Ligation: Theory and Practice

Abstract

Ligation is the process by which NAD DNA ligases catalyse the formation of phosphodiester bonds between juxtaposed 3′‐hydroxyl group and 5′‐phosphate termini in duplex deoxyribonucleic acid (DNA). Ligases use adenosine triphosphate or nicotinamide–adenine dinucleotide (NAD) as cofactors for this covalent joining of DNA.

Keywords: DNA ligases; recombinant DNA; ligase chain reaction; mammalian ligases

Figure 1.

T4 and E. coliDNA ligase activity at single‐stranded breaks or nicks (a) and at double‐stranded breaks with cohesive or sticky ends. (b) T4 DNA ligase activity at blunt ends. (c) ATP, adenosine triphosphate; NAD, nicotinamide–adenine dinucleotide. They catalyse the formation of phosphodiester bonds between juxtaposed 5′ phosphate groups and 3′ hydroxyl termini in duplex DNA. While T4 DNA ligase uses ATP as a cofactor, E.coli ligase uses NAD.

Figure 2.

Reaction mechanisms for T4 DNA ligases. (a) T4 DNA ligase (L) reacts with ATP to form a phosphoramide‐linked AMP with the amino group of the active lysine site. Pyrophosphate (PPi) is released. (b) The 5′‐phosphate at the nick attacks the activated phosphoryl group of the AMP to form an adenylated DNA. (c) The enzyme catalyses joining of the 3′‐OH of the DNA at the nick to the activated 5′‐phosphate to form the phosphodiester bond and concomitant release AMP.

Figure 3.

Ligation of a restriction fragment into a plasmid or vector DNA. (a) Cloning of a restriction fragment into a vector with cohesive ends. The insert can be cloned into the vector in two possible orientations with respect to the vector sequences. Both recombinant molecules are shown and they can be distinguished by restriction mapping. (b) Directional cloning of a restriction fragment into a vector with heterologous ends. The restriction fragment is digested with two different restriction enzymes to generate heterologous ends. The plasmid or vector DNA is also cleaved with the same enzymes to generate heterologous ends. This results in the cloning of the insert DNA in only one particular orientation with respect to the vector sequences. The other orientation results in noncompatible ends for ligation, and hence this recombinant product is eliminated from the ligation reaction mixture. ATP, adenosine triphosphate.

Figure 4.

Diagram depicting DNA amplification and detection by means of the LCR. The target DNA is heat denatured and four complementary oligonucleotides are then hybridized to the target at 65°C. A thermostable ligase is used to link covalently adjacent oligonucleotides that are perfectly matched to the target. Products from one cycle of ligation become targets for the next cycle, and thus the number of products increases exponentially. Oligonucleotides that contain a single base mismatch at the junction do not ligate efficiently, and therefore do not amplify the ligated product. The single base mismatch at the junction is shown in red.

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Further Reading

Barany F (1991) Genetics disease detection and DNA amplification using cloned thermostable ligase. Proceedings of the National Academy of Sciences of the USA 88: 189–193.

Ciarrocchi G, MacPhee DG, Deady LW and Tilley L (1999) Specific inhibition of eubacterial DNA ligase by arylamino compounds. Antimicrobial Agents and Chemotherapy 43: 2766–2772.

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Frank KM, Sekiguchi JM, Seidi KJ et al. (1998) Late embryonic lethality and impaired V(D)J recombination in mice lacking DNA ligase IV. Nature 396: 173–177.

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Gumport RI and Lehman IR (1971) Structure of the DNA‐ligase adenylate intermediate: lysine‐linked adenosine monophosphoramidite. Proceedings of the National Academy of Sciences of the USA 68: 2559–2563.

Kubota Y, Nash RA, Klungland A et al. (1996) Reconstitution of DNA base extension‐repair with purified human proteins: interaction between DNA polymerase β and the XRCC1 protein. EMBO Journal 15: 6662–6670.

Struhl K and Tabor S (2000) Enzymatic manipulation of DNA and RNA: DNA ligases. In: Ausubel FM, Brent R, Kingston RE, et al. (eds) Current Protocols in Molecular Biology, vol. I, chap. 3, pp. 3.14–3.14.3. New York: John Wiley.

Timson DJ and Wigley DB (1999) Functional domains of an NAD+‐dependent DNA ligase. Journal of Molecular Biology 285: 73–83.

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Tomkinson AE and Levin DS (1997) Mammalian DNA ligases. BioEssays 19: 893–901.

Ukai H, Ukai‐Tadenuma M, Ogiu T and Tsuji H (2002) A new technique to prevent self‐ligation of DNA. Journal of Biotechnology 97: 233–242.

Qi X, Bakht S, Devos KM, Gale MD and Osbourn A (2001) L‐RCA (liagation‐rolling circle amplification): a general method for genotyping of single nucleotide polymorphisms (SNPs). Nucleic Acids Research 29: E116.

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Kandavelou, Karthikeyan, Mani, Mala, Reddy, Sekhar PM, and Chandrasegaran, Srinivasan(Sep 2005) Ligation: Theory and Practice. In: eLS. John Wiley & Sons Ltd, Chichester. http://www.els.net [doi: 10.1038/npg.els.0003838]