Methods and reagents for molecular cloning

Abstract

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.

1 . 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.   
     
     
         2 . The method of  claim 1 , wherein the second end of the first ds nucleic acid molecule comprises a blunt end, and the second end, of the second ds nucleic acid molecule comprises a blunt end. 
     
     
         3 . The method of  claim 1 , wherein the second end of the topoisomerase-charged first ds nucleic acid molecule comprises a 3′ thymidine overhang, and the second end of the second ds nucleic acid molecule comprises a 3′ adenosine overhang. 
     
     
         4 . The method of  claim 1 , wherein the topoisomerase-charged, first ds nucleic acid molecule comprises a second 5′ overhang at the second end, and the second ds nucleic acid comprising at the second end, a nucleotide sequence complementary to the second 5′ overhang. 
     
     
         5 . The method of  claim 1 , wherein the first ds nucleic acid molecule is a vector. 
     
     
         6 . The method of  5 , wherein the topoisomerase-charged first ds nucleic acid molecule is a cloning vector. 
     
     
         7 . The method of  6 , wherein the topoisomerase-charged first ds nucleic acid molecule is an expression vector. 
     
     
         8 . The method of  claim 1 , further comprising introducing the directionally-linked recombinant nucleic acid molecule into a cell. 
     
     
         9 . The method of  claim 8 , wherein the cell is a eukaryotic cell. 
     
     
         10 . The method of  claim 9 , wherein the cell is a mammalian cell. 
     
     
         11 . A cell produced by the methods of  claim 8 . 
     
     
         12 . A transgenic non-human organism generated from the cell of  claim 11 . 
     
     
         13 . (canceled) 
     
     
         14 . The method of  claim 1 , wherein the second ds nucleic acid molecule comprises an amplification product. 
     
     
         15 . The method of  claim 1 , wherein the topoisomerase is a type IB topoisomerase. 
     
     
         16 - 55 . (canceled) 
     
     
         56 . 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.   
     
     
         57 . The composition of  claim 56 , 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.   
     
     
         58 . The composition of  claim 56 , 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.   
     
     
         59 - 61 . (canceled) 
     
     
         62 . A kit, comprising
 a) a first double stranded (ds) nucleic acid molecule, which comprises a first topoisomerase covalently bound at a 3′ terminus of a first end, and a second topoisomerase covalently bound at a 3′ terminus of 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, wherein said first 5′ overhang is different from said second 5′ overhang; and   b) a plurality of second ds nucleic acid molecules, wherein each ds nucleic acid molecule in the plurality comprises a first blunt end, and wherein the first blunt end comprises a 5′ nucleotide sequence complementary to the first 5′ overhang of the first ds nucleic acid molecule.   
     
     
         63 . The kit of  claim 62 , wherein the second ds nucleic acid molecules in the plurality comprise transcriptional regulatory elements, translational regulatory elements, or a combination thereof. 
     
     
         64 . The kit of  claim 62 , wherein the second ds nucleic acid molecules in the plurality comprise nucleotide sequences encoding a peptide.