The polymerase chain reaction - PCR meaning is defined as a technique for making a large number of copies of a given segment of DNA rapidly and accurately. The polymerase chain reaction also enables the investigators to obtain the large quantities of DNA required for multiple experiments and procedures in forensic analysis, molecular biology, medical diagnostics, and evolutionary biology.
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In 1983, PCR technique was developed by Kary B. Mullis - an American biochemist who won the Nobel Prize for the Chemistry subject in 1993 for his invention. Prior to the PCR development, the methods used to generate copies of, or amplify, recombinant DNA fragments were labor-intensive and time-consuming. A machine built to carry out PCR reactions, on the other hand, can complete multiple rounds of replication by making billions of copies of a DNA fragment in a matter of hours.
PCR is a technique based on the natural processes of a cell that is used to replicate a new DNA strand. Only some biological ingredients are required for PCR. The integral component is the template DNA. It means the DNA that holds the region to be copied, like a gene. As small as one DNA molecule may serve as a template. The sequence of the two short regions of nucleotides (DNA subunits) at either end of the region of interest is all that is required for this fragment to be repeated.
These two particular short template sequences should be known so that the two primers, the nucleotides’ short stretches that correspond to the template sequences, are synthesized. The anneal or primers bind to the template at complementary sites and act as the copying start point. And, the DNA synthesis present at one primer is directed toward the other by resulting in the replication of the desired intervening sequence. Also required are free nucleotides, which are used to build the new DNA strands, and DNA polymerase - an enzyme that performs building by sequentially adding on the free nucleotides as per the instructions of the template.
PCR amplifies a particular region of a DNA strand (DNA target). Most of the PCR methods amplify the DNA fragments ranging from 0.1 to 10-kilo base pairs (kbp) in length, although a few techniques allow for the amplification of fragments up to 40 kbp. The amount of amplified product is determined by the reaction's available substrates, which become restricting as the reaction progresses.
A basic PCR Set-up Needs Many Several Reagents and Components, as Given below:
It is an enzyme. It polymerizes the new DNA strands; heat-resistant Taq polymerase is particularly quite common, as it is more likely to survive the high-temperature DNA denaturation process intact.
It contains the DNA target region to amplify
Two DNA primers, which are complementary to the 3′ (three prime) ends of every sense and the antisense strands of the DNA target (where DNA polymerase only bind to and elongate from the double-stranded region of DNA; without any primers, there is no double-stranded initiation site, where the polymerase can bind); particular primers, complementary to the DNA target region are chosen ahead of time, and often custom-made in the lab or purchased from commercial biochemical suppliers
Let us PCR Process or Steps of PCR in detail.
PCR is given as a three-step process, carried out in the repeated cycles. The initial step is given as separation or denaturation of two strands of DNA molecule. This can be accomplished by heating the starting material to temperatures of up to 95 °C (203 °F). Every strand is given as a template, where a new strand can be built. In the second step, the temperature can be reduced to up to 55 °C (131 °F) so that the primers can anneal to the template. In the third step, the temperature is raised to 72 °C (162 °F), and DNA polymerase starts adding nucleotides to the annealed primer ends.
At the cycle’s end, which lasts about 5 minutes, the temperature is raised, and again, the process begins—the copy count doubles after every cycle. Generally, 25 to 30 cycles produce a sufficient DNA amount.
In the case of the original PCR procedure, the problem was that DNA polymerase had to be replenished after each and every cycle due to the reason, it is not stable at the high temperatures required for denaturation. in 1987, this particular problem was solved with the discovery of heat-stable DNA polymerase, known as Taq, which is an enzyme isolated from the thermophilic bacterium Thermus aquaticus that inhabits hot springs. Also, Taq polymerase led to the PCR machine invention.
Because DNA can be amplified from a wide range of sources, the technique has been applied to several fields. PCR can be used to diagnose genetic diseases and to detect the viral infection’s low levels. It is used to analyze minute traces of blood in forensic medicine and the other tissues to identify the donor using the genetic “fingerprint.” This technique has also been used to amplify the DNA fragments that are found in preserved tissues, like those of a 7,500-year-old human found in a peat bog or of a 40,000-year-old frozen woolly mammoth.
1. Give the Optimization of Polymerase Chain Reaction?
Answer: PCR, in practice, can fail for multiple reasons, in part because of its sensitivity to contamination, causing amplification of spurious DNA products. Due to this, many procedures and techniques have been developed for optimizing the PCR conditions. Contamination with extraneous DNA can be addressed with lab procedures and protocols that separate the pre-PCR mixtures from the potential DNA contaminants.
2. Give the Advantages of Polymerase Chain Reaction?
Answer: PCR has many advantages. It is very fair to understand and use and produces results in a rapid way. This technique is extremely sensitive, capable of producing millions to billions of copies of a specific product for cloning, sequencing, analysis, and there are several other PCR applications.
3. List a Few Limitations of Polymerase Chain Reaction?
Answer: A major limitation of PCR is, prior information about the target sequence is required to generate the primers that allow its selective amplification. Typically, this means that PCR users should know the exact sequence (s) upstream of the target region on each of two single-stranded templates to ensure that DNA polymerase binds to the primer-template hybrids properly and generates the entire target region during DNA synthesis.
4. Give a Variation of Polymerase Chain Reaction?
Answer: Assembly PCR, also known as Polymerase Cycling Assembly (PCA), is a method of synthesising long DNA sequences by performing PCR on a pool of long oligonucleotides with short overlapping segments. The oligonucleotides alternate between antisense and sense directions, and the overlapping segments define the order of the PCR fragments, thereby producing the final long DNA product selectively.