Gene cloning is the method of producing identical genes through different procedures. Method of gene cloning provides opportunity to the scientists to study the structure and function of genes in detail. For this purpose, gene of interest is inserted into the bacterial cell which acts as a host. The cloned gene can be used for many research purposes like detection of diseases, gene therapy and other medical applications. There are following steps needed to make the cloned genes.
Isolation of the Desired Gene:-
First step in gene cloning is the isolation of the desired part of DNA in which the gene of interest is present. Similarly the bacterial plasmids are also isolated in which the desired gene will be inserted. When the desired region with gene of interest is identified, it is isolated by cutting it with restriction enzymes. As all enzymes are proteins, restriction enzymes are also proteins which play a role of catalysts in a chemical reaction. They cut the phosphodiester bond, a covalent bond of deoxyribose in DNA. There are specific sites on the DNA molecule called restriction sites. These are the locations where gene of interest is isolated from the rest of the DNA molecule by breaking the strand of DNA. Now this gene is ready for the introduction to the vector.
Introduction of the Desired Gene into the Plasmid:-
When the gene of interest is isolated, it now needs a host cell where it can replicate and can make multiple copies. For this purpose, plasmid is used which is a molecule inside the bacterial cell. It has the ability to replicate out of the bacterial chromosomal DNA. Plasmids are the best source of gene cloning as they are able to replicate separately and independently from the bacteria's on genetic material. They are double stranded circular molecules. When the gene of the interest is inserted into the plasmid, the ring of the plasmid opens up giving place to the gene to attach with it. Now the plasmid is ready for the introduction into the host organisms as it contains a foreign gene.
Insertion of Plasmid into the Host cell:-
Most commonly used plasmid is the Escherichia coli, specie of bacteria useful for the human body. The plasmid with foreign gene is now inserted into the host organism's body. Plasmid enters the host cell by two mechanisms; either it is inserted by placing the cell in calcium chloride which will enable the plasmid to enter the host cell or by Electroporation, in which through slight electric current, pores will appear in the host cell's membrane, plasmid will enter through these pores. Now the host cell is genetically modifies because it contains the foreign within it.
Beginning of the Cloning of new Gene:-
As described above that plasmid is free of chromosomal DNA that is why when it is inserted into the bacterial cell, it will start replicating at a very high speed making identical copies of the desired gene. Scientists use E.coli as the host cell for the plasmid, because it has the ability to replicate faster than any other microorganism. It is the best microorganism used in recombinant DNA technology.
Isolation of the Cloned gene:-
Scientists put the bacterial cell along with the culture into a culture, where it replicates. Then the cloned gene is isolated again by using restriction enzymes through the process known as lysis. Through this way, the cloned gene can be isolated without any damage to the gene.
Application of Gene Cloning:-
Gene cloning is applicable in many fields of science such as medicine and agriculture.
In medicine, bacteria play a vital role for the synthesis or production of many vitamins, hormones and antibiotics. It is done by introducing the desired gene into the plasmid and then this plasmid replicates to make multiple copies of this gene. If scientists have to treat some dangerous disease, they clone the healthy gene and then insert it into the organism to replace it with the diseased gene.
Bacteria also are important for the nitrogen fixation. Bacteria along with the desired gene are used to increase the crop productivity and health. They make the farmers free of using expensive fertilizers which can damage the crops.
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