I have eGFP and the gene fragment i assembled by pcr.The next step is that i have to join my fragment fragment into 3'p end of the eGFP.I have three primers FP1 is forward primer for eGFP, FP2 has overlapping between eGFP and my fragment. RP1 is just revcom of my fragment 3 prime sequence. How can I assemble this by pcr. is it possible to mix all the three primers together with GFP and my fragment.enter image description here


1 Answer 1


Well, technically yes but no. Why yes is because on paper this looks simple enough but think about the technicalities. Your FP1 and FP2 will have different TMs, meaning the temp required to make the complete fragment with FP1 and RP1 will be different than to the MP of FP2. Effectively when the temp for FP2 reaches it might cause FP1 and RP1 to detach.Or the opposite might happen, where FP1 and RP1 are attached whereas FP2 might not.
Another problem I could think of is that as the PCR is cycling, the difference will lead to random length amplifications if at all you get any. The maximum amp product you might see will be of Frag-2.

If you want to fuse such fragments, I suggest going for OE-PCR (Overlap extention PCR). This is a highly effective method to join two pieces of DNA using PCR. the concept is similar to what you have proposed. Look at the wiki page- https://en.wikipedia.org/wiki/Overlap_extension_polymerase_chain_reaction OEPCR This image also explains the process...

  • $\begingroup$ Thanks for you explanation. For Overlap extention PCR i need more primers or overhangs in the fragment right. But i don't have the overlap in my fragment instead i have it on my primer. Is it possible to do by Gibson assembly? $\endgroup$
    – Rengaraj
    Commented May 4, 2021 at 19:20
  • $\begingroup$ @Anteres I don't see what you mean by "technically yes". It is simply not possible to achieve the fused fragment by the three primers proposed by the op. $\endgroup$
    – gaspanic
    Commented May 4, 2021 at 22:28
  • 1
    $\begingroup$ @gaspanic - correct, it can't be done by the PCR as designed. It could be done with a fairly simple blunt-ended ligation of the two products. Though in that case I would do the PCRs individually rather than in 1 tube. The OE-PCR is the correct procedure for this, or simply put it all into a GFP-vector in-frame with the GFP. I can't think of many applications where you would need a naked GFP-GOI construct without promoters, RBS etc. $\endgroup$
    – bob1
    Commented May 4, 2021 at 23:54
  • $\begingroup$ The ultimate aim is to integrate my fragment into the 3' end of the gfp plasmid. The plasmid has all the essential features. I thought i can assemble gfp and my fragment by pcr then i can incorporate into the plasmid. Is that wrong? $\endgroup$
    – Rengaraj
    Commented May 5, 2021 at 11:49
  • $\begingroup$ What do you mean by the 3' end of the gfp plasmid? Is GFP already in the plasmid, and all you need is to insert your second fragment downstream of GFP? In that case your primers FP-2 and RP-1 should be sufficient, assuming that you have appropriate restriction sites near the 3' end of GFP. $\endgroup$
    – gaspanic
    Commented May 5, 2021 at 22:29

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