Ligation (molecular biology)

In molecular biology, ligation is the joining of two nucleic acid fragments through the action of an enzyme. It is an essential laboratory procedure in the molecular cloning of DNA whereby DNA fragments are joined together to create recombinant DNA molecules, such as when a foreign DNA fragment is inserted into a plasmid. The ends of DNA fragments are joined together by the formation of phosphodiester bonds between the 3'-hydroxyl of one DNA terminus with the 5'-phosphoryl of another. RNA may also be ligated similarly. A co-factor is generally involved in the reaction, and this is usually ATP or NAD⁺.

Ligation in the laboratory is normally performed using T4 DNA ligase, however, procedures for ligation without the use of standard DNA ligase are also popular.

The mechanism of the ligation reaction was first elucidated in the laboratory of I. Robert Lehman. Two fragments of DNA may be joined together by DNA ligase which catalyzes the formation of a phosphodiester bond between the 3'-OH at one end of a strand of DNA and the 5'-phosphate group of another. In animals and bacteriophage, ATP is used as the energy source for the ligation, while In bacteria, NAD⁺ is used.

The DNA ligase first reacts with ATP or NAD⁺, forming a ligase-AMP intermediate with the AMP linked to the ε-amino group of lysine in the active site of the ligase via a phosphoamide bond. This adenylyl group is then transferred to the phosphate group at the 5' end of a DNA chain, forming a DNA-adenylate complex. Finally, a phosphodiester bond between the two DNA ends is formed via the nucleophilic attack of the 3'-hydroxyl at the end of a DNA strand on the activated 5′-phosphoryl group of another.

A nick in the DNA (i.e. a break in one strand of a double-stranded DNA) can be repaired very efficiently by the ligase. However, a complicating feature of ligation presents itself when ligating two separate DNA ends as the two ends need to come together before the ligation reaction can proceed. In the ligation of DNA with sticky or cohesive ends, the protruding strands of DNA may be annealed together already, therefore it is a relatively efficient process as it is equivalent to repairing two nicks in the DNA. However, in the ligation of blunt-ends, which lack protruding ends for the DNA to anneal together, the process is dependent on random collision for the ends to align together before they can be ligated, and is consequently a much less efficient process. The DNA ligase from E. coli cannot ligate blunt-ended DNA except under conditions of molecular crowding, and it is therefore not normally used for ligation in the laboratory. Instead the DNA ligase from phage T4 is used as it can ligate blunt-ended DNA as well as single-stranded DNA.

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