Selective cloning of homoduplex nucleic acids

Abstract

The subject invention provides for a method of selectively cloning homoduplex nucleic acid molecules, in particular, by using a strain of host cells that contains a conditionally expressed and/or conditionally active mismatch-recognizing enzyme, e.g., a temperature sensitive variant of the gene encoding the endonuclease VII from phage T4. Using this host strain, the invention features a novel cloning method that selects for PCR products that are devoid of PCR-generated mutations.

1 . A host cell for selectively cloning homoduplex nucleic acid molecules, 
 wherein the host cell is:    (a) competent;    (b) contains a gene encoding a resolvase that is conditionally expressed and/or conditionally active; and    (c) can be maintained under conditions under which the resolvase is not expressed or repressed and propagated in inactive form.    
     
     
         2 . The host cell of  claim 1 , wherein said host cell is eukaryotic.  
     
     
         3 . The host cell of  claim 1 , wherein said host cell is prokaryotic.  
     
     
         4 . The host cell of  claim 1 , wherein said conditionally expressed resolvase is selected from the group consisting of: a bacteriophage resolvase, a prokaryotic resolvase, and a eukaryotic resolvase.  
     
     
         5 . The host cell of  claim 1 , wherein said conditionally expressed resolvase is bacteriophage T4 Endonuclease VII.  
     
     
         6 . The host cell of  claim 1 , wherein said conditionally expressed resolvase is a temperature sensitive mutant of bacteriophage T4 Endonuclease VII.  
     
     
         7 . The host cell of  claim 1 , wherein said conditionally expressed resolvase is a temperature sensitive mutant of bacteriophage Endonuclease VII encoded by SEQ ID NO:1.  
     
     
         8 - 29 . (canceled)  
     
     
         30 . A kit comprising a competent host cell containing a heterologous gene encoding a conditionally expressed resolvase, and packaging materials therefore.  
     
     
         31 . A kit comprising a competent host cell containing a heterologous gene encoding a conditionally expressed resolvase, wherein the host cell can be maintained under conditions under which the resolvase is expressed and packaging materials therefore.  
     
     
         32 . A kit comprising a host cell containing an expression vector encoding a temperature sensitive mutant of bacteriophage T4 Endonuclease VII of SEQ ID NO:2, PCR reagents, helper phage and packaging materials therefore.  
     
     
         33 - 38 . (canceled)