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Scientists have been playing with nucleic acids for decades. Now, with so many newly discovered molecules to explore—no longer just genomic DNA and mRNA but mitochondrial DNA, microRNA, small interfering RNA—companies and even individual researchers are coming up with newer and better ways to purify and store nucleic acids for various downstream applications. And some even have technologies that preclude the need for purification altogether. By Diana Gitig Inclusion of companies in this article does not indicate endorsement by either AAAS or Science, nor is it meant to imply that their products or services are superior to those of other companies. Isolating DNA is probably among the most basic things life scientists do. It is likely one of the first protocols they learn—undergraduates, even high school students do minipreps—and they do it constantly, no matter what they are studying. Goals as diverse as generating mutant proteins and organisms, performing microarray experiments, and crystallizing proteins all depend on purified nucleic acids. It is thus perhaps not all that surprising that companies have recognized the need and are providing different kits and technologies to meet the different requirements each application demands. Full AutomationThe immense volume of samples required for "omics" studies demands automation. Taigen Bioscience in Taiwan has addressed that demand with LabTurbo, their fully automated nucleic acid extraction system. According to David Daf, the president of Taigen, they chose to use spin columns because they are "the most popular method—they are traditional, reliable, and maintenance costs are low." So customers are dealing with the same silica membrane chaotropic chemistry they are comfortable with, but Taigen has automated it to save time, labor, and plasticware. Taigen was the first to launch individual spin column automation in 2005; Qiagen followed with its Q system in 2007. LabTurbo's most unique feature is its use of vacuum manifolds in lieu of centrifugation. Daf says, "Centrifuges introduce air turbulence, which can create a cross-contamination problem. And the gravity may damage the membrane, leeching silica into the elution buffer." Moreover, it is extremely difficult for a robotic arm to transfer samples from a linear matrix, like a 96-well plate, to a circular centrifuge chamber. LabTurbo is completely linear and has separate vacuum manifolds to further minimize cross-contamination; one for binding and washing, and another for eluting. Cross-contamination is one of the biggest issues in automated systems, and Taigen has gone to extensive lengths to prevent it. LabTurbo can be used to purify genomic DNA or total RNA, from various sources including blood, urine, viruses, plants, and cultured cells—even cigarette butts. The nucleic acids are then ready for use in any downstream application. Qiagen has been a huge player in nucleic acid manipulation for a long time. They provide many similar products and technologies as some smaller, newer companies mentioned here: the Rotor-Gene Q is like Taigen's LabTurbo, and QIAsafe is similar to Biomatrica's SampleMatrix described below. But as Nicole York, Qiagen's marketing communication manager, points out: "What we bring in addition is integration with our instruments as well as our global service organization." They also have an automated polymerase chain reaction (PCR) system, QIAgility, and machines for automated nucleic acid purification. Playing It SafeOnce DNA has been purified—regardless of how it is done—the question of how to store it remains. DNA has traditionally been stored in solution at -20°C. But freezers use a lot of energy—which is expensive and not particularly green—take up a lot of space, and can fail, with disastrous results for the samples inside and the questions they were destined to answer. Furthermore, DNA often needs to be shipped. Judy Muller-Cohn and Rolf Muller got tired of dealing with these problems, so they invented sample matrix technology, and co-founded Biomatrica. With his high throughput genomic studies, Muller says, "I was generating 10,000 samples every week. How can you stabilize these samples in a manner that enables this workflow?" They looked to nature for inspiration. "Extremophiles live everywhere—dry valleys in the Arctic Circle and the Sahara desert, where there is no moisture in the air. These eukaryotes have DNA, RNA, and proteins that are stable for 125 years outside of a freezer," points out Muller-Cohn. Some of these organisms, like tardigrades and brine shrimp, use anhydrobiosys—life without water—as a mechanism to prevent degradation. And so does SampleMatrix, Biomatrica's proprietary core technology. Samples are preserved through the formation of a protective, thermostable barrier while air-drying. This barrier protects them from being degraded by heat or ultraviolet light. Samples are recovered by rehydration, and can be used immediately for various analyses. Biomatrica has several products that exploit this technology; one for DNA, one for RNA, and one for plasmids. The company can also stabilize proteins and is working on stabilizing cell lines—all using anhydrobiosys. Mike Hogan, the founder and chief scientific officer of Genvault, tells a similar story. He was providing microarray analysis as a service for high throughput screens and quickly realized that clients' shipping their DNA to him was the rate-limiting step. "Not having access to high-quality biological specimens in biobanks has held up the entire field" of genetic screening, Hogan says. He started Genvault to be "involved in all aspects of biosample management and transport. Universal genetic screening will become routine at some point in the near future," he notes. "We are trying to position ourselves for the next era—it is like transitioning from mainframes to the Internet. Things are constantly moving rather than being stored in one spot." Genvault's products are inspired by Guthrie cards, the pieces of Whatman paper that have been used to store and test neonatal blood samples for the past 60 years. GenPlates are little cups of this paper molded into 384-well plates and are used for storing crude nucleic acid material—like bacteria, blood, or plasma—dry and at room temperature for decades. GenSolve is a unique solution that facilitates recovery of samples from the FTA paper. GenTegra is an inert medium, intended to emulate bone, for dry room temperature storage and transport of purified DNA. And GenVault is currently developing a matrix to include the dry-state storage of proteins and biomarkers. All for One and One for AllAccording to Nezar Rghei, a vice president of Norgen Biotek in Ontario, Canada, new trends such as the use of microarrays, the importance of epigenetics, and the advent of systems biology inspired their All-in-One Purification kits. This is "macromolecular fractionation," he says, rather than isolation of one component. "Molecules are interrelated and cannot be studied in a vacuum," according to Rghei. "Once we studied expression using arrays, studying RNA was not sufficient. It became necessary to study protein simultaneously. And when they are isolated separately, there is a normalization problem." Norgen's columns are made of silicon carbide, in contrast to most others that are commercially available, which are just silicon. The difference means that Norgen's matrix can bind to proteins as well as nucleic acids, and each component can be eluted sequentially, even from very small samples. Its technology also meets the challenges posed by other discoveries, such as quantitative PCR and the discovery of miRNAs—namely, the small sizes of the samples and molecules being studied. Rghei notes that RNA species shorter than 200 nucleotides cannot be isolated in other silicon-based spin columns without the use of phenol which, in addition to being toxic, can inhibit sensitive qPCR assays. Norgen's is "the only kit on the market that isolates all RNA species without phenol, because it has the ability to bind total RNA and miRNA in the same sample at equal rates. And these are the only kits that can isolate total RNA [mRNA, miRNA, and biomarkers in plasma, which are often products of apoptosis or other degradative processes and therefore very short] in 96-well plates." miRNAs, large RNAs, genomic DNA, and proteins are thus eluted from a single column, without the use of phenol, chloroform, or acetone, from samples as diverse as blood, yeast, fungi, even soil and water. Going WithoutSometimes, especially when samples are limited, the best purification is no purification at all. Or so thinks Richard Fekete, research and development manager/scientist at Applied Biosystems, a division of Life Technologies. (Life Technologies is the new company formed by the merger in November 2008 of Invitrogen and Applied Biosystems, which had previously acquired Ambion.) Applied Biosystems combined "the knowledge of RNA purification from Ambion with the real-time PCR expertise of Applied Biosystems to create one easy-to-use, validated workflow with maximum performance," explained Fekete. The result is the Cells-to-CT product line. The sample preparation method consists of adding a proprietary lysis buffer to the sample, incubating for five minutes at room temperature, then adding the proprietary stop solution and incubating for another two minutes at room temperature. That's it. It can be done in 96- or 384-well plates, so it is perfect for high throughput screening. Samples are then ready for RT-PCR, and results are equivalent to those obtained with purified RNA. A notable feature of the Cells-to-CT kit is that it is a unique product on the market that has been optimized to prepare single cells for analysis. When starting with the genetic material from only one cell, the losses that are inherent in the washing and eluting steps traditionally used for purification can become a real issue. To avoid it, some researchers have resorted to a "homebrew" method of boiling their samples in buffer or even water. But "boiling does not free RNA from cellular structures and the high temperatures increase the chance of degradation, reducing sensitivity. Moreover, it can change the expression profile," Fekete points out. Cells-to-CT lyses cells while inactivating RNases, which locks the expression profile at the moment of lysis. Since there is no purification, there is no loss due to heating, sample transfer, or irreversible binding to columns. Miltenyi Biotec is known for cell separation. But as Kirt Braun, Miltenyi's marketing manager, says: "We wanted to provide solutions for downstream applications. Our mRNA isolation allows the isolation of highly pure mRNA in 15 minutes, from as little as five cells. So the two variations of the technology [cell separation and molecular separation] work in concert to provide a more comprehensive workflow solution to the research community." Miltenyi's MACS (magnetic assisted cell separation) technology allows for "one-step, in-column RNA isolation and cDNA synthesis or labeling," according to Braun. Because purification is not a separate step in this in-column cDNA synthesis, pipetting of the sample is minimized, and so is loss of mRNA and cDNA. The technology relies on microcolumns, which Braun explains, "are comprised of a plastic housing filled with a uniform steel ball matrix. With the μMACS mRNA Isolation Kit the binding of mRNA in solution takes seconds and does not require mixing since the MicroBeads stay in suspension due to their extremely small size." The sample is eluted with the column still in the magnetic stand to prevent the carryover that is often a problem in other magnetic separation methods.Yet another approach that, as its website states, "makes DNA purification irrelevant" is made by Finnzymes and its name tells just about all one needs to know about it: Direct PCR. Finnzymes makes a whole slew of Direct PCR kits and protocols for use with specific starting materials, allowing PCR directly from blood, mouse ear and tail, formalin-fixed paraffin embedded (FFPE) tissues, plant leaves, bird feathers, or muscle tissues. Typically, a small volume of the sample is simply added to the PCR reaction mixture. However, Netta Fatal, Finnzymes' marketing communications manager, qualifies: "PCR from unpurified starting material often requires more optimization than standard PCR reactions from purified DNA and may not always be suitable for all applications." To minimize the need for optimization the company has protocols for several starting materials available on its website. "And of course we are constantly developing protocols for new starting materials to be able to serve researchers the best we can," says Fatal. Do-It-YourselfBut despite the availability of all of these products, some researchers are still die-hard do-it-yourself types. Carl Batt, the Liberty Hyde Bailey Professor in the Department of Food Sciences at Cornell University, made a PCR-based biosensor to detect microbial pathogens. His goal (which he achieved) was to make a highly sensitive and highly portable detector that could be used anywhere, not just in a laboratory setting. "What we were looking for was a purification component that could be integrated into a chip that would then be able to serve as an amplification chamber for PCR. The challenge is developing a system which would not require manual transfer of the sample from the purification to the amplification process," says Batt. Batt and his colleagues overcame that challenge, creating a biosensor consisting of a microchip with a region of silica-coated pillars to purify DNA linked to a region for real-time PCR. The pillars are etched into the microfluidics channel to increase its surface area. DNA binds to the pillars in the presence of the chaotropic salt guanidinium isothiocyanate, then is washed with ethanol and eluted with a low-ionic strength buffer—just like a miniprep. This lab-on-a-chip is 36 cm x 28 cm x 15 cm, and it can detect 104 Listeria monocyogenes cells in 45 minutes. "What we developed is a more controlled and certainly a more integrated approach," concludes Batt. Personalized medicine seems to be almost upon us. Genetic screens will become mainstream, for biomarkers to predict drug sensitivities, risk assessments, even genealogical studies. And now that the US National Cancer Institute's Office of Biorepositories and Biospecimen Research is setting up a national biobank, like many countries in Europe and Asia have already done, the need to isolate, store, and transport nucleic acids is only going to increase with time. Fortunately, these forward-thinking entrepreneurs have anticipated that need, and developed these products to meet it.
Note: Readers can find out more about the companies and organizations listed by accessing their sites on the World Wide Web (WWW). If the listed organization does not have a site on the WWW or if it is under construction, we have substituted its main telephone number. Every effort has been made to ensure the accuracy of this information. Inclusion of companies in this article does not indicate endorsement by either AAAS or Science, nor is it meant to imply that their products or services are superior to those of other companies. Diana Gitig is a freelance science writer living in White Plains, New York. DOI: 10.1126/science.opms.p0900035
UV-VIS SpectrophotometerThe BioSpec-nano is a dedicated spectrophotometer for the quantitation of nucleic acids that features an automated precision drive mechanism to obtain accurate and reproducible results. The BioSpec-nano requires only 1 µl of sample (for a pathlength of 0.2 mm) or 2 µl (for a pathlength of 0.7 mm), which is pipetted onto the measurement plate. No standard rectangular cell is needed for ultrasmall volumes, although for applications that are not volume-limited, an optional rectangular cell is available. Automatic functions include sample mounting, measurement, and cleaning. The operator does not need to perform the tedious, repetitive, and inconsistent placement of the fiber-optic element or the manual cleaning required by other instruments. Sample measurement time is three seconds. Shimadzu Gel VisualizationSafeCloner enables safe, ultraviolet-free visualization of ethidium bromide, SYBR Green, and SYBR safe gels. SafeCloner allows DNA bands to be seen safely and clearly, using high-efficiency LED illumination and filter glasses. The use of LED illumination removes the dangers of conventional transilluminator use, such as stray ultraviolet light, which can cause retinal damage. LED illumination does not damage DNA even after long exposure times. In addition, the cloning efficiency of DNA fragments eluted from gels exposed to SafeCloner is significantly greater than that of DNA isolated from traditional ultraviolet-illuminated gels. SafeCloner’s blue epi-LED illumination and special filter glass result in greater sensitivity and contrast than longer wavelength 365-nm transilluminators. SafeCloner can be used with inexpensive gel documentation products such as the Cleaver Scientific Microdoc unit. Cleaver Scientific Blood RNA Isolation KitThe BiOstic Stabilized Blood RNA Isolation Kit is for the extraction of total RNA from PAXgene Blood RNA Tubes in one hour with high yields. A specially formulated wash buffer ensures reliable and complete collection of cells at the start of the protocol and a novel homogenization method provides more complete RNA solubilization for increased recovery on spin filter membranes. Deoxyribonuclease I and a high-activity buffer are provided for on-column genomic DNA removal during the procedure. All wash buffers contain ethanol to save time and ensure reagent quality. Mo Bio Laboratories Complete DNA ProfilesA new 16-locus system makes it possible to get complete profiles from inhibited DNA samples. Forensic samples are often compromised by substances like dirt, fabric dyes, or blood, leading to inconclusive results. The robust and sensitive PowerPlex 16 HS System contains optimized buffer that makes it possible to obtain informative results even with challenging samples, achieving full profiles with higher levels of inhibitors than previously possible. In cases with very limited DNA, like touch samples, PowerPlex 16 HS is sensitive, giving complete profiles with 100 pg of DNA and interpretable data with even less sample. The newly designed product comes complete with internal lane standard in addition to hot-start Taq DNA polymerase premixed in the reaction buffer. The protocol requires fewer pipetting steps than previous systems. Promega PCR without DNA PurificationUntil the launch of Direct PCR, it had been virtually impossible to amplify DNA directly from starting material due to the presence of polymerase chain reaction (PCR) inhibitors. A separate DNA purification step had been needed to remove the inhibitors, which increases costs and hands-on time. Direct PCR eliminates the need for DNA purification prior to PCR and allows PCR to be performed directly from different types of starting material, including blood, mouse ear and tails, and formalin-fixed paraffin-embedded tissue. Direct PCR is based on Finnzymes’ unique DNA polymerases featuring fusion protein technology, Phusion High-Fidelity DNA Polymerases and Phire Hot Start DNA Polymerase. These robust polymerases tolerate PCR inhibitors significantly better than conventional polymerases like Taq or Pfu, which can be completely inhibited by minimal amounts of PCR inhibitors. The first Direct PCR kit specifically for blood was recently launched: Phusion Blood Direct PCR Kit. Finnzymes
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Science. ISSN 0036-8075 (print), 1095-9203 (online)
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