Cloning

It is a molecular biology technique wherein a selected sequence of DNA is cloned into multiple copies in a host organism (usually bacteria). A vector is required to introduce the DNA sequence of interest into the host. This vector in most cases is a plasmid, especially containing an antibiotic resistance gene. Plasmids are treated with restriction enzymes which cleave the plasmid DNA at a specific region called as the restriction sites. This cleavage usually leads to the production of single-stranded sticky ends or overhangs which can anneal with the foreign DNA. This annealing process is referred to as ligation/hybridization because it occurs with the action of DNA ligase. The plasmid is now known as a recombinant plasmid. The bacterial cells (such asE. coli) which have been treated with calcium chloride to make them more permeable to DNA molecules are added to the recombinant plasmid mixture. By the process known as transformation, bacterial cells take up the recombinant plasmid. These bacterial cells are then grown on the nutrient agar containing a specific antibiotic. After incubation, this will result in the growth of only those cells which have taken the antibiotic resistance gene in plasmid, suggesting that these cells have taken up the recombinant DNA. Theplasmid replicates by itself, independent of the host replication. However when host cells divide each of the daughter cells receives copies of plasmids. This results in the production of large copies of the recombinant plasmid.

Separate components or Ready-to-use Kits are available toassist plasmid preparation, ligation, transformation, and plasmid purification.