Dear All
How many nucleotides does the 5' exonuclease remove during Gibson Assembly. I mean I was wandering how does the exonuclease recognize the position where it has to stop removing the nucleotides.
I would appreciate your response.
Thank You
Ikram
Hi Ikram,
The number of bases the exonuclease removes is not a specified or determined number in a Gibson assembly reaction. This is a dynamic reaction that is "balanced" by the polymerase activity which is also present in the reaction. Think of these two enzymes constantly removing (exonuclease) and adding (polymerase) bases. Every now and then (hopefully more often than not!) the exonuclease will remove bases and allow 2 of your fragments to anneal, thus allowing the polymerase to use the new fragment of DNA as a template to do its job. The ligase (which is also present in the reaction!) will then repair the nick that is left once the polymerase has done all it can.
NEB has a great website that explains this whole process very clearly (https://www.neb.com/products/e2611-gibson-assembly-master-mix).
Best,
Dan
Dear Ikram,
Another simple reason would be that the exonuclease is from bacteriophage T5, not from a thermophilic organism. Therefore, it is rapidly inactivated at 50˚C, at which the thermostable DNA polymerase is fully active. The short life of the enzyme may determine the extent of digestion.
Best regards, Koreaki
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