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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Venus: You can switch the bases as you wish – there’s no intrinsic need to use A/T.
Kate: Sorry — I didn’t explain that very well. The insert treatment uses dTTP and the vector treatment uses dATP.
am I missing something??? I have been struggling with this for almost an hour. Why do the primers have a “T” right 5′ to the coding seq? Shouldn’t it be an “A” so that there will be a “T” in the complementary strand, which is the one being treated/digested with exonuclease because it’s the 3′ end.
Is this figure wrong or am I missing something?
I wanna use dGTP instead of dTTP to vector.
So, I have to change adapter seq..
Is this OK?
Of course, I will use dCTP in PCR products.
Actually, I wonder if dTTP must be used to vector in the LIC method ..
Dear Nick, can you please put up a real world example with a pET21 vector, the RE for cutting the vector, gene sequence to be amplified, the amplified gene with its start and stop codons and the pair of primers, then it will help me learn and do LIC now, thanks Samuel INDIA
Hey, what happens if you don’t have the right primers? Is it possible to ligate the overlap primers to the cute little protruding As? I remember that there are protocols that ligate adapters to PCR products, I just can’t recall the keywords and can’t find anything on the internet…
With this one would need only one additional primer set per plasmid.