Table 2.

Comparison of two-fragment assembly by using PCR overlap extension or ligase

Assembly by PCR overlapping	Assembly by ligase
Two fragments must have several amino acids overlapping in their terminus.	Two fragments must have one same restriction enzyme site in their terminus.
Two fragments are amplified by PCR. The purified two fragments are assembled by a second PCR step and then subcloned into the vector.	Two fragments are amplified by PCR. The purified two fragments and vector digested by restriction enzymes are assembled sequentially by ligase.
The procedure is complicated, but efficiency is higher.	The procedure is simple, but efficiency is lower.