supercoiled DNA and transfection

[Graham Dellaire]

>   shadow <shadow at medicine.medsch.ucla.edu> writes:
>  It is often said that plasmid DNA should be predominantly
>  supercoiled for it to be expressed efficiently following 
>  transfection.  Does anybody know of any published data on this?
>  Also, in my hands, the apparent amount of the supercoiled DNA 
>  can vary from one agarose gel to the next without any change in
>  transfection efficiency. Have other people noticed that, too?
>  This makes it difficult to draw any conclusions about the relation-
>  ship between supercoiling and transfection efficiency.
>  
>>>>
Well,

I recently read an article  on supercoiling in bacteria (91 trends in Genetics?)
in which there are references going back quite a few years that show that
supercoiling (negative) is required for efficient transcription.  As well, there seems to
be an equilibrium as too much negative super coiling will prevent many reactions involving
this DNA (ex. transcription, recombination and RE digests of the DNA).  Have you ever noticed 
a band of DNA that runs below the regular CC in a plasmid prep when run on agarose... this
band is highly negatively supercoiled DNA and is very difficult to cut.  Just try growing some
bacteria at 30 degrees or in low pH or with very little aeration... all these conditions will produce
highly super coiled DNA (neg) which is refractory to RE digest.  I recently had a problem
with a rearranging plasmid so I tried transfecting the DNA into "sure" cells from Stratagene.
These cells lack gyrase which can relax negative supercoiling, the result that "band" appears
when I run the prepped DNA on an agarose gel... this may be one of the reasons the bacteria
doesn't recombine plasmids as easily as other strains (DH10 a is notorious in our lab for plasmids 
above 12 Kb)....by 1. preventing transcription of potentially leathal products from your insert 
and 2. preventing access to proteins involved in recombination
both through negative supercoiling...... not to mention it has almost every know recombination system
knocked out too....: )

G. 


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Graham Dellaire			Snail Mail:
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