by Laura Lane
In addition to basic research and drug discovery, work in national defense, clinical diagnostics, and other areas have embraced machine-driven nucleic acid purification. Speed isn’t the only reason. “[Automated] workstations not only increase throughput, but also ensure that assay steps are performed consistently, human error- and learning curve-free from run to run,” according to a paper published in the Journal of the Association for Laboratory Automation1.
Moreover, companies now offer more products for lower throughput and space-challenged labs, opening the door to automation for practically every researcher. How do you get started? Set aside any pre-conceived notions. And familiarize yourself with what’s out there. Here, Biocompare sets the record straight on some of the prevailing myths surrounding automated nucleic acid purification.
Myth #1: Automation is suitable only for projects that use 96-well plates.
The notion of automation has become synonymous with high throughput. However, the truth is that automation simply refers to accomplishing a task without the need for manual input. While simultaneously processing 96 samples is indeed efficient, employing automation for just 16 (or any amount of) samples means “virtually zero hands-on time,” says Jeff Briganti, strategic marketing manager in nucleic acid purification for Promega Corporation. “It’s not high throughput, but it’s automated.”
No need to feel sheepish about making your first inquiry on which kits and devices best fit your needs. “The number of people doing single well preparations far outweighs the number doing 96-well,” Briganti says.
Myth #2: The risk of cross-contamination looms large over automation.
Playing it smart can eliminate errant drips or airborne particles from falling into the wrong well. First, choose the sample types that are most compatible with automated techniques. “In general, if a sample needs homogenization to get DNA out of it, then it’s difficult to use the 96-well format,” says Mark Brolaski, chief executive officer of MO BIO Laboratories Inc. Homogenization requires beating beads and shaking that could cause liquid from the well to land on the mat covering the well. “Once you take off the mat, it’s extremely difficult to prevent cross contamination,” he says.
Fortunately, companies have made significant headway in overcoming such a challenge. With MO BIO’s UltraClean-htp 96-Well Plant DNA Isolation Kit, steel beads first provide the rigorous homogenization needed to break down the cell wall. Then, a centrifugation step draws down any aerosol or liquid contaminants back into the well.
On the other hand, purification of nucleic acids from blood samples or PCR reactions doesn’t require rigorous shaking. These were much more logical candidates for the 96-well format and have proven to be successful applications.
In addition, look for liquid handlers that rely on liquid-filled systems, says Wendy Lauber, director of product management for Tecan. “If you have air-filled systems, aerosol could potentially contaminate the channel - can go up into the chamber — and there’s no way to clean outthe aerosol contamination.” She also recommends using software that allows you to program the movement of tips such that they don’t pass over certain samples.
Myth #3: You have to purchase all sorts of expensive equipment to get started.
Not necessarily. For example, with Invitrogen’s magnetic beads, you just need a basic liquid handling system. Unlike conventional magnetic beads that sink, Invitrogen's beads are light enough to stay in solution, circumventing the need for a shaker. The beads “stay in suspension very well,” says Suzanne Kennedy, product development manager at Invitrogen. “They just sit for 30 minutes and remove all the nucleic acids.”
Kennedy is referring to the ChargeSwitch ion exchange mechanism coating the beads. Activated at low pH, the surface chemistry binds to the negatively charged phosphate backbone of nucleic acids at low pH. Once the magnetic beads are segregated, raising the pH releases the nucleic acids. “You can coat it on to virtually anything,” Kennedy says, pointing to plates and other Teflon surfaces.
Myth #4: Using automated methods requires learning complicated new technologies.
Many of the latest products are still using traditional silica binding technology. The difference is how they’re being used. Now, you’ll find silica on beads and plates that are packaged into automation-ready kits.
However, “we’re likely to hit a limit of what silica can do” (such as with smaller sample sizes), says Briganti. In these increasingly prevalent situations, the final elution step faces the challenge of collecting nucleic acids at very low concentrations. “Either we do a concentration step, or we come up with a better system for that final recovery,” he says.
Promega’s effort to bring silica further has resulted in PureYield, which includes an updated version of the silica membrane and newly optimized buffers. “It’s a very efficient binding membrane. It binds quickly. Solution flows through quickly,” Briganti says, explaining that the process takes a mere 30 minutes compared to the three hours required using gravity-driven column techniques. The product is available for both plasmid and RNA isolation in formats for either centrifuge or vacuum.
Myth #5: Cross-reacting reagents could prevent the development of more efficient extraction technologies.
Actually, the one-tube format is very much in use with automated nucleic acid solutions. Most involve advances that eliminated the need for chaotropic salts, which are known to interfere with enzyme action, both in the extraction procedure and downstream applications, such as PCR. Invitrogen’s ChargeSwitch represents one of the options.
Invitek has introduced Invisorb, a patent-pending technology that employs non-chaotropic buffers. Without chaotropic salts, enzymes, such as proteinases, RNAses, or DNAses, can remain active long enough to do a thorough job of eliminating their respective targets. “Enzymes can be used for a longer time, so you can get purer nucleic acids,” says Hans Joos, Invitek’s head of research and development.
With its enzyme-friendly nature, Invisorb also allows for greater efficiency. The company’s RTP (ready to prep) product comes with lysis buffer and lytic enzymes all in one tube, which are otherwise added in separate steps. “We can have every component required for extraction in the mixture. Just add the sample and a bit of water and the system is running,” Joos says.
Even with chaotropic salts in use, companies have come up with other substances aimed at maximizing yield. Promega’s PureYield RNA midi prep contains a resin-like substance that selectively binds genomic DNA, improving the purity of the final RNA product. “DNAse treatments are inefficient,” Briganti says.
MO BIO’s inhibitor removal technology eliminates substances that inhibit PCR reactions. “DNA isolation is one thing, but getting it cleaned up enough for PCR is another,” Brolaski says. One of their anti-inhibitor substances prevents denim dyes from interfering with analyzing blood found in jeans.
References:
1Lorenz MGO, “Liquid-handling robotic workstations for functional genomics” Journal of the Association for Laboratory Automation, 9(4): 262-267, August 2004.
