This year marks 150 years since the discovery of DNA in 1871 by Johann Friedrich Miescher and Felix Hoppe-Seyler . Over the decades, DNA analysis and manipulation has majorly advanced, meaning its applications can be more widespread . 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 . In 1983, Kary Banks Mullis invented PCR after collaborating with a US based company to create the desired components needed for the process . The components needed for PCR include: Taq polymerase, template DNA, primers, dNTPs, MgCl2 and a thermal cycler . The template DNA is arguably the most important component as it is the basis of amplification . The amount required for genomic DNA is 0.1-1µg, but the amount present is irrelevant if it is contaminated with impurities . 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 . 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 . 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 .
There are many different types of PCR, however only a few are commonly used . Simple PCR is perhaps the most common method . This involves heating of the template DNA, so it separates, allowing free nucleotides to bind and this binding is then synthesised by Taq polymerase . 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 . 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 . 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 . This is most commonly achieved using fluorescent dyes that are retained between the double strands of DNA .
PCR testing is a current news focus due to COVID-19 and its success in detecting the virus . RT-PCR, which detects RNA sequences of the SARS-CoV-2 virus has become the “gold standard” diagnostic tool for COVID-19 . Results are available in a few hours, and this currently represents the most rapid method available to monitor the presence and spread of infection . 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 .
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