Fate of plasmid - How to detect plasmid DNA on host chromosome (Mar/05/2008 )
I need to find out what happens to plasmids that seemed to be lost from the host cell (during culturing- that is no plasmids in next generation). They can either be entirely lost, have lost part of their chromosome - the part I use for detection of them, have been incorporated fully or partly onto the host chromosome. How do I detect whether plasmid DNA is on the host chromosome without sequencing the whole genome? And without beeing dependent on extracting huge amounts of DNA.
try a southernblot.
in the most simplest situation:
your host is a yeast/bacteria with a small genome and chromosomes, you may then run your host cell's chromosomes onto a gel via pulsefield gel electrophoresis. You can then directly probe the chromosomes via probes specific to the plasmid.
In more complex situations, you can use a restriction enzyme that cuts your plasmid ones, and the genome a few times. Conduct a southernblot on your (a) sample,( agenomic DNA from a normal host cell, and some ©plasmid DNA. If the southernblot shows a band on your sample that is large then the plasmid, but also absent in the normal genomic DNA, then you have an integration event.
You may create DNA probes for different sections of your plasmid, just incase said probe is not specific enough, or the section it is bound to is absent.
However with all that said, is it really that important to know what happened to the plasmid? Since selection was lost, (I assume selection was maintain through out the culturing), it is quite probably that the plasmid simply fell out. It couldn't replicate well enough in the host.
Can you check the plasmid using PCR? do you have the primers with you? Just a suggestion.
Thanks for the suggestions. I thought of something like that.
I am doing a long term plasmid stability study - and we thought it would be relevant to know the plasmid really is lost or if it just seems to be lost.
yes I have the primers (or will design them) and I will check with pcr if there is a plasmid. But can I use pcr to locate an integrated plasmid on the host chromosome?
Yes, of course. You can detect an integrated chromosome by PCR of a region of the putative integrated plasmid. If you need to locate it, then you need to do inverse PCR, which is straightforward, but different.
Yes ok, it sounds so easy and I did something like that once. But this plasmid is extremely large (120 kb) and I do not really know where it will break up before insertion. I guess I can just use different sets of primers then... Arbitrary primers?
well primers aren't that expensive. Maybe you could get a few sets of primers?