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Methods Enzymol 68: 41-50 (1979)[80164777]

Addition of homopolymers to the 3'-ends of duplex DNA with terminal transferase.

T. Nelson & D. Brutlag

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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