October 1, 2005 (Vol. 25, No. 17)
Productivity Often Hinges on Ability to Purify Sample
From biodefense to drug discovery, nucleic acid purification is critical. New technologies being developed for this arena have been the subject of talks at numerous conferences recently.
“The industry’s desire for robust, high throughput assays continues to grow, and a common theme has emerged: process bottlenecks have shifted to sample preparation,” according to Lorah Perlee, director of scientific applications at Protedyne (www.protedyne.com).
“For laboratories performing genomics, clinical, or diagnostic applications, productivity is heavily influenced by the ability to purify quality genomic DNA from whole blood,” Perlee adds.
Protedyne has developed a high throughput, vacuum-based BioCube system utilizing the Qiagen (www.qiagen.com) QIAamp DNA Blood Kit to purify genomic DNA from whole blood.
Designed as a modular automation system, BioCube can evolve as applications are modified and technology advances, according to Perlee, who notes that the system is vacuum only and provides walkaway capability and a vision system based on CCD technology. The system combines laboratory robotic hardware with a software infrastructure for data management and process tracking of DNA yield, purity, and reproducibility between wells and between plates.
Perlee cites a demonstration in which a Protedyne BioCube provided high-throughput, vaccum-based automated purification of 3-6 µg of genomic DNA per well from plates containing 200 µl samples of whole blood with the Qiagen kit.
The methodology enables performing lysis, washing, and elution steps with Protedyne’s automated vacuum manifold assembly; making tool changes during the run; automatically replenishing reagents with refillable reagent reservoirs; preventing protease degradation with chilled deck positions; optimizing cell lysis with heated deck positions; and ensuring data integrity with positive barcode identification, a dedicated LIMS system, and a fully enclosed work envelope, reports Perlee.
The genomic DNA quantity and quality were confirmed by spectrophotometric analysis and agarose gel analysis. PCR experiments demonstrated that the purified samples were suitable for downstream processing.
“BioCube eliminates a critical sample preparation roadblock and improves the number of genomic DNA samples that may enter downstream high throughput assays,” Perlee concludes.
Successful implementation of efficient biodefense surveillance systems depends on several critical factors, according to Helge Lubenow, Ph.D., director of R&D for nucleic acid purification at Qiagen.
“Effective and reliable methods for sample collection, isolation, detection, and identification of potentially pathological agents are crucial in the determination of the severity of diseases such as anthrax, botulinum, and cholera,” says Dr. Lubenow, who spoke at Cambridge Healthtech’s (www.healthtech.com) Systems Integration in Biodefense Conference in Washington, DC, in August.
In Dr. Lubenow’s opinion, standardized systems characterized by a simple uniform workflow ensure minimal operator training and increase process safety and reliability. Flexible automation systems further increase efficacy, avoid any sample-to-sample variation, and minimize human contact to minimize operator risk.
“A major system requirement in biodefense diagnostics is the absence of any false negative results (failing to detect an agent in a threat scenario) as well as the absence of false positives, [to avoid] provoking an emergency response and false alarm,” adds Dr. Lubenow.
“Biodefense-related research and diagnostics in surveillance therefore requires robust, easy-to-use, standardized, and validated procedures to be able to achieve highest reliability, sensitivity and selectivity required.”
Dr. Lubenow maintains that Qiagen employs a set of technologies and formats that are fully compatible with the requirements defined by the major technology trends in biodefense today: process integration, system integration, and microfluidics to enable preparedness.
These technologies, from preanalytics to analytical reagents (chromatography, selective adsorption, filtration, magnetic particle-based selective adsorption, thin membrane technology, hybrid capture, endotoxin removal, whole genome amplification, and real-time PCR), generate highly efficient, validated processes for sample collection, stabilization, and prepararation for the isolation of pure nucleic acid and protein analytes, according to Dr. Lubenow.
Qiagen has instrumentation for sample purification suiting different throughput needs. For example, a low throughput, portable system (the BioRobot EZ1) is employed in research as well as in confirmation testing during sureveillance.
Higher throughput systems, like the BioRobot universal system, can be used during assay development and validation.
“Our expertise in automation makes us a competent companion for the system integration partners developing highly integrated surveillance systems. Sample preparation technologies are available for the purification of both bacterial and viral agents, in manual as well as automated formats,” notes Dr. Lubenow.
DNA purification incorporating a chaotropic salt has been used extensively across a range of application areas, according to John Leaton, of Valco Instruments (www.vici.com). The DNA of interest can be isolated because of its ability to bind silica in the presence of high concentrations of chaotropic salts.
“Chaotropic” refers to a compound’s ability to disrupt the regular hydrogen bond structures in water, thus affecting the secondary structure of polymers such as DNA, RNA, and proteins, as well as increasing the solubility of nonpolar substances in water. While denaturing proteins, they do not denature DNA or RNA.
Lawrence Livermore National Laboratory has demonstrated the feasibility of rapidly detecting and identifying biological agents using its Handheld Advanced Nucleic Acid analyzer (HANAA) that can produce results in less than 30 minutes, according to Leaton, who talked about the technology at the IBC Conference in Boston.
Prior to introducing DNA into the HANAA, it is necessary to lyse cells and remove interferents that suppress the amplification of DNA fragments in order for the PCR to proceed. Valco has teamed up with Global FIA (www.globalfia.com) to automate this process in a high-throughput robotic system incorporating a flow-through zone fluidics manifold.
The fluid manipulation techniques of Global FIA’s mini-Flo Pro handling system, which incorporates the milliGAT pump, process and facilitate the execution of multiple sequential unit operations as required in this sample cleanup procedure. The column is packed with zirconia/silica beads.
Providing advantages over present approaches to precision pumping such as peristaltic pumps and syringe pumps, the device achieves flow rates covering six orders of magnitude from 0.1 nanoliters/sec to 170 microliters/ sec (10 milliliters/min) without changes to the hardware, according to Leaton. Eliminating the refill cycle and thereby increasing throughput, the pump improves the flow-through DNA purification process.
“The sample cleanup module adequately handles the cleanup of DNA samples using standard purification procedures adapted and automated on a standalone mini-FloPro zone fluidics sample prep module,” says Leaton.
“The milliGAT pump is an integral and powerful component in the sample prep module and has tremendous potential for other fluid handling applications.”
In the last five years, there has been a significant increase in the demand for synthetic oligonucleotides, owing to their potential for therapeutic applications, according to Michael McGinley, liquid chromatography product manager of Phenomenex (www. phenomenex.com). Company scientists made a presentation on “Novel Strategies for the High-Throughput Purification of Synthetic Oligonucleotides” at IBC’s TIDES Conference in Boston earlier this year.
“Reversed-phase high-performance liquid chromatography (RP-HPLC) is the most extensively used technology for the cleanup of synthetic oligonucleotides,” says McGinley.
Phenomenex uses trityl or other protecting groups to separate full-length oligonucleotides from impurities. This approach optimizes solvents for effective washing of contaminants and controlled elution of full-length oligos, while providing a multiplex format for automation applications, notes McGinley.
The Triophilic purification technology provides a universal protocol for DNA and RNA purification applicable to all current 5′-protective chemistries. Ninety-six samples can be purified in 1 hour with 95% purity for synthetic DNA and RNA and 95% recovery of full-length product, claims McGinley.
Phenomenex is introducing three new products. Clarity Purification Cartridges, which McGinley describes as “an entirely new technology for doing trityl-on purification,” include a custom-designed, patent-pending Triophilic phase and 3-in-1 buffer system to purify oligonucleotides in a high-throughput parallel format that delivers purities and recoveries on a comparable level to reversed phase and ion exchange protocols. It works for all types of oligonucleotides, including RNA and thioate modified oligos, adds McGinley.
Another product, Clarity Oligo-RP, is a reversed-phase HPLC column specifically designed for the purification of oligonucleotides. The Oligo-RP phase is based on Phenomenex’ TWIN particle technology and specifically addresses those who can only use trityl-off purification protocols.
Finally, Clarity Desalting Cartridges are reversed-phase SPE cartridges for simple oligonucleotide desaltingfor applications that do not require purified oligonucleotides.