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 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.     

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 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 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.     

A Fully Automated High-throughput Nucleic Acid Purification System Using Silica Coated Magnetic Bead Technology

The design of a fully automated high-throughput system for the purification of sequencing templates is described. Hardware, software, and chemistries have been optimized to suit the needs of high-throughput laboratories involved in genome sequencing projects. Using this system, up to 5760 samples (60 x 96-well plates) can be purified in less than 16 hours during a single unattended run. The system can also be configured to perform sequencing reaction setup for all 60 plates following template purification, extending total run times to < 24 hours. Final sequencing reactions are prepared in 384- well microplates.     

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.     

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     

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.     

DNA sequencing and string learning

In laboratories the majority of large-scale DNA sequencing is done following theshotgun strategy, which is to sequence large amount of relatively short fragments randomly and then heuristically find a shortest common superstring of the fragments [26]. We study mathematical frameworks, under plausible assumptions, suitable for massive automated DNA sequencing and for analyzing DNA sequencing algorithms. We model the DNA sequencing problem as learning a string from its randomly drawn substrings. Under certain restrictions, this may be viewed as string learning in Valiant's distribution-free learning model and in this case we give an efficient learning algorithm and a quantitative bound on how many examples suffice. One major obstacle to our approach turns out to be a quite well-known open question on how to approximate a shortest common superstring of a set of strings, raised by a number of authors in the last 10 years [9], [29], [30]. We give the firstprovably good algorithm which approximates a shortest superstring of lengthn by a superstring of lengthO(n logn). The algorithm works equally well even in the presence of negative examples, i.e., when merging of some strings is prohibited. Some of the results presented in this paper appeared in theProceedings of the 31st IEEE Symposium on the Foundations of Computer Science, pp. 125–134, 1990 [21]. The first author was supported in part by NSERC Operating Grant OGP0046613. The second author was supported in part by NSERC Operating Grants OGP0036747 and OGP0046506.     

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.     

DNA-BASED STEGANOGRAPHY

This paper presents an implementation of steganography using DNA molecules. We first encode a plaintext message into a DNA sequence using a randomly generated single-substitution key. An oligonucleotide containing the encoded message, designated the message strand, is synthesized and mixed with a large amount of background DNA. To retrieve the message, the intended recipient must know the sequences of two primers that anneal to target regions present on the message strand. Polymerase chain reaction (PCR) and sequencing are used to retrieve the encoded sequence, which is decoded into the original plaintext via the single substitution key. This study shows that the steganographically hidden message can be retrieved only by using the two secret primers, meaning that the only applicable cryptanalytic approach is a brute-force search for the two primer sequences. Since each primer can have 4²⁰ different possible sequences, the amount of time required to crack DNA-based steganography is long enough to qualify the technique as essentially unbreakable.     

Automatic detection of Salmonella enterica in sprout irrigation water using a nucleic acid sensor

A nucleic acid sensor capable of automated sample and reagent loading, real-time PCR, automated detection, and sample line cleaning was tested. Real-time PCR reactions were performed with Salmonella enterica in autoclaved and spent alfalfa sprout irrigation water. S. enterica boiled cells were detected over a range of approximately 10⁴ to 10⁸ CFU/reaction (rxn). It was possible to generate enough PCR product to visualize a band on a gel at the expected size over approximately five orders of magnitude from 3.2 × 10³ to 10⁸ CFU/rxn. Automated detection experiments yielded correct identification of 9/9 positive control reactions over a range of 10⁴ to 10⁸ CFU/rxn, correctly identified a negative control reaction, and a sample of 3.2 × 10³ CFU/rxn was incorrectly identified as negative. Primer dimers were not seen in positive or negative control reactions with sprout irrigation water, suggesting that it may be possible to improve the detection limit simply by increasing the number of thermal cycles or by lowering the annealing temperature. The system required no interpretation of real-time PCR data by the operator. The entire process of loading, running the PCR, automated data interpretation, and sample line cleaning was completed in under 2 h and 20 min, significantly faster than it would take to ship a sample and have it tested by an independent laboratory.     

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.     

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).     

Job sequencing with continuous machine operation

This paper presents the methods for minimizing the total processing time of the $\text{n-}\text{job}$, $\text{m-}\text{machine}$ sequencing problem. To commence working it must continue processing consecutive jobs without having to wait between 2 jobs. The problem can be formulated as a linear programming problem which requires a large number of variables and constraints. However, when the same job sequence is to be processed on each machine, a branch-and-bound procedure for solving the problem is proposed. The 2 methods are used to solve a sample problem and the results are given.     

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.     

Improved $${{\ell }^{1}}$$-tracker using robust PCA and random projection

In this paper, we propose an improved \({{\ell }^{1}}\)-tracker in a particle filter framework using robust principal component analysis (robust PCA) and random projection. At first we redesign the template set and its update scheme. Three target templates and several background templates combined with the trivial templates are used to represent the candidate images sparsely. One fixed target template is generated from the image patch in the first frame. The other two are dynamic target templates, called stable target template, and fast changing one used for long time and short time, respectively. Robust PCA is used to generate and update the stable target template, and fast changing target template is initialized by the stable one at certain times. The background templates are used to strengthen the ability of distinguishing background and foreground. Then, the large set of Haar-like features are extracted and compressively sensed with a very sparse measurement matrix for the \({{\ell }^{1}}\)-tracker framework. The compressive sensing theories ensure that the sensed features preserve almost all the information of the original features. Our proposed method is more robust than the original \({{\ell }^{1}}\)-method. Experiments have been done on numerous sequences to demonstrate the better performance of our improved tracker.     

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 digital microfluidic sample preparation for next-generation DNA sequencing

Next-generation sequencing (NGS) technology is a promising tool for identifying and characterizing unknown pathogens, but its usefulness in time-critical biodefense and public health applications is currently limited by the lack of fast, efficient, and reliable automated DNA sample preparation methods. To address this limitation, we are developing a digital microfluidic (DMF) platform to function as a fluid distribution hub, enabling the integration of multiple subsystem modules into an automated NGS library sample preparation system. A novel capillary interface enables highly repeatable transfer of liquid between the DMF device and the external fluidic modules, allowing both continuous-flow and droplet-based sample manipulations to be performed in one integrated system. Here, we highlight the utility of the DMF hub platform and capillary interface for automating two key operations in the NGS sample preparation workflow. Using an in-line contactless conductivity detector in conjunction with the capillary interface, we demonstrate closed-loop automated fraction collection of target analytes from a continuous-flow sample stream into droplets on the DMF device. Buffer exchange and sample cleanup, the most repeated steps in NGS library preparation, are also demonstrated on the DMF platform using a magnetic bead assay and achieving an average DNA recovery efficiency of 80%±4.8%.