An overview of PCR (polymerase chain reaction)

A Nobel prize. One of the highest sophistications that can be given to a scientist working in the fields of physics, chemistry, physiology, or medicine. Marie Curie received one, as well as Albert Einstein and Sir Alexander Fleming, but what does a person have to do to be recognised in such a high manner? Well, transforming the way DNA is analysed could help.

Kary B. Mullis received a Nobel prize in 1985 (NobelPrize.org, 2020) for his invention of the Polymerase Chain Reaction (PCR) in 1983 (Mullis, 1990). PCR means that a small sample of DNA can be replicated millions (even billions) of times in just a few hours, which has had provided great success for the scientific industry (Garibyan and Avashia, 2013).

You might be asking yourself why is PCR so important but I will get to that soon, first I want to try and give a brief explanation of how PCR works, after all the replication of DNA could seem God-like if you look at it objectively, so I think it warrants a deeper explanation if you are unfamiliar with the process.

Firstly, behind all PCR procedures are five materials: a DNA sample (also known as a DNA template), DNA polymerase enzyme, forward and reverse primers¹ , nucleotides (also known as dNTPs), and buffer to stabilise the enzyme (Wellcome Genome Campus Courses and Conferences, 2013).

A video by Dr Cath Arnold (on behalf of the Wellcome Organisation) demonstrates how these components are used, this video can be found in my references below but I will attempt to summarise the process which Dr Arnold describes in depth.

 An important base for a PCR reaction is a sterile environment, the amount of each substance used is measured in micrograms so any small amount of contamination would influence the end analysis. To further prevent contamination of not only the PCR reaction, but of stored materials, a one-way system is used whereby components are added to the mixture in different rooms; in the first room all previously listed components of the PCR, apart from the DNA, are combined in measured amounts in a container. In the next room the DNA sample is added and in the third and final room is where the mixture is introduced to the PCR machine.

Once in the machine the mixture undergoes 30-40 amplification cycles. In each of these cycles, which take 30-60 seconds, there are 3 processes.  First is denaturation, whereby the mixture is heated to 94-95 degrees centigrade which causes the strands of DNA to separate; next is the annealing stage which is when the forward and reverse primers bind to the opposing strands of the DNA, during this stage the mixture is heated to between 50-65 degrees centigrade to allow the binding. The last stage is extension, which occurs when the mixture is heated to 72 degrees centigrade which extends, or “replicates”, the sample of DNA by essentially making it a longer version of itself.

So great! We have replicated a DNA sample! What can we do with this information? Well more commonly, PCR is used to help identify assailants by forensic teams as the DNA can be replicated countless times for analysis procedures which diminish DNA supplies (Cavanaugh and Bathrick, 2017). PCR has a wide range of uses ranging from cloning to medical research to paternity tests (Valones et al.,2009), scientists involved with microbiology are also particularly keen of using PCR to analyse disease.

One example of PCR’s incredible usefulness is in a study by Singh et al. (2008) which was able to correctly identify a previously misdiagnosed species of Malaria! The researchers were able to identify Plasmodium knowlesi in blood samples in Malaysia using PCR, which were previously identified as Plasmodium malariae when microcospy was used; the application of this knowledge could prove to be literally life saving as the treatments for Plasmodium malariae are less complicated and therefore would not treat Plasmodium knowlesi as thoroughly as required for the recovery of a patient.

 

In summary, the uses of PCR can not be overstated for their importance. These days you can find PCR equipment in any old laboratory that can produce a “clean room”, but the  effectiveness of PCR to provide us with a wider understanding of DNA is lightyears from where science was when Watson and Crick first discovered DNA²  and it will be exciting to see where new developments of genetics take us next as the use of tools like CRISPR are on the rise.

Notes

¹ Prior to Mullis’s invention of what we know as PCR today, he had been working on DNA sequencing (attaching nucleotides in order on a DNA template) but could not produce specific binding sites for the nucleotides because he realised that one primer caused unspecific binding, so he employed opposing primers which would bind nucleotides to opposing strands of DNA.

²I mean we could go into the misogynist erasement from of Rosalind Franklin from that discovery, or even Watsons problematic views but we’ll leave it there…

 

 

 

 

 

 

Sources:

Cavanaugh, S. and Bathrick, A., 2017. Direct PCR amplification of forensic touch and other challenging DNA samples: A review. Forensic Science International: Genetics, [Online]. 32, 40-49. Available at: https://www.sciencedirect.com/science/article/abs/pii/S1872497317302119 [Accessed 5 January 2021].

Cox-Singh, J. et al. (2008) Plasmodium knowlesi malaria in humans is widely distributed and potentially life threatening, OUP Academic. Available at: https://doi.org/10.1086/524888 Available at: https://doi.org/10.1086/524888 (Accessed: 07 August 2023).

Garibyan, L. and Avashia, N., 2013. Research Techniques Made Simple: Polymerase Chain Reaction (PCR). The Journal of Investigative Dermatology, [Online]. (Vol 133, part 3), pp.1-4. Available at: https://www.ncbi.nlm.nih.gov/pmc/articles/PMC4102308/ [Accessed 11 November 2020].

Mullis, K., 1990. The Unusual Origin of the Polymerase Chain Reaction. Scientific American, [Online]. (Vol 262, part 4), pp.55-65. Available at: https://www.jstor.org/stable/24996713?seq=1 [Accessed 11 November 2020].

Nobelprize.org. 2020. The Nobel Prize. [ONLINE] Available at: https://www.nobelprize.org/ . [Accessed 9 November 2020].

 

Valones, M. A., Guimarães, R. L., Brandão, L. A., de Souza, P. R., de Albuquerque Tavares Carvalho, A., & Crovela, S. 2009. Principles and applications of polymerase chain reaction in medical diagnostic fields: a review. Brazilian journal of microbiology : [publication of the Brazilian Society for Microbiology], 40(1), 1–11. https://doi.org/10.1590/S1517-83822009000100001

Wellcome Genome Campus Courses and Conferences. 2013. How to set up a PCR. [Online Video]. 17 January 2013. Available from: https://youtu.be/V2JYy6-DE9c. [Accessed: 12 November 2020].