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Feature Articles : Oct 1, 2005 ( )
Manipulation and Analysis of Nucleic Acids
Growth of Molecular Diagnostic Testing Spurs Demand for Isolation Products!--h2>
The growing interest in molecular diagnostics as well as genomic and gene-expression analysis is accelerating demand for products related to manipulation and analysis of nucleic acids.
Since nucleic acid isolation is among the most technically demanding and labor-intensive procedures performed in molecular diagnostic laboratories, the incorporation of automated instrumentation for carrying out high throughput sequencing has eliminated human errors and addressed contamination-related problems while increasing productivity and resource savings.
Manufacturers of such automated systems need to primarily target end users in the clinical diagnostics segments that require extremely pure and highly reproducible separation technologies.
Automated Membrane-based System
The shift in focus from sequencing to functional genomics in the research community and the consequent demand for consistently pure nucleic acid samples are expected to drive the market for automated instrumentation products. Such automated systems are likely to facilitate analysis of gene function on a gene-by-gene basis by allowing a larger number of samples to be processed for downstream applications.
Much of the research effort on the isolation of nucleic acids (DNA and RNA) has been directed toward improvements in the purity and yield of extracted nucleic acids, advanced operation speed and facility, automation, and cost reduction.
For instance, researchers at FujiFilm in Germany recently launched the QuickGene 810 system, which uses a high-performance porous membrane that does not require a centrifuge and can extract nucleic acids using a simple low-pressure filtration method. This automatic system is expected to provide high purity and yield, even as it offers other attributes including compactness, simple handling, and fast operation.
Researchers at the U.K.-based Merck Biosciences designed a DNA kit for rapid, small-scale isolation of genomic DNA from blood leukocytes. The kit is based on a method that involves the capture of blood cells (leukocytes) on magnetic silica particles, lysis of captured cells, and immobilization of large genomic DNA molecules on the surface of the particles.
Since the DNA is recovered in less than 15 minutes and the procedure is easy to automate, the kit provides a low-cost, quick, and efficient approach for large-scale isolation of DNA from whole blood samples in preparation for downstream screening.
Researchers have started using magnetic particles and eliminated liquid handling while performing the high throughput isolation of DNA and RNA. In fact, the advanced magnetic particle processing technologies enable a high rate of recovery of purified targets and excellent reproducibility, and a reliable automation method for larger throughput DNA and RNA purification.
While automated, high throughput nucleic acid isolation products are popular in the US, there is a huge market for low- to mid-throughput instrumentation from smaller hospital labs across European countries.
Some manufacturers have responded to this demand and currently offer products for low throughput sample-processing that are ideal for clinical laboratories. The instruments also offer flexibility as the end user can increase the sample processing capability by upgrading to a higher throughput instrument.
Compact Automated System
Automating the isolation of microbial DNA eliminates many of the time-consuming physical and enzymatic purification steps, which translates into considerable benefits for the laboratory workflow and creates standardized procedures for template DNA preparation.
Many microbiology labs are now demanding automated devices that can process small samples in a cost-effective manner.
Researchers at Roche Diagnostics in Mannheim, Germany developed an instrument that offers flexible and reliable DNA isolation by processing one to eight samples within a period of 2050 minutes. This system is expected to be useful for efficient and automated isolation of bacterial DNA from various sample types.
Magnetic bioseparation-based technologies are gaining in popularity as laboratories demand reduced sample degradation, fewer intricate protocols, and gentler sample handling in applications such as mRNA extraction, separation of DNA from solutions or gels, or isolation of plasmid DNA.
Scientists at Bio-Nobile (Turku, Finland) developed a novel magnetic bioseparation solution that allows for gentle handling of samples; it is fast and provides flexibility and convenience for laboratories working with smaller samples.
This system can obtain good yields with a sample volume of less than 10 microliter and is suitable for a wide range of applications, including the purification of gDNA, plant DNA, or plasmid DNA, as well as the isolation of mRNA from a wide range of materials.
Researchers at Eppendorf (Hamburg, Germany) developed a new and more streamlined plasmid purification technology that is a significant improvement over the standard alkaline lysis technology. The new method works by lysing bacterial cells and completely solubilizing cellular components using a combination of enzymes and detergents. The plasmid DNA is then captured directly from the lysate on a filter device during a quick centrifugation step.
This new technology is much faster than the alkaline lysis method since it requires fewer handling steps; the entire process of isolating the plasmid DNA from the cells takes less than 10 minutes. The highly purified plasmid DNA can be directly used in downstream applications such as transformation, ligation, polymerase chain reaction (PCR) analysis, sequencing, restriction digests, and cloning.
Environmentally Friendly Purification Technologies
There is an increasing demand for cleaner' purification approaches, which minimize the use of materials that affect the performance of the finished product and are a source of harmful contaminants. This not only ensures that the product is safe but also reduces the costs associated with downstream processing.
Researchers working at Invitrogen in Scotland developed a technology that purifies nucleic acids without the use of centrifuge, vacuum, chaotropic salts, solvents, or ethanol that often inhibit downstream enzymatic reactions.
The technology uses a simple linkage that enables the binding and release of nucleic acids, which is mediated by changing the pH of the solution. This method promises to provide performance and process advantages over current purification technologies that involve the use of chemicals. A favorable cost-benefit ratio is expected to push this technology into a variety of applications such as genotyping, expression profiling, cloning, sequencing, short tandem repeat (STR) analysis, and PCR, among others.
Researchers from Boehringer Ingelheim (Ingelheim, Germany) and BIA Separations (Ljubljana, Slovenia) are using an environment-friendly chromatographic technology for the purification of pDNA that allows for the fast processing of large volumes while improving product quality.
Since this method does not require the use of enzymes and organic solvents that are necessary in the conventional alkaline cell lysis method, it avoids quality, patient safety, and environmental-related issues. Apart from purifying pDNA, this technology can also be applied in separation and purification of peptides, proteins, oligonucleotides, and polynucleotides.
Kit to Minimize Endotoxin Contamination
Standard and traditional plasmid purification systems result in high levels of endotoxin contaminating the purified plasmid DNA. Although endotoxin-free plasmid DNA is not essential in several molecular biology experiments, it is essential during the transfection of sensitive mammalian cell lines where the presence of endotoxins could influence the outcome and reproducibility of the experiment.
Sigma-Aldrich researchers in Munich developed a kit that enables DNA purification directly from an overnight culture, which ensures rapid isolation of endotoxin-free plasmid DNA. In this method, the lysate obtained after lysis is mixed with a binding solution, which includes reagents that achieve less than 0.1 endotoxin units per microgram of plasmid DNA without having to sacrifice high plasmid recoveries.
In the future, nucleic acid purification and amplification systems are expected to find numerous opportunities in oncology and personalized medicine. Future developments must therefore be geared toward increasing the throughput of the instruments to enable them to process higher volume of genomic samples in lesser time.
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