Quantitative PCR. PCR can be used quantitatively, by carefully monitoring the progress of a PCR amplification and determining when a DNA segment has been amplified to a specific threshold level. (a) The amount of PCR product present is determined by measuring the level (fluorescence) of a fluorescent probe attached to a reporter oligonucleotide complementary to the DNA segment that is being amplified. Probe fluorescence is not detectable initially due to a fluorescence quencher attached to the same oligonucleotide. When the reporter oligonucleotide pairs with its complement in a copy of the amplified DNA segment, the fluorophore is separated from the quenching molecule and fluorescence results. (b) As the PCR reaction proceeds, the amount of the targeted DNA segment increases exponentially, and the fluorescent signal also increases exponentially as the oligonucleotide probes anneal to the amplified segments. After many PCR cycles, the signal reaches a plateau as one or more reaction components are exhausted. When a segment is present in greater amounts in one sample than another, its amplification reaches a defined threshold level earlier. The “No template” line follows the slow increase in background signal observed in a control that does not include added sample DNA. CT is the cycle number at which the threshold is first surpassed.