What is PCR?
Polymerase Chain Reaction — principle, discovery, ingredients, amplification steps and applications.
The polymerase chain reaction, or PCR for short, is a scientific method for quickly amplifying a specific DNA sequence into millions or billions of copies [1]. PCR selects a section of the genome to be amplified by utilizing short synthetic DNA fragments called primers. Multiple rounds of DNA synthesis are then used to amplify that segment [2]. Today, PCR is a widely used and frequently essential technology in biological and medical research labs for a wide range of applications.
It is remarkable to consider that a technology that served as the foundation for the human genome project and is essential to many molecular biology laboratory protocols was only discovered some 40 years ago by Kary Mullis (1944–2019) who shared his Nobel Prize in Chemistry along with Michael Smith in 1993 [3].
A PCR needs five basic ingredients to be set up. These include:
- Template DNA
- Primers — short stretches of DNA that initiate the PCR reaction
- Deoxyribonucleotide phosphates (dNTPs) containing adenine, guanine, cytosine and thymine
- Taq polymerase enzyme
- Buffers
These components are identified in the supplied source as the five basic ingredients required to set up a PCR [4].
Amplification is accomplished using three principal steps:
- Denaturation: heating double-stranded DNA templates to separate the strands.
- Annealing: binding of short DNA molecules called primers to the flanking sections of the target DNA.
- Extension: DNA polymerase extends each primer's 3′ end along the template strands.
To create precise copies of the target DNA exponentially, these stages are repeated (or “cycled”) 25–35 times [5].
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In the modern world, when many new diseases are in transitional stages of developing and re-emerging, such as COVID-19, there are many uses for PCR. In addition, scientists from all around the world are working together to reduce the incidence of some of the most terrible and deadly diseases, such as cancer.
Detecting changes in gene expression caused by physiological and pathological causes quickly and accurately is one of this method's main uses as a research tool. Measurements of viral or bacterial load or the state of malignancy are made using this method in clinical diagnostics. Numerous advancements in diagnostic pathology have been made possible by PCR, particularly in the areas of hematological, genetic illnesses, and viral infections [6].
For instance, an illustration of amplified products visualized under a UV-transilluminator to detect a gene of Salmonella Typhi has been shown in Figure 2.
For these applications, strict guidelines, high sensitivity, and consistent performance are required. As a result, heat cyclers and reagents need to meet these specifications and be designed especially for them.
Molecular diagnostics encompasses several tests such as genetic analysis, identification of carcinogenic mutations, and examination for infectious agents. Amplification of distinct short tandem repeats (STRs) on genomic DNA is necessary for human identification by PCR in forensics in order to distinguish between people [7]. PCR is essential to agriculture for testing for genetically modified organisms (GMOs), detecting food pathogens, and plant genotyping for breeding [8].
- Garibyan L, Avashia N. Polymerase chain reaction. J Invest Dermatol. 2013 Mar;133(3):1–4. doi: 10.1038/jid.2013.1. PMID: 23399825; PMCID: PMC4102308.
- Genome.gov — Polymerase Chain Reaction
- Nobel Prize — Kary Mullis
- YourGenome — What is PCR?
- Thermo Fisher Scientific — PCR Basics
- DeBiasi RL, Tyler KL. Polymerase Chain Reaction in the Diagnosis and Management of Central Nervous System Infections. Arch Neurol. 1999;56(10):1215–1219. doi:10.1001/archneur.56.10.1215
- Nwawuba Stanley U, Mohammed Khadija A, Bukola AT, Omusi Precious I, Ayevbuomwan Davidson E. Forensic DNA Profiling: Autosomal Short Tandem Repeat as a Prominent Marker in Crime Investigation. Malays J Med Sci. 2020 Jul;27(4):22–35. doi:10.21315/mjms2020.27.4.3. PMID: 32863743; PMCID: PMC7444828.
- Arenas M, Pereira F, Oliveira M, Pinto N, Lopes AM, Gomes V, Carracedo A, Amorim A. Forensic genetics and genomics: Much more than just a human affair. PLoS Genet. 2017 Sep 21;13(9):e1006960. doi:10.1371/journal.pgen.1006960. PMID: 28934201; PMCID: PMC5608170.
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