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.

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 - 20 . (canceled) 
     
     
         21 . A method for generating a directionally linked recombinant nucleic acid molecule, the method comprising contacting:
 a) a first precursor double stranded (ds) nucleic acid molecule comprising a first end, which comprises at the 5′ terminus, a first 5′ target sequence, and at the 3′ terminus, a topoisomerase recognition site; and a second end which comprises at the 3′ terminus, a topoisomerase recognition site;   b) a second ds nucleic acid molecule comprising a first blunt end and a second end, wherein the first blunt end comprises at the 5′ terminus a nucleotide sequence complementary to the 5′ target sequence; and   c) a topoisomerase specific for the topoisomerase recognition site, under conditions that allow topoisomerase activity, and that allow hybridization of the first 5′ target sequence and the nucleotide sequence complementary to the target sequence, thereby generating a directionally linked recombinant nucleic acid molecule.   
     
     
         22 . The method of  claim 21 , wherein the second end of the first precursor ds nucleic acid becomes a blunt end upon cleavage by the topoisomerase, and the second end of the second ds nucleic acid molecule is a blunt end. 
     
     
         23 . (canceled) 
     
     
         24 . The method of  claim 21 , wherein the first precursor ds nucleic acid molecule comprises a second 5′ target sequence located at the second end and the second ds nucleic acid molecule comprises at the second end, a nucleic acid sequence complementary to the second 5′ target sequence. 
     
     
         25 - 27 . (canceled) 
     
     
         28 . The method of  claim 21 , wherein the first precursor ds nucleic acid molecule comprises an expression control element and the second ds nucleic acid molecule comprises an open reading frame, wherein in the directionally linked recombinant nucleic acid molecule, the expression control element is operatively linked to the open reading frame. 
     
     
         29 - 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′ terminus 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 - 38 . (canceled) 
     
     
         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 . (canceled) 
     
     
         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 - 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 - 64 . (canceled)