PCR turns twenty this year, dating from its invention in 1985 by Kary B. Mullis. Since then, applications and adaptations of PCR have proliferated. The advent of real-time PCR couples a modified thermocycler with real-time kinetics, for a highly accurate method of quantitating the template DNA or RNA.
Additionally, manufacturers such as Applied Biosystems (www.appliedbiosystems.com), Stratagene (www.stratagene.com), and Ambion (www.ambion.com) have improved many other aspects of PCR, such as dyes and detection reagents, the speed of the procedure, and enzymes.
Real-time PCR differs from traditional, end-point PCR in one important aspect: The concentration of DNA is measured after each cycle, rather than at the end of the process only. The measurement is made through the use of fluorescent dyes.
When concentration is plotted against time, the result is a curve that is logarithmic in its early phases. This logarithmic plotoften referred to as "linear" because it is a straight line plotted on log scaleyields kinetic information that can be used to calculate initial concentrations of template DNA with a high degree of accuracy.
This method is particularly attractive in the case of gene expression studies using mRNA. By looking at gene expression, researchers can detect changes due to a disease state or detect the presence of a pathogen, for example. Gene expression also provides clues about whether a drug compound is working or not.
Traditional methods for quantitation of mRNA such as northern blotting, in situ hybridization, and nuclease protection assays are falling out of favor due to inferior sensitivity and accuracy compared to real-time PCR.
The 7900HT system manufactured by Applied Biosystems is one of the more widely recognized real-time PCR instruments, and gene expression assays are one of its most popular applications.
Applied Biosystems product manager Chris Grimley, reports that "Gene expression is our biggest application. We're looking for changes in gene expression due to a disease state. A highly quantitative result is important."
The 7900HT offers quantitative PCR with multiple channels for different fluorophores, results in about 35 minutes, and 96- or 384-well plate formats with robotic plate-handling capabilities.
Demand for Tools
A gene expression assay generally begins with mRNA, which is reverse transcribed to cDNA. The cDNA becomes the template for the PCR reaction. The demand for tools for RNA analysis, quantification, and amplification have exploded since Sayda Elbashir and Thomas Tuschle published their seminal paper on the subject in Nature in 2001 (24;411(6836):494-8).
At Ambion, the response was swift. Senior scientist Gary Latham reports that, "Two days after the paper in Nature, tech services was deluged with calls. That really put us on track. We had to act as quickly as we could. We helped to feed the movement."
There are now many reports in the literature of the use of real-time or quantitative PCR (QPCR) for detecting mRNAs in RNA interference experiments. Jost and Wettwer of the University of Technology (Dresden, Germany), report the use of QPCR to reveal reduction of a cardiac potassium channel, Kv4.3, by RNA interference (Biochem Biophys Res Commun 330(2): 555-60).
Likewise, Chen and Steer used QPCR to quantitate downregulation of the huntingtin gene during a study combining RNA interference and gene transfer via a nonviral transposon system (Biochem Biophys Res Commun 329(2)646-52). Alternatively, M.W. Graham, et al. used long-range PCR to actually create an RNA interference construct (Methods Enzymol 392: 405-19).
One of Ambion's major contributions to this movement has been a new reverse transcriptaste called ArrayScript. This is a mutated M-MLV reverse transcriptase engineered to have greater cDNA yield.
Reverse transcription is frequently a bottleneck in the process of quantitating RNA. More efficient and faster enzymes mitigate this problem. Ambion also offers kits for preparing PCR templates directly from cell lysates without labor-intensive intermediate steps.
Although a great deal of interest is focused on real-time PCR applications, researchers are finding new uses for end-point PCR as well. At Schering-Plough (www.schering-plough.com), genotyping by PCR is replacing traditional sequencing for genotyping.
Jason Simon, Ph.D., has been principally using Applied Biosystem's TaqMan product line for genotyping for the past 18 months in his discovery technologies laboratory.
The lab identifies SNPs in a publicly available reference population and tests clinical trial samples for them, either focusing on drug candidate genes or on drug metabolism. This work primarily supports Schering-Plough's programs in inflammation, inflammatory diseases, and allergic rhinitis.
The TaqMan system uses a set of two probes, one for each of two alternate alleles. The probe that matches the target gene will be preferentially amplified and detected in the end product. "We did a lot of validation assays before we went full-bore into TaqMan," explains Dr. Simon. "We did genotyping by sequencing and genotyping by TaqMan. The accuracy was high."
With increasing variety in applications for PCR, and increasing reliance on PCR in laboratories, especially in drug discovery, comes a need for increasing speed. Speed can mean two things. It can mean high throughput, where instruments are adapted to handle 96- and 384-well plate formats or to fit into an automated, robotic system.
Many companies are already offering real-time PCR systems that can work with plates and robotic handlers. Another way to increase throughput is through multichannel detection, where four, five, or six different fluorescent dyes are used in the reaction. This enables a researcher to combine this many reactions in a single vessel, then monitor the different reactions at different wavelengths.
Increasing Velocity of Reaction
There's also pure speed. Most lab workers are accustomed to waiting two hours or more for PCR reactions to finish. But several manufacturers are increasing the velocity of the reaction itself. Stratagene has abandoned taq polymerase entirely and has produced a DNA polymerase engineered from organisms of the kingdom Archea, which are frequently found in extreme environments and have proved to be a rich source of heat-tolerant enzymes.
These enzymes are marketed under the name FullVelocity. Stratagene says that with the FullVelocity enzymes, PCR can be completed in under an hour. However, Ann St. Louis, Stratagene's director of product marketing in gene expression, reports that in-house testing with specialized equipment resulted in total run times of 20 minutes.
"We have a high-speed amplification method that is another way to move the technology forward by increasing throughput," explains St. Louis. "There really is a need for faster instruments because the enzymes are faster than the instrument."
One example illustrating both the successful use of real-time PCR and an urgent need for faster, higher throughput reactions is a study authored by scientists from Komfo Anokye Teaching Hospital, Kumasi, Ghana (Transfusion 45(2):133). In the paper, Jean Pierre Allain and colleagues test a combined triple screen for HIV, hepatitis C, and hepatitis B for viral RNA and DNA in donated blood samples.
The triplex screen was found to be successful in identifying HIV and HCV RNA, but had a 1.55% false negative rate for HBV DNA. By pooling and testing blood ten samples at a time, costs can be kept low enough to screen all blood for infections, whereas under the previous system only some blood received adequate screening.
Another aspect of PCR speed is the physical limitation of the instrumentation. Whereas Stratagene cited mechanical limitations, Roche Applied Sciences (www.roche-applied-science.com) has optimized instrumental speed, but remains limited by its enzymes.
According to John Ogden, Ph.D., marketing manager at Roche Applied Sciences, in order to have good, accurate, sensitive results in PCR, it is important for the instrument to move as quickly as possible between temperature steps.
To that end, Roche designed the LightCycler, a system that controls the temperature of the reaction chamber with heated and cooled air. To maximize heat transfer, samples were contained in glass capillary tubes.
Until recently, this technology did not transfer to a block system. The temperatures of the blocks changed too slowly. However, Roche has solved the problem and is now releasing its new LightCycler 480.
"Now we can provide in a 96- or 384-well format everything people loved about LightCycler," says Dr. Ogden.
In response to the obvious synchronicity between Roche's LightCycler and Stratagene's FullVelocity enzymes, Dr. Ogden comments, "We haven't tried putting high-velocity enzymes with the LightCycler. It might be that somewhere in here there is this match of having fast enzymes and high-quality instrumentation and temperature control," but hastened to add that the LightCycler had only been validated with Roche's proprietary master mixes.
"If you mix and match between brands, it's difficult to know what you would get. I can't vouch or not vouch for that."
In the near future, scientists are looking forward to using rapid and quantitative PCR techniques to build miniature devices designed to give instantaneous results. QPCR may become the basis for a bedside molecular diagnostics tool or a handheld bioterror detection device. Many new applications are as-yet unconceived and will be inspired by the increasing power of the technique.