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By Paul Nicolaus

April 3, 2019 | Imagine a future decades down the road in which polymerase chain reaction (PCR) is viewed as old-fashioned. It might prove challenging considering the method, first developed in the 1980s, transformed the study of DNA and has been called one of the most important scientific advances in molecular biology.

Before its existence, methods used to copy DNA fragments relied on plenty of time and labor. Now, though, it is possible to produce billions of copies in just a few hours or less. PCR made such a splash, in fact, that its creator eventually received a Nobel Prize for Chemistry.

The method is now used in an array of laboratory and clinical techniques, including the detection of bacteria or viruses and the diagnosis of genetic disorders, according to a National Institutes of Health (NIH) PCR Fact Sheet, and it has even been used to copy DNA fragments found in preserved tissues, like those of a 7,500-year-old human pulled out of a peat bog.

PCR has been “absolutely fundamental to the development of molecular biology,” said Jim Huggett, principal scientist at the National Measurement Laboratory (NML) in the UK and senior lecturer at the University of Surrey. It has made possible everything from the sequencing of the Human Genome Project to the development of viral vaccines.

Even so, he says it’s possible to envision a time, say fifty years from now, when PCR may be seen as a “quaint but very necessary method” that sidestepped a problem: our methods to detect DNA are insensitive.

In the meantime, researchers generate billions of copies of the sequence of interest in order to carry out experiments and procedures in fields like molecular biology, evolutionary biology, and medical diagnostics.

Blueprint for Reference Measurement Procedures

The general concept of PCR has remained virtually unchanged since the 80s, but there have been notable developments along the way, like the concept of monitoring DNA amplification in real time through the monitoring of fluorescence.

In real-time PCR (also known as quantitative PCR or qPCR), fluorescence is measured following each cycle, and the cycle at which a given fluorescent signal occurs reflects the amount of DNA in the sample. “If the amplification curve occurs earlier in the reaction, we can conclude there is more DNA at the beginning than if it happens later in the reaction,” Huggett said, “and that allows us to quantify quite precisely.”

This qPCR method is used for a number of applications within the cancer and infectious disease space, he explained, and particularly for viral load testing. HIV viral load is monitored in patients to establish whether therapy is working, for example, or to monitor the development of drug resistance. It works because there are reference materials that are able to allow different laboratories to compare their measurements, and the world of virology can form precise measurements and treat patients depending on how much viral load they measure.

“But it’s safe to say that the examples where quantification is used are quite rare,” he added. This may be due at least in part to the challenge of standardization. One of the difficulties of PCR related research, Huggett explained, and particularly with qPCR is that much of the work is not reproducible.

To tackle that dilemma, he has been involved in the publication of the Minimum Information for Publication of Quantitative Real-Time PCR Experiments (MIQE) guidelines and more recently the digital MIQE guidelines, which outlines the critical information that needs to be included in a paper. That’s at the preclinical research point, he pointed out, but the focus for the NML is supporting molecular diagnostic developers to ensure that their methods are “robust and reproducible” as well.

He and colleague Alison Devonshire, science leader with the NML’s molecular and cell biology team, say they are particularly interested in digital PCR (dPCR). (LGC, formerly the Laboratory of the Government Chemist, was privatized in the ’90s but maintained the government role. Devonshire and Huggett’s work for the NML, hosted at LGC, is funded by the UK government.)

This technique, Huggett explained, is the newest iteration of PCR even though it was actually developed before qPCR. Whereas qPCR looks at an amplification curve to quantify the DNA present, dPCR makes it possible to count individual DNA molecules.

Rather than using a reference (calibration) material that everything is compared to, Huggett and Devonshire envision a reference (calibration) method that could be used to count the DNA molecules. Following a specific standard operating procedure (SOP) could allow different laboratories to make materials which would be value assigned using digital PCR that, in the short term, could then be used to calibrate other methods like qPCR or next generation sequencing.

One reason they and others at National Measurement Institutes across the world are interested in this technology is that it has the potential to be SI-traceable, or traceable to the International System of Units. (Other familiar SI-traceable units include the second, for example, or the kilogram.) “What that means is it’s not traceable to a method, it’s not traceable to a material, but it’s traceable to a constant,” he said. “And in this case, it’s counting.”

At the moment, they are looking at whether these methods can be used to support routine measurements in areas such as infectious diseases and cancer genomics. The genetic testing of tumor tissue and circulating cell-free DNA (cfDNA) for somatic variants guides patient treatment of many cancers, and these types of measurements are needed to advance precision medicine, Huggett, Devonshire, and colleagues explained in a 2018 paper published in Clinical Chemistry (doi: 10.1373/clinchem.2017.285478).

Previously, there weren’t any reference measurement procedures available for nucleic acid quantification that would support the translation of tests for circulating tumor DNA into routine use. So they outlined a process by which a method can be validated and developed the first primary reference measurement procedure with direct SI-traceability for a variant in the KRAS gene which is frequently mutated in colorectal cancer. This is the first method for nucleic acids listed on the Joint Committee for Traceability in Laboratory Medicine’s (JCTLM) database of clinical reference measurement procedure.

“That’s really a world first” in terms of developing that sort of approach, Devonshire said, which is more routine in clinical chemistry where measurements are calibrated to reference materials defined in SI units, or those accepted for use with the SI (e.g. mg/L). In molecular diagnostics and genetics, no one has taken that approach before, she added, “so we hope that’s going to be a blueprint which will enable people to develop other reference measurement procedures in the future for a range of diagnostic measurements.”

Building Upon Existing Technology

Nucleic acid amplification techniques (such as PCR) and sequencing technologies are not mutually exclusive, and the work of Jörg Tost proves that point. In general, the lab he leads is mainly interested in epigenetic modification—looking at DNA methylation and microRNAs in different human complex diseases.

Editor’s Note: Devonshire and Tost are both speaking during a session on new PCR methods and Enabling Technologies for Circulating Biomarkers at Molecular Diagnostics Europe, May 6-9 in Lisbon, Portugal.

As a side project, though, the director of the Laboratory for Epigenetics & Environment at the Centre National de Recherche en Génomique Humaine (CNRGH), CEA – Institut de Biologie Francois Jacob in France, said his lab explored a method for performing sensitive analysis of rare mutations.

This was not the development of a new technology, Tost explained, but rather a tweak to existing technology. He and his colleagues took the ice-COLD-PCR approach initially developed by the laboratory of Mike Makrigiorgos at the Dana Farber Cancer Institute in Boston, Mass., and realized that using chemically-modified blocking oligonucleotides led to improved enrichment efficiencies.

Rather than amplifying a specific mutation, this Enhanced-ice-COLD-PCR approach does the opposite. The use of oligonucleotides (which are complementary to only the unmutated wild-type sequence) along with the COLD-PCR protocol allows researchers to define a temperature where a single mismatch is enough to destabilize this blocking oligonucleotide-DNA duplex. The destabilization makes it possible to selectively amplify the mutated molecule at a higher rate compared to the wild-type molecule.

This method is well-suited for mutation hotspots (such as KRAS, BRAF, and NRAS) when there are many different mutations nearby because “we don’t need any information about the mutation,” Tost said. “We don’t need to prescreen. We don’t have a specific assay for each mutation.”

There is just this enrichment that provides a DNA sequence in which the mutation is present in a large proportion, which can then be detected with traditional sequencing techniques like Sanger, pyrosequencing, or Next Generation Sequencing (NGS). “It’s really just a trick to improve an existing technology but which actually makes a major difference,” he added, that makes it possible to “see the mutations very easily.”

While the initial workflow used pyrosequencing for a short time-to-results, Tost is moving toward an NGS based readout because it is more widespread and may lead to better signal to noise ratios. “One of the other things we have been doing is we have extended this to DNA methylation analysis,” he noted.

Envisioning the Future of PCR

If you look at flu or gastro panels, for example, many of them are handled in a very manual, labor-intensive process that introduces the potential for human error, explained Dan Harma, Chief Commercial Officer at NeuMoDx Molecular, a sample-to-result molecular diagnostic company based in Ann Arbor, Mich.

Currently, many tests are carried out by manually streaking or by systems that automate the plate management process but are large and expensive. True continuous random-access platforms like the NeuMoDx 96 and 288 offer sample to result testing in a multiplex format that is more automated with a faster turnaround time, he pointed out.

Looking ahead, Harma believes molecular testing will be able to replace that with a faster, more efficient, and more accurate test. Molecular testing will become more automated, he said, and the total overall cost of molecular testing will likely decrease as technology continues to improve and the economies of scale increase.

From Tost’s vantage point, the future of PCR molecular diagnostics will involve a simplification of the entire process using a cassette-like device that performs sample preparation as well as mutation detection followed by a standardized, easy-to-interpret output. “Detecting a mutation is not really the tricky part nowadays,” he said. The primary challenge, rather, is that samples are not stored, collected, or processed in a way that allows for the reliable detection of mutations.

As he looks ahead, Huggett points to efforts to simplify both the instruments and the process in order to bring the advantages of PCR closer to healthcare workers and patients. Consider a chlamydia test, for example. “Your patient comes in, you sample them, the test is sent to the centralized laboratory,” Huggett explained. “The patient is not going to wait for the three days it takes, or probably more, to get the sample back. So the patient leaves.”

The problem is that this process creates a risk. You may lose the sample, for example, or the sample could be mislabeled. Or that patient might go back into the community and infect other people. Now imagine a test in the clinic that takes, say, an hour. You ask the patient to stay, give him or her a magazine, and you get the results then and there. “So you’re taking the test to the patient and simplifying that diagnostic step,” he said. “PCR just lends itself to this because it is so fast.”

Although he anticipates that new genomic technologies “will presumably at some point take over so that we don’t have to amplify the molecules a billion times,” there are some challenges involved, such as regulatory issues and the process of gaining acceptance within the clinical community. So even though he can envision a future when this method is thought of as quaint, Huggett thinks “PCR is here to stay for a while.”

Paul Nicolaus is a freelance writer specializing in science, nature, and health. Learn more at www.nicolauswriting.com.