The predictable exponential increase in DNA copy number seen with polymerase chain reaction (PCR) makes it a powerful tool for quantifying the number of copies of a specific target sequence. This technique, known as quantitative PCR (qPCR) or quantitative reverse transcription PCR (qRT-PCR), allows a researcher to determine the number of copies of a specific mRNA (see Transcription) in an experimental sample. qPCR is generally carried out with a cDNA target generated from the mRNA in a sample through a process called reverse transcription. qPCR reactions occur in the same manner as a normal PCR reaction, with a couple of additions and a change in the machine being used.
The additions made to the reaction mixture itself come in the form of a fluorescent marker that is used as the indicator of the number of DNA copies present. There are two types of markers used: intercalating dyes and fluorescently labeled probes. Intercalating dyes are dyes that have a planar, or flat, shape that can fit between the nucleotides of a DNA molecule. When in this position the dye as able to absorb a specific wavelength of light then emit a second, different wavelength of light (see Fluorescent Dyes). This emitted wavelength will get stronger with each PCR cycle due to there being more copies of the target DNA, allowing for more dye to intercalate. The ability to generate the exciting wavelength, and detect the emitted wavelength, of the fluorescent probe is what sets qPCR machines apart from standard thermocylers.
The second type of marker used for qPCR are fluorescent probes. These probes are short DNA sequences that bind to the middle of the target DNA sequence. The probes have two fluorescent dyes conjugated to it, with one quenching the other. When the DNA polymerase carries out the extension step in the PCR cycle it runs into the probe that has annealed to the middle of the target DNA sequence. When this occurs, the polymerase chews up ( using its exonuclease activity) the probe, releasing the two fluorescent dyes. This allows the two dyes to diffuse away from each other, allowing the emitted wavelength of the previously quenched dye to be detected by the qPCR machine. The amount of free dye is proportional to the number of individual PCR extensions that have occurred, because a single dye molecule is released for each DNA strand that is duplicated.. Once the fluorescent reading have been taken for each cycle in a qPCR run, it can be analyzed various ways to determine the amount if initial target transcript that was present in the sample being tested (see qRT-PCR Analysis).