital of Philadelphia, Abramson Research Building, Philadelphia, PA, USA #These authors contributed equally to this work Purpose: To investigate the anchoring of plasmid DNA/anti-DNA antibody/cationic lipid tri-complex (DAC micelles onto bisphosphonate-modified 316 L coronary stents for cardiovascular site-specific gene delivery. Methods: Stents were first modified with polyallylamine bisphosphonate (PAA-BP, thereby enabling the retention of a PAA-BP molecular monolayer that permits the anchoring (via vector-binding molecules of DAC micelles. DAC micelles were then chemically linked onto the PAA-BP-modified stents by using N-succinimidyl-3-(2-pyridyldithiol-propionate (SPDP as a crosslinker. Rhodamine-labeled DNA was used to assess the anchoring of DAC micelles, and radioactive-labeled antibody was used to evaluate binding capacity and stability. DAC micelles (encoding green fluorescent protein were tethered onto the PAA-BP-modified stents, which were assessed in cell culture. The presence of a PAA-BP molecular monolayer on the steel surface was confirmed by X-ray photoelectron spectroscopy and atomic force microscope analysis. Results: The anchoring of DAC micelles was generally uniform and devoid of large-scale patches of defects. Isotopic quantification confirmed that the amount of antibody chemically linked on the stents was 17-fold higher than that of the physical adsorbed control stents and its retention time was also significantly longer. In cell culture, numerous green fluorescent protein-positive cells were found on the PAA-BP modified stents, which demonstrated high localization and efficiency of gene delivery. Conclusion: The DAC micelle

  13. The human cytomegalovirus gene products essential for late viral gene expression assemble into prereplication complexes before viral DNA replication.

    Science.gov (United States)

    Isomura, Hiroki; Stinski, Mark F; Murata, Takayuki; Yamashita, Yoriko; Kanda, Teru; Toyokuni, Shinya; Tsurumi, Tatsuya

    2011-07-01

    The regulation of human cytomegalovirus (HCMV) late gene expression by viral proteins is poorly understood, and these viral proteins could be targets for novel antivirals. HCMV open reading frames (ORFs) UL79, -87, and -95 encode proteins with homology to late gene transcription factors of murine gammaherpesvirus 68 ORFs 18, 24, and 34, respectively. To determine whether these HCMV proteins are also essential for late gene transcription of a betaherpesvirus, we mutated HCMV ORFs UL79, -87, and -95. Cells were infected with the recombinant viruses at high and low multiplicities of infection (MOIs). While viral DNA was detected with the recombinant viruses, infectious virus was not detected unless the wild-type viral proteins were expressed in trans. At a high MOI, mutation of ORF UL79, -87, or -95 had no effect on the level of major immediate-early (MIE) gene expression or viral DNA replication, but late viral gene expression from the UL44, -75, and -99 ORFs was not detected. At a low MOI, preexpression of UL79 or -87, but not UL95, in human fibroblast cells negatively affected the level of MIE viral gene expression and viral DNA replication. The products of ORFs UL79, -87, and -95 were expressed as early viral proteins and recruited to prereplication complexes (pre-RCs), along with UL44, before the initiation of viral DNA replication. All three HCMV ORFs are indispensable for late viral gene expression and viral growth. The roles of UL79, -87, and -95 in pre-RCs for late viral gene expression are discussed.

  14. Remote operations on the furnace assembly and product canister in high-level waste vitrification plant at Tarapur and scope for automation in future such plants

    International Nuclear Information System (INIS)

    Mohandas, J.M.; Samuel, M.T.

    1990-01-01

    Vitrification of high level liquid waste produced in a fuel reprocessing plant is carried out inside a concrete cell with provisons for all operations on the main equipment, that is the induction heated furnace, to be carried out by remote handling gadgets. Since each canister of the vitrified waste product in a batch can contain upto 0.6 million curie of activity, these operations are required to be highly reliable. The present plant has provisions for these remote operations mainly by manual mode using remote handling gadgets like power manipulator, MSMs, incell crane etc. With the advent of advanced technology of automation and possible use of robotics, it is considered that a much more reliable, less time consuming and efficient operation can be carried out using such techniques. This paper describes the present mode of operation in the cell and points out the areas in which automation of these operations can be adopted. (author). 3 figs

  15. Human Assisted Assembly Processes

    Energy Technology Data Exchange (ETDEWEB)

    CALTON,TERRI L.; PETERS,RALPH R.

    2000-01-01

    Automatic assembly sequencing and visualization tools are valuable in determining the best assembly sequences, but without Human Factors and Figure Models (HFFMs) it is difficult to evaluate or visualize human interaction. In industry, accelerating technological advances and shorter market windows have forced companies to turn to an agile manufacturing paradigm. This trend has promoted computerized automation of product design and manufacturing processes, such as automated assembly planning. However, all automated assembly planning software tools assume that the individual components fly into their assembled configuration and generate what appear to be a perfectly valid operations, but in reality the operations cannot physically be carried out by a human. Similarly, human figure modeling algorithms may indicate that assembly operations are not feasible and consequently force design modifications; however, if they had the capability to quickly generate alternative assembly sequences, they might have identified a feasible solution. To solve this problem HFFMs must be integrated with automated assembly planning to allow engineers to verify that assembly operations are possible and to see ways to make the designs even better. Factories will very likely put humans and robots together in cooperative environments to meet the demands for customized products, for purposes including robotic and automated assembly. For robots to work harmoniously within an integrated environment with humans the robots must have cooperative operational skills. For example, in a human only environment, humans may tolerate collisions with one another if they did not cause much pain. This level of tolerance may or may not apply to robot-human environments. Humans expect that robots will be able to operate and navigate in their environments without collisions or interference. The ability to accomplish this is linked to the sensing capabilities available. Current work in the field of cooperative

  16. Automated DNA sequence-based early warning system for the detection of methicillin-resistant Staphylococcus aureus outbreaks.

    Directory of Open Access Journals (Sweden)

    Alexander Mellmann

    2006-03-01

    Full Text Available BACKGROUND: The detection of methicillin-resistant Staphylococcus aureus (MRSA usually requires the implementation of often rigorous infection-control measures. Prompt identification of an MRSA epidemic is crucial for the control of an outbreak. In this study we evaluated various early warning algorithms for the detection of an MRSA cluster. METHODS AND FINDINGS: Between 1998 and 2003, 557 non-replicate MRSA strains were collected from staff and patients admitted to a German tertiary-care university hospital. The repeat region of the S. aureus protein A (spa gene in each of these strains was sequenced. Using epidemiological and typing information for the period 1998-2002 as reference data, clusters in 2003 were determined by temporal-scan test statistics. Various early warning algorithms (frequency, clonal, and infection control professionals [ICP] alerts were tested in a prospective analysis for the year 2003. In addition, a newly implemented automated clonal alert system of the Ridom StaphType software was evaluated. A total of 549 of 557 MRSA were typeable using spa sequencing. When analyzed using scan test statistics, 42 out of 175 MRSA in 2003 formed 13 significant clusters (p < 0.05. These clusters were used as the "gold standard" to evaluate the various algorithms. Clonal alerts (spa typing and epidemiological data were 100% sensitive and 95.2% specific. Frequency (epidemiological data only and ICP alerts were 100% and 62.1% sensitive and 47.2% and 97.3% specific, respectively. The difference in specificity between clonal and ICP alerts was not significant. Both methods exhibited a positive predictive value above 80%. CONCLUSIONS: Rapid MRSA outbreak detection, based on epidemiological and spa typing data, is a suitable alternative for classical approaches and can assist in the identification of potential sources of infection.

  17. DNA nanotechnology

    Science.gov (United States)

    Seeman, Nadrian C.; Sleiman, Hanadi F.

    2018-01-01

    DNA is the molecule that stores and transmits genetic information in biological systems. The field of DNA nanotechnology takes this molecule out of its biological context and uses its information to assemble structural motifs and then to connect them together. This field has had a remarkable impact on nanoscience and nanotechnology, and has been revolutionary in our ability to control molecular self-assembly. In this Review, we summarize the approaches used to assemble DNA nanostructures and examine their emerging applications in areas such as biophysics, diagnostics, nanoparticle and protein assembly, biomolecule structure determination, drug delivery and synthetic biology. The introduction of orthogonal interactions into DNA nanostructures is discussed, and finally, a perspective on the future directions of this field is presented.

  18. The ATP Sites of AAA+ Clamp Loaders Work Together as a Switch to Assemble Clamps on DNA*

    Science.gov (United States)

    Marzahn, Melissa R.; Hayner, Jaclyn N.; Finkelstein, Jeff; O'Donnell, Mike; Bloom, Linda B.

    2014-01-01

    Clamp loaders belong to a family of proteins known as ATPases associated with various cellular activities (AAA+). These proteins utilize the energy from ATP binding and hydrolysis to perform cellular functions. The clamp loader is required to load the clamp onto DNA for use by DNA polymerases to increase processivity. ATP binding and hydrolysis are coordinated by several key residues, including a conserved Lys located within the Walker A motif (or P-loop). This residue is required for each subunit to bind ATP. The specific function of each ATP molecule bound to the Saccharomyces cerevisiae clamp loader is unknown. A series of point mutants, each lacking a single Walker A Lys residue, was generated to study the effects of abolishing ATP binding in individual clamp loader subunits. A variety of biochemical assays were used to analyze the function of ATP binding during discrete steps of the clamp loading reaction. All mutants reduced clamp binding/opening to different degrees. Decreased clamp binding activity was generally correlated with decreases in the population of open clamps, suggesting that differences in the binding affinities of Walker A mutants stem from differences in stabilization of proliferating cell nuclear antigen in an open conformation. Walker A mutations had a smaller effect on DNA binding than clamp binding/opening. Our data do not support a model in which each ATP site functions independently to regulate a different step in the clamp loading cycle to coordinate these steps. Instead, the ATP sites work in unison to promote conformational changes in the clamp loader that drive clamp loading. PMID:24436332

  19. The ATP sites of AAA+ clamp loaders work together as a switch to assemble clamps on DNA.

    Science.gov (United States)

    Marzahn, Melissa R; Hayner, Jaclyn N; Finkelstein, Jeff; O'Donnell, Mike; Bloom, Linda B

    2014-02-28

    Clamp loaders belong to a family of proteins known as ATPases associated with various cellular activities (AAA+). These proteins utilize the energy from ATP binding and hydrolysis to perform cellular functions. The clamp loader is required to load the clamp onto DNA for use by DNA polymerases to increase processivity. ATP binding and hydrolysis are coordinated by several key residues, including a conserved Lys located within the Walker A motif (or P-loop). This residue is required for each subunit to bind ATP. The specific function of each ATP molecule bound to the Saccharomyces cerevisiae clamp loader is unknown. A series of point mutants, each lacking a single Walker A Lys residue, was generated to study the effects of abolishing ATP binding in individual clamp loader subunits. A variety of biochemical assays were used to analyze the function of ATP binding during discrete steps of the clamp loading reaction. All mutants reduced clamp binding/opening to different degrees. Decreased clamp binding activity was generally correlated with decreases in the population of open clamps, suggesting that differences in the binding affinities of Walker A mutants stem from differences in stabilization of proliferating cell nuclear antigen in an open conformation. Walker A mutations had a smaller effect on DNA binding than clamp binding/opening. Our data do not support a model in which each ATP site functions independently to regulate a different step in the clamp loading cycle to coordinate these steps. Instead, the ATP sites work in unison to promote conformational changes in the clamp loader that drive clamp loading.

  20. Desenvolvimento de um sistema automatizado para a fabricação de filmes automontados Development of an automated device for fabrication of self-assembled films

    Directory of Open Access Journals (Sweden)

    Francisco Trivinho-Strixino

    2004-08-01

    Full Text Available In this technical note, we describe the construction of a low-cost computer controlled device for layer-by-layer film fabrication. The software allows to control multiple material deposition, washing and drying steps and the time for each operation. To test the device, we produced and characterized self-assembled films of conductive polymers by alternating poly(o-methoxyaniline (POMA and poly(3-thiophene acetic acid (PTAA via the layer-by-layer technique.

  1. Integrity of chromatin and replicating DNA in nuclei released from fission yeast by semi-automated grinding in liquid nitrogen

    Directory of Open Access Journals (Sweden)

    Givens Robert M

    2011-11-01

    Full Text Available Abstract Background Studies of nuclear function in many organisms, especially those with tough cell walls, are limited by lack of availability of simple, economical methods for large-scale preparation of clean, undamaged nuclei. Findings Here we present a useful method for nuclear isolation from the important model organism, the fission yeast, Schizosaccharomyces pombe. To preserve in vivo molecular configurations, we flash-froze the yeast cells in liquid nitrogen. Then we broke their tough cell walls, without damaging their nuclei, by grinding in a precision-controlled motorized mortar-and-pestle apparatus. The cryo-ground cells were resuspended and thawed in a buffer designed to preserve nuclear morphology, and the nuclei were enriched by differential centrifugation. The washed nuclei were free from contaminating nucleases and have proven well-suited as starting material for genome-wide chromatin analysis and for preparation of fragile DNA replication intermediates. Conclusions We have developed a simple, reproducible, economical procedure for large-scale preparation of endogenous-nuclease-free, morphologically intact nuclei from fission yeast. With appropriate modifications, this procedure may well prove useful for isolation of nuclei from other organisms with, or without, tough cell walls.

  2. Integrity of chromatin and replicating DNA in nuclei released from fission yeast by semi-automated grinding in liquid nitrogen.

    Science.gov (United States)

    Givens, Robert M; Mesner, Larry D; Hamlin, Joyce L; Buck, Michael J; Huberman, Joel A

    2011-11-16

    Studies of nuclear function in many organisms, especially those with tough cell walls, are limited by lack of availability of simple, economical methods for large-scale preparation of clean, undamaged nuclei. Here we present a useful method for nuclear isolation from the important model organism, the fission yeast, Schizosaccharomyces pombe. To preserve in vivo molecular configurations, we flash-froze the yeast cells in liquid nitrogen. Then we broke their tough cell walls, without damaging their nuclei, by grinding in a precision-controlled motorized mortar-and-pestle apparatus. The cryo-ground cells were resuspended and thawed in a buffer designed to preserve nuclear morphology, and the nuclei were enriched by differential centrifugation. The washed nuclei were free from contaminating nucleases and have proven well-suited as starting material for genome-wide chromatin analysis and for preparation of fragile DNA replication intermediates. We have developed a simple, reproducible, economical procedure for large-scale preparation of endogenous-nuclease-free, morphologically intact nuclei from fission yeast. With appropriate modifications, this procedure may well prove useful for isolation of nuclei from other organisms with, or without, tough cell walls.

  3. Orthogonal labeling of M13 minor capsid proteins with DNA to self-assemble end-to-end multiphage structures.

    Science.gov (United States)

    Hess, Gaelen T; Guimaraes, Carla P; Spooner, Eric; Ploegh, Hidde L; Belcher, Angela M

    2013-09-20

    M13 bacteriophage has been used as a scaffold to organize materials for various applications. Building more complex multiphage devices requires precise control of interactions between the M13 capsid proteins. Toward this end, we engineered a loop structure onto the pIII capsid protein of M13 bacteriophage to enable sortase-mediated labeling reactions for C-terminal display. Combining this with N-terminal sortase-mediated labeling, we thus created a phage scaffold that can be labeled orthogonally on three capsid proteins: the body and both ends. We show that covalent attachment of different DNA oligonucleotides at the ends of the new phage structure enables formation of multiphage particles oriented in a specific order. These have potential as nanoscale scaffolds for multi-material devices.

  4. Fast and quantitative differentiation of single-base mismatched DNA by initial reaction rate of catalytic hairpin assembly.

    Science.gov (United States)

    Li, Chenxi; Li, Yixin; Xu, Xiao; Wang, Xinyi; Chen, Yang; Yang, Xiaoda; Liu, Feng; Li, Na

    2014-10-15

    The widely used catalytic hairpin assembly (CHA) amplification strategy generally needs several hours to accomplish one measurement based on the prevailingly used maximum intensity detection mode, making it less practical for assays where high throughput or speed is desired. To make the best use of the kinetic specificity of toehold domain for circuit reaction initiation, we developed a mathematical model and proposed an initial reaction rate detection mode to quantitatively differentiate the single-base mismatch. Using the kinetic mode, assay time can be reduced substantially to 10 min for one measurement with the comparable sensitivity and single-base mismatch differentiating ability as were obtained by the maximum intensity detection mode. This initial reaction rate based approach not only provided a fast and quantitative differentiation of single-base mismatch, but also helped in-depth understanding of the CHA system, which will be beneficial to the design of highly sensitive and specific toehold-mediated hybridization reactions. Copyright © 2014 Elsevier B.V. All rights reserved.

  5. Phase 2. Array automated assembly task, low cost silicon solar array project. Quarterly technical report no. 2, April 1--June 30, 1978

    Energy Technology Data Exchange (ETDEWEB)

    Jones, Gregory T.; Rhee, Sang S.; Allison, Kimberly L.

    1978-01-01

    Data was gathered and analysis continued on seventeen process groups. Five process groups were completed. They include: solar cell test data acquisition, plasma etching of resist, laser trimming and holing operation, wafer surface preparation and wafer printing. Preliminary results to date show very promising results for other task groups. Silicon nitride antireflective coating equipment with modifications has high potential for low-cost automation. An electroless nickel plating system has been designed and is under construction to demonstrate the processing of 1500 wafers per hour. The conceptual design of two fully automated laser trimming and holing lines has been completed and has very promising low-cost, high volume potential. The spray-on dopant model equipment construction is nearly completed. Preliminary spray-on tests have shown very good results. Dopants can be sprayed on without wafer edge overlap and good dopant uniformity can be achieved. SAMICS has been applied to most process groups. It has been very effective in analyzing process steps for low-cost potential for meeting the LSA 1986 goals.

  6. Driving nucleolar assembly.

    Science.gov (United States)

    McCann, Kathleen L; Baserga, Susan J

    2014-02-01

    In this issue of Genes & Development, Grob and colleagues (pp. 220-230) identify the minimal molecular requirements to assemble a fully functional nucleolus in human cells and demonstrate the importance of the nucleolar transcription factor upstream binding factor (UBF) as a mitotic bookmark at the ribosomal DNA (rDNA).

  7. Programme DNA Lattices: Design, Synthesis and Applications

    National Research Council Canada - National Science Library

    Reif, John

    2006-01-01

    .... Self-assembled DNA nanostructures provide a methodology for bottom-up nanoscale construction of highly patterned systems, utilizing macromolecular DNA tiles" composed of branched DNA, self-assembled...

  8. Uracil Excision for Assembly of Complex Pathways

    DEFF Research Database (Denmark)

    Cavaleiro, Mafalda; Nielsen, Morten Thrane; Kim, Se Hyeuk

    2015-01-01

    Despite decreasing prices on synthetic DNA constructs, higher-order assembly of PCR-generated DNA continues to be an important exercise in molecular and synthetic biology. Simplicity and robustness are attractive features met by the uracil excision DNA assembly method, which is one of the most in...... genes into the genome, and a standardized assembly pipeline using benchmarked oligonucleotides for pathway assembly and multigene expression optimization....

  9. Functional annotation, genome organization and phylogeny of the grapevine (Vitis vinifera) terpene synthase gene family based on genome assembly, FLcDNA cloning, and enzyme assays.

    Science.gov (United States)

    Martin, Diane M; Aubourg, Sébastien; Schouwey, Marina B; Daviet, Laurent; Schalk, Michel; Toub, Omid; Lund, Steven T; Bohlmann, Jörg

    2010-10-21

    Terpenoids are among the most important constituents of grape flavour and wine bouquet, and serve as useful metabolite markers in viticulture and enology. Based on the initial 8-fold sequencing of a nearly homozygous Pinot noir inbred line, 89 putative terpenoid synthase genes (VvTPS) were predicted by in silico analysis of the grapevine (Vitis vinifera) genome assembly 1. The finding of this very large VvTPS family, combined with the importance of terpenoid metabolism for the organoleptic properties of grapevine berries and finished wines, prompted a detailed examination of this gene family at the genomic level as well as an investigation into VvTPS biochemical functions. We present findings from the analysis of the up-dated 12-fold sequencing and assembly of the grapevine genome that place the number of predicted VvTPS genes at 69 putatively functional VvTPS, 20 partial VvTPS, and 63 VvTPS probable pseudogenes. Gene discovery and annotation included information about gene architecture and chromosomal location. A dense cluster of 45 VvTPS is localized on chromosome 18. Extensive FLcDNA cloning, gene synthesis, and protein expression enabled functional characterization of 39 VvTPS; this is the largest number of functionally characterized TPS for any species reported to date. Of these enzymes, 23 have unique functions and/or phylogenetic locations within the plant TPS gene family. Phylogenetic analyses of the TPS gene family showed that while most VvTPS form species-specific gene clusters, there are several examples of gene orthology with TPS of other plant species, representing perhaps more ancient VvTPS, which have maintained functions independent of speciation. The highly expanded VvTPS gene family underpins the prominence of terpenoid metabolism in grapevine. We provide a detailed experimental functional annotation of 39 members of this important gene family in grapevine and comprehensive information about gene structure and phylogeny for the entire currently

  10. Array Automated Assembly Task Low Cost Silicon Solar Array Project. Phase 2. Annual technical report, September 20, 1977-December 31, 1978

    Energy Technology Data Exchange (ETDEWEB)

    Rhee, Sang S.; Jones, Gregory T.; Allison, Kimberly L.

    1978-01-01

    This program was conducted to develop and demonstrate those solar cells and module process steps which have the technological readiness or capability to achieve the 1986 LSA goals. Results are reported. Seventeen process groups were investigated. Very promising results were achieved. A laserscribe computer program was developed. It demonstrated that silicon solar cells could be trimmed and holed by laser without causing mechanical defects (i.e., microcracks) nor any major degradation in solar cell electrical performance. The silicon wafer surface preparation task demonstrated a low-cost, high throughput texturizing process readily adaptable to automation. Performance verification tests of a laser scanning system showed a limited capability to detect hidden cracks or defects in solar cells. A general review of currently available thick film printing equipment provided the indication that state-of-the-art technology can adequately transform the capability of current printing machines to the elevated rate of 7200 wafers per hour. The LFE System 8000 silicon nitride plasma deposition system with the inclusion of minor equipment modifications was shown to be consistent with the 1986 LSA pricing goals. The performance verification test of the silicon nitride A.R. coating process provided the result that texturized, A.R. coated solar cells display a 14.1% improvement in electrical performance over identical solar cells without an A.R. coating. A new electroless nickel plating system was installed and demonstrated a low-cost, high throughput process readily adaptable to automation. A multiple wafer dipping method was investigated and operational parameters defined. A flux removal method consisting of a three stage D.I. water cascade rinse system with ultrasonic agitator was found to be very promising. Also, a SAMICS cost analysis was performed. (WHK)

  11. Comparison of automated multiplexed bead-based ANA screening assay with ELISA for detecting five common anti-extractable nuclear antigens and anti-dsDNA in systemic rheumatic diseases.

    Science.gov (United States)

    Kim, Yoonjung; Park, Yongjung; Lee, Eun Young; Kim, Hyon-Suk

    2012-01-18

    A newly developed and totally automated Luminex-based assay, the BioPlex™ 2200 system, is able to detect various autoantibodies simultaneously from a single sample. We compared the BioPlex™ 2200 system with ELISA for the detection of six autoantibodies. A total of 127 serum samples from the patients with systemic rheumatic diseases were collected and assayed with the BioPlex™ 2200 system (Bio-Rad, USA) and conventional ELISA (INOVA Diagnostics, USA) for 5 anti-extractable nuclear antigens. Additionally, relative sensitivity of the BioPlex™ 2200 system for detecting anti-dsDNA was evaluated with 79 specimens from SLE patients, which were positive for anti-dsDNA by ELISA. The concordance rates between ELISA and the BioPlex ranged from 88.1% for anti-RNP to 95.2% for anti-Scl-70, and the kappa coefficients between the results by the two assays were from 0.48 to 0.67. Among the 79 anti-dsDNA positive specimens by ELISA, seventy-eight (98.7%) showed positive results for anti-dsDNA by the BioPlex. The BioPlex™ 2200 system showed comparable results with those by conventional ELISA for detecting autoantibodies, and this automated assay could measure multifarious autoantibodies concurrently in a single sample. It could be effectively used in clinical laboratories for screening autoimmune diseases. Copyright © 2011 Elsevier B.V. All rights reserved.

  12. Automation, Performance and International Competition

    DEFF Research Database (Denmark)

    Kromann, Lene; Sørensen, Anders

    This paper presents new evidence on trade‐induced automation in manufacturing firms using unique data combining a retrospective survey that we have assembled with register data for 2005‐2010. In particular, we establish a causal effect where firms that have specialized in product types for which...... the Chinese exports to the world market has risen sharply invest more in automated capital compared to firms that have specialized in other product types. We also study the relationship between automation and firm performance and find that firms with high increases in scale and scope of automation have faster...... productivity growth than other firms. Moreover, automation improves the efficiency of all stages of the production process by reducing setup time, run time, and inspection time and increasing uptime and quantity produced per worker. The efficiency improvement varies by type of automation....

  13. De novo assembly and annotation of the Asian tiger mosquito (Aedes albopictus) repeatome with dnaPipeTE from raw genomic reads and comparative analysis with the yellow fever mosquito (Aedes aegypti).

    Science.gov (United States)

    Goubert, Clément; Modolo, Laurent; Vieira, Cristina; ValienteMoro, Claire; Mavingui, Patrick; Boulesteix, Matthieu

    2015-03-11

    Repetitive DNA, including transposable elements (TEs), is found throughout eukaryotic genomes. Annotating and assembling the "repeatome" during genome-wide analysis often poses a challenge. To address this problem, we present dnaPipeTE-a new bioinformatics pipeline that uses a sample of raw genomic reads. It produces precise estimates of repeated DNA content and TE consensus sequences, as well as the relative ages of TE families. We shows that dnaPipeTE performs well using very low coverage sequencing in different genomes, losing accuracy only with old TE families. We applied this pipeline to the genome of the Asian tiger mosquito Aedes albopictus, an invasive species of human health interest, for which the genome size is estimated to be over 1 Gbp. Using dnaPipeTE, we showed that this species harbors a large (50% of the genome) and potentially active repeatome with an overall TE class and order composition similar to that of Aedes aegypti, the yellow fever mosquito. However, intraorder dynamics show clear distinctions between the two species, with differences at the TE family level. Our pipeline's ability to manage the repeatome annotation problem will make it helpful for new or ongoing assembly projects, and our results will benefit future genomic studies of A. albopictus. © The Author(s) 2015. Published by Oxford University Press on behalf of the Society for Molecular Biology and Evolution.

  14. Functional Annotation, Genome Organization and Phylogeny of the Grapevine (Vitis vinifera Terpene Synthase Gene Family Based on Genome Assembly, FLcDNA Cloning, and Enzyme Assays

    Directory of Open Access Journals (Sweden)

    Toub Omid

    2010-10-01

    Full Text Available Abstract Background Terpenoids are among the most important constituents of grape flavour and wine bouquet, and serve as useful metabolite markers in viticulture and enology. Based on the initial 8-fold sequencing of a nearly homozygous Pinot noir inbred line, 89 putative terpenoid synthase genes (VvTPS were predicted by in silico analysis of the grapevine (Vitis vinifera genome assembly 1. The finding of this very large VvTPS family, combined with the importance of terpenoid metabolism for the organoleptic properties of grapevine berries and finished wines, prompted a detailed examination of this gene family at the genomic level as well as an investigation into VvTPS biochemical functions. Results We present findings from the analysis of the up-dated 12-fold sequencing and assembly of the grapevine genome that place the number of predicted VvTPS genes at 69 putatively functional VvTPS, 20 partial VvTPS, and 63 VvTPS probable pseudogenes. Gene discovery and annotation included information about gene architecture and chromosomal location. A dense cluster of 45 VvTPS is localized on chromosome 18. Extensive FLcDNA cloning, gene synthesis, and protein expression enabled functional characterization of 39 VvTPS; this is the largest number of functionally characterized TPS for any species reported to date. Of these enzymes, 23 have unique functions and/or phylogenetic locations within the plant TPS gene family. Phylogenetic analyses of the TPS gene family showed that while most VvTPS form species-specific gene clusters, there are several examples of gene orthology with TPS of other plant species, representing perhaps more ancient VvTPS, which have maintained functions independent of speciation. Conclusions The highly expanded VvTPS gene family underpins the prominence of terpenoid metabolism in grapevine. We provide a detailed experimental functional annotation of 39 members of this important gene family in grapevine and comprehensive information

  15. Molecular self-assembly advances and applications

    CERN Document Server

    Dequan, Alex Li

    2012-01-01

    In the past several decades, molecular self-assembly has emerged as one of the main themes in chemistry, biology, and materials science. This book compiles and details cutting-edge research in molecular assemblies ranging from self-organized peptide nanostructures and DNA-chromophore foldamers to supramolecular systems and metal-directed assemblies, even to nanocrystal superparticles and self-assembled microdevices

  16. Synthesis of DNA

    Science.gov (United States)

    Mariella, Jr., Raymond P.

    2008-11-18

    A method of synthesizing a desired double-stranded DNA of a predetermined length and of a predetermined sequence. Preselected sequence segments that will complete the desired double-stranded DNA are determined. Preselected segment sequences of DNA that will be used to complete the desired double-stranded DNA are provided. The preselected segment sequences of DNA are assembled to produce the desired double-stranded DNA.

  17. AutoSeqMan: batch assembly of contigs for Sanger sequences

    Directory of Open Access Journals (Sweden)

    Jie-Qiong Jin

    2018-03-01

    Full Text Available With the wide application of DNA sequencing technology, DNA sequences are still increasingly generated through the Sanger sequencing platform. SeqMan (in the LaserGene package is an excellent program with an easy-to-use graphical user interface (GUI employed to assemble Sanger sequences into contigs. However, with increasing data size, larger sample sets and more sequenced loci make contig assemble complicated due to the considerable number of manual operations required to run SeqMan. Here, we present the ‘autoSeqMan’ software program, which can automatedly assemble contigs using SeqMan scripting language. There are two main modules available, namely, ‘Classification’ and ‘Assembly’. Classification first undertakes preprocessing work, whereas Assembly generates a SeqMan script to consecutively assemble contigs for the classified files. Through comparison with manual operation, we showed that autoSeqMan saved substantial time in the preprocessing and assembly of Sanger sequences. We hope this tool will be useful for those with large sample sets to analyze, but with little programming experience. It is freely available at https://github.com/ Sun-Yanbo/autoSeqMan.

  18. Multi-Robot Assembly Strategies and Metrics

    Science.gov (United States)

    MARVEL, JEREMY A.; BOSTELMAN, ROGER; FALCO, JOE

    2018-01-01

    We present a survey of multi-robot assembly applications and methods and describe trends and general insights into the multi-robot assembly problem for industrial applications. We focus on fixtureless assembly strategies featuring two or more robotic systems. Such robotic systems include industrial robot arms, dexterous robotic hands, and autonomous mobile platforms, such as automated guided vehicles. In this survey, we identify the types of assemblies that are enabled by utilizing multiple robots, the algorithms that synchronize the motions of the robots to complete the assembly operations, and the metrics used to assess the quality and performance of the assemblies. PMID:29497234

  19. Multi-Robot Assembly Strategies and Metrics.

    Science.gov (United States)

    Marvel, Jeremy A; Bostelman, Roger; Falco, Joe

    2018-02-01

    We present a survey of multi-robot assembly applications and methods and describe trends and general insights into the multi-robot assembly problem for industrial applications. We focus on fixtureless assembly strategies featuring two or more robotic systems. Such robotic systems include industrial robot arms, dexterous robotic hands, and autonomous mobile platforms, such as automated guided vehicles. In this survey, we identify the types of assemblies that are enabled by utilizing multiple robots, the algorithms that synchronize the motions of the robots to complete the assembly operations, and the metrics used to assess the quality and performance of the assemblies.

  20. Automation Revolutionize the Business Service Industry

    OpenAIRE

    Marciniak, Róbert

    2017-01-01

    In the last decades significant disruptive changes began with the extended use of automation. Many jobs are changed or disappeared and others were born totally with the automation. Together with the progress of technology, the automation was primarily spread in the industrial sector, mostly in the production and assembly lines. The growth maycontinue in the future further, researchers expect more than 35 million industrial robots globally by 2018.But it shades the situati...

  1. BRET fuel assembly dismantling machine

    International Nuclear Information System (INIS)

    Titzler, P.A.; Bennett, K.L.; Kelley, R.S. Jr.; Stringer, J.L.

    1984-08-01

    An automated remote nuclear fuel assembly milling and dismantling machine has been designed, developed, and demonstrated at the Hanford Engineering Development Laboratory (HEDL) in Richland, Washington. The machine can be used to dismantle irradiated breeder fuel assemblies from the Fast Flux Test Facility prior to fuel reprocessing. It can be installed in an existing remotely operated shielded hot cell facility, the Fuels and Materials Examination Facility (FMEF), at the Hanford Site in Richland, Washington

  2. Genome Sequence Databases (Overview): Sequencing and Assembly

    Energy Technology Data Exchange (ETDEWEB)

    Lapidus, Alla L.

    2009-01-01

    From the date its role in heredity was discovered, DNA has been generating interest among scientists from different fields of knowledge: physicists have studied the three dimensional structure of the DNA molecule, biologists tried to decode the secrets of life hidden within these long molecules, and technologists invent and improve methods of DNA analysis. The analysis of the nucleotide sequence of DNA occupies a special place among the methods developed. Thanks to the variety of sequencing technologies available, the process of decoding the sequence of genomic DNA (or whole genome sequencing) has become robust and inexpensive. Meanwhile the assembly of whole genome sequences remains a challenging task. In addition to the need to assemble millions of DNA fragments of different length (from 35 bp (Solexa) to 800 bp (Sanger)), great interest in analysis of microbial communities (metagenomes) of different complexities raises new problems and pushes some new requirements for sequence assembly tools to the forefront. The genome assembly process can be divided into two steps: draft assembly and assembly improvement (finishing). Despite the fact that automatically performed assembly (or draft assembly) is capable of covering up to 98% of the genome, in most cases, it still contains incorrectly assembled reads. The error rate of the consensus sequence produced at this stage is about 1/2000 bp. A finished genome represents the genome assembly of much higher accuracy (with no gaps or incorrectly assembled areas) and quality ({approx}1 error/10,000 bp), validated through a number of computer and laboratory experiments.

  3. Advanced Prospects for Development of Aircraft Assembly Technology

    Directory of Open Access Journals (Sweden)

    Krivtsov, V.S.

    2015-05-01

    Full Text Available On the basis of theoretical studies carried out by the method of virtual bases the concept of automated assembly of aircraft structures, specialized readjustable devices with CNC and robotic systems was created. The principles of automated assembly of aircraft manufacturing enterprise using robotic system are proved.

  4. Using herbarium-derived DNAs to assemble a large-scale DNA barcode library for the vascular plants of Canada 1

    OpenAIRE

    Kuzmina, Maria L.; Braukmann, Thomas W. A.; Fazekas, Aron J.; Graham, Sean W.; Dewaard, Stephanie L.; Rodrigues, Anuar; Bennett, Bruce A.; Dickinson, Timothy A.; Saarela, Jeffery M.; Catling, Paul M.; Newmaster, Steven G.; Percy, Diana M.; Fenneman, Erin; Lauron-Moreau, Aurélien; Ford, Bruce

    2017-01-01

    Premise of the study: Constructing complete, accurate plant DNA barcode reference libraries can be logistically challenging for large-scale floras. Here we demonstrate the promise and challenges of using herbarium collections for building a DNA barcode reference library for the vascular plant flora of Canada. Methods: Our study examined 20,816 specimens representing 5076 of 5190 vascular plant species in Canada (98%). For 98% of the specimens, at least one of the DNA barcode regions was recov...

  5. Library Automation

    OpenAIRE

    Dhakne, B. N.; Giri, V. V; Waghmode, S. S.

    2010-01-01

    New technologies library provides several new materials, media and mode of storing and communicating the information. Library Automation reduces the drudgery of repeated manual efforts in library routine. By use of library automation collection, Storage, Administration, Processing, Preservation and communication etc.

  6. Illustrating how mechanical assemblies work

    KAUST Repository

    Mitra, Niloy J.

    2010-07-26

    How things work visualizations use a variety of visual techniques to depict the operation of complex mechanical assemblies. We present an automated approach for generating such visualizations. Starting with a 3D CAD model of an assembly, we first infer the motions of individual parts and the interactions between parts based on their geometry and a few user specified constraints. We then use this information to generate visualizations that incorporate motion arrows, frame sequences and animation to convey the causal chain of motions and mechanical interactions between parts. We present results for a wide variety of assemblies. © 2010 ACM.

  7. Illustrating how mechanical assemblies work

    KAUST Repository

    Mitra, Niloy J.

    2013-01-01

    How-things-work visualizations use a variety of visual techniques to depict the operation of complex mechanical assemblies. We present an automated approach for generating such visualizations. Starting with a 3D CAD model of an assembly, we first infer the motions of the individual parts and the interactions across the parts based on their geometry and a few user-specified constraints. We then use this information to generate visualizations that incorporate motion arrows, frame sequences, and animation to convey the causal chain of motions and mechanical interactions across parts. We demonstrate our system on a wide variety of assemblies. © 2013 ACM 0001-0782/13/01.

  8. Automated refueling inventory control system at FFTF

    International Nuclear Information System (INIS)

    Ordonez, C.R.

    1983-10-01

    The Refueling Inventory Control System (RICS) at the Fast Flux Test Facility (FFTF) keeps track of all assemblies that reside in the various refueling facilities. The automated RICS allows the user to obtain information regarding any assembly under its control by displaying the data on a screen. It also provides a simulation mode which allows assembly moves on a duplicated data base. This simulation is used to verify the refueling documentation before it is issued

  9. Interactive visualization for assembly planning

    Science.gov (United States)

    Chen, Jer-Sen; Bao, Guodi; Jiang, Jianli

    1995-04-01

    Assembly planning is an important component for automation in manufacturing. It can help reduce the production cost by avoiding unstable subassemblies and eliminating unnecessary tool changes within the assembly cell. The assembly plan generation process begins with the exploration of the precedence relations due to geometrical and mechanical constraints. After the precedence relations are derived, all feasible assembly sequences are generated. A diamond-shape graph is commonly used to visualize all possible assembly sequences. A dual representation of all assembly sequences is also provided to facilitate the assembly sequence comparison task. Each possible sequence is transformed into a nodal representation and assumes a spatial location in a three-dimensional space. The proximity among all assembly sequence nodes in the dual space is designed to reflect the similarity among the sequences. The user can therefore navigate in the space of all feasible assembly sequences and compare similar assembly sequences that are clustered closely in the dual space. All three visualizations, namely the precedence relation, the diamond graph, and the dual graph, are coupled together so that interactions on one visualization are reflected on the other two.

  10. On Constraints in Assembly Planning

    Energy Technology Data Exchange (ETDEWEB)

    Calton, T.L.; Jones, R.E.; Wilson, R.H.

    1998-12-17

    Constraints on assembly plans vary depending on product, assembly facility, assembly volume, and many other factors. Assembly costs and other measures to optimize vary just as widely. To be effective, computer-aided assembly planning systems must allow users to express the plan selection criteria that appIy to their products and production environments. We begin this article by surveying the types of user criteria, both constraints and quality measures, that have been accepted by assembly planning systems to date. The survey is organized along several dimensions, including strategic vs. tactical criteria; manufacturing requirements VS. requirements of the automated planning process itself and the information needed to assess compliance with each criterion. The latter strongly influences the efficiency of planning. We then focus on constraints. We describe a framework to support a wide variety of user constraints for intuitive and efficient assembly planning. Our framework expresses all constraints on a sequencing level, specifying orders and conditions on part mating operations in a number of ways. Constraints are implemented as simple procedures that either accept or reject assembly operations proposed by the planner. For efficiency, some constraints are supplemented with special-purpose modifications to the planner's algorithms. Fast replanning enables an interactive plan-view-constrain-replan cycle that aids in constraint discovery and documentation. We describe an implementation of the framework in a computer-aided assembly planning system and experiments applying the system to a number of complex assemblies, including one with 472 parts.

  11. Photonic hybrid assembly through flexible waveguides

    NARCIS (Netherlands)

    Wörhoff, Kerstin; Prak, Albert; postma, F; Leinse, A; Wu, K.; Peters, T.J.; Tichem, M.; Amaning-Appiah, B.; Renukappa, V.; Vollrath, G.; Balcells-Ventura, J.; Uhlig, P.; Seyfried, M.; Rose, D.; Santos, Raquel; Leijtens, XJM; Flintham, B.; Wale, M.; Robbins, D.; Vivien, Laurent; Pavesi, Lorenzo; Pelli, Stefano

    2016-01-01

    Fully automated, high precision, cost-effective assembly technology for photonic packages remains one of the main challenges in photonic component manufacturing. Next to the cost aspect the most demanding assembly task for multiport photonic integrated circuits (PICs) is the high-precision (±0.1

  12. Automated extraction of DNA from reference samples from various types of biological materials on the Qiagen BioRobot EZ1 Workstation

    DEFF Research Database (Denmark)

    Stangegaard, Michael; Jørgensen, Mads; Hansen, Anders Johannes

    2009-01-01

    We have validated and implemented a protocol for DNA extraction from various types of biological materials using a Qiagen BioRobot EZ1 Workstation. The sample materials included whole blood, blood from deceased, buccal cells on Omni swabs and FTA Cards, blood on FTA Cards and cotton swabs......, and muscle biopsies. The DNA extraction was validated according to EN/ISO 17025 for the STR kits AmpFlSTR« Identifiler« and AmpFlSTR« Yfiler« (Applied Biosystems). Of 298 samples extracted, 11 (4%) did not yield acceptable results. In conclusion, we have demonstrated that extraction of DNA from various types...... of biological material can be performed quickly and without the use of hazardous chemicals, and that the DNA may be successfully STR typed according to the requirements of forensic genetic investigations accredited according to EN/ISO 17025...

  13. Danger! Automation at Work; Report of the State of Illinois Commission on Automation and Technological Progress.

    Science.gov (United States)

    Karp, William

    The 74th Illinois General Assembly created the Illinois Commission on Automation and Technological Progress to study and analyze the economic and social effects of automation and other technological changes on industry, commerce, agriculture, education, manpower, and society in Illinois. Commission members visited industrial plants and business…

  14. BglBricks: A flexible standard for biological part assembly.

    Science.gov (United States)

    Anderson, J Christopher; Dueber, John E; Leguia, Mariana; Wu, Gabriel C; Goler, Jonathan A; Arkin, Adam P; Keasling, Jay D

    2010-01-20

    Standard biological parts, such as BioBricks parts, provide the foundation for a new engineering discipline that enables the design and construction of synthetic biological systems with a variety of applications in bioenergy, new materials, therapeutics, and environmental remediation. Although the original BioBricks assembly standard has found widespread use, it has several shortcomings that limit its range of potential applications. In particular, the system is not suitable for the construction of protein fusions due to an unfavorable scar sequence that encodes an in-frame stop codon. Here, we present a similar but new composition standard, called BglBricks, that addresses the scar translation issue associated with the original standard. The new system employs BglII and BamHI restriction enzymes, robust cutters with an extensive history of use, and results in a 6-nucleotide scar sequence encoding glycine-serine, an innocuous peptide linker in most protein fusion applications. We demonstrate the utility of the new standard in three distinct applications, including the construction of constitutively active gene expression devices with a wide range of expression profiles, the construction of chimeric, multi-domain protein fusions, and the targeted integration of functional DNA sequences into specific loci of the E. coli genome. The BglBrick standard provides a new, more flexible platform from which to generate standard biological parts and automate DNA assembly. Work on BglBrick assembly reactions, as well as on the development of automation and bioinformatics tools, is currently underway. These tools will provide a foundation from which to transform genetic engineering from a technically intensive art into a purely design-based discipline.

  15. BglBricks: A flexible standard for biological part assembly

    Directory of Open Access Journals (Sweden)

    Dueber John E

    2010-01-01

    Full Text Available Abstract Background Standard biological parts, such as BioBricks™ parts, provide the foundation for a new engineering discipline that enables the design and construction of synthetic biological systems with a variety of applications in bioenergy, new materials, therapeutics, and environmental remediation. Although the original BioBricks™ assembly standard has found widespread use, it has several shortcomings that limit its range of potential applications. In particular, the system is not suitable for the construction of protein fusions due to an unfavorable scar sequence that encodes an in-frame stop codon. Results Here, we present a similar but new composition standard, called BglBricks, that addresses the scar translation issue associated with the original standard. The new system employs BglII and BamHI restriction enzymes, robust cutters with an extensive history of use, and results in a 6-nucleotide scar sequence encoding glycine-serine, an innocuous peptide linker in most protein fusion applications. We demonstrate the utility of the new standard in three distinct applications, including the construction of constitutively active gene expression devices with a wide range of expression profiles, the construction of chimeric, multi-domain protein fusions, and the targeted integration of functional DNA sequences into specific loci of the E. coli genome. Conclusions The BglBrick standard provides a new, more flexible platform from which to generate standard biological parts and automate DNA assembly. Work on BglBrick assembly reactions, as well as on the development of automation and bioinformatics tools, is currently underway. These tools will provide a foundation from which to transform genetic engineering from a technically intensive art into a purely design-based discipline.

  16. Cavendish Balance Automation

    Science.gov (United States)

    Thompson, Bryan

    2000-01-01

    This is the final report for a project carried out to modify a manual commercial Cavendish Balance for automated use in cryostat. The scope of this project was to modify an off-the-shelf manually operated Cavendish Balance to allow for automated operation for periods of hours or days in cryostat. The purpose of this modification was to allow the balance to be used in the study of effects of superconducting materials on the local gravitational field strength to determine if the strength of gravitational fields can be reduced. A Cavendish Balance was chosen because it is a fairly simple piece of equipment for measuring gravity, one the least accurately known and least understood physical constants. The principle activities that occurred under this purchase order were: (1) All the components necessary to hold and automate the Cavendish Balance in a cryostat were designed. Engineering drawings were made of custom parts to be fabricated, other off-the-shelf parts were procured; (2) Software was written in LabView to control the automation process via a stepper motor controller and stepper motor, and to collect data from the balance during testing; (3)Software was written to take the data collected from the Cavendish Balance and reduce it to give a value for the gravitational constant; (4) The components of the system were assembled and fitted to a cryostat. Also the LabView hardware including the control computer, stepper motor driver, data collection boards, and necessary cabling were assembled; and (5) The system was operated for a number of periods, data collected, and reduced to give an average value for the gravitational constant.

  17. Process automation

    International Nuclear Information System (INIS)

    Moser, D.R.

    1986-01-01

    Process automation technology has been pursued in the chemical processing industries and to a very limited extent in nuclear fuel reprocessing. Its effective use has been restricted in the past by the lack of diverse and reliable process instrumentation and the unavailability of sophisticated software designed for process control. The Integrated Equipment Test (IET) facility was developed by the Consolidated Fuel Reprocessing Program (CFRP) in part to demonstrate new concepts for control of advanced nuclear fuel reprocessing plants. A demonstration of fuel reprocessing equipment automation using advanced instrumentation and a modern, microprocessor-based control system is nearing completion in the facility. This facility provides for the synergistic testing of all chemical process features of a prototypical fuel reprocessing plant that can be attained with unirradiated uranium-bearing feed materials. The unique equipment and mission of the IET facility make it an ideal test bed for automation studies. This effort will provide for the demonstration of the plant automation concept and for the development of techniques for similar applications in a full-scale plant. A set of preliminary recommendations for implementing process automation has been compiled. Some of these concepts are not generally recognized or accepted. The automation work now under way in the IET facility should be useful to others in helping avoid costly mistakes because of the underutilization or misapplication of process automation. 6 figs

  18. Workload analyse of assembling process

    Science.gov (United States)

    Ghenghea, L. D.

    2015-11-01

    The workload is the most important indicator for managers responsible of industrial technological processes no matter if these are automated, mechanized or simply manual in each case, machines or workers will be in the focus of workload measurements. The paper deals with workload analyses made to a most part manual assembling technology for roller bearings assembling process, executed in a big company, with integrated bearings manufacturing processes. In this analyses the delay sample technique have been used to identify and divide all bearing assemblers activities, to get information about time parts from 480 minutes day work time that workers allow to each activity. The developed study shows some ways to increase the process productivity without supplementary investments and also indicated the process automation could be the solution to gain maximum productivity.

  19. DNA nanotechnology and its applications in biomedical research.

    Science.gov (United States)

    Sun, Lifan; Yu, Lu; Shen, Wanqiu

    2014-09-01

    DNA nanotechnology, which uses DNA as a material to self-assemble designed nanostructures, including DNA 2D arrays, 3D nanostructures, DNA nanotubes and DNA nanomechanical devices, has showed great promise in biomedical applications. Various DNA nanostructures have been used for protein characterization, enzyme assembly, biosensing, drug delivery and biomimetic assemblies. In this review, we will present recent advances of DNA nanotechnology and its applications in biomedical research field.

  20. Lrp, a major regulatory protein in Escherichia coli, bends DNA and can organize the assembly of a higher-order nucleoprotein structure.

    OpenAIRE

    Wang, Q; Calvo, J M

    1993-01-01

    Lrp (Leucine-responsive regulatory protein) is a global regulatory protein that controls the expression of many operons in Escherichia coli. One of those operons, ilvIH, contains six Lrp binding sites located within a several hundred base pair region upstream of the promoter region. Analysis of the binding of Lrp to a set of circularly permuted DNA fragments from this region indicates that Lrp induces DNA bending. The results of DNase I footprinting experiments suggest that Lrp binding to thi...

  1. Geometric reasoning about assembly tools

    Energy Technology Data Exchange (ETDEWEB)

    Wilson, R.H.

    1997-01-01

    Planning for assembly requires reasoning about various tools used by humans, robots, or other automation to manipulate, attach, and test parts and subassemblies. This paper presents a general framework to represent and reason about geometric accessibility issues for a wide variety of such assembly tools. Central to the framework is a use volume encoding a minimum space that must be free in an assembly state to apply a given tool, and placement constraints on where that volume must be placed relative to the parts on which the tool acts. Determining whether a tool can be applied in a given assembly state is then reduced to an instance of the FINDPLACE problem. In addition, the author presents more efficient methods to integrate the framework into assembly planning. For tools that are applied either before or after their target parts are mated, one method pre-processes a single tool application for all possible states of assembly of a product in polynomial time, reducing all later state-tool queries to evaluations of a simple expression. For tools applied after their target parts are mated, a complementary method guarantees polynomial-time assembly planning. The author presents a wide variety of tools that can be described adequately using the approach, and surveys tool catalogs to determine coverage of standard tools. Finally, the author describes an implementation of the approach in an assembly planning system and experiments with a library of over one hundred manual and robotic tools and several complex assemblies.

  2. A semi-automated magnetic capture probe based DNA extraction and real-time PCR method applied in the Swedish surveillance of Echinococcus multilocularis in red fox (Vulpes vulpes) faecal samples.

    Science.gov (United States)

    Isaksson, Mats; Hagström, Åsa; Armua-Fernandez, Maria Teresa; Wahlström, Helene; Ågren, Erik Olof; Miller, Andrea; Holmberg, Anders; Lukacs, Morten; Casulli, Adriano; Deplazes, Peter; Juremalm, Mikael

    2014-12-19

    Following the first finding of Echinococcus multilocularis in Sweden in 2011, 2985 red foxes (Vulpes vulpes) were analysed by the segmental sedimentation and counting technique. This is a labour intensive method and requires handling of the whole carcass of the fox, resulting in a costly analysis. In an effort to reduce the cost of labour and sample handling, an alternative method has been developed. The method is sensitive and partially automated for detection of E. multilocularis in faecal samples. The method has been used in the Swedish E. multilocularis monitoring program for 2012-2013 on more than 2000 faecal samples. We describe a new semi-automated magnetic capture probe DNA extraction method and real time hydrolysis probe polymerase chain reaction assay (MC-PCR) for the detection of E. multilocularis DNA in faecal samples from red fox. The diagnostic sensitivity was determined by validating the new method against the sedimentation and counting technique in fox samples collected in Switzerland where E. multilocularis is highly endemic. Of 177 foxes analysed by the sedimentation and counting technique, E. multilocularis was detected in 93 animals. Eighty-two (88%, 95% C.I 79.8-93.9) of these were positive in the MC-PCR. In foxes with more than 100 worms, the MC-PCR was positive in 44 out of 46 (95.7%) cases. The two MC-PCR negative samples originated from foxes with only immature E. multilocularis worms. In foxes with 100 worms or less, (n = 47), 38 (80.9%) were positive in the MC-PCR. The diagnostic specificity of the MC-PCR was evaluated using fox scats collected within the Swedish screening. Of 2158 samples analysed, two were positive. This implies that the specificity is at least 99.9% (C.I. = 99.7-100). The MC-PCR proved to have a high sensitivity and a very high specificity. The test is partially automated but also possible to perform manually if desired. The test is well suited for nationwide E. multilocularis surveillance programs where sampling

  3. Liaison concatenation – A method to obtain feasible assembly ...

    Indian Academy of Sciences (India)

    Assembly sequence planning; liaison matrix; liaison concatenation; assembly automation. 1. ... Applications (CATIA) software is a user friendly CAD tool .... bility test. The test will be done for n−2 times, where. “n” is the number of parts. After n−2 feasibility tests, the resulted assembly sequences are completely feasible.

  4. Polymer Directed Protein Assemblies

    Directory of Open Access Journals (Sweden)

    Patrick van Rijn

    2013-05-01

    Full Text Available Protein aggregation and protein self-assembly is an important occurrence in natural systems, and is in some form or other dictated by biopolymers. Very obvious influences of biopolymers on protein assemblies are, e.g., virus particles. Viruses are a multi-protein assembly of which the morphology is dictated by poly-nucleotides namely RNA or DNA. This “biopolymer” directs the proteins and imposes limitations on the structure like the length or diameter of the particle. Not only do these bionanoparticles use polymer-directed self-assembly, also processes like amyloid formation are in a way a result of directed protein assembly by partial unfolded/misfolded biopolymers namely, polypeptides. The combination of proteins and synthetic polymers, inspired by the natural processes, are therefore regarded as a highly promising area of research. Directed protein assembly is versatile with respect to the possible interactions which brings together the protein and polymer, e.g., electrostatic, v.d. Waals forces or covalent conjugation, and possible combinations are numerous due to the large amounts of different polymers and proteins available. The protein-polymer interacting behavior and overall morphology is envisioned to aid in clarifying protein-protein interactions and are thought to entail some interesting new functions and properties which will ultimately lead to novel bio-hybrid materials.

  5. Monothiol glutaredoxin Grx5 interacts with Fe-S scaffold proteins Isa1 and Isa2 and supports Fe-S assembly and DNA integrity in mitochondria of fission yeast

    International Nuclear Information System (INIS)

    Kim, Kyoung-Dong; Chung, Woo-Hyun; Kim, Hyo-Jin; Lee, Kyung-Chang; Roe, Jung-Hye

    2010-01-01

    Mitochondrial monothiol glutaredoxins that bind Fe-S cluster are known to participate in Fe-S cluster assembly. However, their precise role has not been well understood. Among three monothiol glutaredoxins (Grx3, 4, and 5) in Schizosaccharomyces pombe only Grx5 resides in mitochondria. The Δgrx5 mutant requires cysteine on minimal media, and does not grow on non-fermentable carbon source such as glycerol. We found that the mutant is low in the activity of Fe-S enzymes in mitochondria as well as in the cytoplasm. Screening of multi-copy suppressor of growth defects of the mutant identified isa1 + gene encoding a putative A-type Fe-S scaffold, in addition to mas5 + and hsc1 + genes encoding putative chaperones for Fe-S assembly process. Examination of other scaffold and chaperone genes revealed that isa2 + , but not isu1 + and ssc1 + , complemented the growth phenotype of Δgrx5 mutant as isa1 + did, partly through restoration of Fe-S enzyme activities. The mutant also showed a significant decrease in the amount of mitochondrial DNA. We demonstrated that Grx5 interacts in vivo with Isa1 and Isa2 proteins in mitochondria by observing bimolecular fluorescence complementation. These results indicate that Grx5 plays a central role in Fe-S assembly process through interaction with A-type Fe-S scaffold proteins Isa1 and Isa2, each of which is an essential protein in S. pombe, and supports mitochondrial genome integrity as well as Fe-S assembly.

  6. Monothiol glutaredoxin Grx5 interacts with Fe-S scaffold proteins Isa1 and Isa2 and supports Fe-S assembly and DNA integrity in mitochondria of fission yeast

    Energy Technology Data Exchange (ETDEWEB)

    Kim, Kyoung-Dong; Chung, Woo-Hyun; Kim, Hyo-Jin; Lee, Kyung-Chang [Laboratory of Molecular Microbiology, School of Biological Sciences, Institute of Microbiology, Seoul National University, Seoul 151-742 (Korea, Republic of); Roe, Jung-Hye, E-mail: jhroe@snu.ac.kr [Laboratory of Molecular Microbiology, School of Biological Sciences, Institute of Microbiology, Seoul National University, Seoul 151-742 (Korea, Republic of)

    2010-02-12

    Mitochondrial monothiol glutaredoxins that bind Fe-S cluster are known to participate in Fe-S cluster assembly. However, their precise role has not been well understood. Among three monothiol glutaredoxins (Grx3, 4, and 5) in Schizosaccharomyces pombe only Grx5 resides in mitochondria. The {Delta}grx5 mutant requires cysteine on minimal media, and does not grow on non-fermentable carbon source such as glycerol. We found that the mutant is low in the activity of Fe-S enzymes in mitochondria as well as in the cytoplasm. Screening of multi-copy suppressor of growth defects of the mutant identified isa1{sup +} gene encoding a putative A-type Fe-S scaffold, in addition to mas5{sup +} and hsc1{sup +} genes encoding putative chaperones for Fe-S assembly process. Examination of other scaffold and chaperone genes revealed that isa2{sup +}, but not isu1{sup +} and ssc1{sup +}, complemented the growth phenotype of {Delta}grx5 mutant as isa1{sup +} did, partly through restoration of Fe-S enzyme activities. The mutant also showed a significant decrease in the amount of mitochondrial DNA. We demonstrated that Grx5 interacts in vivo with Isa1 and Isa2 proteins in mitochondria by observing bimolecular fluorescence complementation. These results indicate that Grx5 plays a central role in Fe-S assembly process through interaction with A-type Fe-S scaffold proteins Isa1 and Isa2, each of which is an essential protein in S. pombe, and supports mitochondrial genome integrity as well as Fe-S assembly.

  7. America's Assembly Line

    DEFF Research Database (Denmark)

    Nye, David Edwin

    A social history of the assembly line, invented in 1913. Both praised as a boon to consumers and as a curse for workers, it has been satirized, imitated, and celebrated for 100 years. It has inspired fiction, comedy, cafeteria layouts, and suburban housing. It transformed industrial labor...... and provoked strikes and union drives in the 1930s, but became a symbol of victory in the Second World War and Cold War. Reinvented by Japan as "lean production" and then increasingly automated after 1990, it remains a cornerstone of production but no longer employs many workers, even as it evolves toward...

  8. 3D DNA Crystals and Nanotechnology

    Directory of Open Access Journals (Sweden)

    Paul J. Paukstelis

    2016-08-01

    Full Text Available DNA’s molecular recognition properties have made it one of the most widely used biomacromolecular construction materials. The programmed assembly of DNA oligonucleotides has been used to create complex 2D and 3D self-assembled architectures and to guide the assembly of other molecules. The origins of DNA nanotechnology are rooted in the goal of assembling DNA molecules into designed periodic arrays, i.e., crystals. Here, we highlight several DNA crystal structures, the progress made in designing DNA crystals, and look at the current prospects and future directions of DNA crystals in nanotechnology.

  9. Recent progress in DNA origami technology.

    Science.gov (United States)

    Endo, Masayuki; Sugiyama, Hiroshi

    2011-06-01

    DNA origami is an emerging technology for designing defined two-dimensional DNA nanostructures. In this review, we focus on and describe several types of DNA origami-related studies, as follows: (1) programmed DNA origami assembly, (2) DNA origami-templated molecular assembly, (3) design and construction of various three-dimensional DNA origami structures, (4) programmed functionalization of DNA origami and combination with top-down nanotechnology, (5) single molecular observation on a designed DNA origami, and (6) DNA nanomachines working on a DNA origami. © 2011 by John Wiley & Sons, Inc.

  10. Using herbarium-derived DNAs to assemble a large-scale DNA barcode library for the vascular plants of Canada1

    Science.gov (United States)

    Kuzmina, Maria L.; Braukmann, Thomas W. A.; Fazekas, Aron J.; Graham, Sean W.; Dewaard, Stephanie L.; Rodrigues, Anuar; Bennett, Bruce A.; Dickinson, Timothy A.; Saarela, Jeffery M.; Catling, Paul M.; Newmaster, Steven G.; Percy, Diana M.; Fenneman, Erin; Lauron-Moreau, Aurélien; Ford, Bruce; Gillespie, Lynn; Subramanyam, Ragupathy; Whitton, Jeannette; Jennings, Linda; Metsger, Deborah; Warne, Connor P.; Brown, Allison; Sears, Elizabeth; Dewaard, Jeremy R.; Zakharov, Evgeny V.; Hebert, Paul D. N.

    2017-01-01

    Premise of the study: Constructing complete, accurate plant DNA barcode reference libraries can be logistically challenging for large-scale floras. Here we demonstrate the promise and challenges of using herbarium collections for building a DNA barcode reference library for the vascular plant flora of Canada. Methods: Our study examined 20,816 specimens representing 5076 of 5190 vascular plant species in Canada (98%). For 98% of the specimens, at least one of the DNA barcode regions was recovered from the plastid loci rbcL and matK and from the nuclear ITS2 region. We used beta regression to quantify the effects of age, type of preservation, and taxonomic affiliation (family) on DNA sequence recovery. Results: Specimen age and method of preservation had significant effects on sequence recovery for all markers, but influenced some families more (e.g., Boraginaceae) than others (e.g., Asteraceae). Discussion: Our DNA barcode library represents an unparalleled resource for metagenomic and ecological genetic research working on temperate and arctic biomes. An observed decline in sequence recovery with specimen age may be associated with poor primer matches, intragenomic variation (for ITS2), or inhibitory secondary compounds in some taxa. PMID:29299394

  11. Porcine transcriptome analysis based on 97 non-normalized cDNA libraries and assembly of 1,021,891 expressed sequence tags

    DEFF Research Database (Denmark)

    Gorodkin, Jan; Cirera, Susanna; Hedegaard, Jacob

    2007-01-01

    with the greatest number of different expressed genes, whereas tissues with more specialized function, such as developing liver, have fewer expressed genes. There are at least 65 high confidence housekeeping gene candidates and 876 cDNA library-specific gene candidates. We identified differential expression...

  12. The effects of linear assembly of two carbazole groups on acid-base and DNA-binding properties of a ruthenium(II) complex

    Science.gov (United States)

    Chen, Xi; Xue, Long-Xin; Ju, Chun-Chuan; Wang, Ke-Zhi

    2013-07-01

    A novel Ru(II) complex of [Ru(bpy)2(Hbcpip)](ClO4)2 {where bpy = 2,2-bipyridine, Hbcpip = 2-(4-(9H-3,9'-bicarbazol-9-yl)phenyl)-1H-imidazo[4,5-f][1,10]phenanthroline} is synthesized and characterized. Calf-thymus DNA-binding properties of the complex were studied by UV-vis absorption and luminescence titrations, steady-state emission quenching by [Fe(CN)6]4-, DNA competitive binding with ethidium bromide, thermal denaturation and DNA viscosity measurements. The results indicate that the complex partially intercalated into the DNA with a binding constant of (5.5 ± 1.4) × 105 M-1 in buffered 50 mM NaCl. The acid-base properties of the complex were also studied by UV-visible and luminescence spectrophotometric pH titrations, and ground- and excited-state acidity ionization constant values were derived.

  13. CRISPR-CAS9 D10A nickase target-specific fluorescent labeling of double strand DNA for whole genome mapping and structural variation analysis.

    Science.gov (United States)

    McCaffrey, Jennifer; Sibert, Justin; Zhang, Bin; Zhang, Yonggang; Hu, Wenhui; Riethman, Harold; Xiao, Ming

    2016-01-29

    We have developed a new, sequence-specific DNA labeling strategy that will dramatically improve DNA mapping in complex and structurally variant genomic regions, as well as facilitate high-throughput automated whole-genome mapping. The method uses the Cas9 D10A protein, which contains a nuclease disabling mutation in one of the two nuclease domains of Cas9, to create a guide RNA-directed DNA nick in the context of an in vitro-assembled CRISPR-CAS9-DNA complex. Fluorescent nucleotides are then incorporated adjacent to the nicking site with a DNA polymerase to label the guide RNA-determined target sequences. This labeling strategy is very powerful in targeting repetitive sequences as well as in barcoding genomic regions and structural variants not amenable to current labeling methods that rely on uneven distributions of restriction site motifs in the DNA. Importantly, it renders the labeled double-stranded DNA available in long intact stretches for high-throughput analysis in nanochannel arrays as well as for lower throughput targeted analysis of labeled DNA regions using alternative methods for stretching and imaging the labeled long DNA molecules. Thus, this method will dramatically improve both automated high-throughput genome-wide mapping as well as targeted analyses of complex regions containing repetitive and structurally variant DNA. © The Author(s) 2015. Published by Oxford University Press on behalf of Nucleic Acids Research.

  14. DNA Sequencing apparatus

    Science.gov (United States)

    Tabor, Stanley; Richardson, Charles C.

    1992-01-01

    An automated DNA sequencing apparatus having a reactor for providing at least two series of DNA products formed from a single primer and a DNA strand, each DNA product of a series differing in molecular weight and having a chain terminating agent at one end; separating means for separating the DNA products to form a series bands, the intensity of substantially all nearby bands in a different series being different, band reading means for determining the position an This invention was made with government support including a grant from the U.S. Public Health Service, contract number AI-06045. The U.S. government has certain rights in the invention.

  15. Automated External Defibrillator

    Science.gov (United States)

    ... To Health Topics / Automated External Defibrillator Automated External Defibrillator Also known as What Is An automated external ... in survival. Training To Use an Automated External Defibrillator Learning how to use an AED and taking ...

  16. LEGO-like DNA Structures

    DEFF Research Database (Denmark)

    Gothelf, Kurt Vesterager

    2012-01-01

    -dimensional (3D) DNA structures by self-assembly of single-stranded DNA “bricks.” The method opens a new route to complex self-assembled (3D) nanostructures that may serve as addressable templates for placing guest molecules with high precision, with possible applications in biophysics, medicine...

  17. Library Automation.

    Science.gov (United States)

    Husby, Ole

    1990-01-01

    The challenges and potential benefits of automating university libraries are reviewed, with special attention given to cooperative systems. Aspects discussed include database size, the role of the university computer center, storage modes, multi-institutional systems, resource sharing, cooperative system management, networking, and intelligent…

  18. Testing Whether Defective Chromatin Assembly in S-Phase Contributes to Breast Cancer

    National Research Council Canada - National Science Library

    Adams, Peter

    2003-01-01

    .... We used a dominant negative mutant of (chromatin assembly factor-I) CAF1, a complex that assembles newly synthesized DNA into nucleosomes, to inhibit S-phase chromatin assembly and found that this induced S-phase arrest...

  19. Testing Whether Defective Chromatin Assembly in S-Phase Contributes to Breast Cancer

    National Research Council Canada - National Science Library

    Adams, Peter

    2004-01-01

    .... We used a dominant negative mutant of (chromatin assembly factor-I) CAF1, a complex that assembles newly synthesized DNA into nucleosomes, to inhibit S-phase chromatin assembly and found that this induced S-phase arrest...

  20. Characterization of assembled MEMS

    Science.gov (United States)

    Jandric, Zoran; Randall, John N.; Saini, Rahul; Nolan, Michael; Skidmore, George

    2005-01-01

    Zyvex is developing a low-cost high-precision method for manufacturing MEMS-based three-dimensional structures/assemblies. The assembly process relies on compliant properties of the interconnecting components. The sockets and connectors are designed to benefit from their compliant nature by allowing the mechanical component to self-align, i.e. reposition themselves to their designed, stable position, independent of the initial placement of the part by the external robot. Thus, the self-aligning property guarantees the precision of the assembled structure to be very close to, or the same, as the precision of the lithography process itself. A three-dimensional (3D) structure is achieved by inserting the connectors into the sockets through the use of a passive end-effector. We have developed the automated, high-yield, assembly procedure which permits connectors to be picked up from any location within the same die, or a separate die. This general procedure allows for the possibility to assemble parts of dissimilar materials. We have built many 3D MEMS structures, including several 3D MEMS devices such as a scanning electron microscope (SEM) micro column, mass-spectrometer column, variable optical attenuator. For these 3D MEMS structures we characterize their mechanical strength through finite element simulation, dynamic properties by finite-element analysis and experimentally with UMECH"s MEMS motion analyzer (MMA), alignment accuracy by using an in-house developed dihedral angle measurement laser autocollimator, and impact properties by performing drop tests. The details of the experimental set-ups, the measurement procedures, and the experimental data are presented in this paper.

  1. Impact of a Central Scaffold on the Binding Affinity of Fragment Pairs Isolated from DNA-Encoded Self-Assembling Chemical Libraries.

    Science.gov (United States)

    Bigatti, Martina; Dal Corso, Alberto; Vanetti, Sara; Cazzamalli, Samuele; Rieder, Ulrike; Scheuermann, Jörg; Neri, Dario; Sladojevich, Filippo

    2017-11-08

    The screening of encoded self-assembling chemical libraries allows the identification of fragment pairs that bind to adjacent pockets on target proteins of interest. For practical applications, it is necessary to link these ligand pairs into discrete organic molecules, devoid of any nucleic acid component. Here we describe the discovery of a synergistic binding pair for acid alpha-1 glycoprotein and a chemical strategy for the identification of optimal linkers, connecting the two fragments. The procedure yielded a set of small organic ligands, the best of which exhibited a dissociation constant of 9.9 nm, as measured in solution by fluorescence polarization. © 2017 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim.

  2. A pipeline for the de novo assembly of the Themira biloba (Sepsidae: Diptera) transcriptome using a multiple k-mer length approach.

    Science.gov (United States)

    Melicher, Dacotah; Torson, Alex S; Dworkin, Ian; Bowsher, Julia H

    2014-03-12

    The Sepsidae family of flies is a model for investigating how sexual selection shapes courtship and sexual dimorphism in a comparative framework. However, like many non-model systems, there are few molecular resources available. Large-scale sequencing and assembly have not been performed in any sepsid, and the lack of a closely related genome makes investigation of gene expression challenging. Our goal was to develop an automated pipeline for de novo transcriptome assembly, and to use that pipeline to assemble and analyze the transcriptome of the sepsid Themira biloba. Our bioinformatics pipeline uses cloud computing services to assemble and analyze the transcriptome with off-site data management, processing, and backup. It uses a multiple k-mer length approach combined with a second meta-assembly to extend transcripts and recover more bases of transcript sequences than standard single k-mer assembly. We used 454 sequencing to generate 1.48 million reads from cDNA generated from embryo, larva, and pupae of T. biloba and assembled a transcriptome consisting of 24,495 contigs. Annotation identified 16,705 transcripts, including those involved in embryogenesis and limb patterning. We assembled transcriptomes from an additional three non-model organisms to demonstrate that our pipeline assembled a higher-quality transcriptome than single k-mer approaches across multiple species. The pipeline we have developed for assembly and analysis increases contig length, recovers unique transcripts, and assembles more base pairs than other methods through the use of a meta-assembly. The T. biloba transcriptome is a critical resource for performing large-scale RNA-Seq investigations of gene expression patterns, and is the first transcriptome sequenced in this Dipteran family.

  3. Disposable electrochemical DNA biosensor for environmental ...

    Indian Academy of Sciences (India)

    A simple procedure for the voltammetric detection of the DNA damage using a disposable electrochemical DNA biosensor is reported. The DNA biosensor is assembled by immobilizing the double stranded calf thymus DNA (dsDNA) on the surface of a disposable carbon screen-printed electrode. The interaction of ...

  4. EDNA-An expert software system for comparison and evaluation of DNA profiles in forensic casework

    DEFF Research Database (Denmark)

    Haldemann, B.; Dornseifer, S.; Heylen, T.

    2015-01-01

    eDNA is an expert software system for DNA profile comparison, match interpretation and automated report generation in forensic DNA casework. Process automation and intelligent graphical representation maximise reliability of DNA evidence, while facilitating and accelerating the work of DNA experts....

  5. Evaluation of cell lysis procedures and use of a micro fluidic system for an automated DNA-based cell identification in interplanetary missions

    Science.gov (United States)

    Hall, J. A.; Felnagle, E.; Fries, M.; Spearing, S.; Monaco, L.; Steele, A.

    2006-12-01

    A Modular Assay System for Solar System Exploration (MASSE) is being developed to include sample handling, pre-treatment, separation and analysis of biological target compounds by both DNA and protein microarrays. To better design sensitive and accurate initial upstream sample handling of the MASSE instrument, experiments investigating the sensitivity and potential extraction bias of commercially available DNA extraction kits between classes of environmentally relevant prokaryotes such as gram-negative bacteria ( Escherichia coli), gram-positive bacteria ( Bacillus megatarium), and Archaea ( Haloarcula marismortui) were performed. For extractions of both planktonic cultures and spiked Mars simulated regolith, FTA ® paper demonstrated the highest sensitivity, with detection as low as ˜1×10 1 cells and ˜3.3×10 2 cells, respectively. In addition to the highest sensitivity, custom modified application of FTA ® paper extraction protocol is the simplest in terms of incorporation into MASSE and displayed little bias in sensitivity with respect to prokaryotic cell type. The implementation of FTA paper for environmental microbiology investigations appears to be a viable and effective option potentially negating the need for other pre-concentration steps such as filtration and negating concerns regarding extraction efficiency of cells. In addition to investigations on useful technology for upstream sample handling in MASSE, we have also evaluated the potential for μTAS to be employed in the MASSE instrument by employing proprietary lab-on-a-chip development technology to investigate the potential for microfluidic cell lysis of different prokaryotic cells employing both chemical and biological lysis agents. Real-time bright-field microscopy and quantitative PMT detection indicated that that gram positive, gram negative and archaeal cells were effectively lyzed in a few seconds using the microfluidic chip protocol developed. This included employing a lysis buffer with

  6. Fuel assembly

    International Nuclear Information System (INIS)

    Abe, Hideaki; Sakai, Takao; Ishida, Tomio; Yokota, Norikatsu.

    1992-01-01

    The lower ends of a plurality of plate-like shape memory alloys are secured at the periphery of the upper inside of the handling head of a fuel assembly. As the shape memory alloy, a Cu-Zn alloy, a Ti-Pd alloy or a Fe-Ni alloy is used. When high temperature coolants flow out to the handling head, the shape memory alloy deforms by warping to the outer side more greatly toward the upper portion thereof with the temperature increase of the coolants. As the result, the shape of the flow channel of the coolants is changed so as to enlarge at the exit of the upper end of the fuel assembly. Then, the pressure loss of the coolants in the fuel assembly is decreased by the enlargement. Accordingly, the flow rate of the coolants in the fuel assembly is increased to lower the temperature of the coolants. Further, high temperature coolants and low temperature coolants are mixed sufficiently just above the fuel assembly. This can suppress the temperature fluctuation of the mixed coolants in the upper portion of the reactor core, thereby enabling to decrease a fatigue and failures of the structural components in the upper portion of the reactor core. (I.N.)

  7. Smart DNA Fabrication Using Sound Waves: Applying Acoustic Dispensing Technologies to Synthetic Biology.

    Science.gov (United States)

    Kanigowska, Paulina; Shen, Yue; Zheng, Yijing; Rosser, Susan; Cai, Yizhi

    2016-02-01

    Acoustic droplet ejection (ADE) technology uses focused acoustic energy to transfer nanoliter-scale liquid droplets with high precision and accuracy. This noncontact, tipless, low-volume dispensing technology minimizes the possibility of cross-contamination and potentially reduces the costs of reagents and consumables. To date, acoustic dispensers have mainly been used in screening libraries of compounds. In this paper, we describe the first application of this powerful technology to the rapidly developing field of synthetic biology, for DNA synthesis and assembly at the nanoliter scale using a Labcyte Echo 550 acoustic dispenser. We were able to successfully downscale PCRs and the popular one-pot DNA assembly methods, Golden Gate and Gibson assemblies, from the microliter to the nanoliter scale with high assembly efficiency, which effectively cut the reagent cost by 20- to 100-fold. We envision that acoustic dispensing will become an instrumental technology in synthetic biology, in particular in the era of DNA foundries. © 2015 Society for Laboratory Automation and Screening.

  8. Fuel assembly

    International Nuclear Information System (INIS)

    Nakatsuka, Masafumi; Matsuzuka, Ryuji.

    1976-01-01

    Object: To provide a fuel assembly which can decrease pressure loss of coolant to uniform temperature. Structure: A sectional area of a flow passage in the vicinity of an inner peripheral surface of a wrapper tube is limited over the entire length to prevent the temperature of a fuel element in the outermost peripheral portion from being excessively decreased to thereby flatten temperature distribution. To this end, a plurality of pincture-frame-like sheet metals constituting a spacer for supporting a fuel assembly, which has a plurality of fuel elements planted lengthwise and in given spaced relation within the wrapper tube, is disposed in longitudinal grooves and in stacked fashion to form a substantially honeycomb-like space in cross section. The fuel elements are inserted and supported in the space to form a fuel assembly. (Kamimura, M.)

  9. Fuel assemblies

    International Nuclear Information System (INIS)

    Nakamura, Mitsuya; Yamashita, Jun-ichi; Mochida, Takaaki.

    1986-01-01

    Purpose: To improve the fuel economy by increasing the reactivity at the latter burning stage of fuel assemblies and thereby increasing the burn-up degree. Constitution: At the later stage of the burning where the infinite multiplication factor of a fuel assembly is lowered, fuel rods are partially discharged to increase the fuel-moderator volume ratio in the fuel assembly. Then, plutonium is positively burnt by bringing the ratio near to an optimum point where the infinite multiplication factor becomes maximum and the reactivity of the fuel assembly is increased by utilizing the spectral shift effect. The number of the fuel rods to be removed is selected so as to approach the fuel-moderator atom number ratio where the infinite multiplication factor is maximum. Further, the positions where the thermal neutron fluxes are low are most effective for removing the rods and those positions between which no fuel rods are present and which are adjacent with neither the channel box nor the water rods are preferred. The rods should be removed at the time when the burning is proceeded at lest for one cycle. The reactivity is thus increased and the burn-up degree of fuels upon taking-out can be improved. (Kamimura, M.)

  10. Valve assembly

    International Nuclear Information System (INIS)

    Sandling, M.

    1981-01-01

    An improved valve assembly, used for controlling the flow of radioactive slurry, is described. Radioactive contamination of the air during removal or replacement of the valve is prevented by sucking air from the atmosphere through a portion of the structure above the valve housing. (U.K.)

  11. Interfacing DNA nanodevices with biology

    DEFF Research Database (Denmark)

    Vinther, Mathias; Kjems, Jørgen

    2016-01-01

    these nanodevices is molecular self-assembly. In nature, deoxyribonucleic acid (DNA) is inarguably one of the most remarkable self-assembling molecules. Governed by the Watson–Crick base-pairing rules, DNA assembles with a structural reliability and predictability based on sequence composition unlike any other...... complex biological polymer. This consistency has enabled rational design of hundreds of two- and three-dimensional shapes with a molecular precision and homogeneity not preceded by any other known technology at the nanometer scale. During the last two decades, DNA nanotechnology has undergone a rapid...... evolution pioneered by the work of Nadrian Seeman (Kallenbach et al 1983 Nature 205 829–31). Especially the introduction of the versatile DNA Origami technique by Rothemund (2006 Nature 440 297–302) led to an efflorescence of new DNA-based self-assembled nanostructures (Andersen et al 2009 Nature 459 73...

  12. CALM/AF10-positive leukemias show upregulation of genes involved in chromatin assembly and DNA repair processes and of genes adjacent to the breakpoint at 10p12.

    Science.gov (United States)

    Mulaw, M A; Krause, A; Krause, A J; Deshpande, A J; Krause, L F; Rouhi, A; La Starza, R; Borkhardt, A; Buske, C; Mecucci, C; Ludwig, W-D; Lottaz, C; Bohlander, S K

    2012-05-01

    The t(10;11)(p12;q14) is a recurring chromosomal translocation that gives rise to the CALM/AF10 fusion gene, which is found in acute myeloid leukemia, acute lymphoblastic leukemia and malignant lymphoma. We analyzed the fusion transcripts in 20 new cases of CALM/AF10-positive leukemias, and compared the gene expression profile of 10 of these to 125 patients with other types of leukemia and 10 normal bone marrow samples. Based on gene set enrichment analyses, the CALM/AF10-positive samples showed significant upregulation of genes involved in chromatin assembly and maintenance and DNA repair process, and downregulation of angiogenesis and cell communication genes. Interestingly, we observed a striking upregulation of four genes located immediately centromeric to the break point of the t(10;11)(p12;q14) on 10p12 (COMMD3 (COMM domain containing 3), BMI1 (B lymphoma Mo-MLV insertion region 1 homolog), DNAJC1 (DnaJ (Hsp40) homolog subfamily C member 1) and SPAG6 (sperm associated antigen 6)). We also conducted semiquantitative reverse transcriptase-PCR analysis on leukemic blasts from a murine CALM/AF10 transplantation model that does not have the translocation. Commd3, Bmi1 and Dnajc1, but not Spag6 were upregulated in these samples. These results strongly indicate that the differential regulation of these three genes is not due to the break point effect but as a consequence of the CALM/AF10 fusion gene expression, though the mechanism of regulation is not well understood.

  13. Autonomous Systems: Habitat Automation

    Data.gov (United States)

    National Aeronautics and Space Administration — The Habitat Automation Project Element within the Autonomous Systems Project is developing software to automate the automation of habitats and other spacecraft. This...

  14. An Automation Planning Primer.

    Science.gov (United States)

    Paynter, Marion

    1988-01-01

    This brief planning guide for library automation incorporates needs assessment and evaluation of options to meet those needs. A bibliography of materials on automation planning and software reviews, library software directories, and library automation journals is included. (CLB)

  15. Prototype implementation of segment assembling software

    Directory of Open Access Journals (Sweden)

    Pešić Đorđe

    2018-01-01

    Full Text Available IT education is very important and a lot of effort is put into the development of tools for helping students to acquire programming knowledge and for helping teachers in automating the examination process. This paper describes a prototype of the program segment assembling software used in the context of making tests in the field of algorithmic complexity. The proposed new program segment assembling model uses rules and templates. A template is a simple program segment. A rule defines combining method and data dependencies if they exist. One example of program segment assembling by the proposed system is given. Graphical user interface is also described.

  16. Automated Budget System -

    Data.gov (United States)

    Department of Transportation — The Automated Budget System (ABS) automates management and planning of the Mike Monroney Aeronautical Center (MMAC) budget by providing enhanced capability to plan,...

  17. Evaluation of an ethidium monoazide-enhanced 16S rDNA real-time polymerase chain reaction assay for bacterial screening of platelet concentrates and comparison with automated culture.

    Science.gov (United States)

    Garson, Jeremy A; Patel, Poorvi; McDonald, Carl; Ball, Joanne; Rosenberg, Gillian; Tettmar, Kate I; Brailsford, Susan R; Pitt, Tyrone; Tedder, Richard S

    2014-03-01

    Culture-based systems are currently the preferred means for bacterial screening of platelet (PLT) concentrates. Alternative bacterial detection techniques based on nucleic acid amplification have also been developed but these have yet to be fully evaluated. In this study we evaluate a novel 16S rDNA polymerase chain reaction (PCR) assay and compare its performance with automated culture. A total of 2050 time-expired, 176 fresh, and 400 initial-reactive PLT packs were tested by real-time PCR using broadly reactive 16S primers and a "universal" probe (TaqMan, Invitrogen). PLTs were also tested using a microbial detection system (BacT/ALERT, bioMérieux) under aerobic and anaerobic conditions. Seven of 2050 (0.34%) time-expired PLTs were found repeat reactive by PCR on the initial nucleic acid extract but none of these was confirmed positive on testing frozen second aliquots. BacT/ALERT testing also failed to confirm any time-expired PLTs positive on repeat testing, although 0.24% were reactive on the first test. Three of the 400 "initial-reactive" PLT packs were found by both PCR and BacT/ALERT to be contaminated (Escherichia coli, Listeria monocytogenes, and Streptococcus vestibularis identified) and 14 additional packs were confirmed positive by BacT/ALERT only. In 13 of these cases the contaminating organisms were identified as anaerobic skin or oral commensals and the remaining pack was contaminated with Streptococcus pneumoniae. These results demonstrate that the 16S PCR assay is less sensitive than BacT/ALERT and inappropriate for early testing of concentrates. However, rapid PCR assays such as this may be suitable for a strategy of late or prerelease testing. © 2013 American Association of Blood Banks.

  18. Automation 2017

    CERN Document Server

    Zieliński, Cezary; Kaliczyńska, Małgorzata

    2017-01-01

    This book consists of papers presented at Automation 2017, an international conference held in Warsaw from March 15 to 17, 2017. It discusses research findings associated with the concepts behind INDUSTRY 4.0, with a focus on offering a better understanding of and promoting participation in the Fourth Industrial Revolution. Each chapter presents a detailed analysis of a specific technical problem, in most cases followed by a numerical analysis, simulation and description of the results of implementing the solution in a real-world context. The theoretical results, practical solutions and guidelines presented are valuable for both researchers working in the area of engineering sciences and practitioners looking for solutions to industrial problems. .

  19. Marketing automation

    Directory of Open Access Journals (Sweden)

    TODOR Raluca Dania

    2017-01-01

    Full Text Available The automation of the marketing process seems to be nowadays, the only solution to face the major changes brought by the fast evolution of technology and the continuous increase in supply and demand. In order to achieve the desired marketing results, businessis have to employ digital marketing and communication services. These services are efficient and measurable thanks to the marketing technology used to track, score and implement each campaign. Due to the technical progress, the marketing fragmentation, demand for customized products and services on one side and the need to achieve constructive dialogue with the customers, immediate and flexible response and the necessity to measure the investments and the results on the other side, the classical marketing approached had changed continue to improve substantially.

  20. Automated nutrient analyses in seawater

    Energy Technology Data Exchange (ETDEWEB)

    Whitledge, T.E.; Malloy, S.C.; Patton, C.J.; Wirick, C.D.

    1981-02-01

    This manual was assembled for use as a guide for analyzing the nutrient content of seawater samples collected in the marine coastal zone of the Northeast United States and the Bering Sea. Some modifications (changes in dilution or sample pump tube sizes) may be necessary to achieve optimum measurements in very pronounced oligotrophic, eutrophic or brackish areas. Information is presented under the following section headings: theory and mechanics of automated analysis; continuous flow system description; operation of autoanalyzer system; cookbook of current nutrient methods; automated analyzer and data analysis software; computer interfacing and hardware modifications; and trouble shooting. The three appendixes are entitled: references and additional reading; manifold components and chemicals; and software listings. (JGB)