A Study of a High-Throughput Plasmid DNA Purification System

An automated process that incorporates Millipore's Plasmid Miniprep₉₆ Montáge™ Kit with the Apogent Discoveries PlateMate Plus® and Tango™ automated high-throughput dispensing systems has been developed for purifying plasmid DNA. To test the efficacy of this process, parameters such as the reproducibility and consistency of the purified DNA quantity and quality as well as the purification speed were analyzed. The purification time for two plates of the Plasmid Miniprep₉₆ Kit (192 samples) was approximately 60 minutes using a PlateMate Plus equipped with 96 disposable tips and the Tango system equipped with 96 RB (resin bead) syringes. High uniformity and consistency in DNA yields (determined by spectrophotometric analysis) and quality (determined by gel electrophoresis analysis) among the different wells were observed. The purified plasmid DNA samples sequenced at an exceptional level with an average PHRED Q > 20 of 819 ± 25.*Millipore and Montage are the trademarks of Millipore Corporation     

Automated High Throughput Purification of BigDye™ Terminator Fluorescent DNA Sequencing Reactions Using Wizard® MagneSil™ Paramagnetic Particles

We describe a reagent system and robotic methods for the purification of BigDye™ Terminator sequencing reactions prior to automated fluorescent sequence analysis. The methods use MagneSil™ paramagnetic particles to isolate sequencing extension products from unincorporated dye-labeled terminators and exchanges sequencer loading solution for reaction buffer. Processed samples give usable data that is greater than 98% accurate from primer plus 5–15 bases to over 700 bases. Typical Phred greater than 20 quality scores range from 600 to over 700 bases. This process has been adapted to a number of liquid handling robotic platforms in both 96- and 384-well formats. One method using a single POD Beckman Biomek® FX can process up to four plates in approximately 40 minutes.     

Automated Purification of Transfection-Grade Plasmid DNA Using Wizard MagneSil Tfx System

We describe a reagent system and robotic protocol for the isolation of highly purified plasmid DNA from cultured cells. The method is based on the Wizard® MagneSil™ Plasmid Purification System, which purifies sequencing-grade plasmid DNA. Two modifications to the sequencing-grade system were made to create the Wizard MagneSil Tfx™ System. This system provides lower endotoxin and other contaminant levels, giving higher quality plasmid for transfection applications. The Wizard MagneSil Tfx™ System uses MagneSil™ Paramagnetic Particles (PMPs) to clear lysate and bind plasmid, eliminating the need for filtration devices. The endotoxin removal step uses MagneSil™ (PMPs) and a guanidine/isopropanol wash to remove RNA and protein. One 96-well plate may be processed in 45 minutes on the Beckman Biomek® FX robotic workstation. We provide data showing DNA yield, contaminant levels, and transfection efficiency for 5 commonly used cell lines. Comparisons with other systems are also shown.     

MagneSil™ Paramagnetic Particles: Magnetics for DNA Purification

MagneSil™ Paramagnetic Particles are silica-paramagnetic particles with an affinity for nucleic acids under defined conditions. Particle structure and solution composition can be altered to selectively adsorb nucleic acids based upon type and size. These properties have been used to develop purification methods based on a three-step bind, wash, and elute process. The MagneSil™ technology is readily adaptable to robotic platforms, allowing complete automation of the purification process in either 96- or 384- well plate formats. This article introduces the basic physical and performance characteristics of the MagneSil™ Paramagnetic Particles.     

The LabMatrix system

The LabMatrix^™ is a prototyping system designed to give the user a practical and versatile platform for testing microfluidic applications in the fields of health care and life sciences. The LabMatrix^™ system consists of a microfluidic breadboard and cover that align and secure a series of specially designed LabMatrix^™ microfluidic chips. Chips are easily arranged and rearranged into a user-defined fluidic network. The LabMatrix^™ system is designed with maximum flexibility in mind, providing the user with a means to prototype a wide range of microfluidic applications in a short period.     

Plastic Microfluidic Systems for High-Throughput Genomic Analysis and Drug Screening

Genomic analysis and drug discovery depend increasingly on rapid, accurate analysis of large sets of sample and extensive compound collections at relatively low cost. By capitalizing on advances in microfabrication, genomics, combinatorial chemistry, and assay technologies, new analytical systems are expected to provide order-of-magnitude increases in analysis throughput along with comparable decreases in per-sample analysis costs. ACLARA's single-use, plastic LabCard™ systems, which transport fluids between reservoirs and through interconnected microchannels using electrokinetic mechanisms, are intended to address these analytical needs. These devices take advantage of recent developments in microfluidic and microfabrication technologies to permit their application to DNA sequencing; genotyping and DNA fragment analysis, as well as pharmaceutical candidate screening, and preparing biological samples for analysis. In a parallel effort, ACLARA has developed a new class of reporter molecules that are particularly well suited to capillary electrophoretic analysis. These electrophoretic mobility tags, called eTag™ reporters, can be used to uniquely label multiplexed sets of oligonucleotide recognition probes or proteins, thereby permitting traditionally homogeneous biochemical reporter assays to be multiplexed for CE analysis. Biochemical multiplexing is key to achieving new thresholds in analytical throughput while maintaining economically viable formats in many application areas. ACLARA's microfluidic, lab-on-a-chip concept promises to revolutionize chemical analysis, similar to the way miniaturization revolutionized computing, making tools continually smaller, more integrated, less expensive, and higher performing.     

Creating change and driving innovation in highly automated and lean organizations: The Temporal Think Tank™ (T3™)

This paper introduces the concept of the temporal think tank™ (T3™), a temporary in-house research center, based on case studies with a highly automated global manufacturer and in various small-sized manufacture (SME's). The T3™ brings individuals from across the organization together in a team setting in which they are charged to look at the future, develop their decision-making skills, and use entrepreneurial thinking to incubate new processes and/or products. After an appropriate time in the temporal think tank™, the individuals are asked to return to their regular positions to implement the best practices and best ideas and doing so spur the organization forward. At intervals, the temporal think tanks™ can be reconvened, or reconstituted with new participants. This approach prepares technology champions for the SME, who can return to the organization with a fresh outlook and renewed energy to keep the enterprise at the peak of efficiency and effectiveness.     

Dispensing Precision for the SciClone™ Automated Liquid Handling Workstation for 96-Channel Pipetting

One of the first steps in drug discovery involves identification of novel compounds that interfere with therapeutically relevant biological processes.

Identification of ‘lead’ compounds in all therapeutic areas included in a drug discovery program requires labor-intensive evaluation of numerous samples in a battery of therapy targeted biological assays. To accelerate the identification of ‘lead’ compounds, Janssen Research Foundation (JRF) has developed in the past an automated high throughput screening (HTS) based on the unattended operation of a custom Zymark tracked robot system. Automation of enzymatic and cellular assays was realized with this system adapted to the handling of microtiter plates. The microtiter plate technology is the basis of our screening. All compounds within our chemical library are stored and distributed in micronic tube racks or microtiter plates for screening. An efficient in-house developed mainframe based laboratory information management system supported all screening activities. Our experience at JRF has shown that the preparation of test compounds and serial dilutions has been a rate-limiting step in the overall screening process. In order to increase compound throughput, it was necessary both to optimize the robotized assays and to automate the compound supply processes. In HTS applications, one of the primary requirements is highly accurate and precise pipetting of microliter volumes of samples into microplates. The SciClone™ is an automated liquid handling workstation capable of both 96- and 384-channel high precision pipetting. For high throughput applications, the SciClone™ instrumentation is able to pipette a variety of liquid solutions with a high degree of accuracy and precision between microplates (inter-plate variability) and tip-to-tip (intra-plate variability) within a single plate. The focus of this presentation is to review the liquid handling performance of the SciCloneTM system as a multipurpose instrument for pipetting aqueous or organic solutions, and virus suspensions into 96- and 384-well microplates. The capabilities of the system and the resulting benefits for our screening activities will be described.     

Managing the Workflow of a High-Throughput Organic Synthesis Laboratory: A Marriage of Automation and Information Management Technologies

The production of high quality, individually characterized libraries of compounds requires a welldeveloped strategy, along with robust, userfriendly data and automation systems to support this strategy. We developed a workflow process to improve the efficiency of producing greater than 50,000 individual compounds per year. Our workflow process eliminates bottlenecks in traditional procedures and thus ensures a steady, predictable compound production rate. Initially, virtual library creation allowed for the determination of the physio-chemical properties of libraries as well as the ability to calculate the needed amounts of reagents and solvents for a production campaign. At this point, we registered all samples into our library database. Production of libraries began with loading the appropriate linkers onto resins, followed by complete characterization of the resin-linker intermediate. The resins were then loaded into IRORI MiniKans™ and sorted using the IRORI Autosort™ 10Kx sorting workstation. Chemistry was then performed using standard laboratory glassware. Additional sorting and chemistry steps were then performed until the compound was ready for cleavage off of the resin. The MiniKans were then sorted into Bohdan MiniBlocks™ and treated with the appropriate cleavage cocktail. Collection into 48-position racks was followed by removal of cleavage solution through vacuum centrifugation. The concentrate was then dissolved in a solvent mixture that allowed for standard liquid handling automation to create 96 well plates for analysis by high throughput flow inject NMR and LC/MS. Compounds > 80% pure by LC/MS and presenting a > 80% overall yield, as determined by NMR, were automatically flagged “acceptable” in the database. Compounds that failed to pass these analytical criteria were flagged “fail” in the database and were examined for trends that could be corrected in future library production. Finally, acceptable compounds were reformatted from the original source plates into master plates.     

High Throughput Methods for Gene Identification, Cloning and Functional Genomics Using the GeneTAC™ G Robotics Workstation

Using a single robotic platform, the GeneTAC™ G³, we have automated most of the processes involved in the cloning and characterisation of novel disease causing genes by addressing the following; firstly, identifying the BACs of interest and making shotgun libraries. Secondly, automating the set up of sequencing reactions using methodology that eliminates the need for DNA preparation of 384 clones. Thirdly, generating sublibraries using selective re-arraying of library clones to enable the determination of the entire genomic sequence of the gene. Fourthly, determining gene function by combination of differential screening and mini Northerns using microarrays printed using the GeneTAC™ G³ system and hybridised using the GeneTAC™ HybStation (Genomics Solutions, Ann Arbor, USA).     

“Electric” Zip Tips™, Preliminary Results

Induction based fluidics (IBF), a new, simple patented approach for transporting liquids in the micro and the macro world, is discussed. Electric fields are shown to energize liquid/s in a container/s to execute an array of purposes. IBF is shown uniquely to energize N liquids in simple off the shelf devices, inductively. We discuss calibration and other issues, as we demonstrate how simple devices can dispense nanoliters and microliters with high precision and accuracy. Furthermore, we show preliminary single and eight channel data for the Zip Tip™ made by Millipore where the device transports liquids “electrically.” We briefly consider how such new devices, “electric” Zip Tips™, might automate desalting and the placement of digests for MALDI TOF analysis.     

Prostate cancer biomarker discovery using high performance mass spectral serum profiling

Prostate-specific antigen (PSA) is the most widely used serum biomarker for early detection of prostate cancer (PCA). Nevertheless, PSA level can be falsely elevated due to prostatic enlargement, inflammation or infection, which limits the PSA test specificity. The objective of this study is to use a machine learning approach for the analysis of mass spectrometry data to discover more reliable biomarkers that distinguish PCA from benign specimens. Serum samples from 179 prostate cancer patients and 74 benign patients were analyzed. These samples were processed using ProXPRESSION™ Biomarker Enrichment Kits (PerkinElmer). Mass spectra were acquired using a prOTOF™ 2000 matrix-assisted laser desorption/ionization orthogonal time-of-flight (MALDI-O-TOF) mass spectrometer. In this study, we search for potential biomarkers using our feature selection method, the Extended Markov Blanket (EMB). From the new marker selection algorithm, a panel of 26 peaks achieved an accuracy of 80.7%, a sensitivity of 83.5%, a specificity of 74.4%, a positive predictive value (PPV) of 87.9%, and a negative predictive value (NPV) of 68.2%. On the other hand, when PSA alone was used (with a cutoff of 4.0 ng/ml), a sensitivity of 66.7%, a specificity of 53.6%, a PPV of 73.5%, and a NPV of 45.4% were obtained.     

A system for dispensing sub-milligram doses of active pharmaceutical powders for early stage solubility assays

At early stages of drug development, the solubility of compounds is an important screening criterion. However, because scientists lack the automated tools needed to perform comprehensive early stage solubility studies, they are only able to perform a small number of experiments by hand, thus exploring only a fraction of the potential formulation design space. To allow a larger formulation design space to be explored at relatively early stages of pharmaceutical development (when 100 mg of a prototype compound is available), TransForm has created the AquaSFinX^™ and SFinX^™ micro-solubility platforms. In this article, a novel solid deposition system is described which is an enabling component of TransForm's solubility platforms. Given 100 mg of a starting material, the deposition system can dispense over six hundred 50-μg plugs of powder into 384-well or 1,536-well plates with well openings as small as 1.5 mm. Currently no commercial powder deposition system can provide this functionality. Including the time required to characterize and run the system, the dispense times range from around 2 minutes per dose for small runs involving 100 mg of starting material, to 18 seconds per dose for larger runs involving a gram or more of starting material.     

Automated Lab-on-a-Chip Analysis of DNA Fragments

The design of a fully automated system for the analysis of DNA fragments in 96-well plates is described. Microfluidic technology is used to integrate sample loading, electrophoretic analysis, and fragment detection onto a miniature lab-on-a-chip device. The microfluidic chip operates in an instrument platform that automates sample access, data collection, and data reporting. Each microfluidic chip provides sizing and concentration values for more than 1000 DNA samples.     

Effect of post-release sidewall morphology on the fracture and fatigue properties of polycrystalline silicon structural films

Surface properties can markedly affect the mechanical behavior of structural thin films used in microelectromechanical systems (MEMS) applications. This study highlights the striking difference in the sidewall surface morphology of n⁺-type polysilicon films from two popular MEMS processes and its effect on fracture and fatigue properties. The sidewall surface roughness was measured using atomic force microscopy, whereas silicon oxide thickness and grain size were measured using (energy-filtered) transmission electron microscopy. These measurements show that the oxide layers are not always thin native oxides, as often assumed; moreover, the roughness of the silicon/silicon oxide interface is significantly influenced by the oxidation mechanism. Thick silicon oxides (20 ± 5 nm) found in PolyMUMPs™ films are caused by galvanic corrosion from the presence of gold on the chip, whereas in SUMMiT V™ films a much thinner (3.5 ± 1.0 nm) native oxide was observed. The thicker oxide layers, in combination with differences in sidewall roughness (14 ± 5 nm for PolyMUMPs™ and 10 ± 2 nm for SUMMiT V™), can have a significant effect on the reliability of polysilicon structures subjecting to bending loads; this is shown by measurements of the fracture strength (3.8 ± 0.3 GPa for PolyMUMPs™ and 4.8 ± 0.2 GPa for SUMMiT V™) and differences in the stress-lifetime cyclic fatigue behavior.     

A 384-Well, Fully-Automatable, Superparamagnetic-Particle Based Dye Terminator Removal Kit

We have developed a method for the automated purification of DNA sequencing reactions using the RapXtract™ 384 Dye Terminator Removal Kit and the Quadra 3™ Workstation. The process enables purification of 384 reactions in five minutes, significantly impacting the through-put potential of sequencing laboratories. The RapXtract technology utilizes superparamagnetic particles, (i.e., particles that are not themselves magnetic but that respond to a magnetic field) and eliminates the need for centrifugation, vacuum filtration, or modified primers. The Quadra 3 Workstation is a 384-channel liquid handling system, fitted with a retractable magnetic nest designed to incorporate a 384 magnetic separator. The combined technologies result in reduced variability associated with manual methods for sequencing reaction purification.     

Automated Purification of DNA from Large Samples: A Study of Effectiveness and Labor Efficiency

Investigations into the underlying genetic contributions to human disease are transitioning from small family-based traditional linkage analyses to large population-based studies designed to identify genetic factors in more complex and common diseases that have the greatest impact on human health. These types of studies have driven the need for larger numbers of samples for analysis and more efficient and effective methods for DNA purification, especially for large samples that provide sufficient quantities of DNA for extensive analysis. The AUTOPURE LS™ Nucleic Acid Purification Instrument, by Gentra Systems, Inc., a platform capable of high-throughput sample purification from large samples, was developed to meet the demands of these large studies. This article presents data demonstrating the equivalency of DNA purified using the AUTOPURE LS automated instrument and the manual method based on the same purification process. In addition, we present data demonstrating the in-lab time savings realized by automating the purification process.     

Automated Nucleic Acid Purification for Large Samples

Advancements in the fields of genomic screening, molecular pathology and clinical research have resulted in a major increase in the demand for high quality DNA and RNA. This escalating demand has resulted in a sample preparation bottleneck and an emphasis on the development of new technologies to automate the purification process. Gentra has developed the AUTOPURE LS™ nucleic acid purification instrument, a platform capable of high-throughput sample purification from large samples, such as 10 mL whole blood. This article presents data showing the equivalency of DNA purified using manual and automated processing.     

Analysis of utility and use of a web-based tool for digital signal processing teaching by means of a technological acceptance model

This paper presents an exploratory study about the development of a structural and measurement model for the technological acceptance (TAM) of a web-based educational tool. The aim consists of measuring not only the use of this tool, but also the external variables with a significant influence in its use for planning future improvements. The tool, designed with Shockwave™ and Macromedia Director™, is a web-based educational environment that has been applied as a teaching methodology in an undergraduate course involved with modern microprocessors architectures and applications. The proposed methodology may be extensible to similar tools and tries to fill the lack of scientific studies in the validation and acceptance of computer-based educational tools.