The Polymerase Chain Reaction

The polymerase chain reaction (PCR) allows the specific and exponential synthesis of a predetermined DNA region via the use of two small, specifically designed fragments of DNA (primers or oligonucleotides), which form the two termini of the nucleic acid molecule to be amplified. PCR amplification reactions in general are highly specific, specificity being determined by the correct hybridisation of primer specific sequences to complementary sequences present on the target DNA molecule to be amplified. PCR primers comprise specific nucleotide sequences which are designed to hybridise to either the parallel or anti-parallel strand of the target DNA molecule, and as such since primers need to be precisely complementary to their target sequences, some sequence data from the terminal ends of the DNA is required for primer design (Fig. 1.1). Once hybridised to the target DNA, the primers provide the double stranded 3′-hydroxyl terminus required by thermostable DNA dependent DNA polymerases to begin the synthesis of a new DNA strand (complementary to the strand to which the primer has hybridised). Moreover, because PCR uses two primers (one designed for each strand of the DNA molecule to be amplified), repeated cycles of primer hybridisation (annealing) and disassociation allows DNA amplification in the 5′ to 3′ direction on both strands to occur, with the primers effectively acting as Okazaki fragments [Marinus, 1976]. PCR amplification is in fact a cyclical process where the sample DNA is initially denatured in order to unwind and separate the DNA double helix into single strands. This is usually achieved by heating the DNA sample in an aqueous environment, usually at a temperature of 94°C for 30 seconds to 5 minutes. Hybridisation of the specific oligonucleotide primers to each strand is then achieved by lowering the temperature of the reaction mix to the annealing temperature (T_m) which is usually set between 40°C and 65°C (dependent on the design of the oligonucleotide sequences used as primers). After the primer hybridisation step, the temperature is raised to approximately 72°C, (an optimal temperature for thermostable DNA polymerase mediated DNA strand replication), and the whole cycle is then repeated a pre-determined number of times. After each cycle of replication, each newly synthesised double stranded DNA molecule (known as an amplimer or amplicon) contains terminal sequences, which are complementary to the primer sequences used (Fig. 1.2). This process allows each amplimer to serve as a template for replication in subsequent rounds of PCR cycling, resulting in a theoretical doubling (exponential amplification) of the number of target molecules during each cycle. Some of the fundamental principles introduced above are detailed in a large body of international scientific literature (e.g. [Jain, 2002; Lubeck and Hoorfar, 2003; Klein, 2002; Wolk et al., 2001; Foy and Parkes, 2001; Erlich, 1999; Kiechle, 1999; Lisby, 1999]).