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Recombinant DNA technology is the process used for producing new genetic combinations by joining different genetic material (DNA) together and inserting them into host organisms from two different species or sources. These new combinations are of value to medicine, science, industry, and agriculture. The insertion of a gene into the host genome is not an easy task. Firstly, the desired gene needs to be selected for its administration into the host then, a suitable vector is selected to form recombinant DNA by integrating the gene with the vector. In living organisms, recombinant DNA was achieved by Herbert Boyer and Stanley Cohen who insert foreign DNA from plasmids by using E.Coli restriction enzymes.
Tools Used in Process of Recombinant DNA technology
1. Restriction Enzymes:- Its role is to identify the site where the desired gene is introduced into the vector genome
2. Enzyme Ligase: It is used to join two fragments. Sticky ends of the desired gene and the vector is attached with the help of ligase enzyme
3. Vector: It is generally a plastid plasmid used to carry and integrate the desired gene
4. Host: Competent host cell into which the Recombinant DNA is inserted.
Steps involved in DNA Technology
1. Isolation of Genetic Material
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Isolation of desired DNA in its pure form meaning free from other macromolecules.
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In the normal cell, DNA exists along with other macromolecules like proteins, RNA, polysaccharides within the cell membrane.
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It must be separated from other macromolecules and also purified by the help of enzymes which include cellulose, Lysozyme, Chitinase, proteases, ribonuclease.
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Ultimately, DNA is precipitated out as a fine thread by the addition of ethanol and purified DNA is spoiled out which is called spooling.
2. Restriction Enzyme Digestion
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Restriction enzyme digestion is a reaction in which DNA is cut at a specific location by restriction enzymes that act as ‘Molecular Scissors’.
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Purified DNA is then incubated with the selected restriction enzyme at optimal conditions for a certain enzyme.
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Agarose gel electrophoresis is the technique in which Agarose gel is used for running out the DNA. With the help of current, Being negatively charged, DNA travels to the positive electrode and on the basis of the size, they are separated out.
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This allows digested DNA fragments to separate and cut out. Using the same procedure vector DNA can also be used.
3. Amplification using PCR
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PCR or polymerase chain reaction is the process in which multiple copies of DNA sequence can be made in vitro by using the enzyme DNA polymerase.
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Millions of copies of DNA can be produced by amplifying single copy with the help of PCR
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The following components are used to run PCR reactions on Thermal cyclers’.
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Template: DNA to be amplified.
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Primers: They are chemically synthesized and small oligonucleotides that are complementary to a specific DNA region.
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Enzyme: DNA polymerase
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Nucleotides: Needed for the extension of primers by the enzyme.
4. Ligation of DNA Molecules
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The same restriction enzyme is used for cutting the purified DNA and selected vector
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Both the cut DNA fragments and cut vector are given when the process opens.
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Ligation is the method in which these two pieces are joined together using enzyme ligase.
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The resulting DNA molecule is a desired DNA of interest and vector.
5. Insertion of DNA Recombinant Host
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In this step transformation takes place. It is a process in which the Recombinant DNA is inserted into a recipient cell, mainly a bacterial cell which is first made competent, so they can accept new DNA.
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The methods used for making a cell competent are Ca+2 ion treatment, thermal shock, electroporation, etc.
6. Obtaining/Culturing the Foreign Gene Product
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A piece of alien DNA is inserted into a cloning vector and alien DNA gets multiplied by transferring it into a bacterial cell.
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Production of desirable protein expression is the ultimate aim. Recombinant protein is the protein in the encoded gene which is expressed in the heterologous host.
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Large volume or Cell culture is needed to produce a large amount of Recombinant protein that benefits humans. To accomplish, vessels used are known as bioreactors
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Bioreactors are basically large containers and can process about 100-1000 liters of cell culture. A bioreactor offers optimum conditions like pH, temperature to convert raw material biologically into specific enzymes, proteins.
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7. Downstream Processing
This process involves the marketing of protein as a final product after going through a quality control test purification clinical test etc.