Biotechnology and Bioinformatics (1+1)

2.1.4.1. Cloning foreign DNA into the circular DNA of a plasmid

• The cell’s total genome is digested using a restriction endonuclease that produces sticky end s to get the `foreign DNA’.
• The circular plasmid is also cut open with the same endonuclease.
• The thousands of DNA fragments of the total genome and of open plasmids are annealed and ligated to get recombinant plasmids in which at least one or two carry the desired gene .
• The highly heterogeneous mixture of recombinant and parental plasmids so produced is introduced into the competent cells of E. coli by the process called transformation .
• The cells are made competent by treating with high concentration of ca⁺⁺ which increases the permeability of the cell wall.
• The cells were then placed on a selective medium containing an antibiotic to which the plasmid carries the resistance gene.
• The transformed cells grow to give colonies of cells on the plate, each colony being formed by a single transformed cell.