Polymerase Chain Reaction (PCR) Applications & Components | Function of DNA Polymerase

In this write up we will discuss Polymerase Chain Reaction (PCR) Applications & Components.

Alongside this, we will also get to know about the Function of DNA Polymerase and Components of PCR and their functions.

WHAT IS A POLYMERASE CHAIN REACTION (PCR)?

Polymerase Chain Reaction (PCR) can be defined as a method that is used to make millions to billions of copies of specific DNA samples very rapidly.

The copies that are made can be partial or complete copies. It was invented in 1983 by Kary Mullis who was an American biochemist at Cetus Corporation.

PCR is used to make copies of a very small amount of DNA sequences. These sequences are exponentially amplified by the series of temperature changes.

Components of PCR and their functions

The requirements for PCR are as follows:-

PRIMERS

Primers are short single-strand DNA fragments. These are also known as oligonucleotides and are the complementary sequences for the target DNA region.

Taq POLYMERASE

It is an enzyme that is responsible for the formation of the polynucleotide chain. This enzyme can withstand the high temperature of the PCR reaction.

dNTPs

Deoxynucleotide triphosphates (dNTPs) are the nucleoside triphosphates that contain deoxyribose sugar.

These are used to build the DNA. DNA polymerase is responsible for adding these dNTPs to the primers and extending the chain of the polynucleotide.

TEMPLATE DNA

It is the sequence of DNA that needs to be amplified.

One cycle of PCR is represented in the below figure. One cycle contains three steps:

1)  Denaturation.

2) Annealing

3) Extension.

One cycle of PCR reaction
(source: https://blog.praxilabs.com/2021/04/28/what-are-the-three-basic-steps-of-conventional-pcr/)

Function of DNA Polymerase Enzyme

Polymerase as the name suggests is an enzyme. The word that ends with “ase” is the enzyme and performs the catalytic activity.

“Poly” means many. So, from its name itself, it is clear that polymerase is an enzyme that is responsible for forming polymers that are made up of many units of monomers.

The polymerase is the enzyme that is responsible for forming nucleic acids i.e. DNA and RNA. Nucleic acids are the polymers of nucleotides.

There are two types of polymerase on the basis of which polymer they form- DNA polymerase and RNA polymerase.

DNA polymerase works by copying a DNA template strand and uses base-pairing interactions. RNA is formed by half ladder replication.

HALF LADDER REPLICATION means one strand acts as the template.

During the formation of RNA with the use of RNA polymerase only one strand acts as the template. Using that strand the RNA is formed.

Function of RNA Polymerase

Applications of Polymerase Chain Reaction (PCR)

SELECTIVE DNA ISOLATION

PCR is a method that allows selective amplification of the specific region of DNA.

Selectively isolated products of PCR can be used in many ways such as for generating hybridization probes that can be used in southern blotting and northern blotting that are done for DNA and RNA respectively.

The PCR product can also be used in DNA cloning that requires a large amount of DNA of a specific region.

PCR is also responsible for supplying a high amount of pure DNA.

PCR Products that are selectively amplified can also be used for sequencing.

AMPLIFICATION AND QUANTIFICATION OF DNA

If the amount of sample available is small then PCR is used for its amplification and analysis. This property is used in forensic science.

PCR is also used for analyzing the DNA of ancestors that are thousands or more than thousands of years old. DNA of forty-thousand-year-old mammoth is analyzed by using PCR.

Quantitative PCR or Real-Time PCR (qPCR) is used for the quantification of DNA.

This is used to measure the accumulated DNA after each round of PCR amplification.

Quantitative PCR measures concentration when the DNA synthesis is taking place.

IN MEDICAL AND DIAGNOSTIC APPLICATIONS

PCR analysis is also important for the diagnosis of genetic diseases before implantation. In this technique, individual cells of the embryo are tested for mutations.

Prospective parents are also tested for being genetic carriers that can result in disease in children.

It is also used in sensitive testing for tissue typing. Tissue typing is vital for organ transplantation.

PCR- based tests are also used to study the mutations that occur in oncogenes during cancer. This can help in customizing the therapy for individual patients that can help in their recovery.

It also helps in the early diagnosis of leukemia and lymphomas.

It can also be used in detecting translocation-specific malignant cells in a more sensitive way when compared to other methods.

It permits the isolation and amplification of tumor suppressors.

IN DIAGNOSIS OF INFECTIOUS DISEASES.

PCR helps in the specific diagnosis of infectious diseases caused by bacteria and viruses in a very short time.

It also helps in the identification of microorganisms that are slow-growers are not cultivable in media. Example of microorganisms that are slow- growers is mycobacteria, anaerobic bacteria, or viruses.

By using PCR one can also discriminate between pathogenic and non-pathogenic strains of microorganisms with the help of specific genes.

The infectious diseases that are diagnosed by using PCR  are HIV (Human Immunodeficiency Virus), M.tuberculosis, SARS-CoV-2, COVID-19, Bordetella pertussis.

IN FORENSIC SCIENCES

The genetic fingerprinting technique used in forensics is based on the PCR technique. It is useful in discriminating a single person from the entire population of the world.

The samples of DNA that are isolated from a crime scene are very minute so they are amplified and compared with the samples from suspects or from the database of earlier convicts.

DNA PATERNITY TESTING is the testing in which an individual is matched with their close relatives. DNA of unidentified humans is tested and compared with the possible parents, siblings, or children.

It also provides an effective way to the determination of gender in forensic cases and ancient specimens. The PCR AMGX/AMGY is used for this purpose.

IN RESEARCH

Polymerase chain reaction (PCR) is applied in the molecular genetics area of research.

The amplifying ability of PCR permits the production of short pieces of DNA. This ability of PCR is used in the generation of hybridization probes for southern blotting and northern blotting.

PCR is also responsible for supplying pure DNA for these techniques.

PCR also helps in the process of DNA sequencing.

PCR also assists the process of DNA cloning. It can be used in extracting DNA segments for insertion into vectors. It can also be used in analyzing and extracting fragments that are already been inserted in vectors.

PCR is also used in the phylogenic analysis of DNA from ancient sources such as frozen tissues of mammoths or from brains of EGYPTIAN MUMMIES.

PCR can also be used for studying the pattern of gene expression. Cells or tissues can be analyzed for understanding the activation of different genes at different stages of division and development.

Quantitative PCR can be used for knowing the actual levels of expression.

PCR is also used in creating mutant genes with mutations that a scientist wants during his/her research.

The creation of this mutant gene is known as site-directed mutagenesis. These mutations are chosen in such a way that the functioning of proteins can be understood and can be improved.

Also Read:-

Types of Epithelial Tissues

DNA Replication Steps

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