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 DNA replication, replication within Host (biology), host organisms. The use of the word ''cloning'' re ...
that can 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 type of enzyme 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 organisms, but some forms (such ...
. The technique was developed in the early 1990s as an alternative to restriction enzyme/ligase cloning. This allows genes to be cloned without the requirement of a restriction site for cloning that is absent from the gene insert. LIC uses long complementary overhangs on the vector and the DNA insert to create a stable association between them.
Steps in Procedure
#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
{{reflist
Further reading
LIC Primer Design
Cloning