Why does the enzyme reliably replicate DNA at high temperatures during PCR and how has this technology impacted life sciences?

PCR is a polymerase chain reaction, an important life science technique for amplifying DNA. The process consists of DNA denaturation, primer ligation, and DNA elongation, which is efficiently carried out at high temperatures using the polymerase from Taq bacteria. The technique is utilized in a variety of fields, including genetic research, criminal investigation, and genetic disease diagnosis.

 

PCR stands for Polymerase Chain Reaction, which means polymerase chain reaction. This technology is one of the revolutionary discoveries in modern life sciences and is used as an essential tool for massively amplifying DNA. The DNA in our bodies is a long double helix made up of multiple pieces of DNA, and the enzymes that link these pieces of DNA are called polymerases. PCR is the process of replicating long pieces of DNA by inducing a chain reaction of these polymerases to join them together. The introduction of PCR technology has enabled a wide range of genetic research and applications that were not previously possible. Before the development of PCR, there were significant difficulties in analyzing very small amounts of DNA, but thanks to this technology, researchers are able to perform a variety of experiments with small amounts of DNA. How does PCR work in practice?
The PCR process can be broken down into three main steps: DNA denaturation, primer ligation, and DNA elongation. The first step, DNA denaturation, is the process of breaking the hydrogen bonds between the double helix structure of DNA to separate it into two single strands. To keep genetic information safe, DNA has very strong chemical bonds between the two single strands, called hydrogen bonds. Hydrogen bonds play an important role in the structural stability of DNA, which allows genetic information to be protected from the external environment. Because hydrogen bonds are not easily broken by physical changes, the process of DNA denaturation takes place at very high temperatures, around 95 degrees Celsius. The purpose of this step is to separate the DNA into single strands, making it easier for primers and polymerases to bind.
The next step, primer ligation, is literally the binding of the primers to the DNA strand. Primers are short, single-stranded pieces of DNA that serve as the starting point for the DNA replication process. Because the bases contained in the primer limit where the primer can bind to the DNA, DNA replication starts at a specific location rather than at a random location. Primers are designed to target specific genes or DNA sequences, which allows researchers to amplify the specific gene they want. In order for the primers to bind to the DNA strand, the hydrogen bonds must remain formed and unbroken, as opposed to the process of DNA denaturation. That’s why the temperature is lowered to around 55 degrees Celsius during this step. In this cold environment, the primers bind to the DNA strand and prepare to replicate the DNA.
In the final step, DNA elongation, pieces of DNA are attached to the back of the primers to create a long strand of DNA. It’s the polymerase that keeps the DNA pieces together. Polymerase is an enzyme that plays an important role in DNA replication within cells, and in PCR, it is responsible for stretching the DNA after it binds to the artificially synthesized primers. The polymerase attaches a piece of DNA with complementary bases to the DNA strand being created, so that a new strand of DNA is created that is complementary to the original strand of DNA being replicated. In this process, DNA is copied exactly, and the original information is retained. Because DNA polymerization is slow at body temperature, it would take a very long time to replicate meaningfully large amounts of DNA. So scientists have looked for ways to make the DNA elongation step happen at higher temperatures, but it’s not easy, because enzymes denature at high temperatures and stop working properly. This is one of the basic biochemical properties of life, as enzymes can only be active within a certain temperature range. This is why some of our digestive enzymes are denatured and don’t work as well when we have a high fever. So how did we solve this problem? While investigating different organisms that survive in different environments, scientists began to look for organisms that can survive at high temperatures. As a result, they found organisms that have adapted to live in high temperatures, meaning that they have normal enzyme activity even in high temperatures. These organisms are able to survive in extreme environments through unique biochemical adaptations, and their enzymes have evolved to suit the environment. The organisms the scientists discovered are Taq archaea, which live in hot springs. Scientists isolated Taq polymerase from Taq archaea. Taq DNA polymerase can carry out normal DNA polymerization at temperatures as high as 72 degrees Celsius. The scientists increased the temperature to speed up the polymerization reaction. They used Taq polymerase to allow the enzyme to function at high temperatures without denaturing. The development of this technique allowed PCR to be performed faster and more efficiently, a major breakthrough that formed the basis of modern life science research.
These three steps make up one cycle of PCR, and the total amount of DNA doubles with each iteration of the cycle. Thus, after n cycles, the amount of DNA is n times 2 times the initial amount of DNA. In theory, the number of cycles can be increased indefinitely, but in practice, the number of cycles must be controlled to prevent the enzyme from becoming less efficient or depleting the reagents in the sample. After just 10 cycles, the amount of DNA is 1024 times the initial amount, and the total amount of DNA increases exponentially with each additional cycle. Because of this amplification property, PCR can obtain a sufficiently large number of copies from a very small amount of DNA. Because of this, PCR is used to amplify a small sample of DNA to make large amounts of the same DNA. This technology plays an important role in many fields, especially in gene cloning, genetic disease diagnosis, and forensic science. PCR facilitates criminal investigations by amplifying evidence found in a crime scene. PCR is also used in DNA testing to increase the sample size, allowing for multiple tests to be performed with less DNA. It can also be used for genetic testing to determine whether a trait is expressed in advance. PCR is also used to study gene mutations by deliberately creating mutations during the replication process. Research using this technology has contributed to drug discovery, disease treatment, environmental protection, and many other areas. PCR research has an important place in the field of molecular biology because of its wide range of applications.