The present invention provides compositions, methods, and kits for
covalently linking nucleic acid molecules. The methods include a strand
invasion step, and the compositions and kits are useful for performing
such methods. For example, a method of covalently linking double stranded
(ds) nucleic acid molecules can include contacting a first ds nucleic
acid molecule, which has a topoisomerase linked to a 3' terminus of one
end and has a single stranded 5' overhang at the same end, with a second
ds nucleic acid molecule having a blunt end, such that the 5' overhang
can hybridize to a complementary sequence of the blunt end of the second
nucleic acid molecule, and the topoisomerase can covalently link the ds
nucleic acid molecules. The methods are simpler and more efficient than
previous methods for covalently linking nucleic acid sequences, and the
compositions and kits facilitate practicing the methods, including
methods of directionally linking two or more ds nucleic acid molecules.