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.     

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.     

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.     

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 two-stage electrophoretic microfluidic device for nucleic acid collection and enrichment

Sample purification and enrichment is an important and usually time-consuming step for on-chip nucleic acid detection and analysis. This paper presents an electrophoretic DNA focusing method in microfluidic devices to enrich nucleic acid concentration by around 2700-fold. The electrical waveforms applied to five individual electrodes are such designed that DNAs move successively to the collection electrodes at high speed, while the interferences from bubbles due to electrohydrolysis are minimized. In a spiral channel with a total length of 48 cm, 1 ml DNA sample is purified and enriched by 57 times at a flow rate of 30 μl/min at first. The captured DNAs are then released and transported to the second microfluidic chamber where DNAs are collected and concentrated by 49 times. Thus, in about 40 min, the two-stage device can extract DNAs from 1 ml sample volume and enrich its concentration by 2790-fold. A trade-off exists between the process throughput and the DNA collection efficiency. A DNA capture efficiency of 99.7 % is reached when the flow rate is 1 μl/min, and the maximum DNA capture throughput is achieved at a flow rate of 30 μl/min. As a platform technology, the device can be integrated into bio-sensing and genetic analysis assays for DNA extraction and pre-concentration.     

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.     

A magnetic bead-based DNA extraction and purification microfluidic device

This article introduces a novel magnetic bead-based DNA extraction and purification device using active magnetic mixing approach. Mixing and separation steps are performed using functionalised superparamagnetic beads suspended in cell lysis buffer in a circular chamber that is sandwiched between two external magnetic coils. Non-uniform nature of magnetic field causes temporal and spatial distribution of beads within the chamber. This process efficiently mixes the lysis buffer and whole blood in order to extract DNA from target cells. Functionalized surface of the magnetic beads then attract the exposed DNA molecules. Finally, DNA-attached magnetic beads are attracted to the bottom of the chamber by activating the bottom magnetic coil. DNA molecules are extracted from magnetic beads by washing and re-suspension processes. In this study, a circular PMMA microchamber, 25 μL in volume, 500 μm in depth and 8 mm in diameter was fabricated to purify DNA from spiked bacterial cell cultures into the whole blood sample using Promega Magazorb DNA extraction kit. The lysis efficiency was evaluated using a panel of Gram-positive (Bacillus subtilis) and Gram-negative (Escherichia coli) bacterial cells cultures into the blood sample to achieve approximately 100,000 copy levels inside the chip. Manufacturer’s standard extraction protocol was modified to a more simplified process suitable for chip-based extraction. The lysis step was performed using 5 min incubation at 56 °C followed by 5 min incubation at room temperature for binding process. Temperature rise was generated and maintained by the same external magnetic coils used for active mixing. The yield/purity and recovery levels of the extracted DNA were evaluated using quantitative UV spectrophotometer and real-time PCR assay, respectively. Real-time PCR results indicated efficient chip-based bacterial DNA extraction using modified extraction protocol comparable to the standard bench-top extraction process.     

Fabrication of DNA purification microchip integrated with mesoporous matrix based on MEMS technology

This paper presents a novel microfabricated DNA purification microfluidic chip with enhanced performance based on the principle of micro solid phase extraction. The microchip comprises a layer of mesoporous material as solid phase matrix which is fabricated on the internal wall of the channel of microfluidic chip by electrochemical etching Si in an electrolyte. The conditions of electrochemical etching and porosity of the mesoporous matrix have been investigated. The properties of mesoporous matrix have been characterized by scanning electron microscopy and by BET (Brunauer, Emmet, and Teller) nitrogen adsorption analysis. The pore size of the mesoporous matrix is in the range of 10–30 nm, and the surface area is about 300 m²/g. Compared with the microfluidic chips with micropillar array matrix or non-porous matrix, the microchip with mesoporous matrix is able to extract enough polymerase chain reaction-amplifiable DNA from cultures of rat mesenchymal stem cells in 20 min. This highly efficient, effortless, and flexible technology can be used as a lab-on-a-chip component for initial biologic sample preparation.     

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.     

基于纳米孔读取的 DNA 存储发展与展望

现代社会正处于数据爆炸的时代,全球对数据存储的需求已经远远超过了已有的存储能力。DNA 是一种天然的遗传信息载体,可实现稳定、高效、低功耗的数据存储。目前的 DNA 存储过程主要分为 6 个环节：编码、写入、保存、检索、读取、解码。纳米孔测序技术被广泛应用于读取 DNA 中所存储的信息。该文系统性地介绍了纳米孔分子检测技术的原理和发展历史,及其在 DNA 存储中的应用。此外,该文总结了机器学习在纳米孔检测技术中的应用,着重介绍了结合机器学习的新型纳米孔检测技术。该文为纳米孔检测技术的发展提供了新方向,也为新型实用化 DNA 存储系统的发展奠定了基础。     

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.     

Extraction of genomic DNA and detection of single nucleotide polymorphism genotyping utilizing an integrated magnetic bead-based microfluidic platform

This study presents a new magnetic bead-based microfluidic platform, which integrates three major modules for rapid leukocytes purification, genomic DNA (gDNA) extraction and fast analysis of genetic gene. By utilizing microfluidic technologies and magnetic beads conjugated with CD_15/45 antibodies, leukocytes in a human whole blood sample can be first purified and concentrated, followed by extraction of gDNA utilizing surface-charge switchable, DNA-specific, magnetic beads in the lysis solution. Then, specific genes associated with genetic diseases can be amplified by an on-chip polymerase chain reaction (PCR) process automatically. The whole pretreatment process including the leukocytes purification and gDNA extraction can be performed in an automatic fashion with the incorporation of the built bio-separators consisting of microcoils array within less than 20 min. The detection of single nucleotide polymorphism (SNP) genotyping of methylenetetra-hydrofolate reductase (MTHFR) C677T region associated with an increased risk of genetic diseases was further performed to demonstrate the capability of the proposed system. The extracted gDNA can be transported into a micro PCR chamber for on-chip fast nucleic acid amplification of detection genes with minimum human intervention. Hence, the developed system may provide a powerful automated platform for pretreatment of human leukocytes, gDNA extraction and fast analysis of genetic gene.     

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 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     

压电石英晶体传感器在单链及乙肝病毒检测中的应用

建立压电石英晶体传感器检测乙肝血液样品的方法。在石英晶体表面固定单链DNA,构建压电晶体DNA生物传感器,然后,分别与乙肝病毒的特异性片段和处理过的乙肝血液样品进行杂交反应。DNA传感器能较好地诊断出血清中的HBV病毒基因。压电石英晶体传感器操作方便、省时,且具有较好的准确度,在临床诊断中有很好的前景。     

Microfluidic devices for sample pretreatment and applications

With the fundamentals of microscale flow and species transport well developed, the recent trend in microfluidics has been to work towards the devices capable of dealing with ‘real’ crude samples directly. Before the real samples reaching the analytical step, it nearly requires significant pre-treated steps. Development of these microdevices that perform sample preprocessing steps is now underway for a broad range of application areas from on-chip DNA analysis, immunoassays to cytometry. This article provides an overview of the latest developments in microfluidic devices for sample pretreatment. Many of the microchips are being designed for individual preprocessing steps, but integration of multiple sample preparation steps has been shown, along with integration of sample preprocessing and analytical procedures on single microchips.     

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.     

Implementation and development of an automated, ultra-high-capacity, acoustic, flexible dispensing platform for assay-ready plate delivery

Compound management faces the daily challenge of providing high-quality samples to drug discovery. The advent of new screening technologies has seen demand for liquid samples move toward nanoliter ranges, dispensed by contactless acoustic droplet ejection. Within AstraZeneca, a totally integrated assay-ready plate production platform has been created to fully exploit the advantages of this technology. This enables compound management to efficiently deliver large throughputs demanded by high-throughput screening while maintaining regular delivery of smaller numbers of compounds in varying plate formats for cellular or biochemical concentration-response curves in support of hit and lead optimization (structure-activity relationship screening). The automation solution, CODA, has the capability to deliver compounds on demand for single- and multiple-concentration ranges, in batch sizes ranging from 1 sample to 2 million samples, integrating seamlessly into local compound and test management systems. The software handles compound orders intelligently, grouping test requests together dependent on output plate type and serial dilution ranges so that source compound vessels are shared among numerous tests, ensuring conservation of sample, reduced labware and costs, and efficiency of work cell logistics. We describe the development of CODA to address the customer demand, challenges experienced, learning made, and subsequent enhancements.     

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.