PCR And Types Of PCR

Molecular biology studies how the molecules interact with one another to perform the function of life. PCR (Polymerase Chain Reaction) is a laboratory technique used to identify DNA sequences. American Biochemist “Kary Mullis” received the Nobel prize for his work in molecular biology and genetics. It is a common tool used in medical and biological research, which is required in the early stage of processing DNA for sequencing. It helps to generate thousands of copies of a particular section of DNA. The reaction allows a few copies of DNA to replicate into million copies. 

What is PCR?

PCR (Polymerase Chain Reaction) is a technique used in a lab to make millions of copies in a different section of DNA. It was developed in 1980 by American biochemist “Kary Mullis”. It is used in the early stage for the processing of DNA for sequencing; it also helps to detect the presence and absence of genes, which helps to identify pathogens.

PCR steps

The steps of PCR involve amplified DNA synthesis with the aid of different replication polymerases. The process of PCR involves denaturation, annealing, followed by extension.  The two strands of the DNA double helix must be split, much like in DNA replication. The hydrogen bonds between the complementary DNA strands are broken as the temperature of the mixture is raised, causing the separation. Denaturation is the term for this procedure. Annealing involves primer binding and polymerisation. the development of new strands occur in the extension stage using the template DNA. One PCR cycle produces two double-stranded target DNA sequences, each with one freshly synthesised strand and one original strand. Because most PCR techniques require significant amounts of DNA, the cycle is repeated numerous times (typically 20–30). Obtaining a billion or more copies takes only 2–3 hours.

Types of PCR

There are different types of PCR in molecular biology they are as follows:

Real-time PCR: It monitors the amplification of targeted DNA molecules in PCR.

Quantitative Real-Time PCR: It quantifies a targeted DNA molecule.

Reverse Transcriptase PCR: It measures RNA expression level.

Multiplex PCR: It detects multiple targets in a single reaction.

Nested PCR: It is intend to reduce nonspecific binding 

Long-range PCR: It used to fragment more than 5kb by using high fidelity DNA polymerase.

Single-cell PCR: One method used to detect single-cell levels.

Fast-cycling PCR: It helps to reduce cycling time.

Methylation-specific PCR: It helps to analyse DNA Methylation patterns in CpG islands.

Hot start PCR: It is a modified form of conventional polymerase chain reaction.

High fidelity PCR: It results in a high degree of accuracy in the replication of DNA.

In situ PCR: It combines the extreme sensitivity of PCR.

Asymmetric PCR: It is a variation of PCR to amplify one strand of original DNA.

Repetitive sequence-based PCR: A net method differentiates microbes by using complementary primers.

Overlap extension PCR: It is used to monitor and optimise as any other long protocol.

Assemble PCR: It used to assemble two gene- sized pieces of DNA into one piece for easier fusion.

Inter Sequence-specific PCR: It used to analyse the DNA methylation pattern in CPG island.

Post translation process in PCR

The post-translation process in PCR deals with three different translations that are based on the chemical group “Phosphorylation”, “Acetylation”, “Hydroxylation”, “Methylation”. “Phosphorylation” has several implications dealing with several cellular processes that include the cell cycle, growth, and single transaction pathways. The phosphate group converts uncharged pockets of protein into negatively charged protein. “Acetylation” refers to the addition of an acetyl group in a protein that involves several protein stability, location, and synthesis to reduce the positive charge in his tone. “Hydroxylation” adds a hydroxyl group to the protein that aids in converting “Lipophilic compounds” into “Hydrophilic compounds”. “Methylation” refers to the addition of the methyl group to a lysine residue of a protein; it has been performed from a wide study in his tone, which leads to gene activity based on the residue that is “Methylated”.

Benefits of PCR

The benefit is that PCR mainly deals with different sectors such as the reputation cycle of “Denaturation”, “Amplification”, and “Replication” in the segmentation of DNA. Specific DNA helps to indicate the presence of microorganisms that “Inhibit bacterial translation”. PCR throughout the field of molecular biology helps researchers to clone and sequence genes for mutation. In recent mobility, a small sample is required for the analysis, PCR is highly sensitive compared to culture and staining. The ability to test antimicrobial resistance, it quickly performs the activity in 4-8 hrs, more cost-effective for select use than cultural and staining. Increase ability to detect fewer organisms such as viruses.

Applications of PCR

The applications of PCR in microbiology are as follows:

• It helps to analyse clinical specimens for infectious agents that include HIV, malaria, anthrax, etc.
• It provides information to patients regarding resistance to therapy. It is observed that many cancers are characterised by creating genes that are identified by PCR.
• It used to identify mutations that occur in many genetic diseases.
• In a forensic laboratory, PCR is used to identify tiny amounts of DNA obtained from droplets of blood.
• A technique allows the generation of large amounts of DNA to purify tiny amounts of the template and study a particular gene. 
• PCR helps to identify relationships among fields of evolutionary biology. In “Anthropology”, it is used to understand human migration patterns. In “Archaeology” used to spot the human race.

Conclusion

To conclude the discussion on molecular biology, PCR used in different aspects to analyse the inexpensive techniques that were used to amplify the small segment of DNA. DNA is necessary for molecular and genetic analysis, for the study of DNA without PCR implication. The discussion made on the advantages that PCR has, and PCR used to determine the DNA and the application of DNA where it is being used. The microbiology in PCR is used to determine how the living organism performs life.