Ligation-independent cloning (LIC) is a form of
molecular cloning
Molecular cloning is a set of experimental methods in molecular biology that are used to assemble recombinant DNA molecules and to direct their replication within host organisms. The use of the word ''cloning'' refers to the fact that the metho ...
that is able to be performed without the use of
restriction endonucleases
A restriction enzyme, restriction endonuclease, REase, ENase or'' restrictase '' is an enzyme that cleaves DNA into fragments at or near specific recognition sites within molecules known as restriction sites. Restriction enzymes are one class o ...
or
DNA ligase
DNA ligase is a specific type of enzyme, a ligase, () that facilitates the joining of DNA strands together by catalyzing the formation of a phosphodiester bond. It plays a role in repairing single-strand breaks in duplex DNA in living organ ...
. The technique was developed in the early 1990s as an alternative to restriction enzyme/ligase cloning. This allows genes that have restriction sites to be cloned without worry of chopping up the inserted gene of interest.
Protocol
#Design PCR Primers with LIC extension
#Perform PCR to amplify gene
#Purify PCR product
#Create 5' overhangs
#Incubate vector and PCR product to anneal
#Transform
References
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Further reading
LIC Primer Design
Cloning
Molecular biology