Takara Bio USA, Inc., manufactures and distributes products under the Takara™, Clontech®, and Cellartis® brands. These products include kits, reagents, and instruments for life sciences research applications, including NGS, PCR, gene delivery, genome editing, stem cell research, cloning, nucleic acid and protein purification, and automated sample preparation. Our comprehensive cloning portfolio supports both traditional methods and In-Fusion® Cloning, a unique and highly efficient method for seamless cloning. This ligation-free protocol is adaptable to a wide range of applications, including multiple-fragment cloning, site-directed mutagenesis, and automated high-throughput workflows.
Ligation Independent Cloning Primer Design
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Ligation independent cloning (LIC) is an easy and effective method to ensure successful cloning, all without the need for ligation. As easy as the technique is, designing primers can be a bit tricky. In this article, we will present a quick overview on primer design for ligation independent cloning.
The easiest way to start is to look at the treated vector that the insert will be annealed into. In the diagram below, panel 1 shows the treated vector waiting for the insert and panel 2 shows the corresponding insert, which would fit into it. It’s easy to see how those would fit together, but how do you make the insert like that?
Panel 3 shows how the insert is made from the PCR product by T4 DNA polymerase and dTTP. The crucial residues are the ones in red. They are added to the oligos to so that they are the first thymidine residues, going back from the 3′ ends. This allows the T4 DNA polymerase and dTTP mix to generate the long sticky ends.
Just like in the vector treatment, the 3′-5′ exonuclease activity of the polymerase digests the 3′ ends of the insert because no are dNTP’s available. This activity stops once it reaches dTTP, where the polymerase and exonuclease activities cancel each other out, resulting in stable, long sticky ends.
It is crucial to note that the forward primer is designed so that the addition of the extra T does not disrupt the reading frame of the N-terminal fusion leading up to the coding sequence.
Panel 4 shows the primers required to amplify the insert with the ligation independent cloning vectors. Of course, one of the primer sequences – the reverse primer – has to reversed to that it reads 5-3′ before it is sent off for sequencing. A common error is to reverse complement this sequence, which of course is wrong – it only needs to be reversed as it is already the complement.
The good news is that you only have to design these adaptor parts of the primers once. As long as you always use the same ligation independent cloning site, the adaptor parts will always should be the same. The parts of the primer that are specific to the gene to be amplified should be designed as normal, and the adaptors simply added to the ends. Note also that since the start codon is vector-borne, the start codon of the gene itself should be omitted.
If you are still confused (you may well be), the best thing to do is try it yourself – get a pencil and a piece of paper, and sketch out the process to become more comfortable with understanding how it works.
You may also find this tool useful for helping to design LIC primers.
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Thanks for the site its great.
For homework I have been asked to design 3′-end primer for PCR of a gene, to create a LIC-compatible end, assuming a certain RE cut to the vector. I think I’ve designed a 5′ primer successfully for another question but I’m struggling to get my head around designing a 3′ end. Partly because I’m thinking in reverse partly since the primer will surely be 5′ to 3′ which won’t pegged back by the T4 polymerase? Fundamentally I don’t get panels 4 and 5. Ta.
I have been trying to ligate a Sac1 Sal1 adaptor i designed into a vector but have not been successful. The sequence of the adaptor is AGCTGTCGAC. Was i wrong with the adaptor design? any suggestions will be highly appreciated. Thank you very much
Yes, I agree, the primers in panel 4 are wrong. The T’s should be A’s. Sorry for the confusion!!
I see what you’re saying, but I think Kate is right. The primer sequences that you’ve listed in panel 4 aren’t the same sequences in panel 3. Besides, regardless of which nucleotide you use to stall the T4 degradation, the “stop-sign” nucleotide has to be the first one of that flavor in the primer. So if you are stalling with A, then the corresponding T should be the first T (from the 5′ end) in the primer sequence.