I have a plasmid that lacks an ori. But others in the field have seen that in some cells plasmids that lack an ori still undergo some levels of plasmid replication. For my work I want to ensure that this plasmid does not replicate at all and if it does I want to measure how much replication is going on so I can account for it. This plasmid replication assay will have to be carried out on very minute amounts of DNA obtained from a nuclear prep (few thousand molecules). Can anyone suggest a method by which I can measure accurate amounts of replication that this plasmid has undergone?
not clear, what kinds of cells are you working in? Yeast (since you mentioned nuclei)?
Sounds like a challenging assay. Are you planning to transfect your plasmid into cells? If so, you have to be able to distinguish newly replicated plasmids from your very large amount of input plasmid. Detecting very small numbers of newly replicated plasmids will probably require some kind of amplification step. A simple qPCR would probably be overwhelmed by the input plasmid.
You need some way of selectively labeling the input plasmid in such a way that it can be efficiently removed (in vitro biotinylation of plasmid DNA??) or not amplified by your qPCR assay, or a way of selectively labeling the newly replicated plasmid so that it can be efficiently purified away from the input plasmid.
What if you used BrdU to label the newly replicated plasmid, then immunoisolate BrdU-labeled plasmid (so the input DNA would be washed away), then qPCR?
you also need a way of quantitating the number of input plasmid DNA molecules so that you can control for transfection efficiency. Perhaps a reporter gene on the input plasmid?
Hi Andrew, thanks for those ideas. I am working with human fibroblasts.
Kristian: I am not sure why I would do that. I don't want my plasmid to replicate. I just want to see if there are some non-ori levels of replication going on.
see http://www.ncbi.nlm.nih.gov/pmc/articles/PMC201251/ for an example of immunocapture of BrdU-labeled DNA
Hi Aditi,
I would propose the following approach:
1) Use plasmid produced in a normal methylation-positive E.coli strain (i.e. DH5alpha).
2) After recovering your plasmid from the fibroblasts, just perform a digest with a restriction enzyme that digests only methylated DNA, such as DpnI. This is similar to what is done when using site-directed mutagenesis kits.
3) For the quantification use quantitative real-time PCR, since this is perhaps the only method providing sufficient sensitivity. The amplicon should span a restriction site of the enzyme you used to digest the plasmid. If restriction is complete, only plasmids replicated in the fibroblasts resist restriction and therefore produce PCR product.
4) Plasmid that has not undergone replication in the fibroblasts should serve as a negative control, and qPCR of un/mock-digested plasmid should provide a measure for the total amount of plasmid recovered.
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