Ad ITRs and Recombination

[Andy Bailey]

In article <MAILQUEUE-101.950503125520.320 at bmg.bhs.uab.edu>, 
PANGELETTI at bmg.bhs.uab.edu says...
>
>                Hi, just thought I'd add in a few thoughts on the Ad ITR 
recombination 
>problem that Graham described.  I noticed this sort of problem when 
>tried to put Ad 12 ITRs in a plasmid to construct a minichromosome.  It 
>was a very unstable plasmid.  My construct never grew very well.  The 
>thing that I would suggest though is using rec- cells to propagate the 
>DNA. The growing temp. may also influence recomb. If that doesn't work 
>you may be limited to 5 ml cultures.  Certainly, I would be careful 
>about subculturing the clone.  Let us know how it turns out.   
>
>Original post:  
>----------------------------------------------------------------------
>I have prepared a plasmid based vector that contains a large terminal 
>fragment of the adenovirus-2 genome.  The vector is 22 kbp and has the 
>odd and frustrating ability to recombine and rearrange when grown in 
>bacteria in large culture volumes (greater than 5 ml!!)  When I grow the 
>bacteria (Dh5 or10) in 5 ml cultures I get no rearrangment but as soon 
>as I try to grow a larger culture volume I end up with a ladder of 
>plasmids or a new one all together!!!
>----------------------------------------------------------------------

I have repeatedly found that cloning any peice of adenovirus DNA 
containing the E1b 55K region of the genome is highly prone to 
recombination. I know others have experienced this problem also. If this 
particular region is not essential for your plasmid, you might try 
removing it from your plasmid. Unfortunately,despite a number of attempts 
I have never been able to overcome this effect of the E1b region.

I hope this might be of help

Andy Bailey