By Mritika Senthil
Throughout the 21ˢᵗ century, generations are continuously exposed to genetic analysis and testing, whether through depictions in films to solve cold cases, collections of ancestry details using services provided by organizations such as “Ancestry” or “23andMe,” or, most recently, in the tests used for detecting COVID-19. While many have questioned the efficacy of these processes, they have nevertheless influenced the global population from a variety of directions.
Polymerase chain reaction (PCR), a principal genetic practice, is a method developed by biochemist Kary B. Mullis used to make millions of copies of a specific DNA segment. It is ideal in our society, specifically enabling personalized genome testing, identification of criminals using corroboration, improvement of agricultural processes, further detection of microorganisms, and developments in medicine.
DNA initially begins with two strands. In the starting step of PCR, or denaturing, heat is used to separate the DNA, producing distinct strands. This temperature is set to approximately 96°C. In the next stage, annealing, the reaction is cooled to around 55-65°C so primers can bind onto the DNA. Primers are pieces of single-stranded DNA that are roughly 20 nucleotides long. They are designed to connect to the desired section of the original DNA and become extended by a polymerase. In the final step, known as extension, the reaction is heated again to a temperature of 72°C, allowing Taq polymerase to extend the primers, and synthesize new strands of DNA. This DNA polymerase enzyme is vital in the creation of new strands of DNA that emulate the original ones. Taq polymerase, which originates from the bacterium Thermus Aquaticus, is used due to its toleration of acute temperatures.
A factor worth noting is the inapplicability of RNA within PCR, specifically its incompatibility with Taq polymerase. In such circumstances, the enzyme reverse transcriptase (RT) is implemented throughout PCR. Using simple terms, this enzyme generates “complementary” DNA (cDNA) from single-stranded RNA (messenger RNA, or mRNA, and microRNA, or miRNA) by using an RNA template.
The aforementioned process is repeated 25-35 times and takes about 2-4 hours. In this period, billions of copies of the target region can be formed.
Considering this process, why would the amplification of a specific section of DNA be so ideal in our society? Generally, these copies can be further analyzed with gel electrophoresis or potentially with various means of sequencing. It can also amplify genes associated with arrays of disorders and can test for a bacterium or virus in a patient’s body. A greater aggregate of this specific area allows for increased flexibility in specific studies. As stated by science writer Alan Dove, “PCR has become a ubiquitous laboratory tool. Nonetheless, researchers, engineers, and physicians are still finding ways to propel it into new territories.”
Q: How can analysis of RNA be implemented using PCR?
A: The enzyme reverse transcriptase (RT) is utilized throughout the general PCR process. It is used to create complementary DNA (cDNA) synthesized from an RNA template.
Q: What are “primers” and in which step of the PCR process are they introduced?
A: Primers are sections of single-stranded DNA that connect to a specific section of the original DNA and become extended by a polymerase. Basically, through hybridization, they demarcate the region of the DNA that will be amplified. It is initially implemented in the annealing stage.