Method for ligating nucleic acids and molecular cloning

Abstract

The invention provides methods of covalently joining nucleic acid molecules and methods of molecular cloning. The methods provide either sequential or simultaneous ligation of flanking or vector nucleic acid molecules to nucleic acid insert molecules by topoisomerase and DNA ligase. The methods provide for directional and non-directional covalent joining and cloning of nucleic acid molecules.

1 . A method of covalently joining a nucleic acid insert molecule to first and second nucleic acid flanking molecules to form a ligated molecule, the method comprising 
 (A) incubating:    said insert molecule, wherein one end of said insert molecule comprises a 5′-hydroxyl group and the other end comprises a 5′-phosphate group, and    a said first flanking molecule, wherein one end only of said first flanking molecule comprises a covalently bound topoisomerase polypeptide, under conditions which permit their covalent joining to form a ligated nucleic acid comprising a said insert molecule positioned adjacent to a said first flanking molecule,    (B) incubating a said ligated nucleic acid of step (A) with phosphatase under conditions which permit removal of a 5′-phosphate group from said ligated nucleic acid; and    (C) incubating a product of step (B) with a said second flanking molecule, wherein one end only of said second flanking molecule comprises a covalently bound topoisomerase polypeptide, under conditions which permit covalent joining to form a ligated molecule comprising a said insert molecule positioned between a said first and a said second flanking molecule.    
     
     
         2 . The method of  claim 1  wherein a said first and a said second nucleic acid flanking molecules comprise a left and a right vector arm, respectively, such that a said insert molecule is flanked by a said left vector arm and a said right vector arm.  
     
     
         3 . The method of  claim 2 , wherein said left and right vector arms each comprise a free end that is not joined to an insert molecule, said method further comprising the step of: 
 joining the free ends of said vector arms to each other by a method selected from the group consisting of nucleic acid ligase mediated ligation, complementary sequence annealing, topoisomerase mediated ligation, in vitro site-specific recombination, in vivo site-specific recombination, and in vivo homologous recombination.    
     
     
         4 . A method of covalently joining a nucleic acid insert molecule to first and second nucleic acid flanking molecules to form a ligated molecule, the method comprising incubating: 
 said insert molecule and said flanking molecules, wherein one end of said insert molecule comprises a 5′-hydroxyl group and the other end comprises a 5′-phosphate group, wherein one end only of said first flanking molecule comprises a covalently bound topoisomerase polypeptide and wherein one end of said second flanking molecule comprises a ligase substrate site, under conditions which permit their covalent joining to form a ligated molecule comprising a said insert molecule positioned between a said first and a said second flanking molecule.    
     
     
         5 . The method of  claim 4  wherein a said first and a said second nucleic acid flanking molecules comprise a left and a right vector arm, respectively, such that a said insert molecule is flanked by a said left vector arm and a said right vector arm.  
     
     
         6 . The method of  claim 5 , wherein said left and right vector arms each comprise a free end that is not joined to an insert molecule, said method further comprising the step of: 
 joining the free ends of said vector arms to each other by a method selected from the group consisting of nucleic acid ligase mediated ligation, complementary sequence annealing, topoisomerase mediated ligation, in vitro site-specific recombination, in vivo site-specific recombination, and in vivo homologous recombination.