Ligation Independent Cloning (LIC) is a technique developed in the early 1990s as an alternative to restriction enzyme/ligase cloning. Inserts are usually PCR amplified and vectors are made linear either by restriction enzyme digestion or by PCR. This creative technique uses the 3’ → 5’ exo activity of T4 DNA Polymerase to create overhangs with complementarity between the vector and insert. Incorporation of dGTP in the reaction limits the exonuclease processing to the first complementary C residue, and not present in the designed overlap, where the polymerization and exonuclease activities of T4 DNA Polymerase become “balanced”. Joined fragments have 4 nicks that are repaired by E.coli during transformation. This technique allows efficient creation of scarless recombinant plasmids at many, but not all, positions in a vector.
More recently, the technique has evolved to include many useful variations. One in particular, sequence and ligation independent cloning (SLIC), has been adopted by many researchers. In this variation, all dNTPs are initially excluded from the reaction. This allows the exonuclease activity of T4 DNA Polymerase to proceed and generate the complementary overlaps between insert and vector. After the overlap is generated, dCTP is added back to the reaction, shifting the enzyme back into a polymerase, where it stalls due to the lack of a complete set of dNTPs in the buffer. The complementary overlap is retained. The product contains 4 nicks, like the original LIC product, and is repaired by E.coli during transformation. This modification of the protocol allows a scarless and sequence-independent insertion into most any vector to be made.
Other variations on the basic LIC design principles have been published and put into practice in various labs. One of these interesting approaches is the use of nicking DNA endonucleases (NiDE) to create the complementary overlaps used to anneal the vector and insert. This method allows rapid creation of compatible ends and avoids the clean-up steps prior to vector and insert joining common to many successful cloning methods.
- Low cost
- Many different vector choices
- Some types of sequence modifications not possible