DNA ligase is an enzyme which can connect two strands of DNA together by forming a bond between the phosphate group of one strand and the deoxyribose group on another. It is used in cells to join together the Okazaki fragments which are formed on the lagging strand during DNA replication.

In molecular biology, DNA ligase can be used to insert genes of interest into plasmid vectors, or to create fusion genes by joining one gene onto another. This process is called ligation (literally “tying a knot”).

Ligation can be performed on lengths of DNA which have “blunt” or “sticky” ends following restriction digests.

In “blunt end” ligation, the DNA fragments are joined directly together by the DNA ligase. There is less control over the orientation of the resultant insertion, however this can be improved by creating single base overhangs (eg. “A-Tailing” with Taq Polymerase and dATP – see figure below).

A-Tailing

In “sticky end” ligation, overlapping regions of complementary single stranded DNA hydrogen bond to each other, and the DNA ligase enzyme connects the sugar phosphate backbones together. Through careful selection of restriction enzymes to create the sticky ends, a great deal of control can be exercised over the site of ligation (see figure below).

Sticky end ligation