In an era of all sorts of sequencing, sometimes we forget that the nucleic acids must be extracted first. That’s not an easy task, and it gets even more complicated with some samples. Scientists need methods that efficiently extract as much nucleic acid as possible—all without damaging it. Ongoing improvements in tools and techniques make it easier for scientists to obtain more DNA or RNA, but there’s room for improvement.
“Every nucleic-acid extraction first releases nucleic acids from the sample, and then isolates DNA or RNA from the sample, without a lot of additional impurities,” explains Paraj Mandrekar, technical services scientist 3 at Promega. “The most challenging sample types require specialized steps that extract and release nucleic acids or have undesirable compounds that co-purify on the column or the particle.” The biggest challenge, he added, comes in samples “that are simultaneously difficult to release nucleic acids from but co-purify undesirable compounds in the isolation method.”
Stored samples—such as formalin-fixed, paraffin-embedded (FFPE) ones—can make nucleic-acid extraction extra tricky. “Thinking of mammalian samples, FFPE is tough, particularly over-fixed, older samples are a significant challenge still,” says Andrew Gane, genomics and diagnostics solutions strategy & technology manager at Cytiva.
Storage, though, is not the only thing that creates an extraction challenge. “For fresh tissues, fatty tissues can be challenging, such as things like brain and breast tissue, chitinous samples, teeth and forensic samples with trace amounts of nucleic acids,” Gane notes.
The idea of extracting nucleic acids in better ways continues to attract broad interest among many scientists. A search of “DNA extraction” on PubMed on June 13, 2020, for example, returned nearly 8,400 articles. Plus, the number of articles on this topic keeps increasing, with 755 articles noted in 2019. Let’s see what’s causing the latest interest in this area.
Adding automation
Like many other aspects of dealing with DNA or RNA, automation can improve extraction processes. “There are many tedious hands-on operations and time-consuming centrifugation or vacuum-suction steps involved in the nucleic-acid extraction,” says Jeffrey Lai, technical manager at Blue-Ray Biotech. “These operations can easily introduce human error, contamination, and other variations.”
Automation helps, but not just any approach to it. When asked what aspects of nucleic-acid extraction need improvement, Gane points to “attention to automation simplicity—taking the onus off the user to install additional components to their platforms and to create open-source reagents that do not tie in customers to specific platforms.”
Plus, Gane notes that throughput, speed, and cost should be addressed as a combination. As he says, some automation platforms are “very cheap but not easy to automate or require additional plasticware that is not covered in the costs of kits themselves—passing the costs to the user.” In these systems, Gane emphasizes, “Sustainability is also something to consider with a lot of throwaway components and potentially environmentally damaging chemicals.”
To address some of these issues, Gane makes several suggestions, such as using magnetic beads in nucleic-acid extraction. “They have been around a while, but recently, there has been a surge in use for their throughput simplicity,” he says. In thinking of interesting approaches, Gane suggests looking at new technologies that purify a sample “in an electric field held in a disposable device, technologies that bind impurities and not nucleic acid, meaning that the sample just flows through without need for bind-wash-elute cycles.”
Making it easier to extract nucleic acids matters, but so does the method. The sustainability is likely to become increasingly important.
Improving integration
Not long ago, extracting nucleic acids required considerable expertise. For example, Mandrekar notes: “When I started working in the product design of nucleic acid–purification chemistries, the most predominant extraction chemistries were manual purifications that required a lot of hands-on time, and generally required a great deal of skill to perform effectively.” He adds, “Some of these chemistries took several hours to get nucleic acids from a handful of samples, and the operator’s skill was a major factor in getting successful isolation.” Today’s chemistries in this area work faster and require less skill to use.
The steps after extraction should also be considered. “Nucleic-acid extraction is always something that is designed with the downstream steps in a workflow firmly in mind,” Mandrekar explains. “When we started to apply automation to nucleic-acid extraction problems, we were looking to build a series of steps that could go from a sample to an answer with as many steps automated and integrated into the subsequent steps in the process.” Some of the newer methods do link more steps—from a raw sample through amplification to detection.
“It has been very exciting to see these linked steps, how they have made the handoff from one traditional step in the process to the next,” Mandrekar says. “In our own work, we have seen the development of instrumentation and methods that can more seamlessly perform pre-processing, and then hand off a lysate to an automated purification method, which in turn can be quantified and normalized into amplification methods.”
Processing plants
Many scientists explore the nucleic acids in plants for both basic and applied research, and it takes some work to get at this DNA or RNA. “Plant samples are by far the most challenging for nucleic-acid extraction,” according to Lai. “Plant cells have a cellulose cell wall, as well as lignin as structural supporting material, that make them hard to be demolished.”
Getting by a plant cell’s structure takes care of just half the battle. “In addition to a cell wall, plants contain significant quantities of polysaccharides, carbohydrates, tannins, phenolics, and other compounds,” Lai says. “These can negatively impact downstream applications.”
In any sample, a scientist’s journey to extract nucleic acids poses a series of challenges. Beyond overcoming those obstacles, a scientist seeks ways to simplify the process through automation and integration. Ultimately, the connection of steps in a nucleic-acid workflow determines the outcome and how broadly a technique can be applied. These DNA- and RNA-related challenges continue, but scientists keep getting better at dealing with these molecules and their surrounding environment.
Image: To analyze nucleic acids, the molecules must by extracted from cells, and plants create some of the biggest challenges. Image courtesy of Mike May
Hero image: A close-up texture of regular human brain tissue. Image from Albund, Dreamstime