Gene Cloning

Gene manipulation or gene cloning involves separating a specific gene or DNA segment from a larger chromosome, attaching or to a small molecule of carrier DNA or simply vector and then replicating this modified DNA thousands or millions of times through both an increase in cell number and the creation of multiple copies of cloned DNA in each cell. The result is selective amplification of a particular gene or DNA segment. A clone is an identical copy. These methods and related tasks are collectively referred to as recombinant DNA technology or more informally genetic engineering.
Cloning of DNA genes from any organism entails five general steps:
• Isolation of gene of interests.
• Insertion of foreign DNA into a vector.
• Introduction of the recombinant vector into host cells.
• Selection of transformed host cells.
• Cloning or mass culture of transformed host cells.

1. Isolation of gene of interests: The gene of interest can be isolated from variety of sources. The gene is either prepared from the genome by restriction enzymes or may be prepared from mRNA using reverse transcriptase.
2. Insertion of foreign DNA into a vector: The gene is fragmented by using the specific restriction enzyme to develop specific cohesive ends. The cloning vector is also treated with the same enzyme so that cohesive ends generated may have complementary residues similar to foreign DNA.
The fragments are brought together to join by DNA ligase enzyme under optimal condition. This steps provides the recombinant DNA i.e. DNA from sources.
3. Transfer of recombinant DNA into the host: Recombinant plasmid or DNA are introduced into bacterial cells by a process called transformation. The cells and plasmid DNA are incubated together at 0oC in a CaCl2 solution, then subjected to a shock by rapidly shifting the temperature to 37oC to 43oC. By doing this some of cells take up the plasmid. Electroporation can be used alternative to this method.
4. Selection of transformed cells: The transformed cells containing recombinant plasmids are identified. This is usually done by adding ‘marker gene’ in the cloning vector. These marker genes are often for antibiotic resistance.
5. Propagation or Cloning: When the required cells are selected they are cultured in nutrient medium so as to propagate the DNA into high numbers. As the bacteria divides, the recombinant DNA molecules also divide producing high number of clone genes. The cloned genes and bacteria are then used for industrial processes.