Ligation is a process that covalently joins the phosphate backbone of double-stranded DNA. It is carried out by an enzyme called DNA ligase.
The ligation reaction generally consist of two steps:
DNA ends collision. This collision occurs by chance and the occurrence rate is lower in low temperatures. Low temperatures stabilize the hydrogen bonding between the complementary nucleotides. The DNA ligase reaches optimum activity at 25o. A general rule is that the lower the incubation temperature, the longer the incubation time.
Enzymatic reaction. DNA ligase catalyzes the joining between 3'-hydroxyl to 5'-phosphate.
Figure 1: The AMP nucleotide is transferred to the 5'-phosphate. Then the AMP-phosphate bond is attacked by 3'-OH forming a covalent bond while releasing AMP.