Gene cloning Introduction
Word clone refers to a copy, exact replica of a cell, tissue etc. Gene cloning is the process by which exact replica of a gene is manufactured. It provides scientists with the opportunity to study structure and functions of a gene in detail. Gene of interested is incorporated in to bacterial host. Gene is cloned and proves to be useful for many processes like detection of diseases, replacement of defective genes i.e. gene therapy and treatment of various ailments.
Gene cloning is the replication of certain type of genes from a certain part of the cell/ DNA to propagate a certain desirable genetic trait. Gene cloning is also known as DNA cloning. In which fragment of DNA is cloned.
Isolation and obtaining of desired gene
The gene to be isolated is identified in the DNA. The DNA is isolated from the organism and then the gene to be cloned is isolated as follows.
DNA obtained is purified and then fragmented with the restriction endonucleases enzymes. They cut the DNA at specific site and hence produce staggered cuts in DNA at specific sites and generate fragments with cohesive ends. Each fragment has single stranded sequence of nucleotides at its ends that is capable of hybridizing with DNA fragmented using the same restriction enzyme.
Incorporation of desired gene in to vector (plasmid)
DNA fragments are then incorporated in to vector i.e. carriers generally used vectors are plasmids. The type of plasmid used in gene cloning has single restriction site and when cleaved by the same restriction enzymes generate cohesive ends that are present in the DNA fragments to be cloned. The cohesive ends of the plasmids and the DNA fragments now line u and the enzyme, DNA ligase, is used to form phospodiester bonds. The plasmid now carries the gene to be cloned.
Insertion of vector in to host cell
The plasmid is inserted into the host cell using the method of transformation. Each cell contains a different segment of DNA from the original organism. Taken together they represent a DNA library. Plasmids enter the host cell by two methods either by placing the cell in calcium chloride which will enable the plasmid to enter the host cell or by gel electroporation in which slight electricity is given and pores appear on the host cell membrane now the plasmids enter these pores. Now the host is known as transgenic because it contains foreign DNA. Host for plasmids that's most commonly used is E.Coli (ESCHERICHIA coli).
Initiation of cloning
Plasmid is free of chromosomal DNA and when inserted in to bacterial DNA starts replicating at a very fast pace thus making copies of the desired gene. E.Coli is used in this process because of its rapid rate of replication than any other organism.
Separation of cloned gene
Now the bacteria cell containing the plasmid is placed in to a culture where the replication process takes place. Now the desired gene is isolated using restriction enzyme and thus this process is known as lysis. Genes can be isolated without causing any damage.
Applications of gene cloning
Gene cloning plays an important role in the synthesis of vitamins, hormones and antibiotics. Different genes for the treatment of diseases like cystic fibrosis and many other diseases like this can be synthesized.
Cyano-bacteria like nostoc are nitrogen fixing bacteria i.e. they fix the nitrogen in the air thus making them useful for the plants. Nitrogen fixing bacteria along with the desired genes are used to increase the crop productivity and health. It thus decreases the use of fertilizers and hence agro chemical free crops are produced which are better in nutritional value.
The process in which defective gene is removed inserting new and healthy gene into its place is known as gene therapy. Very deadly ailments like leukaemia can be treated using gene therapy in which defected gene is replaced. Sickle cell anaemia can also be treated using this process.
From a gene library, it is possible to identify a clone containing the specific gene. This gene can be multiplied by growing the relevant clone in a culture. The base sequence in this gene can be found, as the methods for this purpose are known. From the base sequence, the sequence of amino acids in a polypeptide can be worked out on the basis of the triplet code
Gene therapy is a revolutionary technique in the field of biotechnology. It has multiple uses, has helped mankind in a number of ways, various diseases are being treated using this technique and hence increasing the life span. Healthy and better life is achieved using the technology. Agriculturally crops are improved thus leading to better nutritional values. This has raised the standard of human living.
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