Annealing of two complementary circular SS DNAs

Chris Boyd chrisb at hgu.mrc.ac.uk
Wed Aug 28 06:13:21 EST 1996


Lloyd Graham (graham at dbe.csiro.au) wrote:
: A procedure I am conducting will generate two closed circular
: single-stranded DNAs which are perfectly complementary to each other. I
: would like to anneal them together to generate a double-stranded DNA (a
: 3.6kb plasmid). Is it simply a matter of heating to 95'C and allowing them
: to cool slowly, or will their circular nature complicate things?

As others have mentioned, at least one of the two circles would have to
be linearized for spontaneous annealing to proceed to completion. It's
just possible you could get away with adding a topoisomerase to the
annealing mix to do this, but that's a pure guess.

: Ultimately I just want to transform E.coli with the resulting plasmid.
: Perhaps it's not even necessary for me to do an annealing step - does
: anybody know if transforming with single-stranded plasmid DNA is
: effective? 

I have a vague recollection that ss M13 DNA will transform E. coli
albeit at a low frequency.  Sorry, no refs for that, but there are
papers describing transformation of yeast, B. subtilis and streptomyces
with circular ssDNA.  It would be easy to try.  Co-transformation with
both circles might work better, even without pre-annealing. I think the
major danger would be that deletions might be promoted, but you should
be able to detect this if it happens.

Best wishes,
--
Chris Boyd                       | from, | MRC Human Genetics Unit
chrisb at hgu.mrc.ac.uk             |  not  |  Western General Hospital
http://www.hgu.mrc.ac.uk/~chrisb |   for |   Edinburgh EH4 2XU, SCOTLAND



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