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The polymerase chain reaction is a rapid and versatile in vitro amplification process. It is used widely in research labs for detecting viral and bacterial infections. The amplification of genes happens within the target DNA of an organism. The PCR technique was formulated by Karry Mullis in 1985. Usually, the whole process of PCR is done to execute the selective permeability of the gene amplification. For this process, prior information about DNA sequencing should be known. The DNA or deoxyribonucleic acid sequences are targeted to design the primers in the PCR.
The primers here are of an oligonucleotide nature. Length should be short to execute the hybridization of the target DNA or deoxyribonucleic acid. Common requirements of the polymerase chain reaction are as follows :
The PCR process is carried on the 10-200 liters small reaction tubes which are made up of Peltier tubes. Each cycle of PCR has three steps: a. Denaturation, b. Annealing, c. Renaturation. The PCR process works on the doubling of DNA copies. The target DNA or deoxyribonucleic acid sequences are hybridized to form the new set of extending DNA sequencing probes.
The polymerase chain reaction is of many types which are used in the labs for various purposes. In the pandemic time, the RT PCR process is done for testing the covid-19 infection in the person. A large number of such machines are working in the country to get everyone tested for the deadly virus.
Here is the list of various types of polymerase chain reactions that are prevailed in the scientific world :
It is a method of polymerase chain reaction, used to amplify, isolate and identify known DNA sequences from tissue or cellular organs. The RNA can be used in this process also. In this case, the PCR step is preceded by the reverse transcriptase enzyme activity. Reverse transcriptase enzyme is used to convert the RNA of cellular organisms into cDNA. Here, the taq polymerase enzyme also works which helps in the denaturation process in the first step of the polymerase chain reaction to amplify DNA sequences. Reverse transcription can happen in the presence of Mncl2 in the medium.
The reverse transcription enzyme used in the polymerase chain reaction has both an extrinsic as well as intrinsic nature. Thermostable polymerase enzyme presence makes the RT PCR process more effective and efficient. Due to the efficiency of the reverse transcriptase enzyme in the whole process of RT PCR, the single enzyme reverse transcription can be seen. It is good for DNA amplification and thus leads to viral DNA detection in a population.
The whole process of RT PCR can be conducted either in a single tube or in a two-step fashion. The different tubes can also be present in the process. However, the one-step process is more effective in the reverse transcriptase-polymerase chain reaction. This one-step process paves out for fewer chances of contamination of the target DNA or deoxyribonucleic acid sequences. RT PCR is used in :
The thermocycler PCR machines are the key component of the laboratory apparatus. The whole process of PCR is done inside the machine. It is used to amplify segments of DNA or deoxyribonucleic acid through the use of polymerase chain reaction. The cyclers are helpful in the rapid diagnostics of any type of disease. The device has thermal holes as blocks present which hold the reaction tubes. The thermal cyclers then raise and lower the temperature of the blocks in pre-programmed steps. In this way, thermal cycler PCR can be executed.
In traditional times, the thermal cyclers are designed for use with the Klenow fragments called polymerase I. This enzyme is destroyed during the heating process of the amplification process., and the new enzyme is added to the mixtures for further PCR process. All modern thermal cycler machines are made up of lids that help in the process of executing the PCR. The lids prevent the condensation of the water from the thermal cyclers.
Digital Polymerase chain reaction comes in the category of quantitative PCR technology. It provides a sensitive and proficient way to measure the amounts of DNA sequences or RNA sequences present in the sample. The initial mix here is divided into a large number of individual wells, where the reaction mixtures are stored. This step is carried on before the amplification process that results in the targeting DNA sequencing.
According to the presence and absence of the fluorescence in the amplified region of the reaction, the calculation is done. The detection of targets is done with this DPCR technique.
Quantitative PCR is based on the general principle of PCR, which is used to amplify the DNA strands. After amplification, the DNA gets quantified to the target DNA. This is also called real-time quantitative PCR, as it allows scientists to view the increase in the amount of DNA as it is amplified.
Real-time quantitative PCR is based on the detection and quantitation of a fluorescent reporter, which signals the increase in the direct proportion of the amount of PCR product. The fluorescent probe used here is SYBR which binds to the dsDNA for making it different from others. These fluorescent probes are sequence-specific normally. SYBR green fluorescent the minor groove of the dsDNA. In the concerned solution, the unbound dye exhibits less fluorescence only. The fluorescence is greatly enhanced when it is bounded with the dsDNA present in the sample.
As the name depicts, multiplex PCR consists of multiple primers, fingerprints, and rapid identification factors. Commonly used for assisting in the determination of a specific cause or species. In multiplex PCR, two or more sets of primers are used for different DNA targets in the same PCR reaction. This technique saves considerable time as in one single PCR reaction; multiple specific causes can be analyzed.
The primers used in multiplex PCR are selected carefully based on their annealing temperatures and they seem complementary to each other. The amplicon sizes used in this should be different enough to form different bands on the strand of DNA or deoxyribonucleic acid. So, multiple bands can be obtained. The DNA bands are then visualized by gel electrophoresis and then the usual PCR reaction executes.
When the PCR method is used to detect the presence or absence of a specific DNA product, it is called qualitative PCR. With this technique, PCR is performed for cloning purposes and the identification of pathogens. It is mainly used for the identification of pathogens. DNA replication also happens in the process.
PCR- restriction fragment length polymorphism is also called cleaved amplified polymorphism sequences (CAPS). The process is famous for its techniques in genetic analysis. Polymerase chain reaction-RFLP is applied to detect interspecies in a pool of genomes and analyze their variations.
The polymerase chain reactions are crucial for the research labs. As a researcher, you need to carry out the polymerase chain reactions to get the amplified DNA out of the target DNA sequences in the organism. Polymerase chain reaction paves the way for extensive research by knowing the DNA sequences of the organism, from their DNA and RNA.