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 - 7 . (canceled) 
     
     
         8 . The method of  claim 1 , further comprising introducing the directionally—linked recombinant nucleic acid molecule into a cell. 
     
     
         9 - 33 . (canceled) 
     
     
         34 . 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 to the 3′ tenninus of a first end of the ds nucleic acid molecule, said first end further comprising a first 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 a 5′ nucleotide sequence complementary to the first 5′ overhang, under conditions such that the 5′ nucleotide sequence of the first blunt end 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.   
     
     
         35 . The method of  claim 34 , further comprising contacting the topoisomerase. charged first ds nucleic acid molecule and the second ds nucleic acid molecule with a third ds nucleic acid molecule, wherein a first end of the third nucleic ds acid molecule comprises a 5′ overhang and a second topoisomerase covalently bound at the 3′ terminus, and
 wherein the second ds nucleic acid molecule further comprises a second blunt end, which comprises a 5′ nucleotide sequence complementary to the second 5′ overhang, and 
 wherein the contacting is performed under conditions such that the 5′ nucleotide sequence of the second blunt end of the second ds nucleic acid can selectively hybridize to the 5′ overhang of the first end of the third ds nucleic acid molecule, 
 whereby the second topoisomerase can covalently link the 3′ terminus of the first end of the third ds nucleic acid molecule with the 5′ terminus of the second blunt end of the second ds nucleic acid molecule. 
 
     
     
         36 . The method of  claim 35 , wherein the first ds nucleic acid molecule is directionally linked to the second ds nucleic acid molecule and, thereafter, the third ds nucleic acid molecule is directionally linked to the second ds nucleic acid molecule. 
     
     
         37 . The method of  claim 34 , wherein the first ds nucleic acid molecule is operatively linked to the second ds nucleic acid molecule. 
     
     
         38 . The method of  claim 34 , wherein the first ds nucleic acid comprises an expression control element and the second ds nucleic acid comprises an open reading frame. 
     
     
         39 . The method of  claim 35 , wherein the first ds nucleic acid molecule comprises an expression control element, the second ds nucleic acid molecule comprises an open reading frame, and the third ds nucleic acid molecule encodes a peptide,
 wherein, in the directionally linked recombinant nucleic acid molecule, the expression control element is operatively linked to the open reading frame, and the second ds nucleic acid molecule is operatively linked to the third ds nucleic acid molecule, and   wherein the second ds nucleic acid molecule is operatively linked to the third ds nucleic acid molecule encode a fusion protein comprising the open reading frame and the peptide.   
     
     
         40 . The method of  claim 39 , wherein the peptide comprises a tag. 
     
     
         41 . An isolated double stranded (ds) nucleic acid molecule, comprising 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. 
     
     
         42 . The ds nucleic acid molecule of  claim 41 , wherein the second end comprises a blunt end. 
     
     
         43 . The ds nucleic acid molecule of  claim 41 , wherein the second end comprises a single 3′ thymidine overhang. 
     
     
         44 . The ds nucleic acid molecule of  claim 41 , wherein the second end comprises a second 5′ overhang. 
     
     
         45 . The ds nucleic acid molecule of  claim 41 , wherein the first 5′ overhang comprises the nucleotide sequence 5′-GGTG-3′. 
     
     
         46 . The ds nucleic acid molecule of  claim 41 , wherein the ds nucleic acid molecule is a vector. 
     
     
         47 - 64 . (canceled)