Hello,
Here is my experiment:
I transfect in a plasmid that has been cut once (a linear piece of dsDNA) into human cultured cells. I wait 48hrs to allow the cells to "repair" the plasmid making it a circular piece of dsDNA. I then use a qiagen kit to prep the plasmids from the cells directly. I now have a tube with a pool of "repaired" circular plasmids all of which are unique repair events. I would like to use asymmetric PCR to amplify this pool so that I could visualize the relative size of the repaired plasmids on an agarose gel. Some repair events cause large deletions in the plasmid, so the pool of repaired plasmids will contain circular DNA ranging in size from 4kb to 12kb. I want to use the Phusion polymerase with only one primer in the reaction to amplify the plasmids so I can compare the size of deletions that occurred in my WT cells vs my KO cell line. (we plan to do Next Gen Sequencing on the pool, but it is SO EXPENSIVE that we are looking for a more cost effective "dirtier" method to complement it)
My questions:
Once the Phusion polymerase copies the entire circular plasmid and ends up at the forward primer again, will it just keep on going around and around amplifying the circle or will it terminate once it reaches the bound primer?
Should I digest my plasmid pool before aPCR?
thank you for your help!
cheers,
Valerie
Dear Valery,
First I have to admit that I have not done such an experiment. Nevertheless I hope that my comments are helpfull.
- The Phusion polymerase just stops. There is no strand displacement activity.
- I would avoid to digest the plasmid pool. It will make your analysis more complicated.
I guess, you are aware that the 12kb asymmetric PCR is ambitous and that you have already tested it on the input DNA.
I would rather try to analyze the extracted DNA directly if the sensitivity of detection allows it. In that case, linearization could make the the analysis easier. If fluorescent detection is not sensitiv enough, a fill-in reaction with radioactive nucleotides is an alternative.
Perhaps this short method paper is helpfull : Modified Hirt procedure for rapid purification of extrachromosomal DNA from mammalian cells DOI: 10.2144/98245bm14
Best,
Uwe
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