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DNA Ligation

T4 DNA ligase is an enzyme encoded by the T4 bacteriophage that "ligates" DNA molecules by covalently joining a 3'-OH to an adjacent 5'-phosphate group. The joined ends may be from a single DNA molecule or from different molecules. Molecules with protruding single strand ends can be ligated together if the ends are compatible (i.e. complementary), so that they can anneal to each other. It is also possible to ligate any two blunt-ended DNA molecules together, although this is considerably less efficient since there is nothing to hold the DNA molecules next to each other. Ligations are used in our experiment to create stable recombinant DNA molecules for use in transformations.

Ligations require planning. Restriction fragments with protruding ends to be ligated must contain compatible complementary sequences. If orientation of an insert is important, two different ends will increase the probability of the correct orientation. The joining of a blunt end to a sticky end can be achieved by converting the sticky end to a blunt end, either by filling in the missing bases of a 5' protruding end using the Klenow fragment or by chewing off a 3' overhang with T4 DNA polymerase (the polymerase has a 3'-5' exonuclease activity used to correct misincorporation of nucleotides).

The components to be ligated are mixed in a ratio determined by the desired product. If recircularization (intramolecular ligation) is the goal, the concentration of fragments is kept low to decrease the probability of two different molecules contacting each other. If a product is to be inserted, such as in a cloning procedure, an excess of insert of 2 to 3 x the vector concentration is used, and the concentration of DNA is higher to increase the occurrences of intermolecular ligation.

 

Copyright, Acknowledgements, and Intended Use
Created by B. Beason (bbeason@rice.edu), Rice University, 18 June 2010
Updated 18 June 2010