PCR and its usages in microbiology

By Jenny Tang

PCR, also known as polymerase chain reaction, is a method involving the use of a small amount of DNA to create large quantities of DNA. There are numerous advantages to using PCR, which makes it ideal for many purposes. PCR requires only a small trace of DNA to work with to create more copies of the DNA, in a short amount of time (Garibvan and Avashia, 2014) . This assists in the usage of PCR when it comes to pathogens that are extremely difficult to culture in vitro, as only a small sample of pathogen is needed. This is another reason why PCR is ideal for pathogens that require a long cultivation period, as it saves time on the assay, which prevents error. In comparison to PCR, cultivation is much more time consuming, as around 40 cycles of PCR (the standard cycle number) requires around 45 minutes to an hour (Bustin, 2017). On the other hand, some pathogens have much longer cultivation time periods. Therefore, generally, PCR is less time-consuming compared cultivating pathogens. 

PCR can also be used to detect mutations, which is crucial for identifying genetic disorders. PCR does not detect the mutation code on the piece of the DNA, but it can indicate the presence of mutations through creation of an amplicon. An amplicon is the product of the replication during the steps of PCR. In real time PCR (also known as qPCR), it identifies single nucleotide polymorphisms (SNPs). The difference between PCR and qPCR is that while the reaction occurs, qPCR is able to collect data, as it is able to identify DNA fragments through their Tm values. Tm values are the values when half the double-stranded DNA changes into single-stranded DNA. 

Some common mutations that occur in the DNA code can be found through many different types of qPCR, including blocker PCR and FLAG PCR (also known as fluorescent amplicon PCR). Although they are all types of qPCR, different methods can help identify different types of mutations, such as SNP deletions, insertions, and missense mutations [Morlan et al., 2009)]. In order to do a genetic mutation test, DNA is to be collected. A single cell from an embryo contains enough of the child’s DNA to carry out the entire testing for genetic mutation disorders. This can help identify genetic disorders caused by mutations, like cystic fibrosis and muscular dystrophy. Most paternity tests are also done similarly, with DNA is obtained through a cheek swab from the child and from both parents. This DNA is then compared in different loci between the parents and child to see if it forms a match. 

Another common usage of PCR is to identify diseases such as HIV, malaria, anthrax and many more. The usage of PCR detects the sequences of DNA that code for the pathogen, which allows for identification of the disease through a small given sample from the patient. For example, in HIV-1, the detection of HIV can be found in blood around 4 weeks to 12 weeks after the patient’s exposure to HIV (Fearon, 2005)].

PCR can be used to detect viral infection, as it can detect infection in the early stages. A modern example of this is the coronavirus. PCR is commonly used for identifying coronavirus in patients, as it is not only cheap and reliable but provides quick results as well. A throat swab is used to collect DNA from the patient, or blood is drawn. These samples of DNA are then analyzed. However, PCR methods are limited in the sense that they are only able to tell us whether the coronavirus is currently in the patient’s system, but cannot provide information as to the presence of anti-CoV-2 antibodies from past infections of coronavirus.

References:


Bustin, S.A. (2017). How to speed up the polymerase chain reaction. Biomolecular Detection and Quantification, [online] 12, pp.10–14. Available at: https://www.ncbi.nlm.nih.gov/pmc/articles/PMC5496742/.

Fearon, M. (2005). The laboratory diagnosis of HIV infections. The Canadian journal of infectious diseases & medical microbiology = Journal canadien des maladies infectieuses et de la microbiologie medicale, [online] 16(1), pp.26–30. Available at: https://www.ncbi.nlm.nih.gov/pmc/articles/PMC2095005/.

Garibyan, L. and Avashia, N. (2013). Polymerase Chain Reaction. Journal of Investigative Dermatology, [online] 133(3), pp.1–4. Available at: https://www.ncbi.nlm.nih.gov/pmc/articles/PMC4102308/pdf/nihms593299.pdf.

Kent, C. (2020). What are the different types of Covid-19 test and how do they work? [online] Verdict Medical Devices. Available at: https://www.medicaldevice-network.com/features/types-of-covid-19-test-antibody-pcr-antigen/.

Morlan, J., Baker, J. and Sinicropi, D. (2009). Mutation Detection by Real-Time PCR: A Simple, Robust and Highly Selective Method. PLoS ONE, 4(2), p.e4584.

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