Steps in Gene Cloning or rDNA Technology

    The manipulation of the genetic makeup of living cells by inserting desired gene through a DNA vector is called genetic engineering.

    The DNA formed by joining DNA segments of two different organisms is called recombinant DNA (rDNA) or chimeric DNA.

    The organism whose genetic makeup is manipulated, is called recombinant or Genetically Manipulated Organism(GMO).

    Steps in Recombinant DNA Technology involves several steps
    • The DNA fragment containing the gene sequence to be cloned (insert) is isolated. 
    • Insertion of these DNA fragments into a vector(carrier DNA molecule) 
    • The rDNA molecules are generated when the vector self replicates in the host cell. 
    • Transfer the rDNA molecules into an appropriate host cell. 
    • Selection of the host cells carrying the rDNA molecule using a marker. 
    • Replication of cells carrying rDNA molecules to get genetically identical cells, population or clone.
      1. Gene cloning
        Tools of rDNA Technology
        Enzymes: Restriction Enzymes, Ligase and Alkaline Phosphatase
        restriction endonuclease
        Gene Cloning Vector are of different types depending on the host. These are bacterial vectors, yeast vectors, animal and plant vectors.

        Making Recombinant DNA (rDNA)
        A foreign DNA molecule, referred to as the insert, is cut with the restriction enzyme and is joined to the vector by a ligation reaction. The resulting vector DNA containing the desired DNA is called recombinant DNA (rDNA).

        Recombinant DNA (rDNA) into Host cells
        The chimera is then introduced into its bacterium(host) by various methods i.e.,
        • Heat shock
        • Electroporation
        • Viruses
        • Microinjection
        • Gene Gun
        • Liposome
          • Vectors carrying the genes must be incorporated into the living cells so that they can be replicated or expressed. The cells receiving the vector is called the host cell and once the vector is successfully incorporated into the host (bacterium) cell is said to be transformed.

            Identification of Recombinants
            Vectors,(such as pBR322) carries a selectable marker gene for resistance to the antibiotic ampicillin. If the plasmid vector is introduced into a plasmid free antibiotic sensitive bacterial cell, the cell becomes resistant to ampicillin. Non transformed cells contain no vector DNA, therefore they will not be antibiotic resistant and their growth will be inhabited on agar containing ampicillin

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