Polymerase Chain Reaction (PCR)

A diagrammatic representation of the basic PCR procedure is shown in Figure 4.1. PCR is a cyclical process of copying DNA, which involves heating and cooling. Each cycle has three steps, the first of which involves the denaturation of double stranded DNA. The DNA double helix comprises two phosphate sugar (deoxyribose) polymers that are connected by hydrogen bonding between the bases adenine (A), guanine (G), thymine (T) and cytosine (C). Guanine pairs only with cytosine and adenine with thymine. The PCR method separates the two strands of the DNA helix by heating at a temperature of 93 to 94°C. The hydrogen bonds between the paired bases are disrupted giving rise to single stranded DNA.

The second step involves cooling to allow synthetic oligonucleotide primers to anneal to the strands of DNA. The specificity of the reaction is based on these primers which are generally 20 to 25 bases long. They are selected such that they flank the region of DNA to be amplified with one derived from each of the complementary strands. Computer programs are available to assist with the design of primers and avoid primers that self-anneal, that is, form dimers. The annealing temperature varies depending on the sequence of the primers but is usually between 50 and 60°C. When the primers come in contact with a complementary sequence they bind and create a binding site for the enzyme DNA polymerase.

The third step involves heating to allow primer extension. Primer extension is the synthesis of a complimentary strand of DNA using a single stranded template but starting from a double stranded region. In the PCR the primers are arranged so that each primer extension reaction directs the synthesis of DNA towards the other. The DNA polymerase catalyzes the extension of the annealed primers, that is,. it initiates DNA synthesis. It is necessary to use a heat-stable DNA polymerase such as Taq polymerase to withstand the repeated heating during multiple cycles. Extension usually occurs at 72°C as Taq polymerase, which was originally isolated from a bacterium Thermus aquaticus that inhabits hot springs, is most active at this temperature. Extension requires the four deoxynucleotides (dATP, dGTP, dCTP and dTTP) and a specific buffer containing magnesium which is essential for Taq polymerase activity.

The PCR cycle of denaturation, annealing and extension is repeated and the product resulting from the extension of one primer serves as the template for the second primer and vice versa. Initially synthesis goes beyond the sequence complimentary to the other primer but with each cycle of denaturation, annealing and primer extension the target, that is, the region of DNA flanked by the primers, increases almost exponentially. The PCR cycle is repeated 30 to 40 times to produce detectable amounts of PCR product or amplicon. The simplest way to detect the PCR product is to load a fraction of the reaction onto an agarose gel (Fig. 4.2). The product should be visible as a sharp band of expected size. Fluorimeter-based real-time detection systems (see below) are replacing conventional PCR and gel-based detection systems in many veterinary laboratories. Zinc-finger proteins, DNA-binding proteins that directly and specifically detect PCR products, have recently been used to detect microorganisms including Salmonella spp. and influenza A viruses.