Amplification of the Gene of Interest by PCR

Even after a gene of interest has been isolated and cut out, the amount of that specific DNA fragment obtained directly from a cell or tissue sample is usually far too small to work with efficiently in subsequent cloning steps. The Polymerase Chain Reaction, or PCR, solves this problem by allowing a chosen segment of DNA to be copied, or amplified, millions of times over in a test tube, entirely outside a living cell.

A PCR reaction requires four key components: the DNA template containing the target sequence to be amplified; two short, chemically synthesised single-stranded DNA primers, one complementary to a short sequence just upstream of the target region on one strand, and the other complementary to a short sequence just downstream of the target region on the other strand; a supply of the four deoxynucleotide building blocks (dNTPs) needed to build new DNA strands; and a heat-stable DNA polymerase enzyme, most commonly Taq polymerase, originally isolated from Thermus aquaticus, a bacterium that naturally lives in hot springs and whose enzymes are adapted to remain active at very high temperatures.

The reaction proceeds through repeated cycles, each consisting of three temperature-controlled steps. In the denaturation step, the reaction mixture is heated to a high temperature, typically around 94 to 96 degrees Celsius, which breaks the hydrogen bonds holding the double-stranded template DNA together and separates it into two single strands. In the annealing step, the temperature is lowered, typically to somewhere between 50 and 65 degrees Celsius depending on the specific primers used, allowing the two primers to bind (anneal) to their complementary sequences on the now single-stranded template, one primer on each strand, flanking the target region on either side. In the extension (or elongation) step, the temperature is raised again, typically to around 72 degrees Celsius, which is the optimal working temperature for Taq polymerase; the enzyme extends each primer by adding complementary dNTPs one at a time, synthesising a new strand of DNA in the 5' to 3' direction and effectively copying the target region.

The reason Taq polymerase specifically is essential, rather than an ordinary DNA polymerase such as the one used by E. coli, is that PCR requires repeatedly heating the reaction to the near-boiling denaturation temperature in every single cycle. An ordinary polymerase, adapted to function inside a bacterial cell at around 37 degrees Celsius, would be irreversibly destroyed (denatured) the very first time the reaction was heated to 94 to 96 degrees Celsius, making it useless for a multi-cycle reaction. Taq polymerase, having evolved to function inside an organism that lives in near-boiling water, remains stable and active even after being repeatedly heated and cooled, which is precisely what allows PCR to be automated as a continuous cycling reaction rather than requiring fresh enzyme to be added by hand after every cycle. …