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.     

Quantitative small molecule bioanalysis using chip-based nanoESI-MS/MS

A fully automated chip-based nanoelectrospray (nanoESI) system, NanoMate^® 100 (Advion BioSciences, Inc., Ithaca, NY), was evaluated for its application on quantitative bioanalysis of small molecules in support of exploratory pharmacokinetic (PK) studies. The NanoMate^® 100 was compared with the conventional autosampler coupled with liquid chromatography-electrospray (LC-ESI) interface. An API^® 3000 triple quadrupole mass spectrometer (Applied Biosystems, Inc., Foster City, CA) was used for the evaluation. The results show that the NanoMate^® 100 performs comparably to LC-ESI in terms of standard curve fitting, low limit of quantitation (LLOQ), dynamic range, accuracy, and precision. Parallel analyses of exploratory PK study samples show high correlation (R² = 0.971) between the NanoMate^® 100 and the LC-ESI. The NanoMate^® 100 exhibits advantages in carryover, sample consumption, sample cycle time, and the ability to be full automated. Despite these advantages, the necessarily rigorous sample preparation process limits the application of the NanoMate^® 100 for quantitative analysis in areas such as exploratory PK studies, which often involve multiple compounds in one study and require rapid turnaround. However, the NanoMate^® 100 has great potential in qualitative work (e.g., metabolite identification) as well as in high-throughput quantitative analysis of compound in the development stage (i.e., a single analyte with a well-established sample extraction method).     

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.     

Single Molecule Detection at Interfaces for Applications in High Throughput Screening

One of the most important issues in bioanalytical science is the reproducible ultra-sensitive detection of biomolecules. At present an increasing demand for routine applications is observed for a lot of tasks in pharmaceutical high throughput screening, medical diagnostics and food and environmental analysis. We developed a detection system based on confocal laser-induced fluorescence spectroscopy. The components consist of an apparatus - the so called LB8®-Counter - and specially coated glass bottom microplates, the LB8®-Plates. Together they generate molecular recognition events of biomolecules even down to the single molecule level.     

Virtual screening of novel reversible inhibitors for marine alkaline protease MP

Marine alkaline protease (MP,² accession no. ACY25898) is produced by a marine bacterium strain isolated from Yellow Sea sediment in China. Previous research has shown that this protease is a cold-adapted enzyme with antioxidant activity that could be used as a detergent additive. Owing to its instability in the liquid state, MP's application in liquid detergents was limited. Therefore, the discovery of reversible MP inhibitors to stabilize the protease was imperative. Here, we used the X-ray structure of MP and recompiled AutoDock 4.2 with refined Zn²⁺ characters to screen the free chemical database ZINC. After completing the docking procedure, we applied strategies including the “initial filter”, consensus scoring and pharmocophore model to accelerate the process and improve the virtual screening success rate. The “initial filter” was built based on the docking results of boronic acid derivatives validated as reversible inhibitors of MP by our previous studies. Finally, ten compounds were purchased or synthetized to test their binding affinity for MP. Three of the compounds could reversibly inhibit MP with apparent K_i values of 0.8–1.2 mmol. These active compounds and their binding modes provide useful information for understanding the molecular mechanism of reversible MP inhibition. The results may also serve as the foundation for further screening and design of reversible MP inhibitors.     

A compiler for exploiting nested parallelism in OpenMP programs

This paper presents the design and implementation of a parallelization framework and OpenMP runtime support in Intel^® C++ & Fortran compilers for exploiting nested parallelism in applications using OpenMP pragmas or directives. We conduct the performance evaluation of two multimedia applications parallelized with OpenMP pragmas and compiled with the Intel C++ compiler on Hyper-Threading Technology (HT) enabled multiprocessor systems. The performance results show that the multithreaded code generated by the Intel compiler achieved a speedup up to 4.69 on 4 processors with HT enabled for five different input video sequences for the H.264 encoder workload, and a 1.28 speedup on an HT enabled single-CPU system and 1.99 speedup on an HT-enabled dual-CPU system for the audio–visual speech recognition workload. The performance gain due to exploiting nested parallelism for leveraging Hyper-Threading Technology is up to 70% for two multimedia workloads under different multiprocessor system configurations. These results demonstrate that hyper-threading benefits can be achieved by exploiting nested parallelism through Intel compiler and runtime system support for OpenMP programs.     

FactSage thermochemical software and databases — recent developments

FactSage^® was introduced in 2001 as the fusion of the F*A*C*T/FACT-Win and ChemSage thermochemical packages. The FactSage package runs on a PC operating under Microsoft Windows^® and consists of a series of information, database, calculation and manipulation modules that enable one to access and manipulate pure substances and solution databases. With the various modules one can perform a wide variety of thermochemical calculations and generate tables, graphs and figures of interest to chemical and physical metallurgists, chemical engineers, corrosion engineers, inorganic chemists, geochemists, ceramists, electrochemists, environmentalists, etc. This paper presents a summary of the recent developments in the FactSage thermochemical software and databases. In the article, emphasis is placed on the new databases and the calculation and manipulation of phase diagrams and complex phase equilibria.     

Mosquito: an accurate nanoliter dispensing technology

Mosquito^® from TTP LabTech Ltd. is an innovative nanoliter dispenser that combines the liquid transfer capability of a fixed head pipette with the elimination of cross-contamination, using disposable tips. For many applications required in genomics, proteomics and drug discovery, Mosquito can reduce assay cost by minimizing reagent and sample usage.     

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.     

Selective Ion Extraction for High Throughput Screening with a Microfluidic Enzyme Assay

This proof-of-concept paper describes the application of selective ion extraction to an assay of protein kinase A on a microfluidic chip platform. Selective ion extraction is a flux balance technique, where a combination of independent pressure control and voltage are used to selectively extract one ion from a mixture. The assay product is completely separated and diverted into a separate channel from the waste stream containing the unconverted substrate and enzyme. By detecting only product, background noise generated by the substrate is removed which increases the signal to noise ratio and assay sensitivity. This technique is intended for adapting kinase or protease assays with low conversion rates to an on-chip reaction format for HTS screening.     

Systematic profiling of substrate binding response to multidrug-resistant mutations in HIV-1 protease: Implication for combating drug resistance

Human immunodeficiency virus 1 (HIV-1) protease (PR) represents one of the primary targets for developing antiviral agents for the treatment of HIV-infected patients. However, a number of multidrug-resistant mutations in the enzyme have been observed over the past decades, largely limiting the application of PR inhibitors in antiviral therapy. A systematic investigation of the intermolecular interaction between the multidrug-resistant mutants of HIV-1 PR and its substrates would help to establish a complete profile of substrate response to PR mutations and to design new antiviral agents combating drug resistance. Here, we describe an integrative method to profile 6 clinical multidrug-resistant PR mutants against a panel of 16 substrate octapeptides that flank 12 distinct PR cleavage sites in viral precursor polyproteins. It is found that most multidrug-resistant mutations have only a modest or moderate effect on substrate peptide binding, although these mutations would cause a large free energy loss in PR inhibitor binding. Structural analysis reveals that the substrate peptides are loosely bound within PR active pocket to form a wide contact interface between them, and thus mutation of just single or few residues seems not to influence PR–substrate binding considerably. In addition, peptides derived from variable cleavage sites are generally more sensitive to the mutations as compared to those derived from conserved sites, supporting the co-evaluation mechanism of HIV-1 PR and its substrates under drug suppression. We also identify 12 functionally conserved key residues around the enzyme’s active site, which play crucial role in substrate recognition. In vitro fluorescence anisotropy assays confirm that wild-type PR can bind substrate peptides ARVL/AEAM and NLAF/PQGE with a moderately high affinity (K_D = 2 and 16 μM, respectively). In contrast, the key residue mutations N25D/D29N can completely eliminate (K_D = n.d.) or largely reduce (K_D = 32 and 120 μM, respectively) the binding capability of the two peptides, suggesting that these PR residues could be the potential target sites for developing resistance-free anti-HIV agents.     

MultiSpec—a tool for multispectral–hyperspectral image data analysis

MultiSpec is a multispectral image data analysis software application. It is intended to provide a fast, easy-to-use means for analysis of multispectral image data, such as that from the Landsat, SPOT, MODIS or IKONOS series of Earth observational satellites, hyperspectral data such as that from the Airborne Visible–Infrared Imaging Spectrometer (AVIRIS) and EO-1 Hyperion satellite system or the data that will be produced by the next generation of Earth observational sensors. The primary purpose for the system was to make new, otherwise complex analysis tools available to the general Earth science community. It has also found use in displaying and analyzing many other types of non-space related digital imagery, such as medical image data and in K-12 and university level educational activities.MultiSpec has been implemented for both the Apple Macintosh^® and Microsoft Windows^® operating systems (OS). The effort was first begun on the Macintosh OS in 1988. The GLOBE (http://www.globe.gov) program supported the development of a subset of MultiSpec for the Windows OS in 1995. Since then most (but not all) of the features in the Macintosh OS version have been ported to the Windows OS version.Although copyrighted, MultiSpec with its documentation is distributed without charge. The Macintosh and Windows versions and documentation on its use are available from the World Wide Web at URL:http://dynamo.ecn.purdue.edu/biehl/MultiSpec/MultiSpec is copyrighted (1991–2001) by Purdue Research Foundation, West Lafayette, Indiana 47907.     

Laboratory Evaluation of Representative Disease State Assays on the Abbott ARCHITECT® i2000® Analyzer

We report here the results of our evaluation of representative disease state assays on the Abbott ARCHITECT® i2000® analyzer. The ARCHITECT i-series is a family of immunoassay analyzers under development by Abbott Laboratories (North Chicago, IL, USA). The first instrument available from this family is the i2000 analyzer. The i2000 analyzer is a random access, modular instrument with a maximum throughput of 200 tests per hour and a time to first result of 29 minutes. It is capable of running two step, one step, and automated pretreatment protocols. Modular instrument design allows multiple i2000 analyzers to be combined to form i4000®, i6000®, and i8000® analyzers with maximum throughputs of 400, 600, and 800 tests per hour, respectively. As either an individual module or multiple integrated modules, the instrument is run by one operator using a single system control center (SCC). The SCC has Windows NT based data management software that is touch screen driven. All assays utilize paramagnetic microparticles for analyte capture, and chemiluminescent detection based on a new class of acridinium compound (sulfonyl acridinium carboxamides) with improved aqueous solubility and stability. The i2000 analyzer has onboard, refrigerated storage for up to 25 reagent kits per module, and reagents are available for a wide range of assays. The purpose of our study was to perform a laboratory evaluation of representative thyroid (TSH, Free T4), fertility (Total β-hCG, FSH, LH, Prolactin, Testosterone) and metabolic (Ferritin) assays on the i2000 analyzer. Our evaluation of these assays included sensitivity, precision, and method comparison testing. In addition, we performed a preliminary evaluation of three i2000 tumor marker assays (Total PSA, Free PSA, and CEA) by comparing specimen correlation to other commercially available methods.     

Prediction of MHC II-binding peptides using rough set-based rule sets ensemble

Peptide binding to Major Histocompatibility Complex (MHC) is a prerequisite for any T cell-mediated immune response. Predicting which peptides can bind to a specific MHC molecule is indispensable to minimizing the number of peptides required to synthesize, to the development of vaccines and immunotherapy of cancer, and to aiding to understand the specificity of T-cell mediated immunity. At present, although predictions based on machine learning methods have good prediction performance, they cannot acquire understandable knowledge and prediction performance can be further improved. Thereupon, the Rule Sets ENsemble (RSEN) algorithm, which takes advantage of diverse attribute and attribute value reduction algorithms based on rough set (RS) theory, is proposed as the initial trial to acquire understandable rules along with enhancement of prediction performance. Finally, the RSEN is applied to predict the peptides that bind to HLA-DR4(B1^* 0401). Experimentation results show: (1) prepositional rules for predicting the peptides that bind to HLA-DR4 (B1^* 0401) are obtained; (2) compared with individual RS-based algorithms, the RSEN has a significant decrease (13%–38%) in prediction error rate; (3) compared with the Back-Propagation Neural Networks (BPNN), prediction error rate of the RSEN decreases by 4%–16%. The acquired rules have been applied to help experts make molecules modeling. An Zeng received the Ph.D. degree in computer applications technology from South China University of Technology in 2005. Nowadays she is a lecturer at the Faculty of Computer of Guangdong University of Technology. Her research interests are data mining, bioinformatics, neural networks, artificial intelligence, and computational immunology. In these areas she has published over 20 technical papers in various prestigious journals or conference proceedings. She is a member of the IEEE. Contact her at the Faculty of Computer, Guangdong Univ. of Technology, University Town, PanYu District, Guangzhou, 510006, P.R. China. Dan Pan received the Ph.D. degree in circuits and systems from South China University of Technology in 2001. He is a senior engineer in Guangdong Mobile Communication Co. Ltd at present. His research interests are data mining, machine learning, bioinformatics, and data warehousing, and applications of business modeling and software engineering to computer-aided business operations systems, especially in the telecom industry. In these areas he has published over 30 technical papers in refereed journals or conference proceedings. As a member of the International Association of Science and Technology for Development (IASTED) technical committee on artificial intelligence and expert systems, he served a number of conferences and publications. He is a member of the IEEE. Contact him at Guangdong Mobile Communication Co. Ltd., 208 Yuexiu South Rd., Guangzhou, 510100, P.R. China. Jian-bin He received the M.E. in computer science from South China University of Technology in 2002. He now is a data mining consultant at Teradata division of NCR (China), supporting telecom carriers to do data mining in data warehouses for market research. His research interests include statistical learning, semi-supervised learning, spectral clustering, multi-relational data mining and their application to social science. Contact him at NCR(China) Co. Ltd., Unit 2306, Tower B, Center Plaza, 161 Linhexi Road, Guangzhou, 510620, P.R. China.     

Book Reviews

Books reviewed: Sutton, John, Technology and Market Structure: Theory and History Price, If and Shaw, Ray, Shifting the patterns: breaching the memetic codes of corporate performance Murphy, Emmett C., Leadership IQ Cook, Peter, Best practice creativity Stern, C.W. and Stalk jr, G. (eds), Perspectives on strategy from the Boston Consulting Group Dingli, Sandra (ed), Creative thinking: towards broader horizons     

Model of high-temperature superconducting coupled microstrip lines on anisotropic sapphire substrate

A semiempirical model is proposed for propagation parameters of high-T_C superconducting (HTS) coupled microstrip lines taking into account anisotropic dielectric permittivity of sapphire substrate and its temperature dependence. The nonhomogeneous current distribution in a cross-section of the lines is considered for attenuation coefficient calculation. The current density distribution in the coupled strips and in the ground plane is found for the even and odd modes. The model is verified by a comparison with full-wave analysis results and experimental investigations of coupled microstrip line HTS resonators. © 1998 John Wiley & Sons, Inc. Int J RF and Microwave CAE 8: 375–385, 1998.     

Modeling the transport of ions in unsaturated cement-based materials

This paper gives the details of the multiionic transport model STADIUM^® intended to describe the degradation of cement-based materials exposed to aggressive environments. The main algorithm is based on an operator splitting approach. The first part of the calculations is dedicated to solving the transport equations without considering the chemical reactions. The concentration profile of each ionic species is calculated by taking into account diffusion, electrical coupling between the ions, chemical activity effects and advection caused by a capillary suction flow. In the second part, a chemical module corrects the concentration profiles to enforce the equilibrium between the pore solution and the various solid phases of an hydrated cement paste. The model is compared to experimental results of hydrated cement pastes exposed to pure water and sodium sulfate solutions. Long term simulations were also performed to analyze the behavior of the algorithm.     

Automation and ISO 17025 test validation of a Toxoplasma Gondii antibodies enzyme-linked immunosorbent assay

The authors automated an enzyme-linked immunosorbent assay to detect porcine serum antibodies to Toxoplasma gondii. Two thousand swine sera can be assayed in two eight-hour shifts using a robotic workstation. The automated-ELISA programming is not complex and the test configuration is flexible. This high-throughput screening (HTS) a-ELISA can achieve a 10-fold increase (100→1000 tests) in test capacity over the manual method. The assay has been validated according to the requirements of the ISO/IEC 17025 standard. These include repeatability, reproducibility, and optimal threshold value studies. Other requirements are proficiency panel testing, analyst training, standard operation procedure, and equipment certifications.     

Electromagnetic design of high-temperature superconducting microwave filters

We present novel approaches to electromagnetic design of high-temperature superconducting (HTS) quarter-wave parallel coupled-line microstrip filters. The desired narrow bandwidths (less than 2%) coupled with the large dielectric constant of substrate materials used in the HTS technology (ε_r ≈ 24) make the design problems difficult to treat accurately for many traditional microwave circuit design software packages with analytical/empirical models. We have successfully performed an HTS filter design with accurate electromagnetic field simulations. We discuss problems related to electromagnetic design of such filters. We describe a look-up table method and a powerful space mapping optimization technique, which dramatically reduce the CPU time needed in the design process. Finally, we address the issue of improved modeling of the HTS filter using analytical/empirical models. © 1995 John Wiley & Sons, Inc.     

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.