disappearing DNA

Dom Spinella dspinella at chugaibio.com
Fri Dec 5 10:20:20 EST 1997


Mark O'Donohue writes:

> Has anyone had trouble with disappearing DNA inserts? We have isolated a
> gene from an archaebacterium by PCR and have tried to clone the DNA into
> various vectors (pGEM-T, pZero II, pUC18). However, we don't get many
> colonies after transformation and the ones we do get give strange
> results: often on dot blot analysis several clones light up but when we
> analyse the plasmid DNA either the insert is missing,or is smaller than
> expected, or worse, not only is there no insert,but bits of the plasmid
> are missing too. Can any suggest a way around this instability problem?
> 
> Thanks in advance.

Mark:

Lots of genes (particularly bacterial genes) are unstable when cloned
into plasmids in E. coli -- often because they encode some protein that
is toxic to the host.  If you select for the recombinant plasmid (with
antibiotic) the bugs go to great lengths to rearrrange and delete the
insert.  The way around this depends on what you are trying to do with
the insert.  If you just need to sequence, you can try direct sequencing
of the PCR product.  Alternatively, I heard in a recent talk by Craig
Venter that "gaps" in several bacterial genome sequencing projects (due
to regions of "unclonable" DNA) at his Institute for Genome Research
(TIGR) have been "closed" by cloning in lytic bacteriophage lambda --
since the bugs lyse anyway, toxicity is not an issue.  There's a good
protocol for direct sequencing from lambda on the TIGR website 
(http://www.tigr.org) if you need it.  Good luck.

-- Dom Spinella



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