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  • 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.

Molecular cloning is a process of isolation of a specific DNA fragment and transfer of

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