Topoisomerase-based reagents and methods for molecular cloning

A method of molecular cloning DNA comprising:

• a) introducing to a donor duplex DNA substrate a sequence specific topoisomerase cleavage site by PCR amplifying the donor duplex DNA molecule with oligonucleotide primers containing the cleavage site;
• b) incubating the donor duplex DNA with a sequence specific topoisomerase, resulting in the formation of a sequence specific topoisomerase-donor duplex DNA complex;
• c) incubating the sequence specific topoisomerase-donor duplex DNA complex with a plasmid vector with a 5′ overhang compatible to the donor;
• d) incubating the sequence specific topoisomerase-donor duplex DNA complex with the plasmid vector;
• and
• e) transforming the plasmid vector of step (c) into a host cell.

A method of synthesizing polynucleotides comprising:

• a) annealing a multiple number of duplex DNA strands to form a branched substrate containing a sequence specific topoisomerase cleavage site at each 3′ end;
• b) cleaving the branched substrate by incubation with a sequence specific topoisomerase to form a branched topoisomerase complex;
• and
• c) incubating the branched topoisomerase complex with complementary monovalent and/or bivalent DNA acceptors.