Polymerase Chain Reaction

The Polymerase Chain Reaction (PCR) is a biochemical process involves in DNA replication in vitro. PCR is capable of amplifying segments of DNA from relatively small amount up to a billionfold in the test tube. The key to amplification in PCR is by utilizing a special thermophilic enzyme, Taq DNA polymerase. Polymerase Chain Reaction involves three main steps: Denaturation, Annealing and Extension (Figure 2) in which the steps are repeating in cycles, consequently resulting in doublings of the amount of original DNA fragments that act as target DNA.

 

Initially, the DNA fragment of interest is identified which then serves as the template for producing the primers that can initiate DNA synthesis. The primers are artificially synthesized, in which they are made of DNA, unlike the primers used by cells which are RNA in nature. One DNA molecule is used to produced two copies, then four, then eight and so on. The continuous doubling is achieved by specific enzymes, known as polymerases that are able to elongate the individual DNA building blocks to form long molecular strands. In order for polymerases to perform their work, it is necessary to provide DNA building blocks, i.e., nucleotides consisting of Adenine base (A), Thymine base (T), Cytosine base (C) and Guanine base (G). Futhermore, a longer DNA molecule that serves as a template strand to synthesize DNA fragments. It is a fact that PCR does not actually copy whole DNA molecules but amplifies up to a few thousand base pairs (which target DNA can be found) from within a larger DNA molecule (template DNA). The basic requirements of a PCR reaction is summarized as in Figure 1.

 

PCR cycling reaction starts with denaturation of template DNA by heating. The double-stranded DNA is denatured to single-stranded by breaking the hydrogen linkages in the DNA molecules. Heating up to 94-96˚C will cause all the enzymatic reactions to stop, i.e., the extension from previous cycle. Next,  temperature is adjusted to approxmately 50-65˚C to allow annealing of primers to the complementary bases at opposite ends of the target sequences (single-stranded DNA sequences resulted by heating).  The primers are moving in Brownian movement, the forward and reverse primers bind to complementary bases respectively. Primers are present in excess to ensure that template strands are bound to primers but not to each other. DNA polymerase then extends the primers using the original DNA as template. The ideal temperature for optimum activity of polymerase is approximately 72˚C whereby it binds at the 3’ end of each primers and perform elongation. Therefore, extension by DNA polymerase will produce complementary strands. After a period of incubation, the mixture is heated again to separate the strands. The mixture is then cooled for hybridization process of primers to complementary regions of newly-synthesized DNA strands and elongation takes place. The whole process is repeated in cycles.