Title: Ligation Independent Cloning
Course: BTEC-1100-301-302-303-F21 > Section: Module 4 - Applied Molecular Biology

LIC Cloning Principle

The aLICator LIC cloning system uses directional LIC cloning technology to streamLine and facilitate cloning into an expression vector. LIC ensures high cloning efficiencies of more than 95% and eliminates the need for ligation and restriction enzyme digestion steps. The LIC method uses T4 DNA polymerase to create specific 14–21 nucleotide single-stranded overhangs on the pLATE vectors and DNA inserts (2). T4 DNA polymerase has two enzymatic activities: 5'–3' polymerase activity and 3'–5' exonuclease activity. The exonuclease activity removes nucleotides from the 3' ends of the DNA while the polymerase activity restores the chain using dNTPs and the complementary DNA strand as a template. In the LIC protocol, only dGTP is included in the reaction, causing the 3'-5' exonuclease and 5'-3' polymerase activities to equilibrate at the first occurrence of cytosine in the complementary strand (Fig. 1). After annealing, the LIC vector and insert are transformed into competent E. coli cells without the use of T4 DNA ligase. Covalent bond formation at the vector-insert junctions occurs within the cell to yield circular plasmid.

Figure 1. Generation of sticky ends on the gene of interest with T4 DNA polymerase and dGTP.

In module 3 we amplified the bglA gene from B. Halodurans using primers specific to that gene.  Those primers were complementary to the 3’ and 5’ ends of our gene in order to specifically amplify our target.  However those primers also contained the LIC ends as shown above.  Specifically the forward primer had the sequence AGAAGGAGATATAACT added to the 5’ end and the reverse primer had the sequence GGAGATGGGAAGTCATTA added to its 5’ end.  By adding and incorporating those sequences onto the ends of our bglA gene we can then use the LIC reaction to create large overhangs complementary to the pLATE31 vector.

Detailed instructions for the LIC procedure can be found in the user manual here.

LIC cloning

Mix the 5X LIC buffer by vortexing before each use.

1. To generate the necessary 5' and 3' overhangs on the purified PCR template, prepare the following reaction mixture at room temperature:

Component Volume

5X LIC Buffer: 2 μl
Purified PCR product 85ng:  x μl (volume varies dependent on concentration of PCR product)
Water: up to 9 μl (volume varies dependent on concentration of PCR product)
T4 DNA Polymerase, (1U/μl): 1 μl
Total volume 10 μl
Vortex briefly and centrifuge for 3-5 s.

2. Incubate the reaction mixture at room temperature (20-25°C) for 5 min.

Note: Do not exceed 5 min.

3. Stop the reaction by adding 0.6 ul of 0.5M EDTA, mix well.

Note: Store the prepared PCR product in the reaction mixture at -20°C if the annealing step with the LIC vector cannot be performed immediately. Thaw and mix carefully prior to performing the annealing reaction.

4. Set up the annealing reaction:

Add 1 μl pLATE, LIC-ready vector (60 ng, 0.02 pmol DNA) to the T4 DNA polymerase
treated PCR product prepared in steps 1–3 of this protocol.

Vortex briefly and centrifuge for 3-5 s.

5. Incubate the annealing mixture at room temperature (20-25°C) for 5 min.

Note: Annealing is complete within 5 min of incubation. Reactions can be incubated up
to 2 hours without affecting results. Longer incubation times do not improve efficiency.

6. Use the annealed mixture directly for bacterial cell transformation.

You will also want to set up a negative control reaction.  This is simply the annealing reaction without any PCR product added.  This control enables us to determine the approximate number of false positive colonies found on the reaction plate.

Learning Objectives:

- Clone a gene using an ligation independent cloning (LIC) reaction.