Hi molecular biologists, I'm wondering if any of you might be able to help me with a question I have.
I am attempting to insert the DNA sequence coding for a protein domain into a plasmid (the plasmid is popinF). The insert DNA (E. coli optimised) was synthesised by Thermo (and it has passed their QA/QC), and I've successfully inserted it into popinF and transformed E. coli stellar cells, before collecting 3 different colonies from a plate to perform minipreps and acquire the plasmid with inserts. The sequencing results came back for all of them, and confirmed that the full (and correct!) DNA sequence had been inserted into one of the 3 plasmids.
However, I found it very peculiar that one of my plasmids appeared to have my DNA insert, but in a degenerated form with regards to the sequence. In the alignment shown attached, I can clearly see that there is very very strong matching of the sequenced result to the DNA from ~230 base onwards, showing that the synthetic DNA has inserted. But the sequence prior to this region does not show a high correlation to my DNA insert, and I'm wondering how this could be, and what could have caused this? I know that the synthesised DNA must be correct because I've successfully put the full length sequence into another identical plasmid - could it be that this particular plasmid showing a degenerate sequence could have undergone mutations within the E. coli or have degenerated in other ways, and if so could anybody please expand on the mechanisms and nature of these mutations? If anybody has any insight into mutation events of DNA inserts in plasmids within bacteria or knows of any good literature that reviews it and how to avoid them during recombination/transformation, I would be very appreciative for the help!
Thanks very much all,
Rob
Hi there,
To check the problem is actually due to the proximity with the sequence primer, run an other sequencing with a reverse primer annealing downstream of the 3' cloning site (if the full insert is less than 1kb) or in the 3' region of your insert (if the insert is bigger than 1kb) in order to get the reverse sequence which would contain the proper sequence of the 5' region.
I suspect the problem may not be the plasmid itself, but the actual sequence reaction. It may be the template was poor and you just got a low quality sequence run, I suggest this because the first 100 nt's clearly has many bases the machine was unable to call, and it is very AT rich after that.
The first 100-200 bases of a Sanger run are always poor quality. Trim off those bases and redo the BLAST analysis. You can also sequence from the other direction and pair the reads together to get a longer consensus.
Send a screenshot of your chromatograph - I'm pretty sure the poor matches are poor quality sequence reads.
Hi there,
To check the problem is actually due to the proximity with the sequence primer, run an other sequencing with a reverse primer annealing downstream of the 3' cloning site (if the full insert is less than 1kb) or in the 3' region of your insert (if the insert is bigger than 1kb) in order to get the reverse sequence which would contain the proper sequence of the 5' region.
Hi There,
Storage conditions of your plasmid may be a possible reason (Usually for point mutations). Try to avoid continuous freezeing and thawing of your plasmids stock . Others factors has already been well answerd by Dr @ Katie A Burnette and Michael J. Benedik .
Hi Michael J. Benedik and Katie A Burnette, thanks a lot for your comments! I'm not an expert, so I've attached the chromatogram associated with this read. It seems the peaks are showing fairly clearly, but I'd love to know your thoughts if possible?
Dominique Liger thanks for your suggestion, as Katie mentioned this could be a good approach, I'll try to do it in the future!
Abdul Wahaab thanks very much for your insight, so repeat cycles would be more likely to induce point mutations in the plasmid? Do you happen to know the reason for why this happens?
Many thanks all!
Rob
There exists a critical region of temperature in which some DNA and evensome proteins are denatured during continuous freezing and thawing. Repeated freeze-thawing of DNA will fragment it. DNA is extremely robust though.
You can store it at 4 C long term to avoid freeze-thawing. I would suggest maybe doing this with an aliquot while you are carrying out your experiment. Do not store at 4 degrees in volumes less than 20uL though, and using screw cap tubes can remove any potential condensation issues. There is no practical requirement to freeze DNA unless it is for long term storage.
Rob Barringer, you are correct that from the sequencing histograms they sequence looks quite clear after about nt 90. I typed in the "wrong" sequence from your histogram and did a Blast search and that section from 90-237 is vector sequence. So either there was a small bit of fragment vector DNA in the prep or that clone had a deletion generated. The thing to realize is that weird stuff does commonly occur during closings, so long as you got one clone whose sequence was correct then just proceed with that. I wouldn't worry much about the other odd things that arise, every one of that has molecular biology experience can regale you with stories of unexplained stuff that happened, which is why we always look at and sequence multiple clones to find a correct one.
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