Directional cloning

DNA insert and vector molecules are digested with two different restriction enzymes to create noncomplementary sticky ends at either end of each restriction fragment. This allows the insert to be ligated to the vector in a specific orientation and prevents the vector from recircularizing. (See Cloning.)

The technique by which a vector and a DNA insert are both digested with two different restriction endonucleases to create non-complementary sticky ends at either end of both molecules, so favouring the insert to be ligated into the vector in a specific orientation, while also preventing the vector from re-circularizing.