Biotechnology has been referred to as a branch of science that delivers products and services to the human society. Therefore, it is considered as a trade.
Genetic engineer (gene manipulation / gene cloning/recombinant DNA technology) is the mainstay of the trade. In this chapter, we shall focus our attention on what is genetic engineering and how it practiced.
The primary objective of this practice is to have as many identical copies of a gene. The copies of the same gene together constitute a clone of genes. A common man thinks cloning as something applied only to Dolly and of late, man. The word has been misunderstood by man in this chapter we make a small Endeavour to explain what is cloning and in what way it is apply to genes.
The process of formation of many identical copies of anything, literally, means cloning and the identical copies together constitute a clone. When the process is applied to cells, it called cell cloning, to human as human cloning and to genes as gene cloning and so on.
The objective is to make as many identical copies of a beneficial gene possible and to make the genes express and form polypeptide products.
Many copies of a gene therefore, will increase number of the same polypeptide product. This feature makes the process viable. There are bases of gene cloning: I. host cell based cloning and II. Polymerase Chain Reaction (I based cloning
I. Host Cell Based Cloning: In this process, a host, a prokaryotic or a eukaryotic cell used as a suitable environment for cloning a gene. Two important tools: (1) enzymes (2) nucleic acids execute this process. The process consists of the following eleven step
1. Isolation of the donor DNA.
2. Cutting of the donor DNA into fragments by restriction end nucleases.
3. Separation of the DNA fragments by agarose gel electrophoresis.
4. Transfer of the separated fragments onto a nitrocellulose filter or nylon membrane blotting and identification of the donor fragment (insert) by molecular hybridization autoradiography.
5. Isolation and cleavage of the vector (carrier) DNA (plasmid).
6. Joining (ligation) of the donor DNA fragment (insert) to a vector DNA resulting! Recombinant/chimerical DNA
7. ' Delivery of the recombinant DNA into a host cell for amplification (transformation).
8. Plating and culture of the transformed host cells.
9. Selection of the correct host cell containing the recombinant DNA.
10. Expression of the desired DNA fragment for a polypeptide product.
11. Screening of the expressed products.
The first step is to break open the cells of a tissue. The disruption of the limiting membrane of the cells should be as gentle as possible so as to prevent a mechanical breaking of the DNA
Several methods are available to accomplish this.
1. If the cell is a plant cell, an enzyme, cellulase digests the cell wall.
2. Following this, the plasma membrane is dissolved in a detergent. The detergent used the purpose is sodium codicil sulfate (SDS).
3. Animal cells are treated with SDS and then spun in a blender gently.
4. The resultant suspension is treated with a proteolysis enzyme (proteinase I) to digs the chromosomal proteins.
5. Then, saturated phenol is added and the resultant is centrifuged. The supernatant! Separated and to it is added an equal volume of phenol and chloroform and isoamy alcohol mixture (24: 1) and then centrifuged again.
6. The clear supernatant is separated and twice as much of ice cold ethanol is added to precipitate the DNA.
In a DNA preparation, the RNA is digested by using rib nuclease (Raze). The isolation of mRNA is discussed in a later section.