Quantitative PCR Update

Courtesy of Applied Biosystems THE TAQMAN PROCEDURE: Applied Biosystems' TaqMan procedure relies on the 5'-to-3' exonuclease activity of Taq polymerase. The TaqMan probe bears a fluoro-phore at the 5' end and a quencher on the 3' end, rendering the molecule non-fluorescent. During amplification, the probe binds to the template between the two PCR primers. When the polymerase encounters the probe, it starts chewing away at the end, releasing the fluorophore into solution, where it is free to fluoresce. In the nearly two decades since its discovery, the polymerase chain reaction (PCR) and its derivative techniques have revolutionized molecular biology. The exponential nature of the technique allows one, in theory, to calculate the amount of starting material from the amount of product at any point in the reaction. In practice, however, reaction conditions can interfere with the exponential amplification and affect product concentration. Standard PCR works best, therefore, as a qualitative technique. But many researchers want to use PCR quantitatively (Q-PCR), to measure gene expression or copy number, or to calculate viral titer, for example. Early attempts at quantitation involved stopping PCRs at various points to generate standard curves--a laborious, low-throughput process. Modern techniques are much friendlier, adding real-time reaction monitoring with dedicated instruments and fluorescent probes. Because these techniques combine amplification, detection, and quantitation in a single step, they are considerably faster than endpoint-driven analyses. And, scientists can run such reactions in microtiter plates, making the method high-throughput, too. "Quantitative PCR has applications in many areas of research," says Erik Parkinson, technical support representative, biochemistry division, Salt Lake City-based Idaho Technology. "For example, in cancer research it's possible to quantify how much of a cancer gene is expressed." Parkinson calls real-time PCR the benchmark in quantitative studies, and Heidi Nelson-Keherly, director of Lark Technologies, concurs. Houston-based Lark Technologies, established in 1997, was the first service company to offer quantitative real-time PCR commercially. Researchers can use Q-PCR for copy-number analysis, gene expression analysis, and SNP genotyping; they can detect alternative splicing events, quantify viral loads, and facilitate micro-biological diagnoses as well. COMPETITIVE VS. REAL-TIME One Q-PCR approach, quantitative competitive PCR, assesses initial template concentration by analysis of the amount of product after the reaction is completed. The technique employs two templates in each reaction: a DNA fragment (the competitor) of known concentration, and the target DNA. The amplification of the two templates occurs simultaneously, with both fragments competing for the same set of primers. By comparing the ratio of the amounts of the two amplified products, scientists can then determine the initial concentration of the target sample.1 Courtesy of Molecular Probes MOLECULAR BEACONS: The simplest of the hairpin probes, molecular beacons consist of a sequence-specific region (the loop) flanked by short complementary sequences that form the hairpin. At either end of the molecule are a fluorescent quencher and dye. In solution, the molecule adopts the hairpin configuration, bringing the quencher and dye into close proximity and extinguishing fluorescence. When the molecule binds to its target, however, it unfolds, freeing the fluoro-phore from quenching. Quantitative competitive PCR suffers from two drawbacks. First, small variations in technique (such as pipetting) can dramatically alter product yield. Second, most protocols require multiple dilutions or additional analysis after amplification, rendering the method low-throughput. In contrast, fluorescence-based assays can be monitored in real-time, either continuously, or at some particular stage (say, the annealing step) in each cycle. The initial template concentration is calculated by determining when the level of fluorescence rises above a particular threshold value. Many real-time reactions employ sequence-specific probes, but some use general DNA-binding probes, such as SYBR Green I and SYBR Gold, which act by binding to double-stranded DNA. As the amount of product increases, fluorescence intensity rises proportionally. This approach is both inexpensive and generic, requiring only a general DNA-binding dye instead of sequence-specific probes for each template to be tested. But these dyes are less specific than sequence-specific probes (they may bind to primer dimers, for example) and also cannot be used in multiplexed assays--that is, in assays in which several distinct sequences are assayed in one reaction tube by labeling the probes with dyes of differing emission wavelengths. THE TAQMAN COMETH Sequence-specific probes have neither of these problems. Researchers have developed several different varieties, including TaqMan®, hybridization, and hairpin-based probes. TaqMan probes, developed by Applied Biosystems of Foster City, Calif., consist of single-stranded oligonucleotides that are complimentary to one of the target strands. A fluorescent dye adorns the 5' end and a quencher is bound to the 3' end. This molecule is chemically modified on the 3' end to prevent the polymerase from using it to prime DNA synthesis; instead, the probe hybridizes to the template between the two amplification primers. When Taq DNA polymerase encounters this oligonucleotide, its 5'-3' exonuclease activity digests the probe, releasing the fluorescent dye from quenching. TaqMan reactions therefore sometimes are called fluorogenic 5' nuclease assays. Idaho Technology developed an alternative approach: hybridization probes. Two sequence-specific probes bind adjacent to each other on the amplicon in a head-to-tail arrangement--one has a fluorescein moiety at the 3' end; the other is labeled on the 5' end with either Red 640 or Red 705. When fluorescein is stimulated by the excitation source, it induces fluorescence of the second dye by fluorescence resonance energy transfer (FRET, see Box). The emission maxima of the red dyes are sufficiently distinct to allow measuring two different targets simultaneously. HAIRPINS, HAIRPINS, HAIRPINS Several probe classes are based on stem-loop hairpin structures. At one end of the short stem is a fluorophore; at the other, a fluorescent quencher (typically DABCYL). The sequence-specific portion of the molecule resides in the loop. This molecule can exist in two states, open and closed. When free in solution, the molecule adopts a closed, hairpin-like conformation, bringing fluorophore and quencher into close proximity and stifling fluorescence. When it is bound to its target, however, the molecule opens, releasing the fluorophore from quenching. The hairpin structure gives these probes added specificity, as the probe-target complex must be thermodynamically more stable than the hairpin structure itself for stable binding to occur. Courtesy of DXS Genotyping DUPLEX SCORPIONS: The benefits of an unary reporter are significant. As the reaction is effectively instantaneous, it occurs prior to any competing or side reactions such as target amplicon reannealing or inappropriate target folding. This leads to stronger signals, more reliable probe design, shorter reaction times, and better target discrimination. Molecular beacons are the simplest hairpin probes, consisting only of the hairpin itself. In theory, they are easily multiplexed, but in practice, few efficient fluorophore/quencher pairs exist that will function at a given wavelength. Sanjay Tyagi and Fred Kramer at the Public Health Research Institute, Newark, NJ, developed wavelength-shifting molecular beacons, which contain a generic harvester fluorophore, a probe-specific emitter fluorophore, and a quencher, specifically to alleviate this problem.2 Scorpion probes covalently couple the stem-loop structure to a PCR primer. From 5' to 3', these probes contain the fluorophore, stem-loop, quencher, a PCR blocker (which prevents read-through by DNA polymerase), and primer. During the PCR annealing step the specific probe sequence folds back on itself to bind its compliment within the same DNA strand, opening up the hairpin loop and separating the fluorophore and quencher; the resulting structure resembles a scorpion's tail. Because the hybridization of probe sequence to amplicon is intramolecular, Scorpion probes are more efficient than binary systems like molecular beacons. One variation on this approach: duplex Scorpions, in which the fluorophore- coupled detection sequence segment and the quencher-coupled fragment exist on separate, complementary molecules. Like stem-loop Scorpions, duplex Scorpions are unary rather than binary probes, but they offer even greater signal intensity, owing to enhanced separation of the fluoro-phore and quencher. And finally, the Amplifluor™ Universal Detection System, developed by Serologicals Corp., Norcross, Ga., also uses the paired fluorophore-quencher hairpin structure. During the first two PCR cycles, an allele-specific tail is incorporated into the 5' end of the amplicon. Amplifluor UniPrimers, used in subsequent cycles, then anneal to this new tail sequence. These UniPrimers contain a stem-loop structure at the 5' end; as extension of the reverse strand proceeds through the UniPrimer sequence, the hairpin opens to yield fluorescence. LET THERE BE LUX Invitrogen of Carlsbad, Calif., recently developed a new class of real-time probes called LUX™ (light upon extension) fluorogenic primers. Like hairpin probes, LUX primers adopt a stem-loop structure in solution, and like Scorpion probes, LUX primers are intended for use as PCR primers. Yet they do not contain a quencher moiety, making them less expensive than dual-labeled primers. Instead, these fluorescent oligonucleotides are designed to self-quench based on sequence context. LUX primers quench when free in solution, fluoresce weakly when denatured, and emit light strongly when incorporated into DNA. As each probe can have a unique fluorophore, LUX primers are easily multiplexed. Recent work demonstrates the broad utility of quantitative real-time PCR. Fred DeGraves of Auburn University, Ala., and colleagues applied the dual hybridization probe approach to develop a protocol for the detection and quantitation of Chlamydia, an intracellular bacterium.3 Kin-Chow Chang, department of veterinary pathology, University of Glasgow, and his group adopted TaqMan probes to validate genes that they found, via microarray analysis, to be differentially expressed during pig skeletal muscle development.4 Virginia Commonwealth University pathologist Shawn Holt and colleagues used Amplifluor primers to detect and quantify telomerase activity in 19 specimens from patients with breast cancer,5 and S.C. Veasey of the University of Pennsylvania School of Medicine and his team used molecular beacons to quantify the expression of eight different serotonin receptor subtypes in individual neurons that they had purified using laser microdissection.6 Finally, Isabelle Richard of Genethon in Evry, France, and colleagues demonstrated the utility of both molecular beacons and Scorpion primers to detect alternative splicing variants of calpain-3.7 Courtesy of Serologicals Corporation AMPLIFLUORS: Serologicals' Amplifluor Universal Detection System has two parts. The first step incorporates a hybridization sequence for the reporter UniPrimer, which is used in the second step. During DNA polymerization, the UniPrimer hairpin structure is displaced, generating a fluorescent signal that correlates directly to the amount of amplification product. THE BOTTOM LINE A recent comparison of TaqMan and molecular beacon chemistries found them to be more or less equivalent in sensitivity, accuracy, and reproducibility.8 Other probe systems are likely similarly qualified, so money could be a deciding factor. The fluorescent probes are expensive, but that price pales in comparison to the thermal cycler itself. A comprehensive review of real-time-capable thermal cyclers has been previously presented in The Scientist.9 Briefly, industry standards include instruments from: Applied Biosystems; Bio-Rad Laboratories of Hercules, Calif.; Cepheid of Sunnyvale, Calif; Corbett Research of Sydney, Australia; MJ Research of Waltham, Mass.; Roche Diagnostics of Basel, Switzerland; and Stratagene of La Jolla, Calif. These instruments offer a variety of options and support a range of chemistries, but they are all pricey, and not every research or clinical laboratory can afford to upgrade to real-time quantitative capabilities. Nevertheless, the technique is not necessarily out of reach. For one thing, some fluorescent systems can be used in endpoint analyses, which require only a fluorescence plate reader. While not as sophisticated or precise as real-time detection, this approach can be useful in studies of melting point and allelic discrimination, for example. Alternatively, service companies such as Lark Technologies, Althea Technologies of San Diego, Commonwealth Biotechnologies of Richmond, Va., and DxS Ltd. of Manchester, UK, can do the work themselves, freeing researchers from the expense of real-time quantitative PCR, and from the learning curve associated with perfecting the technique. Wendy Gloffke (wgloffke@aol.com) is a freelance writer in Allentown, Pa. References 1. E. Zubritsky, R. Soule, "Pinning down PCR," Analytical Chemistry News and Features, 71:191A-5A, 1999. 2. S. Tyagi et al., "Wavelength-shifting molecular beacons," Nat Biotechnol, 18:1191-6, 2000. 3. F.J. DeGraves et al., "High-sensitivity quantitative PCR platform," Biotechniques, 34:106-15, January 2003. 4. Q. Bai et al., "Development of a porcine skeletal muscle cDNA microarray: Analysis of differential transcript expression in phenotypically distinct muscles," BMC Genomics, 4:8, March 1, 2003. 5. L.W. Elmore et al., "Real-time quantitative analysis of telomerase activity in breast tumor specimens using a highly specific and sensitive fluorescent-based assay," Diagn Mol Pathol, 11:177-85, 2002. 6. G. Zhan et al., "Single cell laser dissection with molecular beacon polymerase chain reaction identifies 2A as the predominant serotonin receptor subtype in hypoglossal motoneurons," Neuroscience, 113:145-54, 2002. 7. M. Taveau et al., "Quantification of splice variants using molecular beacon or scorpion primers," Anal Biochem, 305:227-35, 2002. 8. J. Jebbink et al., "Development of real-time PCR assays for the quantitative detection of Epstein-Barr Virus and cytomegalovirus, comparison of TaqMan probes, and molecular beacons," J Mol Diagn, 5:15-20, February 2003. 9. A. Constans, "Some like it hot: A thermal cycler roundup." The Scientist, 15[24]:32, Dec. 10, 2001. DON'T FRET OVER FRET The underlying principle behind most real-time probe systems is fluorescence resonance energy transfer (FRET), which begins when a dye or fluorophore absorbs light energy. Normally, the excited molecule releases its energy via photoemission, or fluorescence. If, however, another molecule exists nearby--within 10 to 100 angstoms or so--that can accept the fluorophore's energy, the energy will be transferred without emission of light. If the acceptor molecule is a fluoro-phore, the result of the energy transfer will be fluorescence from the energy acceptor. The second molecule may, however, be a fluorescent quencher that silences the reporter molecule, releasing the energy as nonradiative heat or vibrational energy. In either case, the excitation spectrum of the acceptor molecule must overlap the emission spectra of the donor

Quantitative PCR Update

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