A technique mainly used to change the phenotype of an organism (host) when a genetically altered vector is introduced and integrated into the genome of the organism. So, basically, this process involves the introduction of a foreign piece of DNA structure into the genome which contains our gene of interest. This gene which is introduced is the recombinant gene and the technique is called the recombinant DNA technology.
Inserting the desired gene into the genome of the host is not as easy as it sounds. It involves the selection of the desired gene for administration into the host followed by a selection of the perfect vector with which the gene has to be integrated and recombinant DNA formed. Thus the recombinant DNA has to be introduced into the host. And at last, it has to be maintained in the host and carried forward to the offspring.
Tools of Recombinant DNA technology
The enzymes which include the restriction enzymes help to cut, the polymerases- help to synthesize and the ligases- help to bind. The restriction enzymes used in recombinant DNA technology play a major role in determining the location at which the desired gene is inserted into the vector genome. They are two types, namely Endonucleases and Exonucleases.
The Endonucleases cut within the DNA strand whereas the Exonucleases remove the nucleotides from the ends of the strands. The restriction endonucleases are sequence-specific which are usually palindrome sequences and cut the DNA at specific points. They scrutinize the length of DNA and make the cut at the specific site called the restriction site. This gives rise to sticky ends in the sequence. The desired genes and the vectors are cut by the same restriction enzymes to obtain the complementary sticky notes, thus making the work of the ligases easy to bind the desired gene to the vector.
The vectors – help in carrying and integrating the desired gene. These form a very important part of the tools of recombinant DNA technology as they are the ultimate vehicles that carry forward the desired gene into the host organism. Plasmids and bacteriophages are the most common vectors in recombinant DNA technology that are used as they have a very high copy number. The vectors are made up of an origin of replication- This is a sequence of nucleotide from where the replication starts, a selectable marker – constitute genes which show resistance to certain antibiotics like ampicillin; and cloning sites – the sites recognized by the restriction enzymes where desired DNAs are inserted.
Host organism – into which the recombinant DNA is introduced. The host is the ultimate tool of recombinant DNA technology which takes in the vector engineered with the desired DNA with the help of the enzymes.
There are a number of ways in which these recombinant DNAs are inserted into the host, namely – microinjection, biolistics or gene gun, alternate cooling and heating, use of calcium ions, etc.
Recombinant DNA Technology steps
Cutting the gene at the recognition sites – The restriction enzymes play a major role in determining the location at which the desired gene is inserted into the vector genome.
Polymerase chain reaction – It is a process to amplify the gene once the proper gene of interest has been cut using the restriction enzymes.
Insertion of the desired recombinant DNA into the host organism – As mentioned in Tools of recombinant DNA technology, there are various ways in which this can be achieved. The effectively transformed cells/organisms carry forward the recombinant gene to the offspring.
Obtaining the product of the recombinant gene mostly in the form of the manufactured protein.
Application of recombinant DNA technology
- DNA technology is also used to detect the presence of HIV in a person.
- Application of recombinant DNA technology in Agriculture – For example, manufacture of Bt-Cotton to protect the plant against ball worms.
- Application of medicines – Insulin production by DNA recombinant technology is a classic example.
- Gene Therapy – It is used as an attempt to correct the gene defects which give rise to heredity diseases.
- Clinical diagnosis – ELISA is an example where the application of recombinant
A clone is a cluster of individual entities or cells that are descended from one progenitor. Clones are genetically identical as the cell simply replicates producing identical daughter cells every time. Scientists are able to generate multiple copies of a single fragment of DNA, a gene which can be used to create identical copies constituting a DNA clone. It takes place through the insertion of DNA fragments into a tiny DNA molecule. This molecule is made to replicate within the living cell, for instance, a bacterium. The tiny replicating molecule is known as the carrier or the DNA vector
Yeast cells, viruses, Plasmids are the most commonly used vectors. Plasmids are the circular DNA molecules that are introduced from bacteria. They are not part of the main cellular genome. It carries genes which provide the host cell with beneficial properties such as mating ability, drug resistance. They can be conveniently manipulated as they are small enough and they are capable of carrying extra DNA which is weaved into them.
Gene Cloning Applications
Listed below are the applications of gene cloning:
- Gene Cloning plays an important role in the medicinal field. It is used in the production of hormones, vitamins and antibiotics.
- Gene cloning finds its applications in the agricultural field. Nitrogen fixation is carried out by cyanobacteria wherein desired genes can be used to enhance the productivity of crop and improvement of health. This practice reduces the use of fertilizers hence chemical-free produce is generated
- It can be applied to the science of identifying and detecting a clone containing a particular gene which can be manipulated by growing in a controlled environment
- It is used in gene therapy where a faulty gene is replaced by insertion of a healthy gene. Medical ailments such as leukaemia and sickle cell anaemia can be treated with this principle.
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