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.
Hm. How about this description:
Design your primers pF and pR with primer3 as usual, write them down in the usual 5?-3? direction. Before ordering, you have to add some basepairs:
Take 15 bp from the left side (pleft) and 15 bp from the right side (pright) of your restriction site in the plasmid. Then:
a) LIC primer left: pleft pF
b) LIC primer right: reverse complement(pright) pR
So: The reverse overlap has to be reverse complemented. Then add your overlapping regions 5? to each primer
Hm. Don’t know if this is easier to understand…
Thanks Max…
Your description simplifies things a bit, but remember that an extra base (the ones I have colored in red) has to be added on both primers to stop the polymerase from eating into the coding sequence.
I think that the best way to learn how to design these primers is to read the background, then sit down with a piece of paper and work it through for yourself.
OK. I’d prefer using one of those new polymerases (like Finnzyme’s Phusion or the very similar PfuUltra Fusion II from Strategen) to get rid of the Ts and remove mutations from your PCR product (if it’s long).
Max,
I meant that the oligos need the extra base to insert a T that stops the T4 DNA polymerase from going any further during the reaction to generate the sticky ends.
Hey, what happens if someone doesn’t have the right primers? Maybe you have 50 primer pairs WITHOUT the overlaps. Is it possible to ligate them later to the PCR products, to these cute protruding As? I remember that there are protocols to add extensions to PCR products using the As. I just cannot remember the keyword and can’t find anything on the internet without them…
This would add one step with the advantage of more flexibility (one primer set per plasmid)
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.
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
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 ..
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?
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.
Nick,
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.
Thanks Jode.
Yes, I agree, the primers in panel 4 are wrong. The T’s should be A’s. Sorry for the confusion!!
Nick.
Dear All,
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