Bio-Rad Ligation and Transformation Module - Manual (Page 19)

DNA ligation with sticky ends — To prepare a cloning vector for ligation
with insert DNA, it is cut with a restriction enzyme within the MCS, opening
it to receive the inserted DNA. If the insert has sticky ends, that is, overhangs
on the end of the DNA strands, then the vector should be cut with the
same enzyme, producing sticky ends that will be complementary to the
ends of the insert DNA. For example, if the insert DNA has been prepared
by cutting it at both ends with Bgl II, then the vector would also be cut with
Bgl II. Having complementary sticky ends improves the efficiency of ligation,
whereas mismatches in the sequences reduce efficiency.
Because the sticky ends on the vector and the insert are complementary,
when they come into contact during the ligation reaction they will base-pair
with each other using hydrogen bonds. (The base-paired sticky ends of the
insert and vector are not stably associated, and they can dissociate prior
to ligation.) While the insert and vector are associated, T4 DNA ligase
forms a phosphodiester bond, covalently linking the two pieces of DNA.
Actually, there are two ligations. The first ligation is intermolecular, between
one end of the vector and one end of the insert, resulting in a linear DNA
molecule. The second ligation is intramolecular, circularizing the molecule.
Sticky end digestion.
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