Recombinational cloning using nucleic acids having recombination sites

Abstract

Recombinational cloning is provided by the use of nucleic acids, vectors and methods, in vitro and in vivo, for moving or exchanging segments of DNA molecules using engineered recombination sites and recombination proteins to provide chimeric DNA molecules that have the desired characteristic(s) and/or DNA segment(s).

What is claimed is:  
     
         1 . An in vitro method of cloning an amplification product comprising: 
 (a) obtaining an amplification product comprising a first recombination site and a second recombination site which do not recombine with each other; and    (b) combining said amplification product in vitro with a vector comprising a third recombination site and a fourth recombination site which do not recombine with each other, under conditions such that recombination occurs between said first and third and said second and fourth recombination sites, thereby producing a product vector.    
     
     
         2 . The method of  claim 1 , further comprising inserting said product vector into a host cell.  
     
     
         3 . The method of  claim 1 , wherein said vector is an expression vector.  
     
     
         4 . The method of  claim 1 , wherein said vector comprises at least one additional nucleic acid sequence selected from the group consisting of a selectable marker, a cloning site, a restriction site, a promoter, an operon, an origin of replication, and a gene or partial gene.  
     
     
         5 . The method of  claim 1 , wherein said vector comprises at least one origin of replication.  
     
     
         6 . The method of  claim 1 , wherein said vector comprises at least one promoter.  
     
     
         7 . The method of  claim 1 , wherein said vector comprises at least one selectable marker.  
     
     
         8 . The method of  claim 1 , wherein said amplification product is linear.  
     
     
         9 . The method of  claim 1 , wherein said first, second, third or fourth recombination sites are lox sites or functional mutants thereof.  
     
     
         10 . The method of  claim 9 , wherein said lox sites are selected from the group consisting of loxP sites and loxP511 sites.  
     
     
         11 . The method of  claim 1 , wherein said first, second, third or fourth recombination sites are att sties or functional mutants thereof.  
     
     
         12 . The method of  claim 11 , wherein said att sites are selected from the group consisting of attB sites, attP sites, attL sites and attR sites.  
     
     
         13 . The method of  claim 1 , wherein said first, second, third or fourth recombination sites are selected from the group consisting of a lox site, an att site, an FRT site, and functional mutants thereof.  
     
     
         14 . The method of  claim 1 , wherein said amplification product and said vector are combined in the presence of at least one recombination protein.  
     
     
         15 . The method of  claim 14 , wherein said recombination protein is Cre.  
     
     
         16 . The method of  claim 14 , wherein said recombination protein is selected from the group consisting of Int, Xis and IHF.  
     
     
         17 . The method of  claim 1 , wherein said amplification product is a polymerase chain reaction product.  
     
     
         18 . The method of  claim 17 , wherein said polymerase chain reaction product is linear.