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Key Steps of Molecular Cloning

Molecular cloning is a process of isolation of a specific DNA fragment and transfer of this fragment into a plasmid vector. As a part of the plasmid vector, the DNA fragment could be easily amplified, sequenced, stored for long periods of time, and used for gene expression and other functional studies. Two starting ingredients of molecular cloning are a plasmid vector and a DNA fragment that has to be inserted in it. The DNA fragment is usually a gene or other functional region from a living cell, or it could be an artificial sequence with properties useful for a researcher. Plasmid vector is circular piece of DNA that could be easily amplified in E. coli, stored for long periods of time, and easily manipulated in a test tube. A typical plasmid vector contains: an origin of replication that allows it to be replicated inside a bacterial cell; a selection marker, for example a beta lactamase gene coding for ampicillin resistance; and a multiple cloning site , which could be cleaved with several restriction enzymes, such as BamHI (G-GATCC), EcoRI (G-AATTC) or NcoI (C-CATGG). In many vectors, the multiple cloning site is surrounded by sequences of promoter and terminator, that guide expression of inserted genes after the vector is introduced inside a cell. To be used for molecular cloning, both vector and insert DNA are treated with restriction enzymes that cleave double stranded DNA molecules producing overhanging single stranded nucleotide tails. After their ends have been prepared with restriction enzymes, vector and insert are combined together, and another enzyme, called a DNA ligase is added to the mix. At the same time as complimentary base pairing of single stranded overhands brings the ends of vector and insert together, the DNA ligase fuses them into one intact DNA molecule. In order to make multiple copies of this molecule, the ligation mixture is introduced inside the E. coli cells in a process called transformation. During the transformation the cell-DNA mixture is kept on ice and then exposed to 42 oC. Such sudden change in temperature drives the DNA inside some of the E. coli cells. Then the cells are plated on a plate with growth medium supplemented with a selective antibiotic. Only the cells that acquired the plasmid have resistance to the antibiotic and are capable of growth on such a medium. After overnight incubation at 37 oC each transformed cell produces a colony of identical cells, oftentimes called a clone. The selected clones are then individually picked, grown even further in a liquid medium, and the DNA is extracted from them. Thus, in the process of molecular cloning, a DNA fragment that represented a tiny fraction of cell genome is integrated into a bacterial plasmid. As a part of a plasmid, this DNA fragment represents a quarter or more of total DNA in a test tube and it could be effortlessly and endlessly amplified in E. coli.