What is a Polymerase Chain Reaction?
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.
[Image will be Uploaded Soon]
About the Polymerase Chain Reaction
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.
Principles
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:
A DNA Polymerase -
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.
A DNA Template -
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
PCR Process or Steps of PCR
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.
FAQs on Polymerase Chain Reaction
1. What is Polymerase Chain Reaction (PCR) and its main purpose?
Polymerase Chain Reaction, or PCR, is a laboratory technique used to make millions to billions of copies of a specific segment of DNA. Its main purpose is to amplify a tiny amount of DNA, making it abundant enough to be easily analysed, studied, or used in other procedures like genetic testing or forensic analysis.
2. What are the three main steps in a single PCR cycle?
A single cycle of PCR involves three key steps that are repeated many times:
- Denaturation: The reaction is heated to a high temperature (around 95°C) to separate the double-stranded DNA into two single strands.
- Annealing: The temperature is lowered to allow short DNA sequences called primers to bind to their complementary sections on the single-stranded DNA templates.
- Extension: The temperature is raised slightly, and an enzyme called DNA polymerase extends the primers, synthesising new strands of DNA.
3. What key components are needed to set up a PCR reaction?
To perform PCR, you need a few essential components in a reaction tube:
- DNA Template: The original DNA sample containing the target sequence to be amplified.
- Primers: Short, single-stranded DNA pieces that define the start and end points of the target region.
- DNA Polymerase: A heat-stable enzyme (like Taq polymerase) that synthesises the new DNA strands.
- Nucleotides (dNTPs): The building blocks (A, T, C, G) that the DNA polymerase uses to create the new DNA.
- Buffer Solution: Provides the optimal chemical environment for the reaction to occur efficiently.
4. Why is a heat-stable DNA polymerase, like Taq polymerase, essential for PCR?
A heat-stable DNA polymerase is crucial because the PCR process requires a high-temperature denaturation step to separate the DNA strands. Most enzymes would be destroyed at this temperature. Taq polymerase, originally isolated from a heat-tolerant bacterium, can withstand these high temperatures and remains active through many cycles of heating and cooling, eliminating the need to add fresh enzyme after each cycle.
5. How is PCR used in real-world applications like medical diagnosis?
PCR is a powerful diagnostic tool. For example, it can detect the presence of viral or bacterial DNA in a patient's sample, even if the pathogen is present in very low numbers. By amplifying the pathogen's specific genetic material, doctors can quickly diagnose infections like HIV, Hepatitis, and COVID-19 (using a variation called RT-PCR). It's also used in forensics to identify individuals from trace DNA samples.
6. What are the main advantages and limitations of using the PCR technique?
The main advantage of PCR is its incredible speed and sensitivity, allowing for the rapid amplification of tiny DNA samples. However, a major limitation is that you must have prior knowledge of the target DNA sequence to design the specific primers. Another challenge is its high sensitivity, which makes it very susceptible to contamination from other DNA sources.
7. How does the 'chain reaction' in PCR lead to exponential amplification?
The term "chain reaction" refers to the fact that the DNA products synthesised in one cycle become the templates for the next cycle. In the first cycle, you double the amount of target DNA (from 1 to 2 copies). In the second cycle, both of these copies are used as templates, resulting in 4 copies. This doubling continues with each cycle (8, 16, 32, etc.), leading to an exponential increase in the number of DNA copies.
