Prime time for real-time PCR

TECHNOLOGY FEATURE Hairpins and more hairpins 308 Standards, standards 310 Box 1: Some like it hot, some fast 306 Box 2: Getting primed and probed 309 Box 3: Thermocyclers go real time 310 © 2005 Nature Publishing Group http://www.nature.com/naturemethods Prime time for real-time PCR Real-time PCR is the favored method for measuring gene expression. Researchers benefit from a vast and growing choice of reagents and instruments for their experiments. Laura Bonetta reports. Real-time PCR combines the amplification of a DNA sequence with the detection of the amplified products during each reaction cycle—in other words, in real time. In comparison to conventional PCR, it can be used to detect a much wider range, over 107 fold, of starting template concentration. It is also less time-consuming, as it does not require analysis of the end products by gel electrophoresis, and can provide a quantitative result. It is no surprise that the technique, first described in the mid-1990s, has quickly grown in popularity. Researchers use it to measure gene expression and copy number, calculate viral titers, and carry out single-nucleotide polymorphism analysis, to mention just a few applications. In particular, real-time reverse transcription (RT)-PCR has become the method of choice to rapidly and quantitatively examine the expression of specific genes. Scientists can readily detect as little as a twofold change in the expression of a target gene in response to different treatments in hundreds of samples per day. Because of the prominence of real-time PCR, companies are flooding the market with newer, improved reagents for every step of the process, from sample preparation to reverse transcription to amplification—all of which promise to make the process even faster, more efficient and more reliable (see Box 1). In addition, the methods and instruments used to measure the amplification products continue to become more sophisticated. As a result, researchers wanting to use real-time PCR are faced with a staggering choice of options. PCR gets real PCR was developed in 1983, a discovery that earned Kary Mullis the Nobel Prize in Chemistry ten years later. The technique, now a staple of every molecular biology Real-time PCR reagents. (Courtesy of Applied Biosystems Group.) lab, uses the thermostable Taq DNA polymerase to extend short single-stranded synthetic primers using the target DNA or cDNA as a template during repeated cycles of heat denaturation, primer annealing and primer extension. With each cycle the amount of template DNA is doubled until one of the reagents becomes limiting, and the reaction reaches a plateau. At the completion of the reaction, amplification products are analyzed by size fractionation using gel electrophoresis. Real-time PCR follows the same course, except that the products are detected as they are made, during the exponential phase of the reaction rather than at the end. Detection reagents now on the market are based on probes and dyes that produce a fluorescent signal each time a doublestranded product is made. The more copies of nucleic acid present at the start of the reaction, the fewer amplification cycles are required to make sufficient product to detect by fluorescence imaging. The cycle in which a significant increase in fluorescence above the threshold is measured—referred to as the C T value—can therefore be used to calculate the quantity of DNA in the sample. Detecting amplicons To carry out real-time PCR, researchers have to choose not only what primers to design (see Box 2) but also what detection chemistry to use. In many cases these decisions will be influenced, if not determined, by the thermocycler (see Box 3) that they have access to and the instrument’s chemistry and dye compatibilities. There are many popular chemistries for real-time PCR. One class uses different fluorescent dyes incorporated in short oligonucleotide probes specific for the amplified target. The second class consists of dyes that bind double-stranded DNA and become fluorescent; the most NATURE METHODS | VOL.2 NO.4 | APRIL 2005 | 305 © 2005 Nature Publishing Group http://www.nature.com/naturemethods TECHNOLOGY FEATURE commonly used of these is SYBR Green I, sold by many companies that provide PCR reagents. As the amount of PCR product increases, more SYBR Green I dye binds to DNA, resulting in a steady increase in fluorescence. The technique is inexpensive and generic, as it requires the same detection reagent for each template to be tested. But detection with dyes like SYBR Green I is less specific than probe-based detection methods. For example, if primers bind to each other, the dye will bind to these socalled primer dimers and generate a signal. In addition, SYBR Green I cannot be used in multiplexed assays—in which several distinct targets are included in a single tube or well—because it will not distinguish among different sequences. Despite these drawbacks, SYBR Green I can be used to quantify the amount of template in a sample if the PCR is fully optimized. But many researchers prefer to use this dye to optimize PCR and check that the primers are working well, before ordering a probe-based assay. TaqMan rules A variety of probes specific for the amplified target (or amplicon) can be used in real-time PCR. By far the favorite, especially among scientists who have just started using the technique, are TaqMan probes. BOX 1 SOME LIKE IT HOT, SOME FAST In the beginning, for those wanting to do PCR, there was Taq. Nowadays thermostable DNA polymerases come in different flavors, each with its own unique capabilities. Researchers also benefit form a variety of kits for amplification, reverse transcription and sample preparation. Some master mixes are optimized for fast reactions, whereas others contain proprietary reagents that prevent mispriming. Many of the commercially available Taq polymerases are special blends, such as Fermentas’ high-fidelity PCR enzyme mix. Stratagene markets the Pfu polymerase, which has a lower error rate than Taq thanks to its proofreading capacity. Takara Mirus Bio, on the other hand, sells Bca BEST, a DNA polymerase with stranddisplacing and template-switching activities that can be used to perform reverse transcription and DNA amplification in a single tube. So-called hot-start polymerases are variations of the naturally occurring enzyme that become active only at high temperatures (typically 95 °C). This property reduces the chances of mispriming—that is, the polymerase extending primers bound to complementary or partially complementary sequences on nontarget DNAs while the reaction is being set up. “Products created during setup decrease the efficiency of PCR,” says Joseph Donnenhoffer of Roche. The company’s FastStart Taq polymerase has a one-base deletion that renders the enzyme inactive at room temperature. The polymerase is sold as part of the LightCycler real-time PCR kits. Sigma Aldrich sells a Taq polymerase that (...truncated)