Plasmid Sequences Mutated :( - Perhaps my insert is too toxic? Recommendations and wisdom welcome. (Jun/07/2011 )

Hi all,

I have been working on a cloning project for the last three months now and about four weeks ago, for the first time, we got positive RT-PCR results for the insert of interest. I subsequently mini-prepped the samples to check size and orientation and then maxi-prepped the three that worked. All three were fully sequenced (forward and reverse), but each sample is mutated... at least 25 non-silent mutations within the 9.8kb insert per sample. There are no insertions or deletions, just a change in the base at random spots. All three samples were from the same transformed ligation product, but the mutations are not consistent from sample to sample, and of course, none of the samples perfectly match the reference sequence (which was obtained prior to ligation). Another thing to note is that the reading frames are okay... no stops within the genes of interest.

Some more information:
PCR fragment insert is 9.8kb with flanking 5' and 3' LTR regions, cut with ZraI.
Vector is pBR322, also cut with ZraI (from NEB) and CIP'd.
Insert and vector were purified and ligated with T4 DNA Ligase from NEB. Ligation was verified via gel image.
My insert was synthesized using the Advantage GC Kit from Clontech with Genomic LA Taq Polymerase.
Ligated product was electroporated with Stbl4 cells (from Invitrogen), shaken at 30C/225rpm and grown at 30C overnight on AMP.
Minis and maxis were prepared with Promega PureYield kits.

After transformation and plating, the insert+vector plates had lots of colonies (upwards of 200, which is why I did a RT-PCR screen), but all of varied sizes (lots of kicking out going on in this sample). The vector-only plate had very few colonies (5 at the most), so I'm confident that the ligation was good, but I'm thinking the sequence is just too toxic for the Stbl4 cells. Does anyone have any recommendations for other electrocompetent or chemically competent cells I might try? What about protocol changes? We're quite desperate to get this insert to work! Any suggestions will be greatly appreciated.

Thanks!

DM

What is your insert? Is it expressed? You might need to switch to a low copy or inducible copy number plasmid. If you are getting transcription from outside the insert, then check out the Lucigen vectors, which have terminators surrounding the cloning site. What are you doing with the plasmid next?

Problem may be also in the polymerase you use to amplify the insert. Taq is not proof-reading enzyme and makes mistakes. You could try some high-fidelity polymerase mix.
I cloned relatively short product (1kb) recently and most of the clones had at least one mutation somewhere. Even though we use our recombinant Taq for sequencing without any false calls, for a single clone there seems to be more important to use hi-fi enzymes.

phage434 on Tue Jun 7 19:42:21 2011 said:

Our insert is HIV, and ideally we want it to be expressed because we do infectivity testing after our plasmids are assembled. The infectious plasmids are prepared and given to other labs. Non-infectious plasmids are discarded and unfortunately, these mutations seemed to have made our plasmids non-infectious. The ENV region of the sample we're working with was tested using TOPO TA and the clones worked in infectivity testing just fine. However, it could be another region of the sequence that is causing issues.

I'm under the impression that pBR322 is a low-copy number plasmid, but I've never heard of inducible copy number plasmids. One of the issues we had at the beginning was finding a plasmid that would have unique restriction sites that would not be found in our insert... I'll forward the Lucigen site to my supervisor and see what she thinks (the whole 'clone the uncloneable!' tag-line looks promising ). Thanks for your suggestion!

Trof on Wed Jun 8 06:14:55 2011 said:

We considered that the polymerase could be a problem, but after we sequenced our PCR product, we no longer think this is the case. The kit we're using includes a higher fidelity polymerase mix, so we're confident that the product we are using to clone has the correct sequence. And of course, we do expect there to be a few mutations each time we try this, but we normally see less than 10 changes in the entire sequence. Thanks for your response!

inserts with LTR are unstable and will cause recombinations when amplified in Ecoli. I had the experience of generating an infectious clone of HTLV-1 and finally I only got 2 minis, I couldnt even get midis because the plasmids were lost when I tried to scale up. So you may try using a control insert without LTR and see what will happen.

gyma on Fri Jun 10 10:21:43 2011 said:

Thanks for the suggestion! LTR is necessary for our end product, but if we get some extra time, producing a product without LTR would be a good test of the cells' interaction with our sample. A representative from NEB also recommended cloning two halves to check if one of the halves is toxic to the cells, although again, it wouldn't be our desired full-length final product.