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PCR M
ore than a decade ago, the polymerase chain reaction (PCR) (1, 2) revolutionized molecular biology by making lab work faster and easier. These days, many researchers expect quantitative PCR to imitate its predecessor's success. Used to determine the concentration of specific nucleic acids, quantitative PCR has been embraced by researchers and clinicians for gene quantitation, pathogen detection, and even process validation in pharmaceutical production. New methods are being published products are being developed and new companies specializing in applying the technique are being formed Despite recent attention focused on this technique, quantitation is an old idea—almost as old as PCR itself. "Quantitative PCR has been happening all along," says Francois
Elizabeth Zubritsky
all the attention now? There are Widespread interest twoWhy main reasons, Ferre and others say. firstis the current emphasis on in gene quantitation The genomics research. As genes are located, functions need to be determined, and and high-throughput their studies of gene expression become the "The next dimension of research is assays are putting focus. to figure out what is expressed and how much and when," says Mike Lucero, prodquantitative PCR backuct marketing manager for PCR at PerkinElmer. "That is just as basic as knowing in the spotlight. what the DNA sequence is." Ferre, who heads the gene quantification company Althea Technologies. In the early 1990s, for example, Michael Piatak, Ferre, and others used quantitative PCR to show that HIV viral loads—the degree of infection—in patients' blood were higher than previously thought (3,4). "Even back in 1989, at meetings focused on PCR quantitation was s aot topic," "erre says.
The other reason is the emergence of kinetic PCR also called real-time PCR In kinetic PCR measurements are recorded as the reaction occurs. In contrast, the standard endpoint analyses are done using electrophoretic gels after PCR is complete. The basic real-time technique dates back to 1993, but Ferre says it really took off in 1995-1996, when fluorescent techniques that monitor the accumulation of product emerged.
Analytical Chemistry News & Features, March 1, 1999 191 A
Focus The need for quantitation
But why go to all that trouble? After all, PCR should be inherently quantitative. The number of template molecules should double with each cycle of heating and cooling, resulting in exponential growth (5). To calculate how much product will accumulate, all you should need to know is how much material you started with and how many reaction cycles were run. You should be able to work backward, too, calculating the initial number of copies by knowing the amount of end product and the number of reaction cycles. That's the theory. In practice, the process is not that straightforward. Amplification eventually reaches a plateau, and it may not be exponential to begin with because of variations in reaction conditions or the presence of inhibitors. Either way, estimates of the number of copies can be wildly inaccurate. Competitive PCR
The earliest quantitative method was to create standard curves by stopping reactions at various points and determining the amounts of product. The process required radioactive labeling for reasonable sensitivity, and it was tedious, often involving repeated runs at various dilutions of the template. So researchers cheered when a technique called quantitative competitive PCR came along in 1990 (6). In competitive PCR, two templates are used in a single reaction. One template is the test sample, the initial quantity of which is unknown. The second is the competitor—an internal control that may have nearly the same sequence. In some cases, the competitor differs by only one base pair (bp), so that it includes a restriction enzyme site that the test template does not. Or the competitor may be slightly different in size, causing the two products to migrate different distances on a gel. In any case, thefinalamounts of both products are compared; and because the initial amount of the competitor is known the initial amount of the test template can be determined Competitive PCR is still widely used, especially for RNA work, but it requires several assumptions that do not always hold true, Ferre says. The target and the standard may not be amplified at the same 192 A
rate, especially if they have very different sequences. Even a difference of 1 bp can lead to a substantial difference in amplification efficiency, he says. In reversetranscriptase PCR (RT-PCR), which begins with RNA instead of DNA, the efficiencies of reverse transcription for the template and the standard also might be different. Finally, if restriction enzymes are used, the analysis can be skewed unless all of the competitor is cleaved. More importantly, individual strands of competitor and test DNA may join together, or hybridize, to form a third DNA species called a heterodimer. This problem, Ferre says, "can seriously complicate the quantitative analysis". Ferre favors the use of external controls, but he's not opposed to competitors. "They can work if you manipulate the system very, very carefully," he says. Another drawback to competitive PCR is low throughput, says Carl Wittwer, a University of Utah researcher who developed a fluorescent instrument for quantitative PCR. "With competitive PCR, you get your best answer when the concentration of your competitor is near the concentration of your target," he says. Because you don't know that concentration, you must run several reactions using different dilutions of target or competitor the equivalent of creating a standard curve. Most competitive PCR techniques also require additional analysis—often by gel electrophoresis—after amplification, says Wittwer. To avoid such extra steps, some researchers are turning to real-time techniques, where quantitation is done without opening the reaction tube. This approach is faster and easier, and it lowers the risk of contaminating the reaction with stray DNA a single molecule of which could ruin an experiment. Early kinetic PCR
When many researchers say "quantitative PCR", they mean kinetic or real-time PCR The technique became well known in 1995— 1996, and, like the original PCR ii was an idea waiting to be discovered. The foundation had been laid years before by Russell Higuchi and colleagues at Roche Molecular Systems. In 1992, they developed a quantitative technique based on a
Analytical Chemistry News & Features, March 1, 1999
well-known property of EtBr: when EtBr is bound to DNA and excited by UV llghtt ,i fluoresces (7). That made EtBr perfect for visualizing bands on electrophoresis gels— something that had been done for years. Higuchi and colleagues realized they could leave the products in the reaction tubes instead and measure the EtBr fluorescence. A year later, the researchers showed that they could continuously monitor EtBr accumulation to obtain kinetic measurements (8). The technique was simple and homogeneous and it did not require a competitor It took about two more years for kineticPCR instruments to hit the market. The first instrument was Perkin-Elmer's ABI Prism 7700 Sequence Detection System, followed by Idaho Technology's LightCycler (now manufactured and sold by Roche Molecular Biochemicals). Both instruments use fluorogenic chemistry instead of EtBr, and both are "basically thermal cyclers with optical detection systems," says Lucero. Given that thermal cyclers, optical detection, and fluorogenic chemistry had all been around for VP3TS why wasn't real-time quantitative PCR possible earlier? "It was " Lucero says. "It's just thtit no one thought of it" Exonuclease probes
There are two major approaches to kinetic PCR. Thefirstis the Perkin-Elmer method, which combines Higuichi's fundamental kinetic technique with exonuclease probes—originally developed in 1991 by Pamela Holland and colleagues at Cetus (now Roche) (9). The probes capitalize on the ability of some DNA polymerases to cleave unpaired nucleotides from DNA this is called exonuclease activity. Because the Thermus aquaticus {Tad) polymerase used in PCR has this ability exonuclease probes are also called TaqMan probes. In Holland's single-stranded probe complementary target DNA was used in addition to the two PCR primers The reaction would begin as usual with the nrimers binding to the target and the Dolvmerase extending the second strand bv adding nucleotides to the end But if the nrobe matched the target it would also bind And if the poly merase ran into the probe during the extension process, the polymerase would ,
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treat it as an obstacle and remove it by
cleaving it into pieces. Because the probe had been labeled with 32P, the radioactive piece could be isolated on a gel, allowing the researchers to determine how much of the desired product had been produced. To get away from radioactivity and make the assay more homogeneous, the Perkin-Elmer researchers used fluorescent dyes (10). They placed a fluorescent donor at the 5' end (the upstream end) of the probe. In the middle of the probe, they placed a fluorescent quencher, which kept the donor from giving off light. (These days, the quencher is at die 3', or downstream, end.) If the probe matched the target, the donor was cleaved just as Holland's radioactive labels had been cleaved separating the donor from the quencher and increasing die fluorescence (see Figure 1) If the probe did not match no cleavage occurred The donor and quencher remained together and there was no increase in donor fluorescence "We had a quantitative system and a
prototype instrument then," says Linda Lee, the Perkin-Elmer researcher who developed the donor-quencher probe. "The next step was to use the system to discriminate between two different alleles [variants of a single gene]." To do that, Lee "multiplexed". She used two donor-quencher probes, one that matched the gene's normal sequence and one that matched a mutated sequence. The two probes used different fluorescent dyes, emitting at different wavelengths, which allowed the signals to be distinguished, and a spectrophotometer measured the fluorescence in real time directly from the reaction tubes. Hybridization probes At about die same time, Wittwer and colleagues were developing a different approach to real-time PCR with the LightCycler. The instrument was based on an earlier machine, the RapidCycler, which ran ordinary PCR reactions in glass capillary tubes. The capillaries had been chosen
to speed up heating and cooling, which shortened the total reaction time. But Wittwer also saw the capillaries as "an invitation to shine some light" at the reactions. In direct analogy to flow cytometry, which passes labeled cells by a laser, the researchers "essentially passed capillaries containing labeled reactions by a light source," he says. The researches also developed new fluorescent probes, called hybridization probes (11). Like exonuclease probes, hybridization probes are used in addition to the PCR primers, increasing the specificity, and they can be used to discriminate among different alleles of genes (12). But unlike exonuclease probes, where the signal increases when the fluorescent donor is separated from the quencher, hybridization probes bring two different labels together to allow resonance energy transfer. This happens when both probes bind or hybridize to the template DNA When either probe fails to bind because of mismatches Hie rptnnnnrp enerirv trpti^fer dnps not occur and the fluorescence is much less intense T h u s the flnnrescenrp i
