Cloning is the process of generating a genetically identical copy of a cell or an organism. In early 1970s naturally occurring plasmids were altered genetically in order to facilitate the insertion of fragments of DNA in cloning procedure.
The first plasmids to be used as vectors for introducing foreign DNA into cells were developed from E coli.
Basic procedure in gene cloning experiment consists of introducing foreign gene into bacterial cells. Then, isolating individual cells and growing colonies from each of them. Plasmid serves as a vector, or carrier molecule, for the gene of interest.
Plasmids constructed for the process of molecular cloning are relatively small, has origin of replication (ori), carry genes specifying resistance to one or more antibiotics, and contain number of conveniently located restriction endonuclease sites into which the DNA to be cloned may be inserted.
Steps involved in cloning with plasmid as a vector (Figure 3,4):
- Fragmentation: The DNA of interest is fragmented to provide a relevant DNA segment of suitable size in the first step. Preparation of DNA fragments for cloning is frequently achieved by PCR, restriction digestion, or fractionation by gel electrophoresis.
- Ligation: Subsequently, a ligation procedure is done whereby the amplified fragment is inserted into a vector. The vector (which is generally circular) is linearised by means of restriction enzymes, and incubated with the fragment of interest under appropriate conditions with an enzyme called DNA ligase. As the sticky ends on the vector and on the gene base pair momentarily, ligase seals the nicks, attaching two DNAs through covalent bonds.
- Transfection: Following ligation, a vector with DNA (insert) of interest is transfected into cells. Commonly, electroporation is employed for transfection but other alternative techniques are available, such as chemical sensitization of cells.
- Screening and Selection: Finally, the transfected cells are cultured. Identification of the cell colonies that are successfully transfected with the vector construct containing desired insertion sequence is done. As cloning vectors contain selectable antibiotic resistance markers, they allow only those cells to grow in which the vector has been transfected. Additionally, the cloning vectors may contain color selection markers, which provide blue/white screening on X-gal medium. PCR, restriction fragment analysis and DNA sequencing is done to further confirm successful cloning.
