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Since the discovery of polymerase chain reaction, it is useful in almost all molecular biology techniques. The PCR technique was developed by Kary Mullis in the late 1980s, to amplify the DNA. PCR amplification is done to generate millions of copies from the concerned target DNA. PCR procedure helps make the copies within a very short time. It does not require any more time for concluding the pcr amplification.
The polymerase chain reaction is effective in many areas, like diagnostics of genetic and infectious diseases. Due to its efficiency in producing millions of amplified DNA copies, it is used in areas of forensics, research, and molecular biology techniques. PCR development in the scientific world has eased many experiments of molecular science. The use of polymerase chain reaction has found many applications. In both ways, qualitative as well as quantitative pcr is used to generate millions of copies of DNA from the target DNA. The quantitative method (qPCR, rt pcr) allows measuring the amount of DNA expressed in the sample taken quantitatively While there are also ways of qualitative PCR that are used for the polymerase chain reaction.
The real-time pcr is also used for diagnosing the pathogen required in the samples. To execute the polymerase chain reactions, reagents are needed, which helps in the pcr amplification of DNA. Without the use of reagents, the steps of pcr cannot be executed. In every step of PCR, the reagents play an important role in generating millions of copies from one target DNA.
The name polymerase chain reaction comes from the enzyme DNA polymerase I used in the reaction cycle. Millions of DNA copies are made in one PCR reaction cycle that gives it its efficiency. Here, the chain reactions include various stages of heating and cooling with different sorts of reagents used in the different types of pcr. Each pcr cycle doubles up the amount of DNA and hence, produces many copies of it. Reagents of pcr play a very vital role in this process of making different copies of DNA.
Generally, the following list of reagents are used in pcr used :
Selecting the appropriate and right template of DNA for the polymerase chain reaction is a very important task. In this DNA template, the whole of the pcr technique is based. So, as a researcher or scientist, you should give full attention to the selection of the DNA templates or pcr templates. In the pcr reaction, the templates are necessary to produce the complementary DNA or cDNA.The importance of template DNA is more in performing the reverse transcription pcr reactions.
It is mandatory to select the DNA templates of high quality and quantity to give out the full optimized results. Moreover, the good quality of the PCR template also helps in optimizing the PCR efficiency. The DNA strands or templates become the base of any polymerase chain reaction technique. In the reverse transcription pcr methods, the DNA templates play a major role in producing the pcr amplification data in quantitative number form. As a researcher, you should always take only that type of DNA template that can give more good results.
All types of pcr reactions need a DNA polymerase enzyme. The enzyme works under a high temperature in the first stage of the polymerase chain reaction. Here, the first step includes the separation of the two strands of DNA at a high temperature of approx. 90 degrees Celsius.
In any type of pcr reaction, taq polymerase is used. This polymerase enzyme is a variant of Thermes aquaticus which can withstand high temperatures. In DNA strand separation, a high temperature is required which results in the separation of two strands. In the scientific world, some DNA polymerases are genetically engineered to tolerate the high temperature of the denaturation step in the pcr. It also ensures that there is no non-specific amplification process at the beginning of the pcr steps or procedure. Moreover, the polymerase also facilitates the binding of primers in the given steps of the polymerase chain reactions. Once you add the DNA polymerase enzyme to the pcr sample, it discards the entry of other samples
In the polymerase chain reaction, the initiation of DNA synthesis requires primers. Here, primers refer to the short strands of nucleotides(DNA or RNA) which are complementary to the template strands of DNA. The primers also serve as the point of synthesis for the DNA synthesis in the steps of pcr. This step requires a lower temperature than the denaturation step of the pcr reaction. As the annealing step is completed, hydrogen bonds start to form between the strands of primers and the template DNA used in the pcr machines.
Deoxynucleotide triphosphates (dNTPs) are an important component of the polymerase chain reaction. Without the use of dNTPs, the primers and polymerase enzymes cannot produce DNA synthesis. So, it should be supplied in the reaction mixture to get the good amplified DNA during the polymerase chain reaction.
The reaction mixture of polymerase chain reaction also includes the buffers at optimal conditions to execute the pcr reaction. PCR buffers ensure that the chain reactions work in the proper sequence and optimal conditions. The commonly used buffers in polymerase chain reaction steps are as follows :
Among the above pcr buffers, the tris- HCl, and KCl are used for maintaining the stable pH during the whole reaction of pcr. The magnesium ions work as the cofactors for the DNA polymerase enzymes to maintain the proper DNA amplification. PCR buffers are usually available at 10X concentrations in the PCR mixture in the pcr machines. Buffers are also good for ensuring the better working of the pcr reaction cycles. It also ensures that there is no other enzyme interacts in the process of pcr. In the pcr reaction mixture, the maintenance of proper pH is a must. And this is maintained by the process of adding magnesium ions to the process.
When you perform the polymerase chain reaction, attention should be given to the side of the reagent more. As they are useful in maintaining the proper pH and optimal conditions throughout the pcr cycles. The primers, DNA polymerases, dNTPs, and other chemicals are useful in the steps of the polymerase chain reactions. It is always recommended to use the reagents for the proper functioning of the polymerase chain reactions.
The DNA polymerase and primers have the most extensive role in the whole process of PCR. This helps to amplify the target genes from an organism with ease. The denaturation, annealing, and renaturation with the synthesis process are executed with the help of taq polymerases and primers, and chemical substances. Other magnesium ions work as cofactors for the polymerases to execute the DNA amplification in the process. All the above-mentioned reagents are a must in the process of polymerase chain reactions. The good quality of templates and primers should be maintained for the normal functioning of the PCR machines. The RT PCR, qPCR all types of PCR also need the reagents for reaction cycles.