The DNA products resulting from restriction digestion forming sticky ends may be joined to any other DNA fragments treated with the same restriction enzyme. Thus, when the two sets of fragments are mixed, base-pairing between sticky ends will result in the annealing together of fragments that were derived from different starting DNA. There will, of course, also be pairing of fragments derived from the same starting DNA molecules, termed reannealing. All these pairings are transient, owing to the weakness of hydrogen bonding between the few bases in the sticky ends, but they can be stabilised by use of an enzyme called DNA ligase in a process termed ligation. This enzyme, usually isolated from bacteriophage T4 and termed T4 DNA ligase, forms a covalent bond between the 5′-phosphate at the end of one strand and the 3′-hydroxyl of the adjacent strand (Figure 1). The reaction, which is ATP dependent, is often carried out at 10 °C to lower the kinetic energy of the molecules, and so reduce the chances of base-paired sticky ends parting before they have been stabilised by ligation. However, long reaction times are needed to compensate for the low activity of DNA ligase in the cold. It is also possible to join blunt ends of DNA molecules, although the efficiency of this reaction is much lower than sticky-ended ligations.
Fig1. Ligation of molecules with cohesive ends. Complementary cohesive ends base-pair, forming a temporary link between two DNA fragments. This association of fragments is stabilised by the formation of 3′→5′ phosphodiester linkages between cohesive ends, a reaction catalysed by DNA ligase.
Since ligation reconstructs the site of cleavage, recombinant molecules produced by ligation of sticky ends can be cleaved again at the ‘joins’, using the same restriction enzyme that was used to generate the fragments initially. In order to propagate digested DNA from an organism it is necessary to join or ligate that DNA with a specialised DNA carrier molecule termed a vector . DNA fragments are thus inserted by ligation into the vector DNA molecule (Figure 2), which allows the whole recombined DNA to then be replicated indefinitely within microbial cells. In this way, a DNA fragment can be cloned to provide sufficient material for further detailed analysis, or for further manipulation. This method can be used to generate a collection of clones (gene library) if the DNA extracted from an organism is digested with a restriction enzyme and all fragments ligated into vector DNA.
Fig2. Outline of gene cloning.
