Ligation Independent Cloning (LIC)
NKI Protein Facility, Netherlands Cancer Institute (NKI)
LIC cloning allows insertion of DNA fragments without using restriction enzymes into specific vectors containing engineered overhangs. The procedure is based on the generation of complementary single stranded DNA overhangs in vector and insert using the proofreading capability of T4 DNA polymerase. The overhang should be preceded at the 5’site by any of the 4 bases (A, C, G or T) which is not present (in a double-stranded configuration after vector cleavage) within the overhang sequence. Advantages of the method are: No restriction enzymes and ligase are required during cloning (cost effective); and (if multiple LIC vectors are available) efficient, versatile cloning into multiple vectors (e.g. with different tags, suited for different hosts, etc.) is possible. To illustrate the method, a vector from the NKI-LIC vector suite (ref) containing an N-terminal his-tag is used in the following example.