PCR: The Progression of DNA Analysis

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PCR: The Progression of DNA Analysis

This year marks 150 years since the discovery of DNA in 1871 by Johann Friedrich Miescher and Felix Hoppe-Seyler [1]. Over the decades, DNA analysis and manipulation has majorly advanced, meaning its applications can be more widespread [2]. An example of this is polymerase chain reaction (PCR), a technique that is now widely used to rapidly make millions to billions of copies of a specific DNA sequence [2]. In 1983, Kary Banks Mullis invented PCR after collaborating with a US based company to create the desired components needed for the process [2]. The components needed for PCR include: Taq polymerase, template DNA, primers, dNTPs, MgCl2 and a thermal cycler [2]. The template DNA is arguably the most important component as it is the basis of amplification [2]. The amount required for genomic DNA is 0.1-1µg, but the amount present is irrelevant if it is contaminated with impurities [2]. Template DNA must be pure as even small traces of substances such as EDTA can inhibit Taq polymerase, but impurities can be removed by ethanol precipitation and washing [2]. Taq polymerase synthesises DNA from nucleotides and was introduced into the PCR process in 1986 as it is thermotolerant and can withstand the high temperatures needed for the PCR process [2]. The amount of Taq polymerase used is usually 1-1.5µl, but if the purity of the template DNA is disrupted, then larger quantities are needed [2].

There are many different types of PCR, however only a few are commonly used [3]. Simple PCR is perhaps the most common method [2]. This involves heating of the template DNA, so it separates, allowing free nucleotides to bind and this binding is then synthesised by Taq polymerase [2]. We have the facilities and capabilities to carry out basic PCR testing at our in-house contract lab and use it to identify bacteria to species level [4]. Reverse transcriptase PCR (RT-PCR) is a technique that combines reverse transcription of RNA into DNA, followed by amplification of specific DNA targets using PCR [3]. Quantitative PCR (qPCR), also known as Real Time PCR, allows the quantification of a specific DNA sequence in real time by measuring DNA concentration while the synthesis process is taking place [5]. This is most commonly achieved using fluorescent dyes that are retained between the double strands of DNA [5].

PCR testing is a current news focus due to COVID-19 and its success in detecting the virus [3]. RT-PCR, which detects RNA sequences of the SARS-CoV-2 virus has become the “gold standard” diagnostic tool for COVID-19 [3]. Results are available in a few hours, and this currently represents the most rapid method available to monitor the presence and spread of infection [3]. Although other tests, such as CT scans of patients, can detect COVID-19 cases quickly and accurately, they become impractical once the number of infected people become large [3].



References:

  1. Dahm R. Friedrich Miescher and the discovery of DNA. Developmental Biology. 2005;278(2):274-288.
  2. Singh J, Birbian N, Sinha S, Goswami A. A critical review on PCR, its types and applications. International Journal of Advanced Research in Biological Sciences. 2014;1(7). 
  3. Long C, Xu H, Shen Q, Zhang X, Fan B, Wang C et al. Diagnosis of the Coronavirus disease (COVID-19): rRT-PCR or CT?. European Journal of Radiology. 2020;126:108961.
  4. de Jong A, Youala M, Klein U, El Garch F, Simjee S, Moyaert H et al. Minimal inhibitory concentration of seven antimicrobials to Mycoplasma gallisepticum and Mycoplasma synoviae isolates from six European countries. Avian Pathology. 2021;50(2):161-173.
  5. Kralik P, Ricchi M. A Basic Guide to Real Time PCR in Microbial Diagnostics: Definitions, Parameters, and Everything. Frontiers in Microbiology. 2017;8.

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