Up to Speed on PCR

Real-time PCR Systems Cepheid's Smart Cycler System PCR--a technique so common in today's laboratories that it is easy to forget its revolutionary impact--enables the specific amplification and detection of as little as a single copy of a particular nucleotide sequence. However, PCR has the potential to be used not just for the detection of specific sequences, but also for their quantification, because of the quantitative relationship between the amount of starting target sequence and the amoun

Nov 27, 2000
Deborah Fitzgerald

Real-time PCR Systems


Cepheid's Smart Cycler System
PCR--a technique so common in today's laboratories that it is easy to forget its revolutionary impact--enables the specific amplification and detection of as little as a single copy of a particular nucleotide sequence. However, PCR has the potential to be used not just for the detection of specific sequences, but also for their quantification, because of the quantitative relationship between the amount of starting target sequence and the amount of PCR product at any given cycle that falls within the reaction's exponential range.

The theory is straightforward, but a number of technical caveats are associated with the use of conventional end-point methodologies for quantitative PCR.1,2 In these techniques, PCR results are monitored after a given number of cycles, by which point factors such as limiting reagent concentrations and side reactions may have played a significant role in affecting final product concentration. Quantitative competitive PCR was developed in response to some of these difficulties. In this approach, the starting amount of target is calculated based on the ratio of target to competitor after amplification. However, quantitative competitive PCR is cumbersome, and it can be associated with a number of drawbacks including a limited dynamic range and the need to screen multiple dilutions.1,2

Some of the limitations of end-point PCR have been assuaged in real-time PCR systems, a number of which are now on the market. These systems offer many general technical advantages, including reduced probabilities of variability and contamination, as well as online monitoring and the lack of need for postreaction analyses. Further, some of these systems were developed with contemporary applications such as quantitative PCR, multiplexing, and high-throughput (HT) analysis in mind.2 In real-time quantitative PCR techniques, signals (generally fluorescent) are monitored as they are generated and are tracked after they rise above background but before the reaction reaches a plateau. Initial template levels can be calculated by analyzing the shape of the curve or by determining when the signal rises above some threshold value.1,2 Several commercially available real-time PCR systems are overviewed in this article and/or summarized in the accompanying table. Each of these systems employs either one of several general types of fluorescent probes2 for detection.

Several different basic types of fluorescent probes are used for real-time PCR applications. Some assays employ general probes that bind preferentially to double-stranded DNA (as opposed to the single-stranded template). Others use target sequence-specific reagents such as exonuclease probes, hybridization probes, or molecular beacons (hairpin probes). Although more expensive, sequence-specific probes add specificity to the assay, and enable multiplexing applications.

First On Line


The iCycler iQ system from Bio-Rad
Applied Biosystems of Foster City, Calif., was the first to commercialize real-time PCR. This company offers the ABI PRISM® 7700 Sequence Detection System, which integrates a closed-tube PCR-based assay with instrumentation and software that enable real-time quantitative PCR. This system is optimized for use of a fluorogenic 5' exonuclease assay that employs the Thermus aquaticus (Taq) polymerase. Applied Biosystems has commercialized the requisite reagents in its well-known TaqMan® product line. The fluorogenic probe is complementary to the target sequence, and initially contains both reporter and quencher moieties. When the probe is not bound to template DNA, its reporter and quencher dyes are in close proximity, and the reporter's fluorescent emission is quenched.

The probe is designed to anneal specifically between the forward and reverse primer sites of the target sequence. If a template bearing the target sequence is present, the probe anneals to it. During PCR, the nuclease activity of Taq polymerase cleaves the reporter dye from the probe. The reporter dye, now separated from the quencher, emits a fluorescent signal. Thus, fluorescent signal is emitted only after the probe binds template DNA and is cleaved during the course of PCR. The fluorescent signal is monitored at every cycle as additional reporter dye molecules accumulate. After the signal rises above background, its rate of increase is tracked during a number of linear cycles before the reaction reaches a plateau. These data are then used to calculate initial template levels.

The ABI PRISM 7700 was designed with HT applications in mind. Reactions are performed in 96-well microplates, and it takes merely 15 seconds for this system to collect one round of fluorescent emission data from each well. The reaction tube's transparent lid allows laser light, which is carried on an array of optical fibers, to be distributed to each well. The laser light excites the reporter dye molecules to fluoresce, and the resultant signals are carried by the optic fibers to a charge-coupled device (CCD) camera for detection. Applications include allelic discrimination, melting curves, and quantification of single or multiplexed targets.

Going Light


The LightCycler System from Roche
The LightCycler from Roche Molecular Biochemicals of Indianapolis harnesses light technology licensed from Idaho Technology Inc. of Idaho Falls, and then adds some additional bells and whistles, such as a built-in microvolume fluorimetric detection system that enables real-time quantitative PCR. This system employs thin-walled glass capillary tubes, and typical PCR experiments can be performed in less than 30 minutes. Currently, this system supports two fluorescence-based methods for the detection of amplification products: the "general" DNA stain SYBR Green I (a product of Molecular Probes of Eugene, Ore.), or sequence-specific hybridization probe pairs.

SYBR Green I exhibits very little fluorescence when free in solution; emission is greatly enhanced when it binds to the minor groove of the DNA double helix. Prior to amplification, the reaction mixture contains the denatured DNA, the primers, and the dye. The low-level background fluorescence signal generated by the unbound dye molecules is subtracted during computer analysis. After annealing of the primers, a few dye molecules can bind to the double strand. During elongation, more and more dye molecules bind to the newly synthesized DNA, resulting in dramatically increased light emission. If the reaction is monitored continuously, this increase in fluorescence can be viewed in real time. After denaturation of the DNA during the next heating cycle, the dye molecules are released and the fluorescence signal falls. A fluorescence measurement is performed at the end of the elongation step of every PCR cycle to monitor the increasing amount of amplified DNA.

The hybridization probe format employs two specially designed, sequence-specific oligonucleotides lab-eled with fluorescent dyes. One oligonucleotide probe carries a fluorescein label at its 3' end; the other probe carries a different label (LC Red 640 or LC Red 705) at its 5' end. The chemical nature of the hybridization probes prevents their extension: one probe contains fluorescein at the 3' end, whereas the 5'-labeled probe contains a 3' phosphate moiety. The sequences of the two oligonucleotides are selected so that they hybridize to the amplified DNA fragment in a head-to-tail arrangement. When the oligonucleotides hybridize in this orientation, the two fluorescent dyes are positioned in close proximity to each other. The first dye (fluorescein) is excited by the LightCycler's light emitting diode (LED) filtered light source, and emits green fluorescent light at a slightly longer wavelength. When the two dyes are in close proximity, the emitted energy excites the dye attached to the second hybridization probe, which subsequently emits red fluorescent light at an even longer wavelength. This energy transfer, referred to as fluorescence resonance energy transfer (FRET), occurs efficiently only when the dyes are in close proximity (a distance between 1-5 nucleotides). Thus, in this type of assay, fluorescent intensity measurements are made after the annealing steps. The increasing amount of emitted fluorescence is proportional to the increasing amount of DNA generated during the linear phase of the ongoing PCR process.

 

Core Module

The "heart" of the Smart Cycler® System from Cepheid of Sunnyvale, Calif., is the I-CORE (Intelligent Cooling/Heating Optical Reaction) module. According to company literature, the I-CORE module incorporates state-of-the-art microfluidic and microelectronic design. Each Smart Cycler processing block contains 16 independently programmable I-CORE modules, each of which performs four-color, real-time fluorometric detection. A wide variety of different multiplex or simplex fluorescent tags can used in conjunction with this system, including FAM, TET, TAM, ROX, SYBR Green, Cy3, Alexa, and Texas Red.

Samples are amplified and measured in proprietary, sealable reaction tubes that are designed to optimize rapid thermal transfer and optical sensitivity. The Smart Cycler software enables single or multiple operators to define and simultaneously carry out

multiple separate experiments, each with a unique set of cycling protocols. In addition, thermal and optical data from each and all sites can be monitored in real time, and graphs of temperature, growth curves, and melt curves can be charted as the data are collected. The Smart Cycler Starter System includes a processing block, Windows-compatible computer and monitor, software, mini-centrifuge, tube racks, and a cooling block specifically designed to accommodate Smart Cycler reaction tubes (25 or 100 µl).

 

Coming Soon

Stratagene of La Jolla, Calif., plans to launch the Mx4000 Multiplex Quantitative PCR System at the end of this year. The Mx4000 combines the capabilities of a microplate fluorescence reader with a PCR thermal cycler into a single real-time detection system and allows detection of multiple fluorescent PCR chemistries. It includes an integrated, proprietary thermal system and a multiple-fluorophore detection system. The Mx4000 Multiplex Quantitative PCR System has extended excitation (350 to 750 nm) and detection (350 to 830 nm) ranges, allowing researchers to choose fluorophores with little or no spectral overlap. Each of the four scanning fiber-optic heads independently excites and detects dyes, reading up to four dyes in a single tube. Optimized interference filters are used to block out unwanted crosstalk from spectrally adjacent fluorophores.

Researchers can choose from FAM, HEX, TAMRA, Cy5, Cy3, Texas Red/ROX, and TET filter sets, and custom sets for other fluorophores also are available. Reactions are performed in 96-well microtiter plates, but res-earchers can opt to analyze reactions in a subset of the wells rather than always reading the entire plate. The system includes PCR application software, including a feature that enables real-time amplification plots. Thus, the user can monitor progress of an experiment at any time during thermal cycling, rather than waiting until the end of the run. Data can be viewed in a variety of forms, including amplification plots, scatter plots, sample value screens, fluorescence intensity screens, melting curves, annealing ranges, and text reports. The system employs Stratagene's new solid-state heating and cooling technology. According to the company's promotional literature, the low thermal mass of the sample temperature control block promotes rapid changes in temperature, resulting in speedy thermal ramp rates and greater temperature uniformity.

The instruments mentioned here enable "closed tube" PCR analysis in real time. Results are available during and after PCR, with no additional purification or analyses. This reduces the likelihood of introducing variability or contaminants and allows researchers to say goodbye to postamplification analytical gels. This article focused primarily on products from companies offering fairly comprehensive quantitative PCR systems that include not just the thermal cycler and optical equipment, but the requisite computer hardware and software as well. The accompanying table contains summary information for these systems, as well as for additional real-time PCR instruments that employ user-supplied computer hardware. S

Deborah Fitzgerald (df@sciwriters.com) is a freelance science writer in Birmingham, Ala.

References

1. PE Biosystems, "DNA/RNA Real-Time Quantitative PCR," www.appliedbiosystems.com/molecularbiology/about/pcr/sds/5700_sds/pdf/dnarna.pdf, 1999.

2. E. Zubritsky, "Pinning down PCR: Widespread interest in gene quantitation and high-throughput assays are putting quantitative PCR back in the spotlight," Analytical Chemistry, 71:191A-5A, 1999.