Real Time PCR Components

As in conventional PCR, real time PCR requires the addition of nucleotides (A, C, G, T), reaction buffer, sequence-specific primers (two oligonucleotides, each generally 18 to 20 nucleotides in length), Taq polymerase, magnesium and water. Real time PCR requires one additional component, either a fluorescent dye that binds double stranded DNA or a fluorescent labeled  probe (Figure 7). A labeled probe is an oligonucleotide that is 25 to 30 nucleotides in length, chemically modified to contain a fluorophore. The fluorophore emits energy at a specified wavelength when excited by a light source, and is detectable by the PCR instrument. Referring back to our flashlight analogy earlier, a fluorophore would be the flashlights which enable us to “see” people from our airplane.

The concept of a “probe” can be difficult for learners to grasp, but it is critical in many molecular biology applications. In some applications of using “probes,” scientists are searching for a small gene that represents only a tiny fraction of all the DNA in a plant’s nuclear genome. In other cases, a “probe” can be used to search out a single RNA sequence or protein produced by a plant or other organism. It is a bit like the saying, “looking for a needle in a haystack”. A probe, then, is a specific sequence, either DNA, RNA or protein, depending upon the experiment, which will react only with the gene/RNA/protein of interest at the exclusion of all others. This probe has been labeled in such a way for it to be detectable by the researcher, such as with a radioactive chemical, a fluorophore, an antibody, etc. It is also designed to be highly specific, such that it reacts with (binds with), only the sequence of interest. A probe is analogous to the flashlights being attached to people, not also to trees, cars, buildings, dogs, etc.