Polymerase chain reaction is commonly known as PCR. It is used to produce multiple copies of a gene or DNA of interest. The steps of polymerase chain reaction are denaturation, primer annealing and extension of primers.
What is Polymerase Chain Reaction?
Polymerase chain reaction or PCR is a reaction that is utilised to amplify a gene or fragment of DNA of interest. It is done in vitro using a primer. This technique is used in labs to make billions of copies of the desired gene for research, diagnostic and therapeutic purposes.
PCR was invented by Kary Mullis in 1983. PCR requires a DNA primer designed for the DNA template and DNA polymerase, preferably a thermostable DNA polymerase. In the polymerase chain reaction, the reaction cycle is repeated many times to get billions of copies.
PCR is an integral part of biotechnology, medical biology, diagnostics, forensic analysis, molecular biology research, etc. The amplified DNA can be sequenced, cloned and visualised by gel electrophoresis.
Polymerase Chain Reaction Steps
Each cycle of polymerase chain reaction has three steps. They are:
- Denaturation: The first step in PCR is denaturation. Denaturation is required to separate the double-stranded DNA sample. It is done at 94-98 ℃ for 20-30 seconds. It breaks the hydrogen bonds present between base pairs. Denaturation leads to the formation of single strands of DNA.
- Annealing: The second step is the annealing of the primer. Here the reaction temperature is lowered to allow the complementary base pairing between the primer and the complementary part of the single strands of the DNA template. A proper temperature needs to be maintained in order to allow highly specific and proper primer hybridisation. Then DNA polymerase binds to the template-primer hybrid and starts the DNA synthesis.
- Extension: A thermostable DNA polymerase is used for this purpose. Taq polymerase is commonly used for this purpose. It is done at a temperature of 75-80 ℃ (72℃). The DNA polymerase adds nucleotides in the 5’-3’ direction and synthesises the complementary strand of the DNA template.
This cycle is repeated 25-30 times and with this, the DNA sample can be amplified a billion times. Gel electrophoresis is used to visualise the result of polymerase chain reaction.
PCR reaction requires a primer, thermostable DNA polymerase, template DNA and nucleotides.
Primers for Polymerase Chain Reaction
Two sets of primers are used, which are short stretches of oligonucleotides. They are chemically synthesised and complementary to the part of the DNA template to be amplified. DNA polymerase requires a primer for initiating replication. DNA polymerase cannot initiate the process, it can only add nucleotides, therefore a primer is used for this purpose to initiate the polymerisation process.
Primers are usually 20 nucleotides long. The two primers used, cover the target region of DNA to be amplified and bind to the opposite edges of the template strand. After binding of primers, the region between them gets copied by the DNA polymerase.
The replication or polymerisation of DNA requires a DNA polymerase enzyme. In PCR, Taq polymerase is used. It is thermostable and is isolated from a heat-tolerant bacterium, Thermus aquaticus. A thermostable polymerase is required so that it can remain active at higher temperatures. Higher temperatures are used in the PCR process for denaturation of the DNA double helix repeatedly for multiple cycles. Taq polymerase is most active at around 70 ℃.
Application of Polymerase Chain Reaction
Polymerase chain reaction has many advantages. It is a highly sensitive technique and gives results rapidly. It is simple and easy to understand. It is highly specific and is an important tool in research and biotechnology. Millions and billions of copies of the desired gene can be produced, which can be utilised for various purposes such as sequencing, analysis and cloning.
Some of the important applications of polymerase chain reaction are given below:
- PCR is used for diagnostics. E.g. A viral genome can be amplified and detected for the presence in the host cell.
- A genetic sequence associated with a bacterial toxin can be diagnosed efficiently using PCR, e.g. Pertussis toxin
- PCR amplification enables the detection of a viral genome early after infection. It can help in diagnosing the infection even before the onset of the disease. It significantly helps in the treatment of the disease.
- Viral load can be detected by PCR based techniques.
- RT-PCR is commonly used to detect the SARS-CoV-2 viral genome. It is a variant of PCR and uses the enzyme reverse transcriptase to produce DNA complementary to the viral RNA.
- Oncogenes mutations can be detected early with PCR. It helps in diagnosing various cancers such as leukaemia and lymphoma.
- PCR is used in forensics. A small sample of DNA collected from the crime scene can be amplified, analysed and compared with the suspects.
- PCR can be used for genetic testing. A gene associated with the genetic disorder can be amplified and detected from the sample taken from patients and also from foetal cells.
- PCR is used readily in molecular biology research. PCR helps in sequencing a genome. The unknown viral genome can be sequenced and studied for a better understanding of disease progression.
- PCR is utilised in recombinant DNA technology. The desired gene is amplified and then can be inserted into a vector for cloning.
- PCR is useful in detecting and analysing ancient DNA samples that are thousands of years old. E.g. forty-thousand-year-old mammoth, Egyptian mummies, etc.
- PCR is also helpful in analysing phylogenetic relationships from ancient samples.
- PCR is used to study gene expression. Alteration of gene expression can be studied in the case of tumours, cancers and other diseases.
- PCR can be used to create mutant genes of choice to study site-directed mutagenesis.
- PCR analysis is an important tool to monitor the spread of diseases. The new mutant forms present in the population can be detected and monitored.
- PCR is useful in prenatal testing for genetic disorders and also to find if a person is a carrier of a certain disease.
- Quantitative PCR or qPCR (Real-Time PCR) helps to determine the quantitative level of DNA or gene expression. Accumulation of DNA products is quantified after each cycle of PCR.
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