Ligation (molecular biology)

Ligation is the joining of two nucleotides, or two nucleic acid fragments, into a single polymeric chain through the action of an enzyme known as a ligase. The reaction involves the formation of a phosphodiester bond between the 3'-hydroxyl terminus of one nucleotide and the 5'-phosphoryl terminus of another nucleotide, which results in the two nucleotides being linked consecutively on a single strand. Ligation works in fundamentally the same way for both DNA and RNA. A cofactor is generally involved in the reaction, usually ATP or NAD⁺. Eukaryotic ligases belong to the ATP type, while the NAD+ type are found in bacteria (e.g. E. coli).^[1]

Ligation occurs naturally as part of numerous cellular processes, including DNA replication, transcription, splicing, and recombination, and is also an essential laboratory procedure in molecular cloning, whereby DNA fragments are joined to create recombinant DNA molecules (such as when a foreign DNA fragment is inserted into a plasmid). The discovery of DNA ligase dates back to 1967 and was an important event in the field of molecular biology.^[1] Ligation in the laboratory is normally performed using T4 DNA ligase. It is broadly used in vitro due to its capability of joining sticky-ended fragments as well as blunt-ended fragments.^[2] However, procedures for ligation without the use of standard DNA ligase are also popular. Human DNA ligase abnormalities have been linked to pathological disorders characterized by immunodeficiency, radiation sensitivity, and developmental problems.^[3]