Since I wrote about the wonders of ligation independent cloning a while back, several people have contacted me to say they were confused about how to design primers for this application. It can be a bit confusing, so here I'll give a quick overview on how to do it.
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 chews back the 3' ends of the insert because no are dNTP's available, until 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… and you may well be, the best thing to do is try it yourself – get a pencil and a piece of paper, sketch out the process and get your head around how it works.
Also in the comments section of my original article, Max suggested this tool on the Clontech site for helping to design LIC primers, which you may also find useful. Thanks to him for that spot.
As ever, if you have any questions, comments or suggestions, be sure to make them known. Happy cloning.