Real Time PCR - Some Basic Principles

Figure 7. Diagram of components needed in real time PCR.

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.