The polymerase chain reaction (PCR) is a laboratory method that allows researchers to produce a significant amount of specific DNA using trace amounts of source DNA, which can be obtained from a variety of organisms and tissues (Garibyan & Avashia, 2013). When discovered in the 1980s, the technique – which can produce billions of copies of specific DNA in an afternoon – had an immediate impact on molecular biology, with machines performing PCR rapidly becoming a common tool used in laboratories (Mullis, 1990).
To perform PCR, researchers need to first create a mixture containing the following elements: the template DNA which they want to amplify; a thermostable DNA polymerase; two appropriate oligonucleotide primers; each of the four nucleotides; as well as a reaction buffer (Coleman & Tsongalis, 2006). A DNA polymerase that is often used for PCR is Taq polymerase, which can withstand high temperatures (Chien, et al., 1976). These components are mixed and subjected to a cycle of three programmed steps repeatedly changing its temperature, allowing for DNA amplification (Weier & Gray, 1988; Kramer & Coen, 2001).
The first step consists in heating the mixture to a high temperature (e.g., 95°C), causing the two complementary DNA strands of the template DNA to separate – a process which is called DNA denaturation (Mergny & Lacroix, 2003). Then, the mixture is rapidly brought to a lower temperature, allowing the two primers to bind to the start and the end of the target DNA segments which the researchers want to amplify. This step is known as primer annealing, and can only take place if the target DNA and the primers are complementary in sequence (Garibyan & Avashia, 2013). The final step is to heat the mixture again to a temperature allowing the DNA polymerase to extend the primers by adding nucleotides to the target DNA strand, in effect duplicating the segment that lies between the two primers; this last process is called extension (Mullis, 1990). PCR is an exponential amplification method: each time this three-step cycle is repeated, the number of DNA doubles.
As researchers usually obtain the DNA sequence for the protein they want to study in a plasmid vector that is not suitable for their requirements, cloning is a common application of PCR. The technique allows them to easily and rapidly clone the DNA sequence into a suitable plasmid vector. First, PCR is used to linearise the target plasmid, using primers that will amplify in opposite directions at the target insertion section. PCR is then applied again to amplify the template DNA from the original plasmid, using primers which half bind to the target DNA, and half mimic the section of the plasmid vector where the sequence needs to be inserted. Finally, an enzyme is used to create single-stranded sections of DNA at the ends of both the gene to be inserted and the new vector, and a DNA ligase closes the phosphodiester bonds, connecting the target DNA to the new plasmid vector, a process known as ligation reaction (Lodish et al., 2000).
PCR is particularly appropriate for cloning because it is a highly sensitive technique producing rapid results, allowing researchers to quickly produce copies of specific DNA to sequence and analyse. For example, quantitative real-time PCR (qRT-PCR) allows researchers to both detect and quantify the produced DNA while it is being synthesised, which can be applied to analyse changes in gene expression levels with great precision in particular disease factors, such as tumors and microbes (Garibyan & Avashia, 2013).
But the sensitivity of PCR also makes it a very specific technique, requiring for sequence details to be available for at least part of the target DNA. The sensitivity of PCR and its ability to produce a large number of copies from trace amounts of DNA is also a source of caution for researchers and practitioners analysing results: a positive result will not necessarily translate to a diagnostic and could be clinically irrelevant (Sellon, 2003). Lastly, the DNA polymerase used in the last step of PCR can be prone to errors, leading to unexpected mutations in the generated fragments (McInerney et al., 2014). This is why it is recommended to sequence the DNA in the new vector plasmids to confirm the target DNA has been correctly amplified and inserted during the PCR reaction.
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