Steps In Polymerase Chain Reaction: Principle, Types, Technique and Applications in Pathology

Polymerase Chain Reaction

Steps In Polymerase Chain Reaction

Polymerase chain reaction (PCR) is one of the most important techniques in molecular pathology. With the help of polymerase chain reaction, the single or the pieces of target DNA can be amplified many folds. This technique is now an integral part of every modern laboratory for both diagnosis and research use.

Steps In Polymerase Chain Reaction

The basic steps of polymerase chain reaction are described as:-

  • Denaturation step, 94 ° C: DNA is heated to 94 ° C to make it single-stranded. Only 1–2 min are given to this heating process in each cycle.
  • Annealing, 54 ° C: The temperature is rapidly cooled. At this lowered temperature, the primer quickly anneals with the respective site of DNA. With the help of Taq polymerase, the reaction starts at the first-DNA template site.
  • Extension, 72 ° C.
  • The complementary nucleotides are attached from the 3 ′ to 5 ′ ends of DNA. There is an exponential increment in the number of genes in each cycle. At least 30 cycles of denaturation-annealing-extension are done in each polymerase chain reaction.

Essential Ingredients of Polymerase Chain Reaction

  • Primers: Those primers are the small pieces of artificially made DNA strands that are actually the complementary strands of the 3 ′ end of each strand of target DNA. Primers usually consist of 20–30 nucleotides.
  • DNA polymerase (Taq polymerase): Taq polymerase is a type of DNA polymerase enzyme that extends the new DNA strand. It combines at the end of the primer and then sequentially adds new nucleotides to the DNA strand at 3 ′ ends complementary to the target DNA. A high temperature (94 ° C) is needed to separate the double-stranded DNA. Ordinary DNA polymerase breaks down at this temperature. However, the Taq polymerase has the unique characteristic to work efficiently in higher temperatures. This Taq DNA polymerase is extracted from the bacteria Thermus aquaticus. These bacteria live in hot springs and can survive there.
  • Deoxynucleotide triphosphates (dNTPs): Deoxynucleotide triphosphates (dNTPs) are dATP, dCTP, dGTP, and dTTP. These are the raw material or the basic building blocks of the new DNA strands. The Taq polymerase captures these dNTPs from the working solution and attaches them to the terminal part of the primers to extend the DNA chain.
  • The target DNA from the sample: The target DNA is extracted from the sample.
  • Buffer solution: It provides the optimum chemical environment for the reaction to occur.
  • Magnesium chloride (MgCl2): Magnesium chloride works as a cofactor of the Taq polymerase enzyme.

What Is Polymerase Chain ReactionAnd How It Works?

Polymerase chain reaction acts like a “molecular photocopying” machine and amplifies the specific target DNA. The basic principles of polymerase chain reaction are:

  • Double-stranded target DNA is made into single-stranded DNA by applying heat.
  • Two oligonucleotide strands or primers are added. The oligonucleotide strand binds with its complementary DNA strand to the 3 ′ ends.
  • The DNA strand is now extended with the help of DNA polymerase (Taq polymerase). This polymerase enzyme incorporates the nucleotides in the DNA to make it elongated.
  • The cycle is repeated.

Basic Precautions Earlier Steps In Polymerase Chain Reaction

  • Always wear gloves to avoid any contamination.
  • Completely thaw all the reagents before doing polymerase chain reaction.
  • Keep all the reagents in an ice basket throughout the time of the polymerase chain reaction experiment.

Equipment Uses Steps In Polymerase Chain Reaction

  • Thermal cycler
  • Polymerase chain reaction tubes and caps
  • Ethanol-resistant marker
  • Micropipettes set

3 Thermal Cycling In Polymerase Chain Reaction

Close the cap of the polymerase chain reaction tubes and then put them in the thermal cycler.

Standard Steps

  • Initial denaturation: At 94 ° C for 1 minute
  • Denaturation: At 94 ° C for 30 seconds
  • Annealing: 50–60 ° C for 30 seconds: The temperature may vary depending on the first used. The temperature of annealing the stage should be 3 to 4 ° C lower than the melting point (Tm) of the primers.

Cycling Time

The polymerase chain reaction thermal cycle rapidly heats and cools the polymerase chain reaction reagent mixture. The cycling time depends on (1) the size of the DNA template and (2) the G-C content of DNA.

The number of the thermal cycler is usually set as 25–30 cycles. If the thermal cycle is increased by more than 35, then too many unwanted DNA products may be produced.

Purification of the Amplified Product

The following measures are taken to purify the polymerase chain reaction products from the reaction solution:

  • Agarose gel electrophoresis of the product: Agarose gel electrophoresis is done from the portion of the polymerase chain reaction product to verify the validity of the test.
  • Cloning of products: In this technique, a further polymerase chain reaction is done to confirm the polymerase chain reaction product. Este is done when the gene is present in a very tiny amount.
  • Sequencing of products: This is done by an automated sequencer machine to analyze the sequence of DNA formed as a polymerase chain reaction product.

Types of Polymerase Chain Reaction

There are different types of polymerase chain reaction methods for diagnostic purposes. These are:

1. Direct PCR: This is the standard PCR technique as has been described in the previous section.

2. Reverse transcriptase PCR (RT-PCR): In the case of RT-PCR, at first, cDNA is prepared from the RNA of the target sample. This cDNA is then amplified by the PCR technique.

3. Asymmetric Polymerase Chain Reaction: In this technique, the unequal concentration of primers is used. A great excess of primers is used for the targeted DNA strand that we need to amplify.

As the reaction proceeds, only an adequate amount of primer in the reaction mixture produces the particular DNA strand in excess. Therefore ultimately single-stranded DNA (ssDNA) is formed as a polymerase chain reaction product.

As the reaction is slow and goes on arithmetically, so many more cycles are needed in this technique. The asymmetric polymerase chain reaction is used for DNA sequencing and hybridization as only one strand is needed in such conditions.

4. Hot start Polymerase Chain Reaction: Normally DNA polymerase acts at room temperature and even in the ice pack. Thereby there always remains
the possibility of spurious products.

In the hot start polymerase chain reaction technique, the DNA polymerase is unreactive at a lower temperature and works only at a higher temperature. This is done by conjugating an inhibitor with the polymerase enzyme, and in the higher temperature, the inhibitor is free from the polymerase enzyme and allows it to work.

5. In situ Polymerase Chain Reaction: In the case of in situ polymerase chain reaction, the reaction takes place within a cell on the glass slide. The polymerase chain reaction product is accumulated within the cell, so it is possible to locate the origin of the amplified DNA.

The specially designed polymerase chain reaction machine is used to put the slide within it. This technique is used to amplify the nucleic acid in the fixed tissue and cell instead of in solution.

6. Inverse Polymerase Chain Reaction: Inverse polymerase chain reaction (IPCR) amplifies anonymous DNA sequences. It helps to identify the flanking DNA sequence of the genome outside the boundary of the known target sequence.

7. Single-strand conformation polymorphism (SSCP): The basic principle of SSCP is that the single-stranded DNA has a specific conformation.

Any alteration of the single base change due to mutation may lead to different migration patterns of the single-stranded DNA, and therefore in electrophoresis one can distinguish wild-type DNA from mutant DNA. The following steps are done in SSCP.

8. Real-time Polymerase Chain Reaction: Real-time polymerase chain reaction is also known as quantitative polymerase chain reaction (qPCR) as it constantly monitors the quantity of the amplified DNA in the reaction process.

In the case of qPCR, the amplified DNA is fluorescently labeled, and the emitted fluorescent is directly proportional to the amount of the amplified fluorescent dye.

Therefore in each cycle, the amount of the product can be directly monitored, and it is also possible to quantitate the initial amount of the target DNA in the sample.

9. Nested Polymerase Chain Reaction: In the case of nested polymerase chain reaction, more than two pairs of primers are used for DNA amplification. The first polymerase chain reaction is a conventional polymerase chain reaction, and the primer is used for the DNA template of the sample.

In a secondary polymerase chain reaction, the product of the first polymerase chain reaction is used as the target of the second set of primers. The DNA sequence of secondary polymerase chain reaction is different, and therefore there is no chance of undesired polymerase chain reaction product formation.

Applications Of Polymerase Chain Reaction

The various applications of polymerase chain reaction in the clinical area are described below:

(a) DNA sequencing: Polymerase chain reaction is often used in the case of DNA sequencing.
(b) Diagnosis of infection: Polymerase Chain Reaction is widely used for the diagnosis of viral, bacterial, and parasitic infection. As mentioned before Q-polymerase chain reaction can quantitate the viral load in the body.
Polymerase Chain Reaction rapidly detects tuberculosis within a few hours, whereas the culture of mycobacteria takes a few weeks to develop. As a sensitive technique, a polymerase chain reaction is able to detect tuberculosis in the early latent phase.

(c) Diagnosis and prognostic information on cancer: PCR technique is extensively used in the field of cancer.
(d) Genetic diseases: The Polymerase Chain Reaction technique is very helpful to detect various genetic diseases such as Down’s syndrome, cystic fibrosis,
Gaucher disease, etc.
The main advantage of the polymerase chain reaction technique is that it can bypass the aggressive placental bed biopsy to detect these inherited diseases. The minute amount of fetal cells collected from the mother’s blood or cervical mucosa is enough to reach a diagnosis.
(e) Forensic pathology: The Polymerase Chain Reaction technique is helpful in forensic pathology in different ways:
  • To detect paternity of the child
  • To identity the corpse or mutilated body
  • To identify the criminal from the crime the site and biological materials of the criminal.
(f) Gene therapy: Polymerase Chain Reaction helps to engineer the specific gene to introduce in the diseased person to cure various diseases.

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