Methods and compositions for generating recombinant nucleic acid molecules

Abstract

A method of generating a double stranded (ds) recombinant nucleic acid molecule covalently linked in both strands by contacting two or more ds nucleotide sequences with a topoisomerase under conditions such that both termini of at least one end of a first ds nucleotide sequence are covalently linked by the topoisomerase to both termini of at least one end of a second ds nucleotide sequence is provided. Also provided is a method for generating a ds recombinant nucleic acid molecule covalently linked in one strand, by contacting two or more ds nucleotide sequences with a type IA topoisomerase under conditions such that one strand, but not both strands, of one or both ends of a first ds nucleotide sequence are covalently linked by the topoisomerase. Compositions for performing such methods, and compositions generated from such methods also are provided, as are kits containing components useful for conveniently practicing the methods.

1 . A method for the in vitro generation of RNA molecules, the method comprising: 
 (a) generating a reaction mixture in which a first double-stranded DNA molecule is contacted with a second double-stranded DNA molecule under conditions which allow for both strands of one end of the first double-stranded DNA molecule to become covalently linked to both strands of one end of the second double-stranded DNA molecule,    (b) incubating the reaction mixture of (a) for a sufficient period of time to allow for the covalent linking of the first double-stranded DNA molecule to the second double-stranded DNA molecule, and    (c) generating an RNA transcript from the product of (b) by in vitro transcription,    wherein the first double-stranded DNA molecule has promoter activity and is operably connected to the second double-stranded DNA molecule in (b),    wherein the first double-stranded DNA molecule and the second double-stranded DNA molecule are covalently linked to each other by a topoisomerase, and    wherein the double-stranded DNA molecule produced in (b) does not contain a nick in either strand at the position where the first double-stranded DNA molecule and the second double-stranded DNA molecule are joined.    
     
     
         2 . The method of  claim 1 , wherein both strands of one end of the double-stranded DNA molecule product of (b) are covalently linked by a topoisomerase to both strands of one end a third double-stranded DNA molecule.  
     
     
         3 . The method of  claim 2 , wherein the double-stranded DNA molecules are covalently linked in the order of the first double-stranded DNA molecule, the second double-stranded DNA molecule, and the third double-stranded DNA molecule.  
     
     
         4 . The method of  claim 2 , wherein the third double-stranded DNA molecule encodes a polyadenylation signal.  
     
     
         5 . The method of  claim 1 , wherein the second double-stranded DNA molecule encodes a polypeptide.  
     
     
         6 . The method of  claim 1 , wherein the second double-stranded DNA molecule is generated by polymerase chain reaction.  
     
     
         7 . The method of  claim 1 , wherein one strand of each end of the first double-stranded DNA molecule and the second double-stranded DNA molecule which are joined are topoisomerase-charged.  
     
     
         8 . The method of  claim 1 , wherein the topoisomerase is a type IB topoisomerase or a catalytic domain of a type IB topoisomerase.  
     
     
         9 . The method of  claim 1 , wherein the transcription product of (c) is translated in vitro to generate a polypeptide.  
     
     
         10 . The method of  claim 1 , wherein the first double-stranded DNA molecule comprises a T7 promoter.  
     
     
         11 . The method of  claim 1 , wherein the first double-stranded DNA molecule comprises a T3 promoter.  
     
     
         12 . A method for the in vitro generation of double-stranded RNA molecules, the method comprising: 
 (a) generating a reaction mixture in which a first double-stranded DNA molecule is contacted with a second double-stranded DNA molecule under conditions which allow for both strands of one end of the first double-stranded DNA molecule to become covalently linked to both strands of a first end of the second double-stranded DNA molecule,    (b) incubating the reaction mixture of (a) for a sufficient period of time to allow for the covalent linking of the first double-stranded DNA molecule to the first end of the second double-stranded DNA molecule, and    (c) generating a reaction mixture in which a first double-stranded DNA molecule is contacted with a second double-stranded DNA molecule under conditions which allow for both strands of one end of the first double-stranded DNA molecule to become covalently linked to both strands of a second end of the second double-stranded DNA molecule,    (d) incubating the reaction mixture of (c) for a sufficient period of time to allow for the covalent linking of the first double-stranded DNA molecule to the second end of the second double-stranded DNA molecule,    (e) mixing the products of (b) and (d),    (f) generating RNA transcripts from the products of (e) by in vitro transcription, and    (g) incubating the RNA transcripts produced in (f) under conditions    which allow for the formation of double-stranded RNA molecules,    wherein each of the first double-stranded DNA molecules in (b) and (d) has promoter activity and is operably connected to each of the second double-stranded DNA molecules in (b) and (d),    wherein the first double-stranded DNA molecule and the second double-stranded DNA molecule are covalently linked to each other by a topoisomerase, and    wherein the double-stranded DNA molecule produced in (b) does not contain a nick in either strand at the position where the first double-stranded DNA molecule and the second double-stranded DNA molecule are joined.    
     
     
         13 . The method of  claim 12 , wherein the RNA transcripts produced in (f) are sense and antisense RNA molecules.  
     
     
         14 . The method of  claim 12 , wherein the second double-stranded DNA molecule encodes a polypeptide.  
     
     
         15 . The method of  claim 12 , wherein the second double-stranded DNA molecule is generated by polymerase chain reaction.  
     
     
         16 . The method of  claim 12 , wherein one strand of each of the ends of the first double-stranded DNA molecule and the second double-stranded DNA molecule which are joined are topoisomerase-charged.  
     
     
         17 . The method of  claim 12 , wherein the topoisomerase is a type IB topoisomerase or a catalytic domain of a type IB topoisomerase.  
     
     
         18 . The method of  claim 12 , wherein the first double-stranded DNA molecule comprises a T7 promoter.  
     
     
         19 . The method of  claim 12 , wherein the first double-stranded DNA molecule comprises a T3 promoter.  
     
     
         20 . A reaction mixture comprising: 
 (a) a first double-stranded DNA molecule which comprises a promoter, and    (b) a second double-stranded DNA molecule,    wherein one strand of one end of the first double-stranded DNA molecule is topoisomerase-charged,    wherein one strand of one end of the second double-stranded DNA molecule is topoisomerase-charged, and    wherein the topoisomerase-charged ends of the first double-stranded DNA molecule and the second double-stranded DNA molecule are capable of hybridizing to each other.    
     
     
         21 . The reaction mixture of  claim 20 , wherein the first double-stranded DNA molecule comprises a T7 promoter.  
     
     
         22 . The reaction mixture of  claim 20 , wherein the first double-stranded DNA molecule comprises a T3 promoter.  
     
     
         23 . The reaction mixture of  claim 20 , wherein the topoisomerase is a type IB topoisomerase or a catalytic domain of a type IB topoisomerase.