Ligation, Insertion into Host, and Selection of Recombinants

Having cut the vector and the gene of interest with the same (or compatible) restriction enzyme, and having amplified the gene fragment if needed, the next steps are to permanently join the two pieces of DNA, get the resulting recombinant molecule into a living host cell, and then identify which host cells actually received it.

Joining, or ligation, is carried out by an enzyme called DNA ligase. When a vector and an insert with matching sticky ends are mixed together, the complementary single-stranded overhangs can temporarily base-pair with each other, holding the two pieces of DNA loosely aligned. However, this base-pairing alone leaves a break, or nick, in the sugar-phosphate backbone of each strand at the junction. DNA ligase seals these nicks by catalysing the formation of a covalent phosphodiester bond between the adjacent 3'-hydroxyl and 5'-phosphate ends of the DNA backbone, permanently joining the vector and the insert into one continuous, closed recombinant DNA molecule. Only after this ligation step is complete does the molecule become a true, stable recombinant plasmid, ready to be introduced into a host cell.

Insertion into the host cell is achieved, as described earlier, most commonly by making the host bacterium competent (through CaCl2 treatment and heat shock) and allowing it to take up the recombinant plasmid from the surrounding solution -- a process broadly referred to as transformation. Depending on the type of host cell and vector being used, other methods of introducing DNA are also used in practice, such as micro-injection (physically injecting DNA directly into the nucleus of an animal cell using a fine needle), the gene gun or biolistic method (coating microscopic gold or tungsten particles with DNA and firing them at high velocity into plant cells or tissues), or using a modified, disarmed version of the bacterium Agrobacterium tumefaciens as a natural 'vector' to deliver DNA into plant cells, since this soil bacterium naturally transfers a piece of its own DNA into plant cells as part of its normal infection process. …