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The development of polymerase chain reaction by Kary Mullis has paved a different and effective way for DNA testing. For this iconic discovery, Kary was also honored with the Nobel prize in 1992. This iconic discovery is not limited to Kary only, but many scientists and researchers came forward to join the quest of DNA detection. One of the scientists was Russel Higuchi who took the initiative of monitoring fluorescent probes in the PCR reactions. It leads to the discovery of quantitative polymerase chain reaction (qPCR).
In addition to this, the reverse transcriptase enzyme discovery has led to the invention of reverse transcription-polymerase chain reaction. These all discoveries and inventions have led to path-breaking revelations in the light of DNA work and detection. The RT polymerase chain reaction and qPCR can be coupled with the usual PCR process, to gain the gene expression. The RT -q- PCR helps execute the quantitation of the messenger RNA extracted from the cellular organism. All these above techniques have transformed molecular biology skills. The clinical diagnosis and research are the areas where the various types of pcr are used effectively.
The polymerase chain reaction is called the relatively simple and widely usable tool in molecular biology. It is used to amplify DNA sequences and detect DNA and RNA samples extracted from the organism. When compared to the traditional methods of DNA cloning and amplification, PCR takes less time than those methods. Because of its time-saving nature, it is the favorite tool for almost all molecular biologists. However, PCR reactions are highly sensitive. It should be executed with utmost sincerity and precision. The researcher or scientists should take more precautions while executing it. PCR is used for executing DNA amplification of the required DNA gene.
People often confuse the terms PCR, RT PCR, and QPCR as the same. But they are not that same. However, the core principle in all three types remains the same. Before going to the differences of all the three PCRs, we should know what they are actually?
This is also called the standard PCR. It requires the DNA polymerase, nucleotides, primers, DNA templates, amplifier, thermocycler, amplified DNA, or deoxyribonucleic acid. The standard PCR procedure consist of the following steps:
These are also called steps of PCR by which the process executes. The PCR cycles are repeated almost 20-40 times and the amplified product is then analyzed using the thermal cyclers. The analysis is done to get the DNA detection in the sample of DNA.
The polymerase chain reaction is called the sensitive method. So, it requires very small amounts of DNA to execute a single cycle of PCR. The single unit after the process of denaturation and annealing is differentiated into many small units to avoid any contaminations.
In reverse transcriptase PCR, RNA acts as the template for the reaction. In addition to all polymerase chain reaction steps, a step of amplification of RNA is done to get the DNA aligned. The reverse transcriptase enzyme transcribes the RNA into complementary DNA (cDNA ). It is absent in the standard polymerase chain reaction. The reverse transcriptase enzyme has the RNase H which functions to degrade the RNA position of the hybrid. After this, the next process is concluded by the DNA polymerase I enzyme. The rest of the process of polymerase chain reaction is the same. However, the RNA strands are very sensitive, so they require more carefulness while handling it. Due to the involvement of the reverse transcriptase enzyme, it is called a reverse transcription-polymerase enzyme. When the pcr reaction is combined with quantitative pcr, then it is called real-time RT PCR.
The quantitative polymerase chain reaction is also called real-time PCR. in this pcr technique, it gives out the idea of how much percentage of DNA is present in the sample. In the above RT PCR technique, it is very cumbersome to detect the amount of DNA or RNA. But with the qPCR technique, it can be quantified.
The standard PCR at one go can amplify the DNA up to 2000 nucleotides. While the QPCR can amplify as well as quantify the DNA samples. Here, the quantification is done to measure how many DNA samples are present in the sample.
The PCR is based on the principle of DNA amplification by the primer designing and annealing. While QPCR works on the principle of fluorescence probes and dyes. The DNA samples can be detected by using fluorescent probes that emit fluorescence during the DNA amplification or pcr amplification.
The primer sets are used in the standard polymerase chain reactions while in the QPCR, the primers are labeled with fluorescent dyes and are used in the real-time polymerase chain reaction assay. The primers used in the standard polymerase chain reaction are not labeled with any probe.
The standard polymerase chain reactions are completed in the three steps: denaturation, annealing, and renaturation or extension. In QPCR techniques, with these steps, an additional exponential phase of amplification is done to quantify the DNA.
To perform the analysis of the standard PCR, the conventional gel electrophoresis is done using the PCR amplicons. While in the case of QPCR, the results are recorded by the PCR machine as per the fluorescence emitted by the dyes.
In a standard polymerase chain reaction, the amplicon bands are observed with the help of gel electrophoresis based on the peaks of bands. In the QPCR, different sizes of amplicons are observed in the peaks of QPCR.
In the standard PCR, the electrophoresis is under the pressure of the electrical current, so the amplicon bands migrate towards the positive pole. While in the RT PCR, the fluorescent dye is used for the detection of DNA samples. The peak in the RT PCR denotes the maximum amplification of DNA samples.
In the above point context, all results of standard PCR are recorded when the process gets completed. But with RT PCR, with fluorescence, the results are observed soon.
The conventional polymerase chain reaction shows low-resolution amplification, while the RT PCR, shows high-resolution amplification methods for DNA detection.
It is seen that the conventional polymerase chain reaction is a time -consuming which takes almost 3 to 4 hours. While the RT PCR technique takes only a less time of 1 to 1.5 hours to DNA amplification.
Conclusion
The PCR, RT PCR, and QPCR are the core techniques of molecular biology tools. A lot of research work is carried on with the help of these PCR techniques. The techniques are based on the same principle of DNA amplification, but the process is different for all three techniques. The results of these techniques also vary at their resolution levels. PCR machines for such devices are all present in the market for executing the polymerase chain reactions.
