Figure 13.3 : Adaptor molecule and its usage in cloning.

2. Ligase- Joining two DNA to generate the chimeric DNA is the basis of cloning. As discussed in Figure 13.1, it is an essential step to generate clone containing foreign DNA in a vector. When cohesive ends are generated by the action of restriction endonuclease on DNA associated with each other, a nick remains to be sealed and give complete circular DNA. What DNA ligase is doing? It is an enzyme requires ATP or NAD + as a cofactor to catalyze ligation reaction. Ligase is processing ATP to generate AMP, and then AMP is making adduct with enzyme to form ligase-AMP complex. This complex is binding to the 3' and 5' of DNA bearing nick and bringing them together. AMP is released and phosphodieaster linkage is formed between 3' and 5' end to seal the nick (Figure 13.4).