Recent developments in polymerase chain reaction - Real-time PCR

Polymerase chain reaction (PCR) has for many years been regarded as the new standard procedure in detecting the genes of microorganisms, plants and animals. Detecting the reacting products is reliant upon agarose gel electrophoresis, ethidium bromide staining, ultraviolet irradiation and comparing the size to a DNA size marker. This has slowed progress in adopting the PCR method in diagnostic laboratories. Developing new methods of PCR product detection without opening a reaction tube has opened new possibilities in applying this technique during routine diagnosis. Monitoring PCR product accumulation as amplification progresses is possible through using labeled primers and/or oligonucleotide probes or DNA binding fluorophores. Real-Time PCR methods can be subdivided into two groups: sequence dependent or sequence independent DNA detection. All of these involve using fluorophores linked to oligonucleotides or their presence in the reaction mixture. Their common feature is measuring fluorescence in each PCR cycle and calculating the threshold cycle that corresponds to the DNA copy number in that sample.