by Caitlin Smith
Many biology students remember the introductory lab in which they “purified” some DNA. Looking at the lump of goo that might have originated in someone’s sinus cavity, they marveled at its existence, clumped there on the tip of a toothpick. Today we can marvel at how much time and labor had to be invested to prepare a sample of nucleic acid that was probably badly contaminated. Read on for a snapshot of today’s progress and challenges in the automated purification of nucleic acids.
Harnessing magnetism for faster purification
Magnetic beads or column materials can greatly speed the purification process. Roche Applied Science
recently introduced their MagNA Pure 96 System, which can purify nucleic acids from 96 standard (up to 1 ml) samples in less than an hour, including set-up time. “The system uses barcoded, prefilled reagent trays and disposables that not only reduce hands-on time, but also combine with the system’s LIMS connectivity to ensure data integrity as samples move, for example, to our LightCycler® 480 Real-Time PCR System for qPCR,” says Mike Leous, group marketing manager for genomics and microarrays at Roche Applied Science. “I think the MagNA Pure 96’s ability to purify 96 samples before 9:00 AM, so that a researcher can work with the DNA, RNA, or viral nucleic acids the rest of the day, is the most exciting development in a long time.”
Life Technologies offers three automated instruments for the purification of nucleic acids: “MagMAX Express 24 and 96 Magnetic Particle Processors, and iPrep Nucleic Acid Purification Instrument,” says Eric Liu, product manager for nucleic acid purification. “These platform technologies are supported by a number of specialized kits for optimized purification from a preferred sample type. The MagMAX system allows our customers to perform their sample prep in high-throughput fashion with the choice of either 24 or 96 samples. The iPrep instrument and reagent kits provide customers with highly convenient pre-loaded, ready-to-use cartridges so that the purification is cross-contamination-free.” Liu believes that the introduction of microfluidic technology is one of the most exciting recent developments in the purification of nucleic acids today. “These developments will enable both smaller processing volumes for micro scale purification, and enhanced workflow integration,” he says.
The advantage of flexibility
When it comes to automated workstations, some labs are concerned about compatibility with their other types or brands of equipment. Aurora Biomed incorporates flexibility into their products; for automated robotic workstations they offer the VERSA Mini NAP, VERSA 1100 NAP-PCR, as well as Nucleic Acid Isolation kits. “Our robotic workstations are open to any plastic labware or kits and thus are not locked in with any particular brand of plastic labware and kits,” says Sikander Gill, application scientist and senior R&D at Aurora Biomed. “This allows the customer to switch among various kits from time to time. We offer complete solutions as we have both the reagent kits and the robot available; however, we have programmed our VERSA series of robotic liquid handling workstations to work with kits from other vendors.”
Interchangeable deck modules in the robots allow you to configure it to meet the needs of your particular protocol or technique: “For example, the systems are applicable to magnetic bead-based kits, or they may be used with vacuum-based kits for both nucleic acid isolation and for centrifugations methods during which the vacuum manifold is used instead of centrifugation step,” says Gill. “The same system also carries out PCR set up after nucleic acid isolation is finished. Applications like NGLP (Next Generation Library Prep) that involve various techniques including nucleic acid isolation, reverse transcription, fragment size selection, DNA end modifications, adaptor ligation and sequencing setup can be completed by the same robot.”
Gill believes that one of the biggest challenges in developing automated nucleic acid purification today is “to isolate these molecules from samples such as soil, fossils and from other formalin- or paraffin-preserved materials,” he says. “Unfortunately, the nucleic acid of interest is in very small amounts in such samples, and scientists are still trying to develop novel techniques that are capable of separating the desired fraction from their contaminants. One approach to achieve this is the development of kits, such as Aurora Biomed’s AB96 Magnetic Bead FFPE DNA and RNA kits, that are more efficient at isolating desired nucleic acids. For example, multi-dimensional electrophoresis is a new technique that has potential to alleviate this problem. We are also seeing a trend in that specific sites of interest for nucleic acid samples can be amplified by PCR in only the lysate of a single cell. Such a process eliminates the need for nucleic acid isolation.
Challenges to tackle
Purifying nucleic acids could still be easier—for example, for researchers working with formalin-fixed, paraffin-embedded (FFPE) tissues. “I wish we had already worked out the complete (i.e., no off-line processing) automation of FFPE samples on the MagNA Pure 96 System, but we don’t have that yet,” notes Leous. “Just as we don’t have full automation of FFPE samples, some manufacturers have tried, but no one else has a great automated system for that yet. As far as I know, researchers are still forced to do some off-line pre-processing before automation (e.g., before adding it to our 32-sample MagNA Pure LC Automated Purification System) or to run manual columns, such as our High Pure FFPE RNA Micro Purification Kit. I hope that there will be an elegant, fully automated solution to FFPE purifications soon so that researchers can more easily analyze all of those archived FFPE samples.”
Another challenge is removal of inhibitors in the wash buffer residuals from the solid (such as magnetic beads or membrane column) used to bind the nucleic acids. “The wash buffer usually contains alcohol and salts for nucleic acid purification, so it is necessary to remove the residual wash buffer from the nucleic acid-bound solid before the elution step, or the residual wash buffer will be co-eluted with nucleic acid and inhibit downstream enzymatic reactions, such as PCR,” says David Daf, president of Taigen Bioscience. “[Taigen’s] LabTurbo uses solid clean technology to remove the wash residual. The innovative formula, CCEB (column clean-elution buffer) contains no alcohol and low salts, effectively dilutes the wash buffer residual in the membrane column, and can be removed by pipette. The solid clean technology used in LabTurbo removes the waste buffer contamination in the automated nucleic acid purification.”
A third challenge in purifying nucleic acids today is the issue of scale—namely, replicating today’s advanced purification chemistries for larger-volume samples. To address this issue, says Eric Vincent, Promega’s product manager for genomics, “Promega is releasing the ReliaPrep™ Large Volume HT gDNA isolation system, for isolation of gDNA from 3-10ml whole blood samples in a high-throughput format,” he says. “[This is] to address the unique challenges faced by biobanks, cancer researchers, and contract research organizations when dealing with large-scale preps.”
One reason that scaling processes to larger volumes is challenging is that “the process of adding and removing large volumes of reagents can be time consuming, error prone, and often results in low-quality, low-concentration DNA,” says Vincent. “We have integrated novel chemistry and hardware on the Hamilton Robotics MICROLAB® STARplus to address the bottlenecks. The resulting product, ReliaPrep™ Large Volume HT gDNA isolation system, provides walk-away purification of gDNA from 1-96 large volume samples in under 8 hours while other available systems require 3-4 cycles of processing and re-loading samples and consumables.”
Promega will soon expand the ReliaPrep™ gDNA purification line to other volumes and sample types, with downstream clinical applications a possibility, too, in the early detection of diseases. “For some testing, such as infectious disease, biobanking, or the early detection of cancer, the sample size is dictated by the sensitivity or number of downstream assays, requiring large input volumes to detect rare events earlier,” says Vincent, “while in other instances, small samples are all that are available for research.”
Daf also notes that the purification of rare nucleic acids can have a large impact on human health. “Circulating DNA in the blood can be used for early cancer discovery and cancer gene-type therapy,” he says. “But unfortunately, the circulating DNA is rare in the blood for detection [if] traditional manual or automated nucleic acid protocols are used. Taigen’s recently released LabTurbo LS automated system purifies virus DNA/RNA, miRNA, circulating DNA, genomic DNA, and total RNA, with large input sample volume (up to 2 ml) and small volume elution (down to 60 ul). The LS system concentrates the sample, increasing the yield of target DNA/RNA by 5 to 10 times from different kinds of clinical and research samples.” Today’s nucleic acid purification technology is promising for exciting advances in research, clinical, and diagnostic applications.