Methods Enzymol 68: 41-50 (1979)[80164777]
The linkage of two DNAs in vitro to form recombinant molecules
first became possible with the discovery of DNA ligases. These
enzymes, which seal nicks in DNA, can covalently join two DNAs that
have complementary sticky ends such as the short, staggered ends
generated by many restriction endonucleases. Lobban and Kaiser' and
Jackson et al. showed that complementary ends could be added to DNA
molecules in vitro with terminal transferase. thus allowing any two
DNAs to be linked. These workers added complementary single-stranded
homopolymers to two DNA molecules, annealed the homopolymer regions,
and covalently closed the resulting hybrid in vitro with DNA
Polymerase I and DNA ligase from Escherichia coli. The DNA polymerase
was necessary to trim any excess unpaired nucleotides at the 3'-ends
or to fill in gaps generated by unequal lengths of the complementary
homopolymer regions. Wensink et al.,.' simplified this procedure by
showing that the annealed recombinant molecules were infectious and
that they would be covalently closed in vivo during transfection.
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