Probe Systems - Taqman
The Taqman system utilizes a labeled probe that contains a reporter molecule (FAM) attached at the 5’ end of the oligonucleotide (yellow ball in Figure 13). This molecule gives off fluorescence when excited at a specific wavelength of light from the Taqman machine. Adjacent to the reporter molecule, at the 3’ end of the oligonucleotide probe is a quencher molecule (TAMARA)(cloud in Figure 13). When the quencher is located near the reporter, it prevents the reporter from fluorescing. This is the state of the probe in the beginning of the PCR experiment, no fluorescence is detected.
When the PCR reaction is in the cooling stage of a cycle, the probe binds to the DNA template along with the primers in the typical complementary nature of cytosine (C) pairing with guanine (G), and adenine (A) pairing with thymine (T) (Figure 14). The DNA polymerase enzyme, Taq polymerase, binds first to the 3’ end of the primers and then systematically begins adding new complementary DNA nucleotides in a 5’ -> 3’ direction to the growing copy of the target gene(Figure 15). Taq polymerase also has an exonuclease activity as a normal function, which allows it to remove DNA nucleotides if mistakes are made during the DNA replication process. In real time PCR experiments, when Taq polymerase reaches the bound probe, this exonuclease function enables it to remove the probe nucleotide by nucleotide as it is also adding new nucleotides to the new DNA strand. As a result, this breaks apart the reporter on the 5’ end from the quencher molecule on the 3’ end, and fluorescence is given off (Figure 16). The more copies of the target DNA present, the more probe which can bind and therefore, the more fluorescence detected.
Watch the video below to where Dr. Louis Busjaeger demonstrates how to run a PCR experiment using the Taqman probe system.