More often than desired (about 75 % of the time), when building plasmids via Gibson Assembly (~ 5 kb plasmids; from a maximum of 2 fragments) we obtain clones with several random point mutations all along the plasmid (from 1–3 point mutations to 6+ depending on the day, without specific region targetted).
Since we use the NEBuilder HiFi DNA Assembly kit and since there doesn't seem to be any specificity as of where those mutation are found, I suspect that they arise during the PCR reaction when producing the amplicons.
We use the Phusion High-Fidelity polymerase to generate the amplicons from ~1 pmol of template in 50 µL of PCR reaction (or split in 5 x 10 µL since working with reduced volumes seems to improve the efficiency of amplification) and 30 cycles.
Is this a normal/common issue or does this result indicate that something wrong is happening in our experiments?
And more importantly, any idea of how to prevent such problems?
I have in mind:
- Reducing the number of cycles (how low can one go?) and/or increasing the concentration of template (related).
- Changing polymerase (but for which one and shouldn't Phusion High-Fidelity be one of the best suited?).
- Changing other parameters or conditions (but which ones?)
Any bullet-proof experience on that? Thanks
Changing the DNA polymerase from a Phusion HF to the Q5 suggested in the NEBuilder kit manual (suggestion that I had overlooked) completely solved the problem.
Rem 1: Mind that the elongation time must be slightly increased to amplify "long" fragments with Q5—probably a side effect of its higher proofreading ability. Whereas 20–30 s/kb is recommended, 120 s where not enough to amplify a fragment of 3.5 kb; an elongation time of 240 s—i.e. 60 s/kb—did work though.
Rem 2: In the past, amplification with the Phusion HF DNA polymerase did result in correct sequences in most of the cases, so I was surprised to have issues here. A possible factor that came to my mind afterwards and that could explain the apparition of mutations could be a low purification quality of the DNA preparation used as the template (leftover chemical contaminants). Although I have not (yet) tested and confirmed this possible explanation in my case.
I marked the answer from @markur below as accepted, as his/her suggestions (along with the accompanying discussion underneath) prompted me to go and read the original articles and then to read again more carefully the manual of the NEBuilder kit where the solution was.