3.3 Joining DNA: DNA Ligase
The second key enzyme for cloning is DNA ligase. This enzyme will rejoin DNA strands that are close together and that have a free -3'-OH facing an adjacent 5'-phosphate. This will come about naturally if the sticky ends of DNA molecules that have been cleaved by the same nuclease are allowed to anneal with one another. The most commonly used DNA ligase is one coded for by bacteriophage T4. It may be purified from E. coli cells that are infected by T4.
DNA ligase is activated by ATP which adds an adenylate group to the enzyme. The adenylate group is transferred to the 5' terminal phosphate of the DNA:
The 3' OH now can perform a nucleophilic attack on the phosphate linkages and form the phosphodiester bond to seal the stands:
The enzymatic reaction is run at 5-15o (which is below its temperature optimum of 37o) so that the hydrogen bonds which hold the sticky ends together will not dissociate. Sometimes the ligase will simply reseal the original plasmid back together, and often it may seal together the two ends of the DNA gene that we wish to insert instead of inserting it into the plasmid. We will see how to deal with these problems when we screen for transfomants. Since there is a binding site for both strands of the DNA on the ligase enzyme, it is possible to bind and ligate blunt-ended DNA molecules, but with much lower efficiency.