These plasmids have been used to develop
a method to subclone DNA by sequence exchange. In the standard subcloning
procedure, the recipient plasmid is cleaved with one or two restriction
enzymes and the DNA fragments dephosphorylated to prevent direct ligation
of the backbone or re-insertion of any fragments which may have been released.
The donor plasmid is digested with the same enzymes, and the DNA fragment
which is to be transferred is purified on an agarose gel, to prevent its
re-insertion into the donor backbone upon ligation. This purification step
is time consuming and can be difficult if the DNA fragment is small or
if the donor plasmid is only available in small quantities. The pXC plasmids
overcome these problems. By using a pXC plasmid as a recipient vector,
the digested, dephosphorated recipient pXC DNA can be mixed directly with
unpurified, digested donor pUC plasmid DNA and ligated. After the resultant
constructs are transformed into E. coli, clones are selected on
media containing kanamycin. Any construct not containing the recipient
(pXC) backbone will fail to grow. It is, of course, possible for the donor
(pUC) backbone to be inserted into the recipient rather than the intended
fragment, but such clones occur infrequently and can be easily detected
by virtue of their resistance to ampicillin. All of our sequence exchanges
using these new plasmids have been successful. Exchanged fragment sizes
have ranged from 51 up to 3000 base pairs in size. Furthermore, some fragments
have been exchanged that were only available in such small quantities that
they were barely visible on an agarose gel. Even in cases where the donor
construct was available in abundance and the fragment to be transferred
was large, this approach saves hours, and sometimes a full day. Of course,
the roles of the pXC and pUC plasmids as recipient and donor can be reversed,
and this has allowed us to make sequential transfers, simply by alternating
their use.
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