The Polymerase Chain Reaction

Abstract

It is difficult to underestimate the impact of the polymerase chain reaction (PCR) and related DNA amplification techniques on modern molecular biology and applied molecular medicine. PCR represents a rapid, sensitive, and specific method for amplification of nucleic acid sequences and is the basis for numerous molecular techniques that have become the mainstay of the basic research laboratory, as well as the clinical diagnostics laboratory. The concept of PCR was first described in 1985 (1), and the modern technique emerged a few years later (2). Since that time, the technology has evolved into a reliable and affordable method that is performed in laboratories worldwide. When reduced to its essence, PCR is a molecular technology that facilitates the amplification of rare copies of specific nucleic acid sequences to produce a quantity of amplified product that can be analyzed. In early descriptions of PCR (1,3,4), the Klenow fragment of Escherichia coli DNA polymerase I was used for DNA synthesis during each amplification cycle. However, Klenow fragment is not thermal-stable. Therefore, after each denaturation step the samples were quickly cooled before the addition of enzyme to avoid heat denaturation of the polymerase enzyme and it was necessary to add a fresh aliquot of Klenow fragment enzyme after each denaturation cycle. In addition, the primer hybridization and DNA synthesis steps were carried out at 30°C to preserve the activity of the poly-merase enzyme, resulting in hybridization of primers to nontarget sequences and considerable nonspecific amplification (4). Even with these drawbacks, the original PCR methodology was successfully applied to gene cloning and molecular diagnostic experiments (1,3,4). The major technological breakthrough in development of PCR came with the introduction of a thermostable polymerase to PCR (2). Thermus aquaticus is a bacterium that lives in hot springs and is adapted to the variations in ambient temperature that accompany its environment. The DNA polymerase enzyme expressed by T. aquaticus (known as Taq polymerase) exhibits robust polymerase activity that is relatively unaffected by rapid fluctuations in temperature over a wide range (5). Introduction of Taq to PCR improved the practicality of this methodology. Because Taq polymerase can survive extended incubation at the elevated temperatures required for DNA denaturation (93–95°C), there is no need to add a new enzyme after each cycle. In addition, by using a heat block that automatically changes temperatures (a thermocy-cler), the PCR cycles becomes automated. Incredibly, the basic PCR technique has not changed that much since 1988 (2), although new developments in commercially available molecular reagents have made the technique easier to perform.