Polymerase Chain Reaction (PCR): Principle, Steps, Types, Applications & Limitations

Polymerase Chain Reaction (PCR) is a technique used to amplify a specific segment of DNA or to generate a large number of copies. It is a simple but very useful procedure in Molecular Biology; developed in 1983 by Kary Mullis, an American biochemist. In other words, PCR allows you to create millions of copies of a specific DNA sequence from a small sample – sometimes as little as a single copy. PCR is a critical process for a variety of genetic technologies, and it has actually enabled the development of a suite of new technologies.

Key Terms: Polymerase Chain Reaction, PCR, Molecular Biology, DNA, DNA Polymerase, Deoxyribonucleotide Triphosphate, Enzymes

Principle of PCR

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PCR amplifies a specific region of a DNA strand (DNA target). The majority of PCR methods amplify DNA fragments ranging in length from 0.1 to 10-kilo base pairs (kbp), though a few techniques allow for the amplification of fragments up to 40 kbp. The amount of amplified product is determined by the available substrates in the reaction, which become limited as the reaction progresses.

Read More: MCQs on PCR (Polymerase Chain Reaction)

Components of PCR

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Following are the components of the PCR: 

• DNA Template: In this, the DNA of interest forms the sample.
• DNA Polymerase: Taq Polymerase is used which is thermostable and it does not denature at very high temperatures.
• Oligonucleotide Primers: Oligonucleotide Primers are the short stretches of single-stranded DNA that are complementary to the 3 ends of sense and anti-sense strands.
• Deoxyribonucleotide Triphosphate: They provide energy that is required for polymerization and are the building blocks for the synthesis of DNA. 
• Buffer System: Magnesium and Potassium offer the minimal conditions for DNA denaturation and renaturation. This is also essential for polymerase activity, fidelity, and stability.

Components of PCR

Steps of PCR

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There are three major cyclic reactions that are involved in PCR: 

Denaturation: When the reaction mixture is heated to 94°C for about 0.5 to 2 minutes, denaturation occurs. The hydrogen bonds between the two strands of DNA are broken, resulting in single-stranded DNA. The single strands of DNA are now used as a template for the creation of new strands of DNA. To ensure the separation of the two strands, the temperature should be maintained for a longer period of time.

Annealing

• For about 20-40 seconds, the reaction temperature is reduced to 54-60°C. The primers bind to their complementary sequences on the template DNA at this point.
• Primers are single-stranded DNA or RNA sequences of 20 to 30 bases in length.
• They act as the starting point for DNA synthesis.
• Because the two separated strands run in opposite directions, there are two primers: a forward primer and a reverse primer.

Read More: Molecular Basis of Inheritance: DNA, RNA and Genetic Code

Elongation

• The temperature is increased to 72-80°C at this point. The Taq polymerase enzyme adds the bases to the 3' end of the primer.
• This causes the DNA to lengthen in the 5' to 3' direction. Under ideal conditions, the DNA polymerase adds about 1000bp per minute.
• Taq Polymerase can withstand extremely high temperatures. It binds to the primer and adds bases to the single strand of DNA. As a result, a DNA molecule with two strands is formed.
• These three steps are repeated 20-40 times in order to obtain a large number of DNA sequences of interest in a short period of time.

Steps of PCR

Types of PCR

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The various types of PCR are listed below: 

• Real-time PCR: the DNA amplification is detected in real-time with the help of a fluorescent reporter.
• Quantitative Real-Time PCR (Q-RT PCR): It uses the DNA amplification linearity to detect, characterize and quantify a known sequence in a sample.
• Reverse Transcriptase PCR (RT-PCR): It is a technique used to synthesize double-stranded complementary DNA from mRNA that can be isolated from either blood or tissue samples.
• Multiplex PCR: It amplifies several different DNA sequences differently.
• Nested PCR: It is designed to improve specifity and sensitivity. 
• Arbitrary Primed PCR: It is a DNA fingerprinting technique based on PCR. It uses primers the DNA sequence of which is chosen arbitrarily.

Applications of PCR

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Here are the major applications of PCR: 

• PCR is used to test clinical specimens for the presence of infectious agents such as HIV, hepatitis, malaria, anthrax, and others.
• PCR can predict response or resistance to therapy and provide information on a patient's prognosis. Many cancers are characterised by small mutations in specific genes, which PCR is used to detect.
• PCR is used to analyse mutations found in many genetic diseases (e.g. cystic fibrosis, sickle cell anaemia, phenylketonuria, muscular dystrophy).
• PCR is also used in forensic laboratories and is particularly useful because only a small amount of original DNA is required, such as a droplet of blood or a single hair.
• PCR is an important technique in the cloning procedure because it allows for the generation of large amounts of pure DNA from a small amount of template strand and the study of a specific gene.
• The Human Genome Project (HGP) relied heavily on PCR to determine the sequence of the human genome's 3 billion base pairs.

Limitations of PCR

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The PCR reaction begins to generate copies of the target sequence in an exponential fashion. Only during the exponential phase of the PCR reaction can the starting quantity of the target sequence contained in the sample be determined. 

Because of polymerase reaction inhibitors found in the sample, reagent limitation, pyrophosphate molecule accumulation, and self-annealing of the accumulating product, the PCR reaction eventually ceases to amplify the target sequence at an exponential rate and a "plateau effect" occurs, rendering endpoint quantification of PCR products unreliable. This is the property of PCR that necessitates Real-Time Quantitative RT-PCR.

Read More: DNA Polymerases

Things to Remember

• Kary Mullis, an American biochemist, invented the polymerase chain reaction (PCR) in 1983.
• PCR is a common tool in medical and biological research labs. 
• It is used in the early stages of DNA processing for sequencing, to detect the presence or absence of a gene to aid in pathogen identification when infected, and when creating forensic DNA profiles from tiny DNA samples.
• Many research labs use PCR, and it has practical applications in forensics, genetic testing, and diagnostics. 
•  PCR can also be used to detect a bacterium or DNA virus in a patient's body: if the pathogen is present, amplify regions of its DNA from a blood or tissue sample.
• Many infectious diseases can be identified using PCR tests, which are both accurate and reliable. PCR tests are very essential in disease prevention as they can often make diagnoses before symptoms of infection appear.