Principles of Biotechnology
* Biotechnology is a field of applied biology which utilises organisms or their enzymes to produce useful products for human beings.
* Modern biotechnology arose from two core techniques, namely, genetic engineering and chemical engineering processes.
* Genetic engineering techniques help isolate and select only desirable genes and avoid the transfer of undesirable genes in target organisms.
* Some genetic engineering techniques are gene cloning, recombinant DNA and gene transfer.
* Gene cloning refers to the production of a lineage of cells which contains one type of DNA fragment of interest derived from a population of many types of DNA fragments.
* Recombinant DNA is artificial DNA created from two or more sources and incorporated into a single recombinant molecule.
* Gene transfer involves the insertion of unrelated genetic information in the form of DNA into cells.
Restriction Enzymes
* Restriction enzymes are used to cut DNA to produce restriction fragments.
* They are assumed to provide a defence mechanism against several deadly viruses such as bacteriophage.
* Restriction enzymes belong to a higher class of enzymes called nucleases, which are categorised as exonucleases and chain.
* Endonucleases make cuts at specific points within the DNA and are thus known as ‘molecular scissors’.
* Hind II was the first restriction endonuclease whose functioning depended on a specific DNA nucleotide sequence.
* Restriction endonuclease recognises a specific recognition sequence or palindrome, so it binds to the DNA and cuts the double-stranded DNA helix at specific points.
Cloning Vectors
* A cloning vector is a DNA molecule which has an origin of replication and is capable of replicating a bacterial cell.
* The first artificial plasmid, which is now commonly used as a cloning vector, is Pbr322.
* The origin of replication or ori is a sequence of DNA at which replication gets initiated in a plasmid.
* Selectable markers help identify and eliminate non-transformants and exclusively permit the growth of transformants.
* A cloning site is an artificially constructed region within a vector molecule containing a number of closely spaced recognition sequences.
* Selectable markers have been developed to differentiate between recombinants and non-recombinants by producing colour in the presence of a chromogenic substance.
* Scientists have transformed pathogens such as Agrobacterium tumifaciens into useful vectors.
* Foreign DNA fragment can also be introduced into host cells using methods such as micro-injection and biolistics or a gene gun.
Process of Recombinant DNA Technology- I
* In recombinant DNA technology, the first step is to select the piece of DNA of interest to be inserted into a vector.
* DNA can be isolated by treating bacterial cells, plant cells and fungus with enzymes such as lysozyme, cellulase and chitinase.
* The second step in recombinant DNA technology is fragmentation of DNA by restriction endonucleases.
* Fragmented DNA can be separated according to its molecular weight by using a technique known as gel electrophoresis.
* The third step in recombinant DNA technology is amplification of the gene of interest.
* The extracted DNA sample can be amplified by using technique known as Polymerase Chain Reaction or PCR.
Process of Recombinant DNA Technology- II
* Before inserting recombinant DNA into a host cell, the cell has to be made competent to receive the DNA.
* Selectable markers such as ampicillin identifies and eliminates nontransformants and permits the growth of tansformants.
* If any protein-encoding gene is expressed in many hosts such as plant cells, bacterial cells and fungal cells, it is called a recombinant protein.
* Cultures such as an ampicillin medium may be used to extract the desired protein.
* Biomass can be produced in larger quantities with the help of a bioreactor, which is a vessel used to carry out a chemical process.
* Downstream processing refers to the recovery and purification of biosynthetic products before they are brought to the market as finished products.