This blog post explores the principles behind PCR technology, which functions stably even at high temperatures, and examines its impact on the field of life sciences.
PCR stands for Polymerase Chain Reaction. This technology is one of the revolutionary discoveries in modern life sciences and serves as an essential tool for amplifying DNA in large quantities. The DNA within our bodies consists of multiple DNA fragments linked together to form a long double-stranded structure. The enzyme that connects these DNA fragments is called a polymerase. In essence, PCR induces a chain reaction of this polymerase enzyme to join DNA fragments together, replicating long DNA strands. The introduction of PCR technology has enabled various genetic research and applications that were previously impossible. Before PCR was developed, analyzing extremely small amounts of DNA was quite difficult. However, thanks to this technology, researchers can now perform various experiments even with minimal DNA samples. What exactly is the specific process of PCR?
The PCR process can be broadly divided into three stages: DNA denaturation, primer annealing, and DNA extension. The first stage, DNA denaturation, involves breaking the hydrogen bonds between the DNA double helix strands to separate them into two single strands. DNA forms extremely strong chemical bonds between its two single strands to securely store genetic information; these strong bonds are called hydrogen bonds. Hydrogen bonds play a crucial role in the structural stability of DNA, protecting genetic information from external environments. Since hydrogen bonds are not easily broken by ordinary physical changes, the DNA denaturation process occurs at a very high temperature, around 95 degrees Celsius. The significance of the DNA denaturation step is to separate DNA into single strands, making it easier for primers and polymerase to bind.
The next step, primer binding, is literally the stage where primers attach to the DNA strand. Primers are short single-stranded DNA fragments that serve as starting points in the DNA replication process. The specific bases contained within the primer limit where it can bind to the DNA, meaning DNA replication does not start at random locations but at specific sites. Primers are designed to target specific genes or DNA sequences, allowing researchers to amplify the specific gene of interest. For primers to bind to DNA strands, hydrogen bonds must remain intact, unlike during the denaturation process. Therefore, the temperature is lowered to approximately 55°C during this step. In this low-temperature environment, primers bind to DNA strands, preparing for DNA replication.
In the final step, DNA extension, DNA fragments are added to the back end of the primers to create a long DNA strand. The enzyme responsible for continuously linking these DNA fragments is polymerase. Polymerase is an enzyme that plays a crucial role in DNA replication within cells. In the PCR process, this enzyme binds to the artificially synthesized primers and then extends the DNA. The polymerase enzyme attaches a newly synthesized DNA fragment with complementary bases to the growing DNA strand. Consequently, a new DNA strand complementary to the original template strand is created. This process ensures DNA is replicated accurately, preserving the original information. At body temperature, the rate of DNA synthesis is slow, requiring an extremely long time to replicate a significant amount of DNA. Scientists sought ways to perform the DNA elongation step at higher temperatures, but this proved challenging. The reason is that enzymes denature at high temperatures and lose their function. This is one of the fundamental biochemical characteristics of living organisms, as enzymes can only be active within a specific temperature range. Similarly, when we have a high fever, digestion becomes difficult because some digestive enzymes denature and cannot properly facilitate digestion. So, how could this problem be solved? While investigating various organisms surviving in diverse environments, scientists began searching for life forms capable of surviving in high temperatures. As a result, they discovered organisms that maintain normal enzyme activity even in high-temperature environments—organisms adapted to live in such conditions. These organisms survive in extreme environments through unique biochemical adaptations, and their enzymes have evolved to suit those conditions. The organism scientists discovered was Taq thermococcus, a microbe inhabiting hot springs. Scientists isolated the Taq polymerase enzyme from Taq thermococcus. This DNA polymerase can perform normal DNA synthesis even at high temperatures around 72°C. Scientists increased the temperature to accelerate the polymerization reaction. They utilized the Taq polymerase to ensure the enzyme remained functional without denaturing at high temperatures. The development of this technology enabled PCR to be performed more rapidly and efficiently, marking a crucial breakthrough that forms the foundation of modern life science research.
These three steps constitute one cycle of PCR, and with each cycle repetition, the total DNA quantity doubles. Therefore, after n cycles, the DNA amount becomes 2^n times the initial DNA quantity. While theoretically the number of cycles could be increased indefinitely, in practice, the number of cycles must be appropriately controlled due to factors like enzyme efficiency decline or depletion of reagents within the sample. Even after just 10 cycles, the DNA quantity increases to 1024 times the initial amount, and the total DNA quantity grows exponentially with each additional cycle. This amplification characteristic allows PCR to generate a sufficient number of copies even from very small amounts of DNA. Because of this feature, PCR is used to amplify small DNA samples, producing large quantities of identical DNA. This technology plays a crucial role in various fields, including gene cloning, genetic disease diagnosis, and forensic science. PCR facilitates criminal investigations by amplifying detected evidence. It is also utilized in DNA testing, enabling multiple types of tests with small DNA samples by increasing the sample size. Furthermore, genetic testing allows for the prediction of whether certain genetic traits will manifest. PCR is also used to intentionally induce mutations during replication for research on genetic mutations. Research utilizing this technology makes significant contributions in various fields, including new drug development, disease treatment, and environmental protection. Given its vast range of applications, PCR research occupies a crucial position in the field of molecular biology.
