What is LIC ?

Ligation Independent Cloning of PCR products (LIC-PCR).

Ref : Aslanidis C, de Jong PJ. Nucleic Acids Res. 1990, 18:6069-6074.

Pubmed
LIC is a cloning method that does not require restriction enzymes.
LIC can also be used to introduce tags or protease cleavage sites into your vectors, and to choose the site of insertion.
Small cohesive fragments are introduced both into the insert and into the vector to generate complementary ends
that will ligate without ligase.
These sequences are introduced by PCR into the insert and into the vector. 5'overhangs are then generated using
the exonuclease activity of T4 DNA polymerase that is blocked with a given dNTP, absent from the LIC sequence.
At the 3' end, since the extra sequence is after the stop codon, its nature has little importance(at least for simple LICs)


LIC-schema.png
This requires careful design of the primers, to make the cohesive ends fit well

A tutorial to render LIC simple

This web-based software offers at once many LIC possibilities and ends with the design or primers to order.
Codon usage was considered in the LIC proposals (rare codons were avoided)
What you have to provide is your insert sequence in the correct reading frame (the 30 first and 30 last nucleotides will be used.
The immediately flanking sequences of your vector must also be provided which means that you can choose the cloning site.

Nucleotides at the LIC junctions

In order to stop the T4 exonuclease activity, you need a given nucleotide at the end of the LIC sequence.
On the other hand these nucleotides must be completely avoided at the extremities of the primers where they would stop digestion.
The LIC generator automatically deals with the junction problem.

LIC protocols

You will find here what has worked for us.
Good luck with your primer design

Bug report:marie-claire dot dagher at ujf-grenoble dot fr

Don't forget to acknowledge this site in your publications :
http://noxtoolbox.ujf-grenoble.fr/LICgenerator/

Back to LIC generator
Author: Marie-Claire Dagher, Institute of Structural Biology, December 2007