Introduction

Kary Mullis originally invented PCR in 1985. PCR is best defined as DNA replication in vitro. PCR or the polymerisation reaction is something that we all have heard about in recent times in the covid-19. The polymerase chain reaction is a reaction in which we use special polymerase enzymes to create a chain reaction to replicate the amount of input that could be a living organism such as a bacteria,  virus or it could be a genetic material such as RNA or DNA.

Types of PCR

There could be various types of polymerase chain reactions depending upon the range, the time and the quantity at which the polymerase chain reaction occurs.

Some of the types of polymerisation reactions are

  • Real-time PCR
  • Quantitative real-time PCR (Q-RT PCR)
  • Reverse Transcriptase PCR (RT-PCR)
  • Multiplex PCR
  • Nested PCR

Real-Time Polymerase Chain Reaction

As mentioned by the name, this monitors the real-time reaction and the quantity of DNA which is being looked for. It is a very advantageous laboratory process, and it monitors the amplification of the DNA molecule at a great level. 

The real-time polymerase chain reaction is all about calculating and finding out about the chain reaction and time with respect to the polymerase enzyme working out there. 

Quantitative Real-Time PCR

Quantitative Real-Time PCR is also basically similar to normal real-time PCR. Still, it differentiates or basically works on the basis of the quantity of the sample being examples.

The first applications of PCR were rather impractical due to the usage of thermolabile Klenow fragments for amplification, which needed to be added to the reaction after each denaturation step.

Reverse Transcription PCR

The reverse transcription polymerase chain reaction usually takes place for the detection of Ultra microparticles such as viruses and more. In this type of polymerase chain reaction, reverse transcriptase is used to reverse the entire process of transcription and increase the number to monitor the presence of such organisms.

There are other PCR, such as the multiplex PCR or the nested PCR, depending on their usage for their technique. However, these techniques are not often used when we compare them to the names mentioned above.

Procedure of PCR

As mentioned, PCR is a very complicated technique, and it needs knowledge of microbiology and medicine. If we summarise the procedure of PCR, then one can divide it into three different parts. These are-

  • Denaturation of the template- Denaturation is a term used to denature or destroy a biological structure’s original form. Denaturation could be related to protein, RNA, DNA, and a lot more. This denaturation takes place at the template strand, which is one of the two strands of the DNA. The template strand and the non-template strand are denatured and are opened. Hence the DNA gets converted into a single strand. In the denaturation step, the target DNA is heated to a high temperature (usually 940C-690C) for the separation of the strand
  • Annealing- The literal meaning of the word annealing is joining. Joining the DNA primer with the single strand of DNA is a very important process in the polymerase chain reaction. The reaction temperature is the lower temperature to 40–60 °C allowing the annealing of the primers to the single-stranded DNA template
  • Extension- The last process of the polymerase chain reaction is the extension of the DNA strands. The temperature at this step depends on the DNA polymerase used. Taq polymerase(obtained from the thermophilic bacterium Thermus aquaticus) has its optimum activity temperature at 75–80 °C. Since the main aim of polymerase chain reaction is to multiply the amount of DNA in this case, a continuous loop of reaction takes place until a certain amount of DNA gets prepared, which could be easily isolated and used for other diagnostics and processes. Even the DNA primer is removed at last, and the two strands again get in contact so as to make a perfect DNA or the exact replica of the DNA model inserted in the PCR

The efficiency of PCR is, therefore, a major factor for the basic quantification of the DNA in targets in samples that are of unknown origin. The dependence of the curve showing calibration is responsible for making the quantification, and thus this is determined by specific serial dilutions spacing. 

We have mentioned a lot about this special chain reaction above in the article. Still, due to the increase of usage of this procedure in recent times. Searching for the virus is a very small and nearly undetectable particle in the blood or the sample; it is always advised to go for a polymer chain reaction. 

This will ultimately increase the amount of virus or the other organism inside the test tube sample, and the detection becomes very easy. It is known as reverse transcriptase for a reason as the special enzyme reverse transcriptase has the power to reverse the entire process of transcription.

RT-PCR uses a special RNA as the initial material as a sample for so-called in vitro amplification regarding nucleic acid. The initial concept of retroviral reverse transcriptase was made in the 1970s. This ultimately resulted in the invention of RT-PCR. Reverse transcriptase is a special enzyme and is an RNA-dependent DNA polymerase that acts on RNA in the presence of DNA. The main role of the enzyme is to catalyse the DNA synthesis in which RNA is used as the template. The final product is known as cDNA.

Conclusion

We tried to mention everything about the polymerisation reaction above in the article. Different types of polymerase chain reactions are used worldwide according to their usage and their demand. On the end note, we can say that PCR is very important in medicine and the detection of microbes these days. There is a huge problem in processing and capturing the exact microbes without the actual process. 

In the RT-PCR technique, the main RNA molecules are initially converted into their complementary DNA (cDNA) sequences by the enzyme reverse transcriptases, which is further accompanied by the amplification of the freshly synthesised cDNA by the so-called PCR procedure. This is the main way to study gene expression, and this is also called RT-PCR. The name is denoted due to the reverse transcriptase (RT) function. It is a 2 step process overall.