Can I use just a primer and PCR to join the cutted plasmid like this ? without using any further enzyme like ligase.

Image made by me

After the linear dsDNA plasmid annealed into 2 ssDNA, primer will bind to middle. And Taq Polymerase will bind to the 3' and start extend the DNA to join the plasmid. So I don't need a ligase enzyme, is that possible?

Edit: I think the plasmid will be nicked at 2 position with this method but will this plasmid still work in e.coli cell?

  • $\begingroup$ Your diagram shows two single-stranded pieces of DNA that are annealed together to form two short regions of dsDNA, but in your description you mention linear dsDNA (which is not shown here). Assuming that you intend to denature the linear dsDNA, and then add the shorter primer, then yes, adding a suitable DNA polymerase enzyme, proper buffer and reaction conditions, and dNTP substrates, will yield some relaxed (nicked) circular DNA molecules. If there is a selectable marker on the plasmid then you should be able to transform competent E. coli cells. NOTE: There is no PCR involved. $\endgroup$ – mdperry Dec 15 '18 at 17:21
  • $\begingroup$ @mdperry In all protocol I found. They usually use some extra enzyme. For example Ligase, or exonuclease (Gibsom Assembly), or T4 polymerase. Is there anyway I can use only Taq Polymerase? $\endgroup$ – joe Dec 16 '18 at 4:55
  • $\begingroup$ It is not clear to me which protocols you are referring to. T4 DNA ligase, Exonuclease (type unspecified), and T4 DNA polymerase are three distinctly different enzymes (and could not be substituted for each other). The main difference between Taq polymerase and T4 polymerase is the temperature of the reaction. So I think they can easily be substituted if you change the reaction temperature. It is quite clear that a dsDNA plasmid does not need to be covalently closed in order to transform E. coli. The cells will repair the nicks after they take up the DNA. $\endgroup$ – mdperry Dec 16 '18 at 18:25
  • $\begingroup$ @mdperry "The exonuclease activity of T4 phage DNA polymerase will create overhangs at the ends of your PCR product and within the homologous site of your destination vector, thus allowing your PCR product and vectors to anneal to create a final cloned piece of DNA" - I found it on "Method : Sequence and Ligase Independent Cloning (SLIC)". I may though like you too but I can't find any evidence on the internet, so it still uncertainty to me. $\endgroup$ – joe Dec 17 '18 at 1:35
  • $\begingroup$ Okay, yes, that is true, IF: you add T4 DNA polymerase IN THE ABSENCE OF dNTPs then there is a 3’ -> 5’ exonuclease activity that we used to use to turn a 3’-overhang into a blunt end. As I recall, this is not a very processive activity, and difficult to control, but maybe there are newer applications I a, not aware of. Anyway, you don’t need it for the picture you drew above. What you described is if you have the same short sequence on both ends of the two blunt fragments (based on the quotation). $\endgroup$ – mdperry Dec 17 '18 at 5:41

Your Answer

By clicking “Post Your Answer”, you agree to our terms of service, privacy policy and cookie policy

Browse other questions tagged or ask your own question.