Real time PCR

Another method for the amplification and detection of PCR products is the real time PCR. Different from other methods of detection this is not an end-point analysis but the measurement is performed during the phase of PCR when a logarithmic amplification of PCR products occurs. This allows an exact quantification of the target sequences in the material which is assessed. The method is based on the addition of fluorescence-conjugated probes to the specific primers required for the PCR. These probes hybridize during the running PCR with the continuously increasing amplification products and release fluorescence signals which are detected in an optical device specifically constructed for this approach. Thus, an increase of fluorescence intensity occurs during PCR. The time point (PCR cycle) at which a fluorescence higher than baseline is detected for the first time in a sample correlates with the number of targeted molecules in the sample. The fluorescence intensity of the targeted gene is normalized to a constantly present gene or transcript based on which the number of malignant cells present in the sample can be calculated. Also for this method of detection the application of specific PCR machines equipped with optical devices is needed. Using this machines it is also possible to conventionally detect PCR products, however, the strength of real time PCR is the capability of an exact quantification for follow-up analyses.