Polymerase Chain Reaction (PCR)
Description
The polymerase chain reaction (PCR) is a method used to amplify a small amount of DNA in order to allow scientists to study target genes or specific DNA sequences in detail [1]. DNA is extracted from sample tissues or cells and converted into complimentary DNA (cDNA) [4]. In some cases, RNA is extracted and converted to cDNA with the use of a reverse transcriptase. Target primers identify the location of the DNA sequence(s) in the sample, in which DNA polymerases recognize and begin to synthesize the complementary strands of targeted sequences. With each cycle of the reaction, cDNA begins to amplify exponentially at a point known as the cycle threshold value in which the abundance of cDNA can be measured using an instrument known as a thermocycler.
Primers
PCR primers are single strands of RNA that recognize and attach to the targeted sequence of DNA in the sample tissue. Once the targeted sequence has been located, a DNA polymerase attaches to the primer and begins synthesizing cDNA strands, amplifying the target sequence. For bacteria and archaebacteria, primers that are ubiquitous to the 16s ribosomal RNA (rRNA) are used [1,2,3,5,6]
Methods
There are several essential steps when conducting Standard PCR.
1. Purifying DNA from specific tissues or cells [2]
2. Amplifying cDNA copies of the purified DNA [2]
3. Analysis of copied DNA sequences [2]
There are also a number of different types of polymerase chain reactions including endpoint, quantitative real time, reverse transcription, multiplex, and more. These have been modified to analyze different types of data, such as end point PCR where analysis of cDNA takes place after the plateau phase, or RT-PCR where cDNA is synthesized from RNA through the use of a reverse transcriptase enzyme.
Stages of PCR
1. Denaturing stage: During this phase, the purified sample containing the double stranded (ds) DNA and reaction mixture is heated to a temperature of 94C-95C, breaking the hydrogen bonds and separating the strands to allow for future amplification [7].
2. Annealing stage: During this phase, the reaction mixture is cooled to a temperature of 50C-65C, allowing specific target primers (forward and reverse) to attach to the complementary target DNA sequence through hydrogen bonding. This step is necessary, as DNA polymerases cannot extend the primers to create new copies of the DNA without a section of dsDNA to begin with [7].
3. Extension stage: During this phase, the temperature is increased to 72C to enable to attachment and activity of the DNA polymerase. This specific DNA polymerase comes from the heat-loving bacteria Thermus aquaticus, which is stable at higher temperatures needed for the initial denaturing of double stranded DNA from sample tissues. Once this binds to the forward or reverse primer of the target DNA sequence, it begins to synthesize new strands or copies of the sequence through addition of dNTPs in the 5' to 3' direction [7].
These phases are repeated roughly 20-40 times, producing potentially billions of copies of the targeted sequence in a short period of time.
Uses
Polymerase chain reactions are helpful for scientists as they enhance specific target sequences within the genome of an organism. This can allow scientists to determine the temporal and/or spatial expression of genes throughout an organism, or even the differences between mutant and wild-type plants and animals. Today, PCR is commonly used in identifying pathogens among samples, such as COVID-19 and many other life-threatening viruses [1,4,6].
References
1. Bruce, K.D., Hiorns, W.D., Hobman, J.L., Osborn, A.M., Strike, P., Ritchie, D.A., 1992. Amplification of DNA from native populations of soil bacteria by using the polymerase chain reaction. Applied and Environmental Microbiology 58, 3413–3416. https://doi.org/10.1128/AEM.58.10.3413-3416.1992
2. Henson, J.M., French, R.C., n.d. THE POLYMERASE CHAIN REACTION AND PLANT DISEASE DIAGNOSIS 30.
3. Picard, C., Ponsonnet, C., Paget, E., Nesme, X., Simonet, P., 1992. Detection and enumeration of bacteria in soil by direct DNA extraction and polymerase chain reaction. Applied and Environmental Microbiology 58, 2717–2722. https://doi.org/10.1128/AEM.58.9.2717-2722.1992
4. Schochetman, G., Ou, C.-Y., 2021. Polymerase Chain Reaction 5
5. Tsai, Y.L., Olson, B.H., 1992. Detection of low numbers of bacterial cells in soils and sediments by polymerase chain reaction. Applied and Environmental Microbiology 58, 754–757. https://doi.org/10.1128/AEM.58.2.754-757.1992
6. WILSONl, K.H., Blitchington, R.B., Greene, R.C., 1990. Amplification of Bacterial 16S Ribosomal DNA with Polymerase Chain Reaction. J. CLIN. MICROBIOL. 28, 5.
7. “What Is PCR (Polymerase Chain Reaction)?” Facts, The Public Engagement Team at the Wellcome Genome Campus, 25 Jan. 2016, www.yourgenome.org/facts/what-is-pcr-polymerase-chain-reaction.