Methods and reagents for molecular cloning
The present invention provides compositions, methods, and kits for covalently linking nucleic acid molecules. The methods include a strand invasion step. 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.
1. A composition, comprising: a) a first ds nucleic acid molecule comprising a first end and a second end, wherein the first end comprises a 5′ overhang and a topoisomerase covalently bound at the 3′ terminus, and b) a second ds nucleic acid molecule comprising a first blunt end and a second end, wherein the first blunt end comprises a first 5′ nucleotide sequence, which is complementary to the first 5′-overhang, and a first 3′ nucleotide sequence complementary to the first 5′ nucleotide sequence.
2. The composition of claim 1 , wherein the first 5′ nucleotide sequence of the first blunt end of the second ds nucleic acid molecule is hybridized to the first 5′ overhang of the first end of the first nucleic acid molecule, and the first 3′ nucleotide sequence of the first blunt end of the second ds nucleic acid molecule is displaced.
3. The composition of claim 1 , wherein the first ds nucleic acid molecule further comprises a second 5′ overhang at the second end, wherein the second end of the second ds nucleic acid molecule further comprises a second 5′ nucleotide sequence, which is complementary to the second 5′ overhang, and a second 3′ nucleotide sequence complementary to the second 5′ nucleotide sequence.