In this blog post, I’ll summarize the background, principles, and process behind the invention of PCR, as well as its impact on genetic engineering and its various applications.
In modern society, technologies such as GMOs and human cloning have become major topics of debate due to the ethical issues and safety concerns they raise. Since these technologies are all based on genetic engineering, the widespread discussion surrounding them has led to a greater public awareness of the field. As a result, most people have come to understand that DNA is extremely small and located deep within cells, making it difficult to extract.
If we stop to think about it for a moment, the crux of the problem becomes clear. DNA is difficult to extract and very small. However, conducting scientific research requires experimental and control groups, which in turn necessitates a significant amount of identical DNA. Extracting DNA from the human body is both technically difficult and costly, so this approach has its limitations. Therefore, a method is needed to artificially produce DNA in large quantities and with ease. PCR is the technology that emerged to meet this need—and its very emergence sparked a revolution in genetic engineering and drove significant advancements. PCR is a technique that amplifies the number of specific DNA molecules by a power of two.
In 1953, James Watson and Francis Crick discovered the double-helix structure of DNA, and it was subsequently confirmed that DNA is the genetic material. Scientists naturally began to ask, “How can we manipulate this genetic material, DNA?” and attempts followed to remove parts of the DNA or add new segments. These experiments required large quantities of DNA.
While modern technology offers various methods for obtaining specific DNA in large quantities, given the state of technology immediately following Watson and Crick’s discovery, these methods were either practically impossible or extremely difficult and costly. For example, chemically synthesizing the entire required sequence was incredibly costly and time-consuming, with a high probability of failure. Similarly, using cells that naturally replicate DNA was not straightforward due to various constraints, such as the need to process the DNA into a form usable by the cells. Amid these limitations, the principles of DNA replication were gradually uncovered.
DNA replication proceeds as follows. First, enzymes unwind the double helix, separating the DNA into two strands. Next, an RNA molecule called a primer binds to each single strand to prepare for replication. Once the primers are attached, DNA polymerase recognizes the single strand to which the primer is bound and synthesizes a complementary sequence, replicating it into a double helix. Finally, replication is completed when the RNA primers are removed. Through this process, a single strand of DNA is duplicated.
It was also discovered that chemical reactions within living organisms are catalyzed by enzymes. Based on these two findings, scientists began to wonder, “If we mix the enzymes, DNA, and other substances necessary for replication, might replication occur even without cells?” This idea proved feasible, and Kary Mullis, the inventor of PCR, discovered that by carefully controlling the conditions under which enzymes react and repeatedly cycling the replication process, replication could occur repeatedly even after the initial cycle. Based on this, the PCR technique was developed in 1984.
PCR, developed by Kary Mullis, stands for Polymerase Chain Reaction. Simply put, it refers to a chain reaction of DNA polymerase—that is, the replication process repeating in a chain-like manner. PCR consists of three stages: denaturation, annealing, and extension. During the denaturation stage, the double helix structure separates into two strands; during the annealing stage, primers bind to the target sequence; and during the extension stage, DNA polymerase binds and synthesizes DNA.
To explain this process in a way that’s easy to understand without a diagram: PCR sequentially performs three steps—denaturation (heating to separate the double helix), annealing (lowering the temperature to allow primers to bind), and extension (DNA polymerase synthesizes a new strand)—while repeatedly adjusting the temperature required for each step. By repeating these temperature changes and enzymatic reactions, the original DNA is doubled, and repeating this process multiple times results in exponential amplification.
The PCR process differs from DNA replication that occurs within cells in several ways. First, within cells, an enzyme called DNA helicase unwinds the double helix, whereas in PCR, the temperature is raised to separate the double helix for a faster reaction. Since helicase denatures and becomes inactive at high temperatures, PCR utilizes the property of the double helix to naturally unwind at temperatures of approximately 90 degrees or higher.
Once the denaturation process is complete, the temperature is lowered to induce primer binding. While RNA primers are used for binding within cells, DNA primers are used in PCR to ensure the integrity of the replication. Next, during the extension phase, DNA polymerase must function; however, standard polymerases denature at high temperatures and cannot be used. To solve this problem, a polymerase stable at high temperatures was needed, and researchers solved this issue by using Taq polymerase derived from Thermus aquaticus, a thermophilic prokaryote.
Once a cycle of denaturation, annealing, and extension is complete, the amount of DNA doubles. If sufficient amounts of DNA polymerase, DNA primers, and nucleotides are added, the PCR process can be repeated indefinitely. In other words, if the PCR process is repeated n times starting from a single initial DNA molecule, the DNA is theoretically amplified by 2 to the power of n.
The development of PCR technology has brought about major changes in the field of genetic engineering. This technology, which allows the amplification of desired genetic information by powers of 2, has become an essential tool in biotechnology. Since then, the technology has continued to advance through discoveries such as enzymes that correct incorrectly synthesized sequences and polymerases that offer higher accuracy.
In addition, PCR has seen its range of applications expand as various modifications have emerged alongside the basic form. There are several forms of PCR, including reverse transcription PCR, which amplifies cDNA—the complementary form of DNA—to determine gene expression levels; real-time PCR, which measures DNA quantity in real time; and multiplex PCR, which amplifies not only specific known sequences but also multiple sequences simultaneously. In particular, multiplex PCR is widely used in DNA fingerprinting for criminal investigations and paternity testing.
PCR, which revolutionized genetic engineering, is, quite literally, a technology for amplifying DNA. Before this technology was developed, its application was limited due to the difficulty of obtaining the necessary DNA, but genetic engineering advanced rapidly following Kary Mullis’s invention. As a result, diabetes patients who had suffered from the high cost of insulin were able to access it more affordably thanks to advances in genetic engineering.
If PCR and the advancements in genetic engineering that stem from it continue to progress, the possibility of treating diseases once considered incurable through stem cell research or cloning technology will also increase. Perhaps the day will come when humans are able to conquer disease far more effectively.
