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PCR: The Amplifier Heard 'Round The World
As PCR approaches its 25th birthday, researchers continue to improve it and adapt it to new uses, but users still need to pay attention to the technique's peculiarities.
One night in 1983, the story goes, a scientist named Kary Mullis was taking the scenic drive from Berkeley to Mendocino, California, when he had a flash of inspiration: under the right conditions, purified DNA polymerase might catalyze a chain reaction of replication cycles in a test tube.
SanAir employees checking the DNA sequences of bacteria in a sample. |
As drive-time musings go, this one was a humdinger. Besides tranforming Mullis into a Nobel laureate and his employer, Cetus, into a global player in biotechnology, the polymerase chain reaction has, of course, spawned a revolution in laboratory technology. The most recent evolution of the technique, quantitative real-time PCR, has added a new level of precision and reproducibility, and is fast becoming the standard DNA amplification method in many laboratories.
In traditional PCR, researchers run the amplification for a predetermined number of cycles, then check the products on an agarose gel. In quantitative real-time PCR, experimenters instead monitor the reaction as it proceeds, measuring the amount of double-stranded DNA product continuously and generating quantitative plots of the amplification's progress. It is a sensitive and powerful technique, but beginning and experienced users alike must beware of several potential pitfalls.
Cranking it up to "11"
One can do quantitative PCR without real-time monitoring, but researchers with access to real-time systems usually prefer to use them for all of their PCR work. That's a good reason to shop carefully before buying a real-time PCR system. Indeed, Octavian Henegariu, PhD, Associate Research Scientist in the Section of Immunobiology at Yale University (New Haven, CT) recommends that newcomers to the technique test the products themselves before making a choice.
"You can call the companies and ask for a demo," says Henegariu, adding that when he was shopping for real-time PCR systems a few years ago, "I tested 5 machines, and one of the most expensive ones ... was performing actually much more poorly than I expected by comparison with the other vendors." A typical real-time PCR system includes a standard PCR thermal cycler, plus a fluorescence reader and associated hardware and software to generate real-time amplification curves.
Real-time PCR result for a colony screening experiment. Rising curves correspond to bacterial colonies carrying the target sequence, while flat traces show negative colonies and blank squares indicate empty reaction wells. Image courtesy Tavi Henegariu. Click to enlarge. |
Besides the machine, real-time PCR requires special reagents, such as the fluorescent markers that detect the PCR products. The markers fall into two general categories: fluorescent dyes such as SYBR green, which is activated by any type of double-stranded DNA, and labeled sequence-specific primers, which light up only when they bind their target sequence. SYBR green is considerably cheaper, but sequence-specific primers are less prone to generating background noise from primer dimers and other artifacts.
Conveniently, equipment manufacturers often offer reagent kits to accompany their gear, but Henegariu suggests approaching these skeptically. "One of the vendors... was saying 'our machine requires primarily our kits,' so that once you buy the machine, you're sort of tied to the respective kits," he says.
Knowledgeable researchers commonly buy their PCR equipment and chemical kits separately, shopping for the best price and performance on both. Others prefer to mix their own reagents, an approach Henegariu both advocates and abets on an instructional Web site about PCR.(1) Major suppliers such as Applied Biosystems (Foster City, CA), Bio-Rad (Hercules, CA), Stratagene (La Jolla, CA) and others offer complete lines of real-time PCR kits, and chemical companies are happy to sell fluorescent dyes and buffers to home-brewers.
Regardless of the type of dye, every real-time PCR reaction needs to be optimized. "When you design primers, you should make them so that when you run the reaction itself, you get a unique product, [not] a smear or 2 products or 5 products," says Henegariu.
But while careful primer design can improve the odds, even a well- designed primer pair may sometimes yield artifacts, especially when the primers dimerize with each other and get amplified before the reaction tube reaches the template's melting point. To address that problem, researchers can choose from a long list of "hot start" strategies.
"Most people are using some sort of hot start technique, or they're having to redesign primers," says Lisa Olivier, sales manager at TriLink Biotechnologies (San Diego, CA). Hot starting can be as simple as placing the PCR enzyme atop a layer of wax in the reaction tube, separating the enzyme from the primers and templates until the tube has heated completely. For more reliable results, though, most researchers turn to commercially prepared hot start enzymes, such as those offered by Promega (Madison, WI) and Sigma-Aldrich (St. Louis, MO). In these systems, the polymerase is either sequestered on wax beads or linked to a heat- sensitive inactivating antibody.
This instrument extracts bacterial DNA from water samples for amplification. |
Hot start enzymes are more expensive than regular thermostable polymerases, though, and the cost can add up quickly for researchers doing large-scale studies. To address that market, TriLink recently introduced a less expensive alternative, in the form of hot-start oligonucleotides. "We have a protecting group on the primer itself that the customer would order, and this protecting group will be released from the primer in the initial stages of PCR, when it gets heated up," says Olivier.
That still means ordering a pricier-than-normal primer set for each reaction, though, so the company is also experimenting with hot-start dNTPs. By linking its proprietary hot-start protein inhibitor to the dNTPs, TriLink hopes to sequester these essential building blocks until the reaction tube heats up. If that strategy works, researchers might soon be able to convert any PCR reaction into a hot-start reaction just by using the modified dNTPs.
Good for what ails you
Besides becoming a standard basic research tool in the past few years, real-time PCR has also caught on for practical applications, such as clinical diagnosis and environmental monitoring. "Virtually all of the [PCR] assays that we use now are based on the real-time platform," says Paul Klapper, MD, consultant clinical scientist at the Manchester Royal Infirmary (Manchester, UK).
For clinicians like Klapper, real-time PCR offers an attractive mix of quantitation, precision, and containment. "The real-time PCR system is an enclosed system, so the amplification takes place within a closed tube, [compared to] conventional PCR, where you have to open a tube, transfer it to an agarose gel, and do analysis on that," says Klapper, whose laboratory currently uses the Applied Biosystems TaqMan 96-well system. Keeping the reactions sealed prevents amplification products from cross-contaminating other tests in the lab.
Another new technique, multiplex PCR, is also useful for some types of diagnostic tests, but Klapper cautions that the method is finicky. In multiplex real-time PCR, different colors of fluorescently labeled primers track the amplification of as many as 4 different target sequences simultaneously in one tube. "It sounds nice, but ... there's quite a lot of work to optimize the assay so you don't get preferential amplification of one target or another," says Klapper.
Nonetheless, the Manchester team uses multiplex reactions for some tests, including a broad-spectrum screen for respiratory viruses.(2) "In our case, we're usually looking for a single target, so this cross-competition usually doesn't apply ... if there's influenza A present, it's unlikely influenza B will also be present," says Klapper.
Multiplex PCR may be more troublesome for basic researchers, whose experiments usually offer plenty of room for ambiguity. "When one of the products increases too much, so one gene is amplified a lot more than a second gene, it would artificially decrease the second [gene's amplification]," says Henegariu, adding that for his own multi-gene analyses, "I run them separately. It's a little more work, but it's cost-effective."
Environmental testing, one of PCR's newest frontiers, may also lend itself to the simplicity of simplex reactions. In one cutting-edge project, for example, researchers at SanAir (Powhatan, VA) and Gene Systems (Bruz, France) are testing two new real-time PCR assays for Legionella bacteria, the causative agents of Legionnaire's disease.
One test amplifies a structural gene conserved among all Legionella species, while the other targets a sequence found only in Legionella pneumophila.
"According to the literature, 90% of Legionnaire's disease was caused by the species pneumophila, and only 10% was caused by other species," explains Richard Zhang, director of special pathogens at SanAir. Knowing which species is in a given water supply is critical for identifying the sources of outbreaks.
While the general design of the test is straightforward, environmental analysis requires some controls that clinical and basic researchers seldom ponder. "There is an inhibitor control that's run with each batch of samples, to make sure that any [unidentified] compounds in the sample aren't altering your results in any way," says Bill Kokolis, a senior analyst for SanAir.
If the new real-time system works, though, the payoff could be substantial. "We could drastically reduce the turnaround time on testing. If you can take it from the current 10 to 14 days down to 2 to 3 days, the sooner you treat a possible source [of Legionella], the less risk to the public," says Kokolis.
Indeed, as PCR continues to evolve, the technique seems poised to continue finding useful applications across a wide range of fields. It's certainly enjoyed a scenic ride so far.
References
[1. http://info.med.yale.edu/genetics/ward/tavi/Guide.html]
2. Elnifro et al., "Multiplex PCR: Optimization and Application in Diagnostic Virology," Clinical Microbiology Reviews, 13:4 559-570 (2000).
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