Analyzing DNA gets easier every year, but not every sample makes life simple for scientists. Degraded samples, as discussed here, create challenges in sample preparation. This article explores the key challenges with those samples and how to handle them.
Exposing a DNA sample to the elements for a long time breaks down the molecules. A variety of environmental factors—temperature, pH, ultraviolet light, and more—come into play. “In the ground, soil or dyes can leach into the cells and disrupt them, leaving DNA open to DNases and UV from the sun, which can also nick the DNA and fragment it,” says Susan Walsh, assistant professor in the forensic and investigative sciences program at Indiana University-Purdue University Indianapolis. “All types of samples can potentially suffer from degradation as the DNA is nicked randomly.”
Clinical labs cannot tolerate degraded samples while forensic laboratories have no choice. “For example, a clinical lab’s priority is to protect a DNA sample from environmental conditions to support an accurate patient diagnosis,” says Sha Liao, market segment leader, genetic screening, Hamilton Robotics. For a forensic lab, Kevin Miller—market segment leader, government and regulated laboratories, Hamilton Robotics—says that it “encounters challenges when working with DNA samples from post-mortem or post-depositional environments, which may include the presence of fungus or bacteria, as well as mixtures of DNA from multiple sources.”
The structure of DNA protects itself to a certain extent. Up to lengths of about 150 base pairs, histones fend off damage to the DNA, “but over time pieces get smaller if left to the elements,” Walsh explains. “This would mean amplification of certain regions gets more difficult due to size.”
When asked about the most useful method of preparing degraded DNA, Brian Kemp, associate professor of anthropology and co-director of the laboratories of molecular anthropology and microbiome research at the University of Oklahoma, says, “I don’t think there is a ‘most useful’ method.” He adds, “There are probably about as many methods as there are labs working with degraded DNA.”
Particular problems
When asked about some of the top applications that suffer from degraded-DNA samples, Miller says, “Any biological application is impacted if the DNA is degraded, with the potential for significant downstream consequences.” He adds, “This is why appropriate steps must be taken to preserve DNA at the time of collection. Otherwise, environmental factors are free to alter the sample over time.” That ticking clock can add more and more damage to DNA. “The effects could start small but every change is meaningful,” Miller explains.
Any biological application is impacted if the DNA is degraded, with the potential for significant downstream consequences.
To suspend that clock, scientists should start sample preparation as soon as possible. This can include putting the sample in buffers. This kind of step can “basically stop any action on the sample,” Walsh says. “As for prep, some design steps cater to small fragments.” In next-generation sequencing, for example, steps can be taken to add adapters to the ends of the DNA. Then, “some bit of information can be gleaned, but then it becomes an issue of accurate alignment and bioinformatics,” as Walsh describes it.
Manufacturers provide solutions to many of these problems. For example, the FastPrep bead-beating homogenizer and combined extraction kits from MP Biomedicals can be used to address two key challenges, which product manager Véronique Karsten describes as “identifying species using DNA barcodes in processed and digested food samples harboring degraded DNA and the metagenomic analysis of archaeological and ethnographic samples.” For the first, the FastPrep System can be used with a mini-barcoding approach to analyze 100–200 basepair fragments of DNA, even in degraded samples. FastPrep products have also been used with ancient samples of DNA that were highly degraded and still enough DNA was extracted for analysis.
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Extracting the DNA is a key step to preparing it for analysis. In the Kemp lab, scientists use two methods of DNA extraction. They tend to use their newer method as much as possible, because the older one includes phenol:chloroform, which Kemp says, “I’d rather avoid.”
Hamilton developed automated solutions for DNA extraction that vary by application. “Forensic DNA samples are often degraded, but forensic workflows account for this and allow users to characterize the fragments that they have,” Miller explains. Then, Liao adds, “Our automated workstations can be used and specifically programmed according to the lab’s workflow needs, or users can take advantage of assay-ready workstations that are developed for a specific workflow or assay chemistry, such as nucleic-acid extraction, pre-PCR sample preparation, or genotyping.”
Within long pieces of DNA, Walsh says, short tandem repeats (STRs) “give us valuable info.” But when they are fragmented, she says, the STRs “cannot be fully amplified, and we lose out on this unique information.” In those cases, scientists work with smaller fragments, which are protected by the histones in chromosomes. “But over time and with constant UV damage, even small fragments degrade,” Walsh says.
Ultimately, no single approach solves all problems in working with degraded DNA. The key challenge to a one-size-fits-all method, Kemp says, arises from “all the unknowns when it comes to working with highly degraded and otherwise compromised DNA sources.”
Sample storage
Once DNA is collected, how it’s stored matters. For instance, formalin-fixed and paraffin-embedded (FFPE) methods create crosslinks in DNA samples, which makes “molecular analysis extremely difficult,” Liao says. “Sample storage conditions help to protect samples or minimize further degradation, but if the environment isn’t ideal for that sample, storage may actually exacerbate degradation.”
Sometimes, chemistry helps. As Walsh notes, “The addition of stabilizers can assist in storage, because they prevent DNases from chewing on the DNA and degrading it.”
Other features of storage are also important. “DNA does not like the cold, wet conditions in a refrigerator,” Liao explains. “So, it is usually better off in a freezer, because frozen and dry overcomes wet.” But being in a freezer doesn’t ensure that DNA will not sustain more damage, especially if the temperature varies. “A Hamilton Storage automated storage system removes those risks with controlled and documented climate—temperature and humidity—conditions, and through hands-free retrieval that protects all samples while automatically picking the samples of interest for delivery to the user,” as Liao describes the technology. These units also protect DNA samples from bacteria and particulates that can contaminate a sample.
Improper use of degraded DNA goes beyond just getting the wrong results. In healthcare, a sample of degraded DNA could trigger the wrong diagnosis or indicate an incorrect treatment. “In forensic applications,” Miller notes, “it could mean the difference between the identification of the victim or perpetrator of a crime or not.” So, it pays to get sample preparation right when dealing with degraded DNA. Maybe even more important, the cost of getting sample preparation wrong could be deadly.