UV Laser Micromachining of Polymers for Microfluidic Applications

We have recently begun to explore the use of UV laser ablation micromachining to construct microfluidic devices in polymers. This technique can create microchannels rapidly and modify the resulting polymer surface in a single step. By ablating under different atmospheres, it is possible to alter both the surface chemistries and physical surface morphologies of the microchannels. We have employed electroosmotic flow measurements, chemical mapping, and optical microscopy to characterize the microfluidic devices. In addition, we have studied the parameters affecting the ablation, such as the laser wavelength, laser fluence, laser firing repetition rate, and the material being ablated.     

Automated Lead Discovery and Development in HTS Datasets

The analysis of biological screening results has traditionally been a labor intensive process. Scientists familiar with the biological data under investigation would visually inspect the results, evaluate the quality and promise of active data points and identify leads. The introduction and widespread use of high throughput screening systems has increased the size of biological datasets immensely thus pushing the traditional analysis method to its limits. In this presentation we describe a new automated approach that emulates the decision making process of human experts. This approach combines knowledge-based techniques with human expertise to enable rapid identification, characterization and prioritization of lead candidates.     

Sample Processing with Hydrophobic Microfluidics

Hydrophobic microfluidics is a method for controlling fluid flow in microfluidic systems using short restrictions in channel diameter that act as passive valves. Systems designed using hydrophobic microfluidics have the advantage of easily interfacing with external hardware and integrating with external analysis equipment. This allows it to take advantage of both the micro and macro realms, whichever is most suited for the application, as well as allowing for an inexpensive integration of microfluidics into a company's sample analysis protocols. This method of fluid control is excellent for highly parallel sample analysis, such as DNA processing.     

Detection of Cellular Parameters Using a Microfluidic Chip-Based System

Lab-on-a-chip technology achieves a reduction of sample and reagent volume and automates complex laboratory processes. Here, we present the implementation of cell assays on a microfluidic platform using disposable microfluidic chips. The applications are based on the controlled movement of cells by pressure-driven flow inside networks of microfluidic channels. Cells are hydrodynamically focused and pass the fluorescence detector in single file. Initial applications are the determination of protein expression and apoptosis parameters. The microfluidic system allows unattended measurement of six samples per chip. Results obtained with the microfluidic chips showed good correlation with data obtained using a standard flow cytometer.     

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.     

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.     

Multiplex SNP genotyping using Locked Nucleic Acid and microfluidics

Locked Nucleic Acid's or LNA are a new class of bicyclic DNA analogues that have a high affinity and specificity towards complementary nucleic acids. LNA containing oligonucleotides were used to develop a multiplex SNP genotyping assay based entirely on hybridization between capture probe and target. The approach incorporates a polymer microarray platform, photochemistry for immobilization of oligonucleotides onto microarrays, and a dedicated software tool to aid primer and capture probe design for highly multiplex genotyping. Furthermore, these technologies are combined in an integrated microfluidics platform for simple, highly multiplex and robust SNP genotyping.     

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.     

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.     

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.     

Two-Color Capillary Electrophoresis with On-Column Excitation and Wave-Guide Based Fluorescent Detection

An optical wave-guide based two-color capillary electrophoresis laser induced fluorescence (CE-LIF) instrument is described. The wave-guide based approach allows for on column excitation and detection with two-color discrimination. The instrument is designed to allow either electrokinetic or hydrodynamic injections. In its present configuration, the attainable limit of detection (LOD, S/N = 3) was 50 X 10⁻²¹ moles of fluorescein with a 488-nm excitation source. This study was designed to test the instrument design for applications in protein analyses. Fluorescent dyes with two different wavelengths were simultaneously separated and detected as were complexes formed by labeled antibodies to NFκB p65 and cdc2p34. Quantification of both proteins in THP-1 cell lysates performed using this approach illustrates a rapid screening application of this instrument.     

The First Automated High Content Screening System

With biological discovery and technology advancing in parallel, AstraZeneca (AZ) announced that it would soon take delivery of what is thought to be the first fully automated high-throughput high-content screening system. This custom designed assay platform from RTS Life Science International (RTS) automates the IN Cell Analyzer 3000 sub cellular analysis system from Amersham Biosciences. RTS has integrated its advanced scheduling system with the imaging tool to enable AZ to evaluate the effect of drug compounds on cellular processes.     

“The Spotting Accelerator™”, Customizable Head Assembly For Advanced Microarraying

Microarraying technologies have emerged as key tools for determining genomic expression. The quality of gene arrays is reliant on spotting pins for production of uniform spots, which are consistent in volume, shape, size and alignment.Point Technologies, Inc., has developed an advanced spotting technology. Emphasis has been placed on producing the “perfect spots” customized to each microarraying application. Uniform surface texturing such as highly polished or matte finishes, and hydrophilic/hydrophobic zonal coating is produced using Point Technologies' proprietary micro-machining technology. Microfabrication also permits the production of advanced pin designs, and high density, high throughput print-head assemblies of 1536 pins and greater. Point Technologies specializes in the micro-electrochemical machining of spotting pins in any size, shape and configuration.Presented data will demonstrate spotting pin design solutions, such as: variety of tip dimensions, surface finishes, specialized coatings, and material options instrumental in producing the ideal spot size, shape and spot density during extended usage.     

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 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 RNA Isolation Using the Ambion RNAqueous™-96 Kit with the Packard MutiPROBEII HT Liquid Handling System

Automated isolation of total RNA from cultured cells was accomplished by adapting Ambion's solid-phase total RNA isolation kit, RNAqueous-96, for use with the MultiPROBE II liquid handling system from Packard Instruments.Cultured cells (HeLa S3 adherent cells and K562 cells in suspension) were lysed, DNase treated, washed, and then eluted from the 96-well filter plate.Total RNA recovered was analyzed for yield, intactness, purity, and ability to serve as a substrate for real-time RT-PCR.