Polymerase Chain Reaction (PCR)

Classical analytical biotechnology typically requires hundreds of thousands of molecules as substrates for analysis. In the case of the deoxyribonucleic acid (DNA), the in vivo molecular cloning has been used to generate virtually unlimited number of molecules for downstream processing. Yet, that is usually a labor-intensive task. These limitations can be overcome with in vitro amplification strategies, which are in fact cloning approaches from the functional (amplification) point of view. The keystone of these methodologies is the exponential amplification of DNA, allowing to generate millions of molecules from a single molecule, in just a few minutes. The polymerase chain reaction (PCR) is the most popular in vitro nucleic-acid amplification methodology. In fact, it can be said that the PCR has represented an inflexion point in science and technology, changing the way biotechnology is done by anyone working with organic entities (viroids, virusoids, prokaryotes and eukaryotes), parts of them or derived products, both current and ancient ones. To carry out optimized PCR amplifications, some mathematics, thermodynamics and bioinformatics should be taken into account. The human factor is also needed most of the times if optimal PCR performance is required. That is why the PCR design process is indeed an art, where the manual optimization of the researcher may make the difference and determine the success of a PCR amplification experiment. This chapter reviews the relevance of PCR in biotechnology from a methodological and applied point of view, highlighting the optimization process, as well as the troubleshooting of the technology, which has a significant impact in the biomedical sciences.