The polymerase chain reaction (PCR) has revolutionized the detection of dna and RNA. As little as a single copyof 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 quantitation of PCR products.|
History of Real-Time PCR Techniques
Higuchi et al.1,2 pioneered the analysis of PCR kinetics by constructing a system that detects PCR products as they accumulate. This “real-time” system includes the intercalator ethidium bromide in each amplification reaction, an adapted thermal cycler to irradiate the samples with ultraviolet light, and detection of the resulting fluorescence with a computer-controlled cooled CCD camera. Amplification produces increasing amounts of double-stranded DNA, which binds ethidium bromide, resulting in an increase in fluorescence. By plotting the increase in fluorescence versus cycle number, the system produces amplification plots that provide a more complete picture of the PCR process than assaying product accumulation after a fixed number of cycles.
Chemistry Developments For Real-Time PCRFluorogenic Probes
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,3,4 provides a real-time method for detecting only specific amplification products. Holland et al. 3 were the first to demonstrate that cleavage of a target probe during PCR by the 5' nuclease activity of Taq DNA polymerase could be used to detect amplification of the target-specific product. In addition to the components of a typical amplification, reactions included a probe labeled with 32P on its 5' end and blocked at its 3' end so it could not act as a primer. 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. After PCR, Holland et al. measured cleavage of the probe by using thin layer chromatography to separate cleavage fragments from intact probe
The development of fluorogenic probes by Lee et al.5 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.6
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 andtarget is required to generate fluorescent signal. Thus, with fluorogenic probes, non-specific amplification dueto 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 fluorogenicprobes is that different probes must be synthesized to detect different sequences.