The Experts below are selected from a list of 32850 Experts worldwide ranked by ideXlab platform

James P Landers - One of the best experts on this subject based on the ideXlab platform.

  • an integrated microfluidic device for DNA Purification and pcr amplification of str fragments
    2010
    Co-Authors: Joan M Bienvenue, Jerome P Ferrance, Lindsay A Legendre, James P Landers
    Abstract:

    This work presents the integration of DNA extraction from complex samples and PCR amplification of STR fragments in a valveless, glass microdevice, using commercially available kits and instrumentation. DNA extraction was performed using a microchannel packed with a silica solid phase and a standard syringe pump as a single pressure source driving the extraction process, followed by integrated, online microchip amplification of STR fragments in a total volume of 1.2 microL. Reported characteristics important to this work include the capacity of the device for Purification of DNA from a complex biological sample (whole blood) and the timing of DNA elution from the silica solid phase for successful downstream PCR amplification by placement the microdevice into a conventional thermocycler. Potential application of this microdevice to forensic genetic analysis was demonstrated through the preliminary extraction of DNA from semen, followed by an integrated, multiplexed, on-chip amplification that yielded detectable STR amplicons. By utilizing conventional laboratory equipment, the device presented exploits the benefits of microfluidic systems without complex control systems.

  • microchip based cell lysis and DNA extraction from sperm cells for application to forensic analysis
    2006
    Co-Authors: M Joan M S Bienvenue, B Natalie S Duncalf, B Daniel A Marchiarullo, Jerome P Ferrance, James P Landers
    Abstract:

    The current backlog of casework is among the most significant challenges facing crime laboratories at this time. While the development of next-generation microchip-based technology for expedited forensic casework analysis offers one solution to this problem, this will require the adaptation of manual, large-volume, benchtop chemistry to small volume microfluidic devices. Analysis of evidentiary materials from rape kits where semen or sperm cells are commonly found represents a unique set of challenges for on-chip cell lysis and DNA extraction that must be addressed for successful application. The work presented here details the development of a microdevice capable of DNA extraction directly from sperm cells for application to the analysis of sexual assault evidence. A variety of chemical lysing agents are assessed for inclusion in the extraction protocol and a method for DNA Purification from sperm cells is described. Suitability of the extracted DNA for short tandem repeat (STR) analysis is assessed and genetic profiles shown. Finally, on-chip cell lysis methods are evaluated, with results from fluorescence visualization of cell rupture and DNA extraction from an integrated cell lysis and Purification with subsequent STR amplification presented. A method for on-chip cell lysis and DNA Purification is described, with considerations toward inclusion in an integrated microdevice capable of both differential cell sorting and DNA extraction. The results of this work demonstrate the feasibility of incorporating microchip-based cell lysis and DNA extraction into forensic casework analysis.

  • evaluation of silica resins for direct and efficient extraction of DNA from complex biological matrices in a miniaturized format
    2000
    Co-Authors: Huijun Tian, Andreas Huhmer, James P Landers
    Abstract:

    Abstract For DNA Purification to be functionally integrated into the microchip for high-throughput DNA analysis, a miniaturized Purification process must be developed that can be easily adapted to the microchip format. In this study, we evaluate the effectiveness of a variety of silica resins for miniaturized DNA Purification and gauge the potential usefulness for on-chip solid-phase extraction. A micro-solid-phase extraction (μSPE) device containing only nanograms of silica resin is shown to be effective for the adsorption and desorption of DNA in the picogram–nanogram mass range. Fluorescence spectroscopy as well as capillary electrophoresis with laser-induced fluorescence detection is employed for the analysis of DNA recovered from solid-phase resins, while the polymerase chain reaction (PCR) is used to evaluate the amplifiable nature of the eluted DNA. We demonstrate that DNA can be directly recovered from white blood cells with an efficiency of roughly 70%, while greater than 80% of the protein is removed with a 500-nl bed volume μSPE process that takes less than 10 min. With a capacity in the range of 10–30 ng/mg of silica resin, we show that the DNA extracted from white blood cells, cultured cancer cells, and even whole blood on the low microliter scale is suitable for direct PCR amplification. The miniaturized format as well as rapid time frame for DNA extraction is compatible with the fast electrophoresis on microfabricated chips.

Joao A Queiroz - One of the best experts on this subject based on the ideXlab platform.

  • minicircle DNA Purification performance of chromatographic monoliths bearing lysine and cadaverine ligands
    2019
    Co-Authors: Ana Margarida Almeida, Joao A Queiroz, Fani Sousa, Ana C A Sousa
    Abstract:

    Abstract Minicircle DNA (mcDNA) technology is in the vanguard of vectors designed for gene therapy, since the absence of prokaryotic sequences confers to mcDNA higher biosafety in comparison to other DNA vectors. However, the presence of other isoforms and non-recombined parental molecules hampers the isolation of supercoiled (sc) mcDNA with the chromatographic methods already established for plasmid Purification. In this work, two monolithic supports were modified with lysine and its decarboxylated derivative, cadaverine, to explore their performance in the sc mcDNA Purification. Increasing NaCl gradients and different pH values (from 6 to 9) were tested in both modified monoliths. In general, cadaverine modified support established stronger interactions with mcDNA than lysine modified monolith, at acidic pH. For instance, at pH 6.0 the retention time for RNA and DNA molecules in lysine modified monolith was 11.58 and 14.59, respectively, while for cadaverine modified monolith was 20.32 and 27.12, respectively. The lysine modified monolith was able to successfully isolate sc mcDNA from the lysate sample. However, recovery yield was significantly sacrificed to guarantee high purity levels of sc mcDNA. The cadaverine modified monolith showed better selectivity than the previous monolith, achieving the successful sc mcDNA isolation from the lysate sample. The final sc mcDNA sample, obtained by the column that showed the best performance, was characterized by real-time PCR, presenting 98.4% purity and 78.6% recovery yield. The impurities content, namely genomic DNA, proteins and endotoxins, was found within the criteria established by regulatory agencies. Overall, a simple and practical chromatographic strategy to purify sc mcDNA was for the first time implemented by exploring a modified monolithic column, with no significant reduction on the purity and recovery and without resorting to backbone modification or specific enzymatic digestion. Such features will surely be crucial in the industrial scale-up of this chromatographic strategy since it will not be associated with significant cost-increase.

  • minicircle DNA Purification using a cim deae 1 monolithic support
    2016
    Co-Authors: Tatiana Diamantino, Joao A Queiroz, ângela Sousa, Patricia Pereira, Fani Sousa
    Abstract:

    Minicircle DNA is a new biotechnological product with beneficial therapeutic perspectives for gene therapy because it is constituted only by the eukaryotic transcription unit. These features improve minicircle DNA safety and increase its therapeutic effect. However, being a recently developed product, there is a need to establish efficient Purification methodologies, enabling the recovery of the supercoiled minicircle DNA isoform. Thus, this work describes the minicircle DNA Purification using an anion exchange monolithic support. The results show that with this column it is possible to achieve a good selectivity, which allows the isolation of the supercoiled minicircle DNA isoform from impurities. Overall, this study shows a promising approach to obtain the minicircle DNA sample with adequate quality for future therapeutic applications.

  • advances in chromatographic supports for pharmaceutical grade plasmid DNA Purification
    2012
    Co-Authors: ângela Sousa, Fani Sousa, Joao A Queiroz
    Abstract:

    Chromatographic technology is undoubtedly one of the most diverse and powerful Purification methods for downstream process applications. The diversity and quantity of biomolecules present in crude extracts as well as the similarities between impurities and the target biomolecule are considered the critical challenges in the extraction and Purification steps. Thus, it is important to optimize the Purification protocol to achieve maximum recovery of the target sample. The structure of chromatographic supports has been continuously developed to afford rapid and efficient separations, as well as, the application of specific ligands to improve the selectivity for the target molecule. The present review discusses the structural progress and evolution of the chromatographic supports that have been used for plasmid DNA Purification. Nowadays, the most desirable form of plasmid for gene therapy and DNA vaccination is the supercoiled isoform, due to its stability and higher transfection efficiency over other plasmid topologies. However, the main challenge is not only to produce high quantities of supercoiled plasmid DNA but also to preserve its quality, meeting the strict requirements recommended by the regulatory agencies. Therefore, this review will focus on the chemical and structural classification of the different media and on some of the specific ligands used for plasmid DNA bioseparation.

  • impact of lysine affinity chromatography on supercoiled plasmid DNA Purification
    2011
    Co-Authors: Ana C A Sousa, Fani Sousa, Joao A Queiroz
    Abstract:

    Abstract Gene therapy and DNA vaccination cover a variety of applications using viral and non-viral vectors as vehicles of choice for treatment of genetic or acquired diseases. Recently, most therapeutic applications have been performed with non-viral biological agents preparations highly enriched in supercoiled plasmid molecules and it has been concluded that this isoform is more efficient at gene transfection than open circular isoform. This work describes for the first time a new strategy that uses lysine-chromatography to efficiently eliminate Escherichia coli impurities as well as other ineffective plasmid isoforms present in a complex clarified lysate to purify and obtain pharmaceutical-grade supercoiled plasmid DNA. The quality control tests indicated that the levels of impurities in the final plasmid product were below the generally accepted specifications. Furthermore, the delivery of the purified product to eukaryotic cells, the cell uptake and transfection efficiency were also analyzed. The results showed that the transfection efficiency reached with the application of the supercoiled plasmid conformation, purified with lysine-agarose, was higher than the values achieved for other plasmid topologies. Therefore, this study presents a new enabling technology to obtain the completely purified non-viral vector, able to act with good efficiency as gene therapy delivery vehicle in several diseases like cancer.

  • binding and elution strategy for improved performance of arginine affinity chromatography in supercoiled plasmid DNA Purification
    2009
    Co-Authors: Fani Sousa, D M F Prazeres, Joao A Queiroz
    Abstract:

    New interesting strategies for plasmid DNA (pDNA) Purification were designed, exploiting affinity interactions between amino acids and nucleic acids. The potential application of arginine-based chromatography to purify pDNA has been recently described in our work; however, to achieve higher efficiency and selectivity in arginine affinity chromatography, it is essential to characterize the behaviour of binding/elution of supercoiled (sc) isoforms. In this study, two different strategies based on increased sodium chloride (225-250 mm) or arginine (20-70 mm) stepwise gradients are described to purify sc isoforms. Thus, it was proved that well-defined binding/elution conditions are crucial to enhance the Purification performance, resulting in an improvement of the final plasmids yields and transfection efficiency, as this could represent a significant impact on therapeutic applications of the purified sc isoform.

Fani Sousa - One of the best experts on this subject based on the ideXlab platform.

  • minicircle DNA Purification performance of chromatographic monoliths bearing lysine and cadaverine ligands
    2019
    Co-Authors: Ana Margarida Almeida, Joao A Queiroz, Fani Sousa, Ana C A Sousa
    Abstract:

    Abstract Minicircle DNA (mcDNA) technology is in the vanguard of vectors designed for gene therapy, since the absence of prokaryotic sequences confers to mcDNA higher biosafety in comparison to other DNA vectors. However, the presence of other isoforms and non-recombined parental molecules hampers the isolation of supercoiled (sc) mcDNA with the chromatographic methods already established for plasmid Purification. In this work, two monolithic supports were modified with lysine and its decarboxylated derivative, cadaverine, to explore their performance in the sc mcDNA Purification. Increasing NaCl gradients and different pH values (from 6 to 9) were tested in both modified monoliths. In general, cadaverine modified support established stronger interactions with mcDNA than lysine modified monolith, at acidic pH. For instance, at pH 6.0 the retention time for RNA and DNA molecules in lysine modified monolith was 11.58 and 14.59, respectively, while for cadaverine modified monolith was 20.32 and 27.12, respectively. The lysine modified monolith was able to successfully isolate sc mcDNA from the lysate sample. However, recovery yield was significantly sacrificed to guarantee high purity levels of sc mcDNA. The cadaverine modified monolith showed better selectivity than the previous monolith, achieving the successful sc mcDNA isolation from the lysate sample. The final sc mcDNA sample, obtained by the column that showed the best performance, was characterized by real-time PCR, presenting 98.4% purity and 78.6% recovery yield. The impurities content, namely genomic DNA, proteins and endotoxins, was found within the criteria established by regulatory agencies. Overall, a simple and practical chromatographic strategy to purify sc mcDNA was for the first time implemented by exploring a modified monolithic column, with no significant reduction on the purity and recovery and without resorting to backbone modification or specific enzymatic digestion. Such features will surely be crucial in the industrial scale-up of this chromatographic strategy since it will not be associated with significant cost-increase.

  • minicircle DNA Purification using a cim deae 1 monolithic support
    2016
    Co-Authors: Tatiana Diamantino, Joao A Queiroz, ângela Sousa, Patricia Pereira, Fani Sousa
    Abstract:

    Minicircle DNA is a new biotechnological product with beneficial therapeutic perspectives for gene therapy because it is constituted only by the eukaryotic transcription unit. These features improve minicircle DNA safety and increase its therapeutic effect. However, being a recently developed product, there is a need to establish efficient Purification methodologies, enabling the recovery of the supercoiled minicircle DNA isoform. Thus, this work describes the minicircle DNA Purification using an anion exchange monolithic support. The results show that with this column it is possible to achieve a good selectivity, which allows the isolation of the supercoiled minicircle DNA isoform from impurities. Overall, this study shows a promising approach to obtain the minicircle DNA sample with adequate quality for future therapeutic applications.

  • advances in chromatographic supports for pharmaceutical grade plasmid DNA Purification
    2012
    Co-Authors: ângela Sousa, Fani Sousa, Joao A Queiroz
    Abstract:

    Chromatographic technology is undoubtedly one of the most diverse and powerful Purification methods for downstream process applications. The diversity and quantity of biomolecules present in crude extracts as well as the similarities between impurities and the target biomolecule are considered the critical challenges in the extraction and Purification steps. Thus, it is important to optimize the Purification protocol to achieve maximum recovery of the target sample. The structure of chromatographic supports has been continuously developed to afford rapid and efficient separations, as well as, the application of specific ligands to improve the selectivity for the target molecule. The present review discusses the structural progress and evolution of the chromatographic supports that have been used for plasmid DNA Purification. Nowadays, the most desirable form of plasmid for gene therapy and DNA vaccination is the supercoiled isoform, due to its stability and higher transfection efficiency over other plasmid topologies. However, the main challenge is not only to produce high quantities of supercoiled plasmid DNA but also to preserve its quality, meeting the strict requirements recommended by the regulatory agencies. Therefore, this review will focus on the chemical and structural classification of the different media and on some of the specific ligands used for plasmid DNA bioseparation.

  • impact of lysine affinity chromatography on supercoiled plasmid DNA Purification
    2011
    Co-Authors: Ana C A Sousa, Fani Sousa, Joao A Queiroz
    Abstract:

    Abstract Gene therapy and DNA vaccination cover a variety of applications using viral and non-viral vectors as vehicles of choice for treatment of genetic or acquired diseases. Recently, most therapeutic applications have been performed with non-viral biological agents preparations highly enriched in supercoiled plasmid molecules and it has been concluded that this isoform is more efficient at gene transfection than open circular isoform. This work describes for the first time a new strategy that uses lysine-chromatography to efficiently eliminate Escherichia coli impurities as well as other ineffective plasmid isoforms present in a complex clarified lysate to purify and obtain pharmaceutical-grade supercoiled plasmid DNA. The quality control tests indicated that the levels of impurities in the final plasmid product were below the generally accepted specifications. Furthermore, the delivery of the purified product to eukaryotic cells, the cell uptake and transfection efficiency were also analyzed. The results showed that the transfection efficiency reached with the application of the supercoiled plasmid conformation, purified with lysine-agarose, was higher than the values achieved for other plasmid topologies. Therefore, this study presents a new enabling technology to obtain the completely purified non-viral vector, able to act with good efficiency as gene therapy delivery vehicle in several diseases like cancer.

  • binding and elution strategy for improved performance of arginine affinity chromatography in supercoiled plasmid DNA Purification
    2009
    Co-Authors: Fani Sousa, D M F Prazeres, Joao A Queiroz
    Abstract:

    New interesting strategies for plasmid DNA (pDNA) Purification were designed, exploiting affinity interactions between amino acids and nucleic acids. The potential application of arginine-based chromatography to purify pDNA has been recently described in our work; however, to achieve higher efficiency and selectivity in arginine affinity chromatography, it is essential to characterize the behaviour of binding/elution of supercoiled (sc) isoforms. In this study, two different strategies based on increased sodium chloride (225-250 mm) or arginine (20-70 mm) stepwise gradients are described to purify sc isoforms. Thus, it was proved that well-defined binding/elution conditions are crucial to enhance the Purification performance, resulting in an improvement of the final plasmids yields and transfection efficiency, as this could represent a significant impact on therapeutic applications of the purified sc isoform.

Darrell P Chandler - One of the best experts on this subject based on the ideXlab platform.

  • renewable microcolumns for automated DNA Purification and flow through amplification from sediment samples through polymerase chain reaction
    2002
    Co-Authors: Cindy J Brucknerlea, Toyoko Tsukuda, Brian P Dockendorff, James C Follansbee, Mark T Kingsley, Catherine O Ocampo, Jennie R Stults, Darrell P Chandler
    Abstract:

    Abstract There is an increasing need for field-portable systems for the detection and characterization of microorganisms in the environment. Nucleic acids analysis is frequently the method of choice for discriminating between bacteria in complex systems, but standard protocols are difficult to automate and current microfluidic devices are not configured specifically for environmental sample analysis. In this report, we describe the development of an integrated DNA Purification and polymerase chain reaction (PCR) amplification system and demonstrate its use for the automated Purification and amplification of Geobacter chapellei DNA (genomic DNA or plasmid targets) from sediments. The system includes renewable separation columns for the automated capture and release of microparticle Purification matrices, and can be easily reprogrammed for new separation chemistries and sample types. The DNA extraction efficiency for the automated system ranged from 3 to 25%, depending on the length and concentration of the DNA target. The system was more efficient than batch capture methods for the recovery of dilute genomic DNA even though the reagent volumes were smaller than required for the batch procedure. The automated DNA concentration and Purification module was coupled to a flow-through, Peltier-controlled DNA amplification chamber, and used to successfully purify and amplify genomic and plasmid DNA from sediment extracts. Cleaning protocols were also developed to allow reuse of the integrated sample preparation system, including the flow-through PCR tube.

Roland Zengerle - One of the best experts on this subject based on the ideXlab platform.

  • Centrifugal LabTube platform for fully automated DNA Purification and LAMP amplification based on an integrated, low-cost heating system
    2014
    Co-Authors: Melanie M. Hoehl, Thomas Nesch, Arne Dannenberg, Nils Paust, M Weissert, Alexander H Slocum, Felix Von Stetten, Roland Zengerle, Juergen Steigert
    Abstract:

    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.

  • a versatile deployable bacterial detection system for food and environmental safety based on labtube automated DNA Purification labreader integrated amplification readout and analysis
    2014
    Co-Authors: Melanie M. Hoehl, Juergen Steigert, Nils Paust, Roland Zengerle, Eva Schulte Bocholt, Arne Kloke, Felix Von Stetten, Alexander H Slocum
    Abstract:

    Contamination of foods is a public health hazard that episodically causes thousands of deaths and sickens millions worldwide. To ensure food safety and quality, rapid, low-cost and easy-to-use detection methods are desirable. Here, the LabSystem is introduced for integrated, automated DNA Purification, amplification and detection. It consists of a disposable, centrifugally driven DNA Purification platform (LabTube) and a low-cost UV/vis-reader (LabReader). For demonstration of the LabSystem in the context of food safety, Purification of Escherichia coli (non-pathogenic E. coli and pathogenic verotoxin-producing E. coli (VTEC)) in water and milk and the product-spoiler Alicyclobacillus acidoterrestris (A. acidoterrestris) in apple juice was integrated and optimized in the LabTube. Inside the LabReader, the purified DNA was amplified, readout and analyzed using both qualitative isothermal loop-mediated DNA amplification (LAMP) and quantitative real-time PCR. For the LAMP-LabSystem, the combined detection limits for Purification and amplification of externally lysed VTEC and A. acidoterrestris are 102–103 cell-equivalents. In the PCR-LabSystem for E. coli cells, the quantification limit is 102 cell-equivalents including LabTube-integrated lysis. The demonstrated LabSystem only requires a laboratory centrifuge (to operate the disposable, fully closed LabTube) and a low-cost LabReader for DNA amplification, readout and analysis. Compared with commercial DNA amplification devices, the LabReader improves sensitivity and specificity by the simultaneous readout of four wavelengths and the continuous readout during temperature cycling. The use of a detachable eluate tube as an interface affords semi-automation of the LabSystem, which does not require specialized training. It reduces the hands-on time from about 50 to 3 min with only two handling steps: sample input and transfer of the detachable detection tube.

  • centrifugal gas phase transition magnetophoresis gtm a generic method for automation of magnetic bead based assays on the centrifugal microfluidic platform and application to DNA Purification
    2013
    Co-Authors: Oliver Strohmeier, Roland Zengerle, Alexander Emperle, Gunter Roth, Daniel Mark, Felix Von Stetten
    Abstract:

    Transportation of magnetic beads between different reagents plays a crucial role in many biological assays e.g. for Purification of biomolecules or cells where the beads act as a mobile solid support. Therefore, usually a complex set-up either for fluidic processing or for manipulation of magnetic beads is required. To circumvent these drawbacks, we present a facile and automated method for the transportation of magnetic beads between multiple microfluidic chambers on a centrifugal microfluidic cartridge “LabDisk”. The method excels by requiring only one stack of stationary permanent magnets, a specific microfluidic layout without actively controlled valves and a predefined frequency protocol for rotation of the LabDisk. Magnetic beads were transported through three fluidically separated chambers with a yield of 82.6% ± 3.6%. Bead based DNA Purification from a dilution series of a Listeria innocua lysate and from a lambda phage DNA standard was demonstrated where the three chambers were used for binding, washing and elution of DNA. Recovery of L. innocua DNA was up to 68% ± 24% and for lambda phage DNA 43% ± 10% compared to manual reference Purification in test tubes. Complete Purification was conducted automatically within 12.5 min. Since all reagents can be preloaded onto the LabDisk prior to Purification, no further hands-on steps are required during processing. Due to its modular and generic character, the presented method could also be adapted to other magnetic bead based assays e.g. to immunoassays or protein affinity Purification, solely requiring the adjustment of number and volumes of the fluidic chambers.

  • controlled counter flow motion of magnetic bead chains rolling along microchannels
    2011
    Co-Authors: Marc Karle, Nils Paust, Roland Zengerle, Gunter Roth, Johannes Wohrle, Junichi Miwa, Felix Von Stetten
    Abstract:

    We demonstrate controlled transport of superparamagnetic beads in the opposite direction of a laminar flow. A permanent magnet assembles 200 nm magnetic particles into about 200 μm long bead chains that are aligned in parallel to the magnetic field lines. Due to a magnetic field gradient, the bead chains are attracted towards the wall of a microfluidic channel. A rotation of the permanent magnet results in a rotation of the bead chains in the opposite direction to the magnet. Due to friction on the surface, the bead chains roll along the channel wall, even in counter-flow direction, up to at a maximum counter-flow velocity of 8 mm s−1. Based on this approach, magnetic beads can be accurately manoeuvred within microfluidic channels. This counter-flow motion can be efficiently be used in Lab-on-a-Chip systems, e.g. for implementing washing steps in DNA Purification.