DNA cloning using a plasmid vector: Molecular cloning using a plasmid vector involves five major steps as already shown in Fig. 1 and Fig. 2.

1.  Construction of a recombinant DNA molecule.

In the above figure, vector DNA (the plasmid pUC18) and the foreign DNA insert are cleaved with Eco RI and mixed together in a ligation reaction containing DNA ligase. pUC18 carries the ampicillin resistance gene and has a large number of restriction sites comprising a multiple cloning site within a selectable marker gene.

2.  Transfer of ligation reaction products to host bacteria.

Competent E. coli are transformed with ligation reaction products

3.  Multiplication of plasmid DNA molecules .

Within each transformed host bacterium, there is autonomous multiplication of plasmid DNA. Each bacterium may contain as many as 500 copies of pUC18. Some bacteria in the mixture will be untransformed (not carrying either recombinant or non-recombinant plasmid DNA).

4.  Division of host cells and selection of recombinant clones by blue-white screening.

Bacterial cells are plated on a selective agar medium containing the antibiotic ampicillin and X-gal. If foreign DNA is inserted into the multiple cloning site, then the lac Z' coding region is disrupted and the N-terminal portion of β-galactosidase is not produced. Since there is no functional β-galactosidase in the bacteria, the substrate X-gal remains colorless, and the bacterial colony containing recombinant plasmid DNA appears white, thus allowing the direct identification of colonies carrying cloned DNA inserts. If there is no insertion of foreign DNA in the multiple cloning site, then the lac Z' gene is intact and enzymatically active β-galactosidase is produced and X-gal is degraded. The bacterial colonies containing non-recombinant plasmid DNA thus appear blue.

5.  Amplification and purification of recombinant plasmid DNA.

A recombinant colony is used to inoculate liquid growth medium. After growing the bacteria overnight, the culture is harvested, bacterial cells are lysed, and the plasmid DNA is purified from other cellular components.

Few applications of gene cloning

•  Production of recombinant proteins: Gene of a given sequence may be expressed in bacteria. Desired affinity tag may be added with protein for simpler purification (please recall our discussion during affinity chromatography).

•  Agricultural utility : Making transgenic crop (expression foreign gene) to boost food production.

•  Transgenic organisms: Cloned genes may be inserted into organisms, generating transgenic species, producing pharmaceuticals and other commercially useful compounds.

•  Gene Therapy: Gene therapy involves supplying a functional gene to cells lacking that function for correcting a genetic disorder or acquired disease.