In a field that changes as rapidly as this one, it's hard to imagine any technology that, after 20 years, would remain essentially unchanged and still be used by a vast majority of scientists. But PCR has managed to do precisely that — to the extent that today, two decades after its invention by Kary Mullis, biologists still refer to it as "the gold standard."
The truly impressive thing is that the use of PCR has been on the rise ever since it was first introduced. "It's been growing every year," says Mike Finney, founder of PCR supply firm MJ Research, who left the company last year. "What's amazing actually is that thermal cyclers ... are still selling big," he adds. (These long-term machines, he notes, last 10 to 15 years.) "You would think that eventually the world would have enough thermal cyclers. … That means that people are planning to do a lot more conventional, old-fashioned PCR." Thanks to advances such as real-time PCR and specialty enzymes that continually open new fields — diagnostics, gene expression, and so on — PCR shows no sign of slowing down.
Indeed, industry watchers predict that the recent expiration of Roche's core PCR patents will cause enzyme prices to drop, more suppliers to enter the field, and use of the technology to grow even faster — especially in markets like diagnostics and environmental studies.
Does that leave any room for competitor amplification technologies that don't rely on PCR? Scientists agree that there are plenty of limitations to PCR — but the inability of any proposed alternative technology to gain significant market share over the years could indicate a widespread sentiment in the community that the devil you know is better than the one you don't.
In the making
When PCR was first dreamed up, it was the only way to reliably and exponentially amplify specific regions of DNA — and, for the most part, the same holds today. That's one of the reasons it has gained such popularity over the years, and one of the reasons it often goes unnoted: "It becomes kind of unremarkable and humdrum," Finney says, "but only because it's just so thoroughly everywhere."
Elaine Mardis, co-director of the Genome Sequencing Center at Washington University, says her group has in the past two years significantly increased its use of PCR. Before that, it was mainly used in genome finishing work, she says. "That all changed with the onslaught of doing lots and lots of human resequencing — comparing disease genomes to control samples, that sort of thing. ...It's definitely the best way to go in and sample a specific gene."
One of the ways PCR has maintained its grip on the amplification throne has been through years and years of advances — a few breakthroughs, but primarily incremental, plodding improvements as biology itself has evolved. "The biggest advance ever was just finding thermostable polymerase," Mardis says. That single change helped make PCR a lot more practical for labs to use. Other early improvements came in basic understanding of how to optimize PCR, Finney says — things like figuring out the best concentration for each component, and getting the right length of olignucleotide.
More recent progress has come in the form of new ways to use PCR, such as real-time PCR, says Ernest Mueller, a principal investigator at Sigma-Aldrich. The engineering of specialty enzymes that give higher fidelity or read length has also gone a long way to keeping PCR up with the times, he adds.
The way things are
New enzyme chemistries and new applications for PCR explain why what could be considered a dinosaur technology is still a mainstay for scientists in large-scale biology. Real-time PCR, a means of performing quantitative research, has been one of the biggest breakthroughs. Andy Felton, a product manager at Applied Biosystems who specializes in real-time PCR, says his company has seen growth of PCR slow. "Regular PCR is … very low-growth — kind of static," he says. "It's the real-time boxes that are pretty much driving the growth for that market."
For real-time PCR (also known as qPCR), the major market is gene expression analysis. "Measuring changes in gene expression" is the driver for this field, Felton says. The technology is often also used for detecting pathogens, so the forensics industry is also starting to heat up in its demand for real-time PCR.
In fact, demand is growing so fast that other companies are beginning to see space to compete. Mats Malmqvist, CEO of three-person AlphaHelix Molecular Diagnostic, says the qPCR arena "is a market window that just has begun to open."
Other forms of PCR continue to come online, such as reverse-transcription PCR, a technology that a Roche spokesperson says "has facilitated the use of PCR in handling RNA-based specimens." The extension of the PCR market into new applications will be a business model that many vendors will pursue. ABI's Felton says with real-time PCR, for example, more and more modes of research will be enabled as the company develops and commercializes reagents allowing scientists to perform an increasing range of experiments, all based on the same instrumentation.
Using PCR on microRNAs, for instance, is one application that many vendors see as the next major wave. Felton says ABI has a new set of products to enable this research, but they're not yet on the market. Other vendors are also in the process of bringing PCR to RNAi customers.
But as biology as a whole improves, some say PCR is showing its age as limitations become more intrusive and problematic. "We probably weren't asking a lot of questions as scientists five years ago that required" technology more complex and sensitive than PCR, notes Stephen DeFalco, CEO of US Genomics, who says that "the standard practice in biology is, if you want to see a needle in a haystack, you make a billion needles." The increasingly evident intricacies of systems biology, he adds, "have driven a need for leading-edge tools."
There are plenty of limitations to PCR, and everyone knows it. That's part of the reason the technology has stuck around: it may not be perfect, but so long as everybody's aware of the flaws, it's still a tool that will have its place in the lab. "The strength of conventional PCR," says Doug Storts, director of genetic analysis in R&D at Promega, "is that people have history with it. ... Most researchers are comfortable with the technology."
Some of the limitations are ones that scientists have been working over time to improve. Elaine Mardis cites read length and high fidelity as two problem areas where she would like to see progress. "I don't think you'll ever be able to make a long enough PCR product for the market," observes Bill Jack, a senior scientist at New England Biolabs.
Customers are also looking for faster reactions and better specificity, says Jun Lee, associate director of research and development at Invitrogen. Generic PCR lacks a quantitative component without time-consuming titration steps, adds Promega's Storts. He says scientists consider assay design another stumbling block for the tool.
One complaint — the need for a thermal cycler — may be on its way out, if certain promising technologies come through. Isothermal enzymes have been in the literature lately and are beginning to make a limited appearance in labs, notes Jack at New England Biolabs.
Despite these limitations, PCR is recognized as a good tool for what it does. But new applications may demand more. "PCR did fine for gene-by-gene and gene families," says Joe Don Heath, director of technical services at NuGen. But with the advent of high-throughput technologies like microarrays, which facilitate not only gene expression analysis but now SNP and comparative genomic hybridization studies as well, there's "a real need" for other technology, he adds.
Paving the path
That need has manifested itself in the form of several little shops working on alternative amplification technologies — some that incorporate PCR, but many that avoid it entirely. Aside from technological hurdles, these newcomers have the additional challenge of proving to scientists who are happily familiar with PCR that their tool is worth a try.
"For the last 20 years people have been banging their heads" to come up with a substitute for PCR, says Mike Finney, pointing out that many alternatives have come along that handle little pieces of what PCR can do but nowhere near all of it. "There just isn't anything."
That's not to say there won't be anything, but Finney's point is a good one: there have been so many attempts at a PCR alternative over the years that the community is understandably reluctant to buy into a new tool that, in all likelihood, may go the way of its predecessors.
Still, it's a battle that many consider worth the hassle. For some, it's already paid off — Molecular Staging's rolling circle technology, for one, became respected on its own, and was lent further credibility when it was incorporated into then-Amersham Bioscience's TempliPhi product, based on the phi29 enzyme. Introduced just a few years ago, phi29 has done so well that WashU's Mardis says, "I don't consider that to be so niche anymore. Everybody's using it." But TempliPhi doesn't do the primer-specific work for which so many people rely on PCR.
TempliPhi and its cousin, GenomiPhi, are meant to work in concert with conventional PCR, says GE Healthcare's Andy Bertera, head of marketing for gene and protein discovery. He sees PCR as a tool so entrenched that "it's a tough challenge for any technology to really uproot it," he says. "At GE, our approach is to look at where new products or new technologies may work alongside PCR."
At Promega, scientists aim to improve the speed issue by multiplexing qPCR and RT-PCR. Their Plexor technology, which can assay multiple targets in a single well, is expected to be released next month, according to Kyle Hooper, genomics product manager.
AlphaHelix, meanwhile, claims to have so accelerated the PCR process that it can now keep pace with capillary DNA sequencing machines in a large-scale production facility, says Allan Asp, vice president of business development. CEO Mats Malmqvist says the company has two prototypes in the works: one for qPCR, and one for high-throughput standard PCR, both of which aim to complete a run in less than 15 minutes. Speed is accomplished by using a centrifuge with the reaction vessels while cycling temperature — a process they call superconvection.
Still, many competitors are looking to unseat PCR. NuGen's Heath says his company has a technology that performs linear, rather than exponential, amplification. Because of the way PCR works, he says, small biases for a gene or a handful of genes that have a particularly high binding affinity can skew a sample. "It's been shown that linear amplification has significantly less bias," he adds. But whether scientists, who already complain that PCR is too slow, will be willing to try out a non-exponential platform is a gamble.
Meanwhile, 454 Life Sciences has developed a technique called emulsion-based clonal amplification — due to be published soon — designed to "clonally amplify each fragment of the genome in a single mix," says Kent Lohman, senior director of molecular sciences. The reaction, which occurs on beads suspended in a water-in-oil emulsion, uses amplification reagents optimized by 454, Lohman adds. The technology was engineered to feed directly into the company's PicoTiter sequencing instrument.
Other people are simply trying to avoid using PCR — and, with the growth of single-molecule techniques, the industry is sure to see more and more of this. Stephen DeFalco at US Genomics says directly counting individual molecules is the most reliable and precise way to get the job done. "I really do think that the future of life sciences research ... takes place at the single-molecule level," he adds.
Still, the fact remains that PCR is king and the alternatives don't rank much above poor relations. That's one reason so many people eagerly awaited an event that with any other technology may have gone unnoticed: the expiration of three core PCR patents, which happened at midnight on March 28.
To keep it in perspective, Roche's PCR portfolio contains more than 800 patents, including plenty of continuations on some of the early patents. Still, experts say these particular patents could free up the market enough for new suppliers to jump in the ring and drive prices down. Whether that price drop would be led by the major vendors voluntarily dropping their prices (which may not happen immediately), or be driven by an upstart entrant is anybody's guess.
Mike Finney says a potential price cut could be significant. "Roche has been charging recently 15.5 cents a unit for combination enzyme and PCR rights," he says. "People have been selling the enzyme for down to maybe three or four cents a unit above that cost." So conceivably, he points out, companies could keep their existing profit and drop the price all the way down to three or four cents. Finney says not only does he expect to see such a nosedive in price, but he believes it will fuel a significant rise in the use of PCR.
If that's the case, expect to see major changes in PCR use among diagnostic labs and environmental research, says GE Healthcare's Andy Bertera. "Cost is more of an issue in [these fields," he says, "because you've got a larger number of samples." Price drops will be most freeing for fields like those, where PCR use may have been artificially low due to expense of reagents.
While it will take time to reveal the answers about price drops and alternative technologies, one thing is for sure: PCR isn't going anywhere. Quite simply, Finney says, "it's still just as useful for all the stuff it used to be useful for."
In 1985, calling a technique "PCR" would have been sufficient to let someone know what kind of experiment you were performing. Today, the original PCR family has spawned a number of cousins — making it that much more confusing for people to know which PCR someone has in mind.
Developed in the mid-'80s by Kary Mullis, then at Cetus, polymerase chain reaction began as a means of exponentially copying specific segments of DNA using primers and an amplification enzyme. Used to clone genes, identify genetic fingerprints, and diagnose disease, among other things, PCR is still done today in a manner very similar to its original conception.
Over the years, of course, variants of PCR have come along. Real-time, also known as quantitative, PCR was designed to quantify mRNA. Being able to get a quantity was a vast improvement over conventional PCR for certain applications and ushered the PCR technology into areas like gene expression analysis.
Sometimes called RT-PCR instead of qPCR, real-time PCR can be confused with that other RT-PCR ¯ reverse transcription PCR, a reaction based on an RNA strand in which both complementary and antisense DNA strands are produced using a reverse transcriptase enzyme.
Glimpse Into a Patent Portfolio
Roche's enviable PCR patent estate comprises more than 800 patents, but three that expired in late March were what Roche called its "foundational patents," acquired initially from Cetus. (The European equivalents will expire next year.) Here's a quick look at the ties that no longer bind.
US Patent 4,683,202: Process for amplifying nucleic acid sequences. Inventor: Kary Mullis. Filed: October 25, 1985. The original deal, this was the method that made Mullis famous — using two primers and enzyme.
US Patent 4,683,195: Process for amplifying, detecting, and/or cloning nucleic acid sequences. Inventors: Kary Mullis, Henry Erlich, Norman Arnheim, Glenn Horn, Randall Saiki, and Stephen Scharf. Filed: February 7, 1986. This patent expanded the PCR technology to include cloning a sequence into a vector.
US Patent 4,965,188: Process for amplifying, detecting, and/or cloning nucleic acid sequences using a thermostable enzyme. Filed: June 17, 1987. This patent covers what is still seen as the biggest advance in PCR — finding a thermostable enzyme.
The Good, the Bad, and the Potential
What's the true measure of a technology? Here's a look at where PCR has been, and where it's likely going — based on current challenges people see in it. The following lists were compiled by polling PCR experts on these topics.
Improvements over the years:
• Finding a thermostable enzyme
• Identifying optimal concentrations and oligo length
• Reverse-transcription PCR
• Quantitative PCR
• Specialty enzymes for higher fidelity and longer read length
• Increased speed of the PCR process
• Hot start PCR
• High-throughput PCR
Current challenges (and where you can look for efforts from vendors):
• Still a need for higher fidelity, longer read length, and faster reactions
• Better specificity with primers
• Reliance on thermal cycler (isothermal enzymes look like promising solutions for this problem)
• Need for more multiplexing
• Potential for contamination
• Amplification when the DNA sample is damaged, impure, or from an unknown source
• PCR technology that can handle many templates
• Costs need to be lower for fields like diagnostics, where sheer volume of samples makes PCR prohibitively expensive
• Better/easier assay design
• Higher sensitivity to detect smaller amounts of message
Will the Patents' Expiration Affect You?
In the complicated landscape of PCR patent enforcement — thermal cyclers, for instance, have been free and clear in Europe but heavily protected in the US, while the reverse has been true for a broad thermostable enzyme restriction — no wonder everyone's asking what the core PCR patents' expiration will mean for them. (One thing to remember: the European equivalents of the core PCR patents won't expire until next year.)
There's no simple answer. As patent owner Roche points out, the company still holds 800-odd PCR-related patents, so it's not like the field is suddenly up for grabs. If you happen to be working with a particular enzyme that was patented and using a particular method that was patented, the event means that you can continue to do that research without having to pay the extra money that Roche used to collect for its licensing fee — approximately 15.5 cents per unit of enzyme, according to Michael Finney, former president of PCR instrument and reagent vendor MJ Research. For those scientists fortunate enough to be in that group, prices for reagents could fall to just three or four cents per unit, predicts Finney. He notes that it may take longer for major players — Sigma-Aldrich, Roche, GE Healthcare, Promega, and Applied Biosystems — to lower prices, and that a smaller entrant to the field may lead the price-cutting trend.
But scientists whose research has evolved over the years to include specialty enzymes or to newer applications of PCR (such as qPCR or RT-PCR) won't benefit. Those enzymes and methods are covered by more recent patents and will remain protected for quite some time. Many people are simply hoping for the best and waiting to see if their vendors lower prices.
For vendors, the field is incredibly complicated, and the ensuing confusion will no doubt continue to affect their customers. On one hand, Mats Malmqvist, CEO of AlphaHelix, says the change means "it will be a much easier ocean to navigate. … There will be a tremendous difference — definitely in technology as well as in applications — due to the expiry of the core patents."
But others are not so enthusiastic. "There are a lot of other application patents," points out one vendor representative who preferred not to be named. "Some patents have run out, but it's not like now the world is open."
A spokesperson in the legal department of Applied Biosystems, which gets a cut of PCR royalties for certain uses of the tool, says the company expects vendors to stick with the licenses they already have "and that licensees will continue to honor the terms of their contracts." In fact, a spokesperson for Invitrogen notes that it intends "to remain licensed under Roche's amplification patents, and we assume other vendors will do the same."
Up Next: MicroRNA
Always eager to keep up with researchers at the vanguard, PCR vendors agree that the hottest new application in the next few years will be targeting microRNAs. Many of the vendors GT spoke with acknowledged that they've got products in the works for this particular research field. At this point, it looks like it will be a race to commercialization for most of these new tools.
"MicroRNA — everybody agrees that's the next big thing," says Henrik Pfundheller, director of sales and marketing at Exiqon, which sells qPCR technology. "We are developing real-time PCR assays for detection and silencing of microRNAs. We're going to be launching several assays here in early summer."
At PCR giant Applied Biosystems, product manager Andy Felton says his company has "a new set of products to quantitate microRNAs using TaqMan" but notes that those products are still being tested and haven't hit the market yet.