Biology Protocols

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Ligation of DNA molecules

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This is the joining of neighbouring 3’-hydroxyl and 5’ phosphate ends of a DNA molecule to produce a phosphodiester bond. The reaction is catalysed by DNA ligases. There are several commonly available DNA ligases, of which two are predominantly used in the laboratory environment:

E. coli DNA ligase
This enzyme requires NAD as a co-factor and can catalyse the joining of either nicked DNA or the complementary sequences produced by restriction endonuclease digestion.
T4 DNA ligase
This enzyme requires ATP, but has a wider range of substrate specificity. As well as the types of ligation carried out by E. coli DNA ligase, T4 DNA ligase can also catalyse blunt-ended ligation and the joining of DNA-RNA hybrids. This ability to perform both blunt-end and stick-end ligation makes T4 DNA ligase particularly important in DNA cloning.The main factors affecting ligation are ATP concentration and DNA concentration. ATP concentration affects the ability of T4 DNA ligase to ligate substrates. Increasing the ATP concentration inhibits blunt-end ligation and DNA-RNA formation. This enables the sequential ligation of protruding and blunt ended fragments in the same reaction simply by altering ATP concentration.
When a fragment of DNA ligates it has a choice between inter and intra molecular ligation3. The preference is dependent upon both the length of the fragment and the concentration of the fragment. For a constant length circularisation increases with decreasing concentration. It is possible to describe a concentration j for a substrate where the rate of formation of the inter-molecular ligation is equal to the rate of formation of the intra-molecular ligation:
j(g/L)=51.1 x Mr-1/2
DNA concentrations lower than j will drive towards circularisation (intra molecular ligation). In the biomolecular reaction represented by the production of a typical recombinant plasmid, the required reaction is favoured by an equimolar or higher ratio of “foreign” DNA to cloning vector. It is, therefore, necessary to calculate the “j-value” for both vector and “foreign” DNA molecules and then to define concentrations at which the relative ratios are equimolar or higher and also above their respective j-values.

Vector = pGEX-2T (Mr=3.3 x 106)
j-value =  = 0.028 g/L = 28ng/µL
Insert /”Foreign DNA” = hsdR (Mr=2.1 x106)
j-value = = 0.036 g/L = 36ng/µL
Therefore concentrations in the ligation should be above 28ng/µL for the vector and above 36ng/µL for the insert in order to avoid intra molecular ligations occurring.
For an equimolar ratio:
The concentration of vector DNA required is therefore 57ng/µL, which is above the calculated j-value, therefore intra molecular ligation will not occur. The subsequent ligation for equimolar ratios would be the addition of 36ng/µL “foreign” DNA to 57ng/µL vector DNA with the addition of a DNA ligase. Generally transformation of E.coli requires a maximum of 500ng of DNA in total so only small volume ligations are generally conducted.



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