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.     

Purifying genomic DNA from whole blood on automated, high-throughput, and moderate-throughput platforms

We describe three new automated methods for purifying genomic DNA from whole blood. The MagneSil^® Blood Genomic, Max Yield System uses MagneSil^® paramagnetic particles (PMPs) in a 96-well format to purify the maximal amount of DNA from a 200-μL blood sample. In contrast, the MagneSil^® ONE, Fixed Yield Blood Genomic System uses MagneSil^® Fixed Yield PMPs to purify a normalized amount of DNA from 60 μL of blood in a 96-well format. These methods are implemented on the Beckman Coulter Biomek^® FX automated workstation. The MagneSil^® KF Genomic System uses MagneSil^® PMPs to purify DNA from 1 to 15 samples of 200-μL blood using the moderate-throughput Thermo Electron KingFisher^® mL instrument.The MagneSil^® Blood Genomic System typically yields > 4 μg per 200 μL of whole blood, depending on the white blood cell content. The MagneSil^® ONE System is best suited where there is a requirement for purification of a narrow concentration range of DNA. This system purifies 1 μg (±50%) of DNA from 60 μL of blood. The MagneSil^® KF System purifies 2 to 6 μg of DNA from 200 μL of blood. DNA purified using all of these methods is suitable for PCR, STR, READIT^® SNP genotype analysis, and multiplexed PCR analysis.     

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.     

Automated Purification of Plasmid DNA Using Paramagnetic Particles

We describe a reagent system and robotic method for purifying plasmid DNA for restriction digestion, PCR, and fluorescent sequencing applications. The method uses two types of Wizard® MagneSilTM paramagnetic particles. Following lysis and neutralization procedures, the first particle type binds and removes cell debris; the second type is then used to bind plasmid DNA from the cleared lysate. The particles are then washed to eliminate unwanted contaminants. Purified plasmid DNA is then eluted from the particles with nuclease free water. When using a cell mass of approximately 4 O.D.₆₀₀, the yield is 10–12μg of DNA when using high copy number plasmid. When used in BigDye® terminator sequencing, these DNA templates typically yield read lengths greater than 700 bases and Phred 20 scores of 600 to 750 bases. This purification method has been adapted for use on several robotic platforms in a 96-well format.     

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     

A Demonstration of High-Throughput Electrophoresis

Automating electrophoresis significantly reduces the time required for loading a large number of samples, increases the speed of electrophoresis analysis, and maximizes the resolution power (clear separation of fragments) of this technique. In addition, automation increases the precision of electrophoresis analysis. Here we demonstrate an automated, high-throughput method of loading 96 samples simultaneously onto an electrophoresis gel, using the Apogent Discoveries Tango™ system and the Invitrogen™ E-Gel® 96 system.     

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).     

A Fully Automated Process Using a Magnetic Particle Based Kit for Removal of Dye Terminators from Sequencing Reactions

Prolinx,® Inc. of Bothell, WA has developed the RapXtract™ 384 Dye Terminator Removal Kit for full automation of DNA sequencing reaction purification. The RapXtract product line is based upon proprietary superparamagnetic particle technology that eliminates the need for centrifugation, vacuum filtration, or modified primers to achieve purification of sequencing reactions. The kit described here is pre-dispensed in a 384-well microtiter plate and run on the TECAN GENESIS Workstation 150 (Tecan U.S. Inc., Research Triangle Park, NC). This system enables rapid purification of up to 384 sequencing reactions in a single run.As the completion of the Human Genome Project nears, it is imperative for biotechnology and pharmaceutical companies to increase throughput of DNA sequencing in order to be competitive in the drug discovery and validation process. The “race to market” requires a shift from standard DNA sequencing processes-including DNA sequencing reaction purification-towards complete walk-away automation for all steps.Existing sequencing reaction purification methods (Table 1) require considerable resources including: plastic and other laboratory consumables; specialized equipment, such as high-speed centrifuges or vacuum filtration apparatus; and labor-intensive protocols requiring large amounts of technician time. As a result, walk-away automation of standard purification methods is difficult and expensive.     

Automated purification of dye terminator sequencing reactions: an approach to high-throughput capillary electrophoresis sequencing of large templates

Demands for higher quantity and quality of sequence data during genome sequencing projects have led to a need for completely automated reagent systems designed to isolate, process, and analyze DNA samples. While much attention has been given to methodologies aimed at increasing the throughput of sample preparation and reaction setup, purification of the products of sequencing reactions has received less scrutiny despite the profound influence that purification has on sequence quality. Commonly used and commercially available sequencing reaction cleanup methods are not optimal for purifying sequencing reactions generated from larger templates, including bacterial artificial chromosomes (BACs) and those generated by rolling circle amplification. Theoretically, these methods would not remove the original template since they only exclude small molecules and retain large molecules in the sample. If the large template remains in the purified sample, it could understandably interfere with electrokinetic injection and capillary performance. We demonstrate that the use of MagneSil^® paramagnetic particles (PMPs) to purify ABI PRISM^® BigDye^® sequencing reactions increases the quality and read length of sequences from large templates. The high-quality sequence data obtained by our procedure is independent of the size of template DNA used and can be completely automated on a variety of automated platforms.     

Automated High-Throughput Purification of PCR Products Using WizardMagneSil™ Paramagnetic Particles

Here we describe a reagent system and robotic methods for the purification of PCR^(a) fragments from other contaminating amplification reaction components. The methods use the MagneSil™ paramagnetic particle chemistry^(b) to isolate double stranded DNA fragments from 150bp to 23kbp. Purified fragments are eluted in water ready for downstream applications such as cloning, fluorescent DNA sequencing and microarray printing. This method has been adapted to a number of liquid handling robotic platforms, including the Biomek^® FX and Biomek^® 2000 Laboratory Automation Workstations, in both 96 and 384-well formats.     

Linguistic properties based on American Sign Language isolated word recognition with artificial neural networks using a sensory glove and motion tracker

Sign language (SL), which is a highly visual–spatial, linguistically complete, and natural language, is the main mode of communication among deaf people. Described in this paper are two different American Sign Language (ASL) word recognition systems developed using artificial neural networks (ANN) to translate the ASL words into English. Feature vectors of signing words taken at five time instants were used in the first system, while histograms of feature vectors of signing words were used in the second system. The systems use a sensory glove, Cyberglove™, and a Flock of Birds^® 3-D motion tracker to extract the gesture features. The finger joint angle data obtained from strain gauges in the sensory glove define the hand shape, and the data from the tracker describe the trajectory of hand movement. In both systems, the data from these devices were processed by two neural networks: a velocity network and a word recognition network. The velocity network uses hand speed to determine the duration of words. Signs are defined by feature vectors such as hand shape, hand location, orientation, movement, bounding box, and distance. The second network was used as a classifier to convert ASL signs into words based on features or histograms of these features. We trained and tested our ANN models with 60 ASL words for a different number of samples. These methods were compared with each other. Our test results show that the accuracy of recognition of these two systems is 92% and 95%, respectively.     

Automated genomic and proteomic applications on the Biomek NX laboratory automation workstation

This technical paper describes the utilization of a new automated liquid handler from Beckman Coulter, Inc., the Biomek^® NX Laboratory Automation Workstation, for genomic and proteomic applications. For genomic applications, methodology for plasmid DNA purification using Promega Wizard^® SV 96 reagents was developed for the Biomek NX. A single plate of bacterial pellets can be processed to purified plasmid DNA without user interaction after initial setup. DNA quantity and quality were assessed by spectrophotometric analysis, restriction digestion, PCR (The PCR process is covered by patents owned by Roche Molecular Systems, Inc., and F. Hoffman La Roche, Ltd.), and capillary sequencing. Additionally, the plasmid preparation method was used to purify plasmid DNA from bacterial clones isolated in a bacterial two-hybrid screening procedure. In this case, the system quickly and efficiently prepared clones for rapid identification of target sequences. For proteomic applications, His-tag proteins were purified from bacterial cultures in a 96-well plate format. Following purification, a Bradford assay was used to determine the quantitative yields of the His-tag protein products in each of the aliquots from the purified samples. The AD 340 Automated Labware Positioner (ALP), an integrated absorbance reader, was used for absorbance measurements in the Bradford assay. Given the placement of this ALP on the deck of the Biomek NX, the entire process of protein purification and quantitation was performed in a complete walk-away automated format. Results obtained when purifying proteins, from both uninduced and induced bacterial cultures, on the worksurface of the Biomek NX will be described.     

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.     

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.     

THERMIC: a 2-D finite-element tool to solve conductive and advective heat transfer problems in Earth Sciences

We present a C-language program, THERMIC, that solves the 2-dimensional (pseudo 3D for axi-symmetric cases) conductive and advective heat-transfer equation. THERMIC uses a finite-element method that takes into account realistic geometries, heterogeneous material properties and various boundary and initial conditions. As it also allows for latent heat (heat production due to crystallisation) and for thermal properties, such as thermal conductivity, to be dependent on temperature, it is particularly suited to heat transfer problems encountered in the Earth Sciences. We present sample applications from the various problems already treated by THERMIC (cooling of magma chambers and dykes, the study of a granitic magma ascent or of pore water flow in sedimentary basins).Successfully tested on SUN® and SGI® UNIX workstations and on Microsoft Windows 95®, 98® and NT® 4.0 system based PCs, the THERMIC package can be downloaded from the web (THERMIC home page: http://www.ipgp.jussieu.fr/UFP/thermic/html/Thermic_home.html) and contains source files, makefiles and environment files as well as executable files for both systems and an html directory with help and example files.     

Parking Can Get You There Faster: Model Augmentation to Speed up Real-Time Model Checking

We present an approximation technique, that can render real-time model checking of safety and universal path properties more efficient. It is beneficial, when loops lead to repetition of control situations. Basically we augment a timed automata model with carefully selected extra transitions. This increases the size of the state-space, but potentially decreases the number of symbolic states to be explored by orders of magnitude.We give a formal definition of a timed automata formalism, enriched with basic data types, hand-shake synchronization, urgency, and committed locations. We prove by means of a trace semantics, that if a safety property can be established in the augmented model, it also holds for the original model.We extend our technique to a richer set of properties, that can be decided via a set of traces (universal path properties). In order for universal path properties to carry over to the original model, the semantics of the timed automata formalism is formulated relative to the applied augmentation.Our technique is particularly useful in systems, where a scheduler dictates repetition of control over elapsing time. As a typical example we mention translations of LEGO® RCX™ programs to Uppaal models, where the Round-Robin scheduler is a static entity. We allow scheduler and associated tasks to “park”, until some timing or environmental conditions are met.We apply our technique on a brick-sorter model for a safety property and report run-time data.     

Simlab 1 - A Laboratory Workflow Recorder for Process Assessment

Process management and workflow optimisation have become key issues of good laboratory management and now seem to be predominant over traditional challenges such as the quality of analytical testing. The first author of this paper has conducted several workflow optimisation studies for clinical laboratories in Europe (Universities of Zurich and Amsterdam, Manchester Royal Infirmary and others) and in the US (University of Virginia), using a laboratory-specific simulation software called Simlab™ for realistic computer modeling of laboratory scenarios. Since data input and calibration of the models against reality have always been a challenge in these studies, we looked into possibilities of using automated data extraction to extract as much information as possible from the laboratory information system. In this paper we describe our first experience with a Microsoft Excel™-based software program called Simlab 1, which uses the ASCII file transfer protocol to extract data from the LIS.     

Borehole Optimisation System (BOS)—A GIS based risk analysis tool for optimising the use of urban groundwater

Urban groundwater is generally an underused resource, partially due to the perceived risk of pollution and the strategic difficulties in placing boreholes in built-up areas. The development of a probabilistic risk based management tool that predicts groundwater quality at potential new urban boreholes is beneficial in determining the best sites for future resource development. The Borehole Optimisation System (BOS) is a custom Geographic Information System (GIS) application that has been developed in the ArcView 3.1 environment with the objective of locating the optimum locations for new boreholes in urban areas. It couples three component models, the Catchment Zone Probability Model (CZPM), the Land-use Model (LM) and the Pollution Risk Model (PRM). The CZPM produces probabilistic catchment zones for a user-defined abstraction borehole location under uncertain and variable hydrogeological parameters. The LM identifies current and historical industries located within the selected probabilistic catchment zone. The PRM uses these industrial and the associated hydrogeological and contaminant data to predict probabilistic contaminant concentrations in a particular analysis year. This paper outlines the methodologies employed in the development of BOS and attempts to validate the approach by presenting a simulation that forecasts PCE concentrations at an actual borehole location in the Nottingham urban aquifer. The results predict contaminant levels in the abstracted water that are in agreement with observed values, both being above the UK Drinking Water Standard of 10 μg/l. These demonstrate the applicability of BOS as a tool for informing decision-makers on the development of urban groundwater resources.     

“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.     

Incorporating Automation in Undergraduate Laboratories

Automated techniques and philosophies, which have become increasingly important in modern laboratories, are not traditionally covered in undergraduate chemistry curricula. To this end, we have been incorporating automated sample preparation methods using standard robotic workstations in our undergraduate analytical chemistry laboratory. Using a Benchmate™ II Workstation, an automated method has been developed and implemented for the solid phase extraction of capsaicins from commercial hot pepper sauces prior to liquid chromatographic analysis. This paper reports on pedagogical aspects of incorporating automation into the undergraduate curriculum as well as results obtained for manual and automated extractions conducted by students.