Real Time PCR Handbook. The University of Illinois at Chicago; 2003. Used with permission.
In real-time PCR, amplification and detection occur simultaneously, within the same reaction tube. Amplification occurs in the presence of a reporter: a probe specific for the target to be amplified, which is typically bound to some type of fluorogenic compound. Although the principles of real time reactions vary depending on the type of probe utilized, the basic premise is that the instrument platform combines a thermocycler with a fluorimeter, and utilizes light of specific wavelengths to excite the reporter molecules. If the target is present, probes bind to the target during each cycle of amplification. A fluorescent signal of increasing intensity is generated as the target is amplified, which is measured by the real time instrumentation. This signal intensity is directly proportional to the amount of amplified nucleic acid. When the signal exceeds a threshold, amplification of the target can be demonstrated.
Once the sample is prepared and placed on the instrument, results are available in approximately 1 to 2 hours, depending on the assay and platform. In addition to reduced turnaround times, real time methodologies are more adaptable to automation and require significantly reduced handling of amplicons, and reduced risk of cross contamination. In some ways, the introduction of real time PCR accelerated the integration of molecular methods into the routine clinical diagnostic laboratory setting. Melt curve analysis
Real time PCR methods can also specifically identify amplified products through melt curve analysis. The melting temperature of double stranded DNA depends on its base composition and length. All PCR products for a specific primer pair should have the same melting temperature, unless there is contamination, primer-dimer pairs, or some other problem. Real time instruments can be programmed to perform an analysis of the melting temperature of the PCR product. When hybridization probes are utilized, after the last PCR cycle, the samples are denatured, and then cooled to a temperature approximately 10o C below the expected melt temperature (Tm), as determined by previous testing of known samples. (This cooling protocol maximizes the formation of probe-target duplexes.) Then the temperature is incrementally raised while the fluorescence is continually monitored. At the melting point, the probe separates from the target strand, and the fluorescence rapidly decreases. The instrumentation software plots the rate of change in fluorescence with time on the Y axis, versus temperature on the X axis. The temperature at which the peak rate of change occurs is the Tm, and can be compared to known controls or established ranges to identify the PCR product.