Polymerase Chain Reaction

By Mukta Khanolkar | 31 August 2024

Polymerase Chain Reaction (PCR) is a process used to replicate or ‘amplify’ genes of interest. It repeats a cycle multiple times to produce a large number of DNA/RNA copies. It is a rapid automatic process with high specificity. PCR is particularly useful when working with small quantities of DNA samples, to avoid risking the whole sample in the experiment in case it fails. Thus, any synthetic biology experiment working with DNA relies heavily on PCR. It is no surprise that Kary Mullis was awarded the Nobel Prize in 1993 in recognition of his invention of PCR.

The PCR setup consists of a reaction mixture containing the desired DNA segment, nucleotides, primers, necessary enzymes and more in a device called a ‘thermal cycler’.

PCR makes use of the enzyme DNA polymerase to replicate DNA. Since it is difficult for the enzyme to initiate replication de novo, PCR makes use of primers, which are short sequences of DNA complementary to the gene we are trying to amplify. Once the primers bind to the correct site on DNA, DNA polymerase can extend the primers and facilitate replication.

A single PCR cycle involves the following steps:

  1. Denaturation: The DNA segment is heated to high temperatures (94-98°C) to separate its two strands.
  2. Annealing: The strands are cooled down to 48-72°C. This allows primer molecules present in the reaction mixture to bind with them.
  3. Extension: DNA polymerase adds complementary nucleotides to the single strands of DNA. Thus, two copies of DNA are obtained at the end of the cycle.

gene

With each cycle, the number of DNA copies doubles – that is, it exponentially increases (2n after n cycles). One cycle takes around three minutes. Thus, a billion gene replicas can be produced in a few hours. After amplification, the DNA obtained is purified and then used for other processes.

Fun fact: PCR uses a special type of polymerase called Taq polymerase which is functional even at the high temperatures of the thermal cycler (while normal human enzymes would get denatured). It is isolated from the bacterium Thermus aquaticus.

References:

  1. Khehra N, Padda IS, Swift CJ. Polymerase Chain Reaction (PCR) [Updated 2023 Mar 6]. Treasure Island (FL): StatPearls Publishing; 2024 Jan-.
  2. Image: Britannica

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