Cloning and Mapping

DNA Ligation

The process of joining the ends of two strands of DNA to form one, single, de novo strand is known as ligation. In the field of recombinant DNA, with the synthesis and joining of multiple fragments to form novel molecules, an understanding of the biology of DNA ligation is essential. DNA ligase forms a phosphodiester bond between the 5’ phosphate group and the 3’ hydroxyl group of juxtaposed DNA molecules. This covalent bond is formed in three stepwise reactions: 1) DNA ligase is activated by AMP, releasing pyrophosphate 2) The AMP is transferred to the 5’ phosphate, forming a pyrophosphate bond 3) Once in close proximity (promoted by the pyrophosphate bond), a phosphodiester bond is formed between the 3’ hydroxyl and 5’ phosphate.

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Protocols for DNA Ligation