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.     

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 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     

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.     

IQ technology: an automated, high-throughput screening assay for kinase, phosphatase, and protease targets

IQ^® Technology, a homogeneous, universal-detection platform, originally designed for high-throughput screening (HTS) of kinases and phosphatases, has now been applied to protease screening. Representative enzymes from the major classes of proteases have been assayed in the IQ^® format. Enzyme activity and compound inhibition data are presented for such enzymes as Trypsin, Matrix Metalloproteinase 3 (MMP-3) and Calpain 1. The technology has been tested in 96- to 384- to 1536-well microplate formats and is universally suited for automated screening. IQ^® Technology is a direct, noncompetitive assay that does not require antibodies or radioisotopes. Fluorophore-labeled peptides are used as enzyme substrates. Kinase or phosphatase activity is quantified by direct measurement of the phosphorylation state of the substrate. For protease activity, cleavage is quantified with a peptide substrate containing a phospho-residue distal to the fluorphore. Cleavage of the substrate liberates the fluorphore-labeled terminus from the terminus containing the phospho-residue. Protease activity is measured by the change in fluorescence intensity that occurs when a proprietary compound binds specifically to phosphoryl groups on peptides and quenches the fluorescence. IQ^® Technology can be used with any peptide sequence and is insensitive to high concentrations of ATP and substrate. The IQ^® Technology has been validated against a large number of detergents, organics, and other reagents found in reaction mixtures and has been optimized for HTS applications exhibiting representative Z' values of 0.7.     

A simple method for validation and verification of pipettes mounted on automated liquid handlers

We have implemented a simple, inexpensive, and fast procedure for validation and verification of the performance of pipettes mounted on automated liquid handlers (ALHs) as necessary for laboratories accredited under ISO 17025. A six- or seven-step serial dilution of OrangeG was prepared in quadruplicates in a flat-bottom 96-well microtiter plate, manually using calibrated pipettes. Each pipette of the liquid handler (1-8) dispensed a selected volume (1-200 μL) of OrangeG eight times into the wells of the microtiter plate. All wells contained a total of 200 μL liquid. The absorbance was read, and the dispensed volume of each pipette was calculated based on a plot of volume and absorbance of a known set of OrangeG dilutions. Finally, the percent inaccuracy (%d) and the imprecision (%CV) of each pipette were calculated. Using predefined acceptance criteria, each pipette was then either approved or failed. Failed pipettes were either repaired or the volume deviation was compensated for by applying a calibration curve in the liquid-handler software. We have implemented the procedure on a Sias Xantus, an MWGt TheONYX, four Tecan Freedom EVO, a Biomek NX Span-8, and four Biomek 3000 robots, and the methods are freely available. In conclusion, we have set up a simple, inexpensive, and fast solution for the continuous validation of ALHs used for accredited work according to the ISO 17025 standard. The method is easy to use for aqueous solutions but requires a spectrophotometer that can read microtiter plates.     

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 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.     

Biomek 3000: the workhorse in an automated accredited forensic genetic laboratory

We have implemented and validated automated protocols for a wide range of processes such as sample preparation, PCR setup, and capillary electrophoresis setup using small, simple, and inexpensive automated liquid handlers. The flexibility and ease of programming enable the Biomek 3000 to be used in many parts of the laboratory process in a modern forensic genetics laboratory with low to medium sample throughput. In conclusion, we demonstrated that sample processing for accredited forensic genetic DNA typing can be implemented on small automated liquid handlers, leading to the reduction of manual work as well as increased quality and throughput.     

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.     

Droplet enhanced microfluidic-based DNA purification from bacterial lysates via phenol extraction

Microfluidic platforms have been developed to demonstrate DNA purification via liquid extraction techniques at the microscale using an aqueous phase containing either protein, DNA, or a complex cell lysate and an immiscible receiving organic (phenol) phase. Initially, a serpentine device was used to investigate protein partitioning between the aqueous and organic phase, and DNA purification when both protein and DNA were mixed in the aqueous phase and infused conjunctly with the phenol phase. This two-phase system was studied using both stratified and droplet-based flow conditions. The droplet-based flow resulted in a significant improvement of protein partitioning from the aqueous phase into the organic phase due to the convective flow recirculation inside each droplet improving material transport to the organic–aqueous interface. A second device was designed and fabricated to specifically extract plasmid DNA from bacterial lysates using only droplet-based flows. The plasmid recovery using the microdevice was high (>92%) and comparable to the recovery achieved using commercial DNA purification kits and standard macroscale phenol extraction. This study presents the initial steps toward the miniaturization of an efficient on-chip DNA sample preparation using phenol extraction which could be integrated with post-extraction DNA manipulations for integrated genomic analysis modules.     

A completely automated sample preparation instrument and consumable device for isolation and purification of nucleic acids

Molecular diagnostic analysis and life science studies are dependent on the ability to effectively prepare samples for analysis. We report the development of a system that enables robust sample preparation of nucleic acids. To enable completely automated sample preparation, a consumable cartridge and consumable module system were developed to emulate every step of the sample preparation process. This included enzyme and reagent addition, temperature-controlled incubations, noncontact mixing of enzymes and reagents, buffer exchanges, and sample elution. Using this system, completely automated methods were developed for the purification of viral RNA and DNA from plasma and whole blood and of bacterial genomic DNA from water and whole blood. Extracted nucleic acids were detected and quantified using real-time PCR. The data indicate that automated viral DNA extraction was more efficient than sample extractions performed using a manual process, whereas automated total RNA extraction from the same samples was equivalent to controls. Additionally, we found that the process for bacterial genomic DNA extraction from either water or whole blood was equivalent to the manual extraction processes. We conclude the instrument, consumable cartridge, and reagent system enables easy, cost-effective, and robust sample preparation regardless of the experience of the operator.     

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.     

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.     

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.     

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).     

Developing an Automated Enzyme Immunoassay for High-Throughput Screening of Bovine Serum Antibodies to Neospora Caninum

An enzyme-linked immunosorbent assay (ELISA) for Neospora caninum antibodies was automated with a robotic workstation, the Beckman Coulter Biomek 2000, to screen 200 bovine sera. Comparing these results with manually run ELISA data, a 95.92% agreement (K = 0.9592) between the two assays was obtained. The automated assay was specific and sensitive with excellent positive and negative predictive values. The results were repeatable and reproducible. The automation flexibility was high and the operation complexity was minimal. High-throughput screening (HTS) for bovine antibodies to Neospora caninum was achieved. The assay was developed according to the internationally recognized ISO17025 standard requirements.     

High Throughput Primer Walking of cDNA Clones

Primer walking of cloned DNA is a standard research tool. It has been used in the past to determine the sequence of individual clones of interest. With the expansion of DNA sequencing capacity the need to be able to walk larger numbers of clones has become necessary. Our laboratory is a mid-sized genomics facility. In conjunction with the Advanced Biomedical Computing Center (ABCC) we have developed methods for automating the primer selection, DNA sequencing, contig assembly and sequence analysis for clones arrayed in microtiter format. This approach has allowed us to walk 475 clones (five microtiter plates) selected from a cDNA library.     

Assay on FAQ''s on Total Lab Automation

The purpose of the introduction of an automated laboratory system in one format or another is reducing the costs of the laboratory by cutting staff. Also speeding up the performance often is a goal. At the same time it is planned to increase the quality of the work done by using automated labelling and reading of documents. The use of vacuum tubes and the handling of these by the apparatus are raising the safety of the labwork.

In this paper many aspects of the daily routines when using a TLA system are discussed. The aspects are collected during the discussions before and after the implementation of the Roche CLAS system in our laboratory. Some consequences were foreseen, and others came unexpectedly. But still the subjects form a point of discussion around the TLA systems. The most important message to be picked up is that one really has to consider all possible problems that could arise from the implementation of TLA.