Cloning Techniques
Different techniques are used for cloning genes, cells and organisms.
1. Cell Cloning
Meaning of Cell Clone. Cell clone is a group of genetically identical cells derived from a single parent cell by mitotic divisions.
Procedure for Cell Cloning. Animal tissue is broken into separate cells by mechanical agitation or by immersing in a mixture of (a) proteases, such as trypsin and collaginase, and (b) calcium-binding agents. The proteases breakdown the extra cellular materials that bind the cells together. The calcium-binding agents remove calcium ions required for adhesion between cells.
This procedure gives cell suspension containing many types of cells. A small number of cells are added to a glass dish and covered with a nutrient medium. The latter provides the optimum environment such as pH, osmotic pressure, etc. and the chemical materials which the cells cannot synthesize. The nutrient medium may be natural (serum, chick embryo extract; liver extract) or artificial (solution containing salts, amino acids, vitamins, carbohydrates, etc.). The cells often adhere to the bottom of the dish a little distance away from each other. The isolated cells proliferate and produce distinct colonies or clones of identical cells in about 7-10 days. All the cells in a clone are derived from a single parent cell by mitotic divisions. The clones may be isolated and propagated.
Most microorganisms, both prokaryotic and eukaryotic, which live in nature as single cells, can be easily cloned in vitro, usually on top of agar.
Application of Cell Cloning. Cell cloning enables us to prepare multiple copies of desired recombinant DNA and to store them in gene libraries. A clone of a bacterium containing r-DNA has this DNA segment in each cell as it replicates each time the bacterium divides.
2. Gene Cloning
Meaning of Gene Cloning. Gene cloning refers to the production of a large population of a DNA fragment of interest in pure form.
Technique (Procedure) of Gene Cloning. Gene cloning involves the formation of a recombinant DNA and its introduction into an appropriate host. The recombinant DNA, as stated earlier is the DNA formed by combining DNAs from two different organisms. A DNA fragment of one organism is transferred into another organism (host) by combining it with another DNA. The former DNA is called passenger DNA and the latter is termed vehicle DNA. Plasmid DNA or bacteriophage DNA may be used as the vehicle DNA. The passenger or vehicle DNA is isolated from the cell by lysing it with the enzyme lysozyme. The DNA is separated from the other cell contents by ultracentrifugation and purified. Both passenger and vehicle DNA are cleaved by using the same restriction endonuclease enzyme. They are mixed under suitable conditions. Their complementary sticky ends pair and their ends are sealed with ligase enzyme. This produces a recombinant DNA.
The recombinant DNA is now introduced into a host, which may be Escherichia coli or Bacillus subtilis. The host used should be without plasmids. The walls of host bacteria are made permeable by treatment with calcium chloride or lysozyme. The recombinant DNA is added to the culture in which such host bacteria are growing. The recombinant DNA is taken up by the bacteria along with the nutrients. It replicates when the bacteria divide. The bacterium E. coli divides every 22 minutes under favorable conditions, giving rise to a billion cells in less than 11 hours. This will produce a large clone of recombinant DNA, one copy in each bacterium.
In some cases, the bacterial cells also transcribe the recombinant DNA molecules into mRNA, which is, in turn, translated into polypeptide chains. Hence, cloning can be employed not just to produce large quantities of an individual gene's DNA sequence, but large amounts of the gene's protein product as well.
If a bacteriophage is used as a vector, many identical copies of r-DNA will be formed in a single bacterium.
Applications of Gene Cloning. The process of gene cloning can be used for producing eukaryotic proteins in the bacterial cells, and also for establishing gene library.