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Teaching this “old dog” new tricks helps everyone
A PCR party may be in order, as the polymerase chain reaction turns 30 this year. Although three decades sounds young to me these days, it’s old enough to make this technology a bit of a senior citizen in the world of molecular biology. As Sharon Rouw, PCR product manager at St. Louis-based Sigma-Aldrich, says, “PCR is a little bit of an older technology, so to speak, but people are demanding more from it.” As this method settles ever deeper into biology labs as a go-to technology, however, scientists keep finding ways to put PCR to work.
As Rouw surveys the field, she sees three trends in the application of PCR. First, she points out the use of increasingly crude samples. These include human blood, hair and saliva, as well as plant extracts or seeds. “There are naturally occurring inhibitors in these substances that make it difficult for traditional PCR mixes, which require nucleic-acid purification,” Rouw explains. Today’s more advanced PCR mixes, however, eliminate that purification step, which simplifies the workflow, even with complex samples. From California-based Affymetrix, Jay Brock— associate director, applications development and support in the life sciences reagents business unit—agrees: “From a sample perspective, we are seeing increasing interest in analyzing limited sample types, such as clinical samples, as well as direct sample analysis without a purification step.” He adds, “The new VersaTaq Direct PCR Polymerase from Affymetrix is a novel DNA polymerase that displays resistance to common PCR inhibitors, enabling direct sample analysis without a purification step.”
As a second trend in the application of PCR, Rouw points to higher throughput. “You can do that with miniaturization or speed or both,” she says. “Bigger labs go through many samples as quickly as they can.”
Rouw also sees next-generation sequencing (NGS) driving trends in the application of PCR. Other experts agree. For example, Brock mentions that PCR is being used in “library preparation in targeted NGS workflows, qualification of samples upstream of microarray analysis and NGS and orthogonal technology validation downstream of microarrays and NGS.”
Like many scientific techniques, the applications drive improvements in the technology and the technology allows new applications. For example, using PCR with heterogeneous samples spawned the need for mixes based on inhibitor-resistant enzymes, such as Sigma-Aldrich’s Inhibitor Resistant Genotyping PCR ReadyMix.
Likewise, the interest in increasing PCR throughput drives an ongoing decrease in the size of samples. Scientists need 20–50 μL to run PCR in 96-well plates, but just a few microliters for 1536-well plates. Microfluidic-based PCR chips run the reaction on nanoliter-size samples. The platforms that run smaller samples also tend to run PCR faster. “This works well for human diagnostics,” says Rouw. To make PCR better suited for creating NGS libraries, scientists also want longer and more accurate reads of DNA to simplify downstream bioinformatics. In short, better data in leads to better data out.
To broaden the applications of PCR, scientists often turn to significant changes in the technology. As an example, Brock mentions digital PCR (dPCR), saying that it can be used “to partition samples for more accurate analysis.” Getting the most from a technology today often depends crucially on the balance of reagents and bioinformatics. As an example, Brock says, “We have demonstrated optimal correlation between microarray and real-time PCR data using our software and VeriQuest qPCR reagents in efficient conformational assays downstream of our genomic and transcriptomic arrays.”
Advancing oncology
The application of PCR and how it is changing often depends on the specific field, such as oncology. At GE Healthcare’s California-based Clarient, a reference lab for oncology testing, chief medical officer Kenneth Bloom says, “In my setting, the biggest trends in PCR are to drive down the level of detection of key analytes while increasing the fidelity of the PCR product.” He adds, “The main drivers for this are tumor heterogeneity, rare event detection and sample preparation for NGS.”
Like other areas of application, PCR plays a key role in NGS testing in oncology. “Although the principles of PCR are the same,” says Bloom, “the chemistry and the workflow are modified to generate amplification products with improved sequence quality.” In particular, modifications to the PCR chemicals and enzymes provide more robust and accurate data. As Bloom explains, this provides “a much lower error rate than previously available.”
Tatiana Travis, a microbiologist at the U.S. Centers for Disease Control and Prevention, sets up PCR reactions in a 96-well plate to study drug-resistant pathogens. (Image courtesy of James Gathany)In healthcare, clinicians want to find evidence of cancer as soon as possible, and PCR can be used here. As Bloom says, “Rare event detection is one of the most prominent trends in PCR applications, primarily driven by the promise of liquid biopsies.” In brief, an oncologist uses a liquid biopsy to analyze blood for mutations and cancer-related gene expression. Bloom says, “We have adopted digital PCR as our primary technology for this application.” Here, he says, “In essence, a separate PCR reaction is executed in tiny droplets of solution that contain a single fragment of DNA and RNA, and then each of these droplets is interrogated one at a time.”
Scientists perform PCR on increasingly complexsamples, from saliva to seeds, and that requiresextra steps or advanced reagents. (Image courtesyof the author.)Consequently, Bloom’s team can “examine millions of PCR reactions per sample and markedly increase our level of detection and analytic sensitivity,” he says.
In oncology-related PCR, turning this technology into a diagnostic or treatment-related tool depends fundamentally on developing the easiest workflow—from sample to result—that produces accurate information. “Improvements in chemistry, hardware and software have allowed for the workflow to be seamlessly integrated with no negative impact to quality,” Bloom says, adding, “There is also a reduction in processing time by minimizing the hands-on time and eliminating some steps in the workflow.”
Experimental applications
Although PCR’s 30 years of exploration turned many experiments into applications, researchers continue to investigate new uses of the technology. For example, a team of researchers from the Scientific Institute of Public Health in Belgium described an application of PCR to indoor-air quality in a 2015 issue of Applied Microbiology and Biotechnology. As the scientists wrote, “Currently, contamination of indoor environment by fungi and molds is considered as a public health problem.” They added that the time to grow a sample, which is part of the classical approach to monitoring, delays results and reduces accuracy of the data, because some species can be difficult to grow and identify. As an alternative, this team developed a technique based on SYBR green real-time PCR to detect Aspergillus versicolor, a common indoor fungus that is allergenic. They reported: “The limit of detection was determined to be 1 or 2 copies of genomic DNA of A. versicolor.”
Advanced applications of PCR also enhance safety in the food and beverage industry. As an example of such work, Miyo Nakano of the Toyo Institute of Food Technology in Japan published the details and the sensitivity of his bacterial detection assay based on real-time PCR. He designed the assay to detect and quantify Moorella thermoacetica and M. thermoautotrophica in canned coffee drinks. Testing his assay against a pure culture of M. thermoacetica DNA, it could pick up as little as 15 femtograms—that’s one quadrillionth of a gram. In addition, Nakano found M. thermoacetica in two of 30 drinks tested, which makes this test seem worth the while, especially to coffee drinkers.
Studies like these and the other applications of PCR mentioned above make it easy to understand why one study—Polymerase Chain Reaction (PCR) Market-Global Industry Analysis, Size, Share, Growth, Trends and Forecast 2014– 2020—reported over U.S. $4 billion in sales for the PCR market in 2013. This report added: “Pharmaceutical and biotech industries, clinical diagnostic laboratories, hospitals, academics and public research organizations are the major end-users of the global PCR market.” Some of the work mentioned in this article indicates that even more industries and end users will benefit from PCR in the future.
Today’s mature PCR makes life easier for many scientists, providing more sensitivity than ever in a comparatively easy-to-use workflow. Moreover, this technology takes on more complex samples than ever, and it still provides accurate output with relative ease. Although many biologists think of PCR as an old horse in the stable of increasingly young molecular tools, scientists keep adding attributes that lock PCR in the winner’s circle when it comes to go-to technology. In fact, I’ve been hearing for years that one technology or another will soon replace PCR, but that clearly has not happened yet, and I don’t expect it to happen anytime soon. More than plodding along, PCR runs faster and smoother every year. Happy birthday, PCR!
Mike May is a freelance writer and editor living in Ohio. He can be reached at [email protected].