Patent ID: 6916632

Claim:
A method for generating a directionally linked recombinant nucleic acid molecule, the method comprising contacting: a) a topoisomerase-charged first double stranded (ds) nucleic acid molecule, comprising a first topoisomerase covalently bound at or near a first end, and a second topoisomerase covalently bound at or near a second end, said first end further comprising a first 5′ overhang, and said second end further comprising a blunt end, a 3′ thymidine overhang, or a second 5′ overhang; and b) a second ds nucleic acid molecule, comprising a first blunt end and a second end, wherein the first blunt end comprises at its 5′ terminus, a nucleotide sequence complementary to the first 5′ overhang, under conditions such that the nucleotide sequence complementary to the first 5′ overhang can selectively hybridize to the first 5′ overhang, whereby the first topoisomerase can covalently link the 3′ terminus of the first end of the first ds nucleic acid molecule with the 5′ terminus of the first end of the second ds nucleic acid molecule, and whereby the second topoisomerase can covalently link the 3′ terminus of the second end of the first nucleic acid molecule to the 5′ terminus of the second end of the second ds nucleic acid molecule, thereby generating a directionally linked nucleic acid molecule.