Real-Time or Kinetic PCR

The DNA Facility houses the “real-time” or kinetic PCR instrument, the Applied Biosystems Model 7700 sequence detection system (the TaqMan instrument). The polymerase chain reaction (PCR) has revolutionized the detection of DNA and RNA. As little as a single copy of a particular sequence can be specifically amplified and detected. Theoretically, there is a quantitative relationship between amount of starting target sequence and amount of PCR product at any given cycle. In practice, though, it is a common experience for replicate reactions to yield different amounts of PCR product. The development of real-time quantitative PCR has eliminated the variability traditionally associated with quantitative PCR, thus allowing the routine and reliable quantification of PCR products. This instrument, therefore, now provides investigators with the ability to perform very sensitive, accurate, and reproducible measurements of levels of gene expression. In addition, this instrument can be used in other applications such as measuring viral load, performing allelic discrimination studies, and optimizing PCR conditions.

A. Real-Time PCR Chemistry

Real-time systems for PCR were improved by probe-based, rather than intercalator-based, PCR product detection. The principal drawback to intercalator-based detection of PCR product accumulation is that both specific and nonspecific products generate signal. An alternative method, the 5' nuclease assay, provides a real-time method for detecting only specific amplification products. During amplification, annealing of the probe to its target sequence generates a substrate that is cleaved by the 5' nuclease activity of Taq DNA polymerase when the enzyme extends from an upstream primer into the region of the probe. This dependence on polymerization ensures that cleavage of the probe occurs only if the target sequence is being amplified.

The development of fluorogenic probes made it possible to eliminate post-PCR processing for the analysis of probe degradation. The probe is an oligonucleotide with both a reporter fluorescent dye and a quencher dye attached. While the probe is intact, the proximity of the quencher greatly reduces the fluorescence emitted by the reporter dye by Förster resonance energy transfer (FRET) through space. Probe design and synthesis has been simplified by the finding that adequate quenching is observed for probes with the reporter at the 5' end and the quencher at the 3' end. Figure 1 diagrams what happens to a fluorogenic probe during the extension phase of PCR. If the target sequence is present, the probe anneals downstream from one of the primer sites and is cleaved by the 5' nuclease activity of Taq DNA polymerase as this primer is extended. This cleavage of the probe separates the reporter dye from quencher dye, increasing the reporter dye signal. Cleavage removes the probe from the target strand, allowing primer extension to continue to the end of the template strand. Thus, inclusion of the probe does not inhibit the overall PCR process. Additional reporter dye molecules are cleaved from their respective probes with each cycle, effecting an increase in fluorescence intensity proportional to the amount of amplicon produced.

The advantage of fluorogenic probes over DNA binding dyes is that specific hybridization between probe and target is required to generate fluorescent signal. Thus, with fluorogenic probes, non-specific amplification due to mis-priming or primer-dimer artifact does not generate signal. Another advantage of fluorogenic probes is that they can be labeled with different, distinguishable reporter dyes. By using probes labeled with different reporters, amplification of two distinct sequences can be detected in a single PCR reaction. The disadvantage of fluorogenic probes is that different probes must be synthesized to detect different sequences.

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B. Instrumentation

The ABI PRISM 7700 Sequence Detection System is a flexible system designed to take full advantage of the benefits of fluorogenic probe detection. The 7700 system has a built-in thermalcycler and a laser directed via fiber optic cables to each of the 96 sample wells. The fluorescence emission travels back through the cables to a CCD camera detector. Because each well is irradiated sequentially, the dimensions of the CCD array can be used for spectral resolution of the fluorescent light. Because the 7700 instrument detects an entire fluorescence spectrum, the system is capable of distinguishing and quantitating multiple fluorophores in each sample well. The software analyzes the data by first calculating the contribution of each component dye to the experimental spectrum. Each reporter signal is then divided by the fluorescence of an internal reference dye (ROX) in order to normalize for non-PCR related fluorescence fluctuations occurring well-to-well or over time. The use of this internal reference dye, enabled by the ability to distinguish fluorophores, increases the precision of the data obtained with the 7700 system. The fluorescence emissions of SYBR Green I dye and ROX dye are well resolved, so the benefit of using an internal reference dye is obtained for SYBR Green I dye detection of PCR on the 7700 system. The other advantage of distinguishing fluorophores is that probes labeled with different reporter dyes can be used so that more than one PCR target can be detected in a single tube.

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C. Real-Time PCR Quantitation

The ability to monitor the real-time progress of the PCR completely revolutionizes the way one approaches PCR-based quantification of DNA and RNA. Reactions are characterized by the point in time during cycling when amplification of a PCR product is first detected rather than the amount of PCR product accumulated after a fixed number of cycles. The higher the starting copy number of the nucleic acid target, the sooner a significant increase in fluorescence is observed. Figure 2 shows a representative amplification plot and defines the terms used in the quantification analysis. An amplification plot is the plot of fluorescence signal versus cycle number. In the initial cycles of PCR, there is little change in fluorescence signal. This defines the baseline for the amplification plot. An increase in fluorescence above the baseline indicates the detection of accumulated PCR product. A fixed fluorescence threshold can be set above the baseline. The parameter CT (threshold cycle) is defined as the fractional cycle number at which the fluorescence passes the fixed threshold. A plot of the log of initial target copy number for a set of standards versus CT is a straight line. Quantification of the amount of target in unknown samples is accomplished by measuring CT and using the standard curve to determine starting copy number. The entire process of calculating CT s, preparing a standard curve, and determining starting copy number for unknowns is performed by the software of the 7700 system.

The polymerase chain reaction (PCR) has revolutionized the detection of DNA and RNA. As little as a single copy of a particular sequence can be specifically amplified and detected. Theoretically, there is a quantitative relationship between amount of starting target sequence and amount of PCR product at any given cycle. In practice, though, it is a common experience for replicate reactions to yield different amounts of PCR product. The development of real-time quantitative PCR has eliminated the variability traditionally associated with quantitative PCR, thus allowing the routine and reliable quantification of PCR products.

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D. Primer and Probe Design

Primer Express software uses a set of default parameters to automatically ｓｅｌｅｃｔ primer and probe sets. A summary of the primer and probe design guidelines is shown in Table 1. Even though no probe is required for SYBR Green I dye detection, it is still a good idea to use Primer Express software to ｓｅｌｅｃｔ a primer and probe set when designing a SYBR Green I assay. Although no probe will be used, the primers will meet all the required criteria and if, in the future, there is the need to convert the assay to TaqMan assay chemistry to obtain higher specificity, the probe can immediately be found in the original Primer Express software document.

The Primer Express software is loaded on a Macintosh computer located in the DNA Facility. In order to use the program, investigators will need to bring their target sequence, in a text file format, on a 1.4 Mb floppy disk. DNA Facility personnel are available to provide advice on the use of the software.

An important default parameter in Primer Express software is the selection of amplicons in the 50150 base pair range. Small amplicons are favored because they promote high-efficiency assays that work the first time. In addition, high-efficiency assays enable relative quantification to be performed using the comparative CT method (DDCT). This method increases sample throughput by eliminating the need for standard curves when looking at expression levels of a target relative to a reference control.

Whenever possible, primers and probes should be selected in a region with a G/C content of 2080%. Regions with a G/C content in excess of this may not denature well during thermal cycling, leading to a less efficient reaction. In addition, G/C-rich sequences are susceptible to non-specific interactions that may reduce reaction efficiency and produce non-specific signal in SYBR Green I assays. For this same reason, primer and probe sequences containing runs of four or more G bases should be avoided. A/T-rich sequences require longer primer and probe sequences in order to obtain the recommended Tms. This is rarely a problem for quantitative assays; however, probes approaching 40 base pairs can exhibit less efficient quenching and produce lower synthesis yields.

Table 1. Primer and Probes Selection Guidelines for Quantitative Assays.

Selecting primers and probes with the recommended Tms is one of the factors that allows the use of universal thermal cycling parameters. Having the probe Tm 810 °C higher than that of the primers ensures that the probe is fully hybridized during primer extension.

Primer Express software does not ｓｅｌｅｃｔ probes with a G on the 5´ end. The quenching effect of a G base in this position will be present even after probe cleavage. This can result in reduced normalized fluorescence values (DRn), which can impact the performance of an assay. Having G bases in positions close to the 5´ end, but not on it, has not been shown to compromise assay performance. Another empirical observation is that probes with more C than G bases will often produce a higher DRn. Since Primer Express software does not automatically screen for this feature, it must be checked manually. If a probe is found to contain more G than C bases, the complement of the probe selected by Primer Express software should be used, ensuring that a G is not present on the 5´ end.

The last five bases on the 3´ end of the primers should contain no more than two C and/or G bases, which is another factor that reduces the possibility of non-specific product formation. Under certain circumstances, however, such as a G/C-rich template sequence, this recommendation may have to be relaxed to keep the amplicon under 150 base pairs in length. It should, however, be followed as often as possible, and even when it is not possible, primer 3´ ends extremely rich in G and/or C bases should be avoided.

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E. Thermal Cycling Parameters

All quantitative assays designed using Applied Biosystems’ guidelines can be run using the same universal thermalcycling parameters. This eliminates any optimization of the thermal cycling parameters and means that multiple assays can be run on the same plate without sacrificing performance. This benefit is critical when combining two assays into a multiplex TaqMan assay system, in which the option to run the assays under different thermal cycling parameters is not available. Table 2 shows the universal thermal cycling parameters for quantitative TaqMan or SYBR Green I assays when using DNA or cDNA as the substrate.

F. Sample Preparation

The user is responsible for setting up their own reactions. The samples should be supplied to the DNA Facility in tubes or plates that are ready to be placed into the instrument. In other words, the user is responsible for acquiring the reaction reagents, primers, probe, and reaction vessel. Applied Biosystems TaqMan reagents can be obtained from the Enzyme Core at the Hybridoma Facility (2nd Floor EMRB). The DNA Facility will be responsible for setting up the instrument, data collection, and preliminary data analysis.

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G. Data and Analysis

Results will be available in two forms. The first is a hard copy of the Experimental Report as provided by the Sequence Detection System Analysis Software v. 1.7. The output is presented in a tabular form in which each row corresponds to information generated for each well position. For each well, information regarding the sample name, CT value, and quantity or copy number is provided. If a standard curve was run, its slope, fit (R), and y-intercept is provided in the header information. Alternatively, these data can be provided in an electronic format as a Microsoft Excel workbook file. Additional data analysis, such as DDCT calculations, is the responsibility of the user. However, core personnel are available to assist and/or advise users in performing these additional calculations.

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H. Supplemental Information

• RealtimeRevarticle.pdf )
• Real-time PCR review article (2002) ( Bustin_realtimereview_2002.pdf )
• Real-time PCR overview ( realtimeoverview.pdf )
• Basics of using Real-time PCR ( realtimePCRbasics.pdf )
• Gene quantification calculations ( genequant.pdf )
• Relative quantitation of gene expression ( Compar_Anal_Bulletin2.pdf )