Annealing of two complementary circular SS DNAs

Ned Mantei mantei at neuro.biol.ethz.ch
Sat Aug 17 08:08:56 EST 1996


In article <graham-1608961305340001 at tastiger.dbe.csiro.au>,
graham at dbe.csiro.au (Lloyd Graham) 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?

The two strands can't completely basepair because one strand is limited in
winding around the other if both are circular. I think you will get
something with right-handed helical regions and left-handed supercoils. If
you want to be sure, look at a paper in J. Mol. Biol. somewhere between
about 1975 and 1980, one of the authors being Elizabeth Di Capua. I would
guess that the transfection efficiency would be very low. It might go up
if you were to very lightly nick the DNA (average of maybe 1 break per
circle?).

-- 
Ned Mantei
Dept. of Neurobiology, Swiss Federal Institute of Technology
CH-8093 Zurich, Switzerland
mantei at neuro.biol.ethz.ch   Fax: +41-1-633-1046



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