Compositions and methods for synthetic gene assembly

Abstract

Methods and compositions are provided for assembly of large nucleic acids where the assembled large nucleic acids lack internal sequence modifications made during the assembly process.

What is claimed is: 
     
         1 . A nucleic acid assembly method comprising:
 a) obtaining at least two double-stranded DNA fragments, wherein the at least two DNA fragments comprise ends that can selectively hybridize with one another; and   b) contacting the at least two double-stranded DNA fragments with:
 (i) a DNA polymerase; 
 (ii) a flap endonuclease; and 
 (iii) a DNA ligase, 
   
       under reaction conditions that promote hybridization of the at least two DNA fragments and support the activities of components (i), (ii) and (iii), to produce a product comprising an assembled nucleic acid comprising at least a portion of each of the at least two DNA fragments. 
     
     
         2 . The method of  claim 1 , further comprising performing at least one thermal cycling incubation step after the contacting step (b). 
     
     
         3 . The method of  claim 2 , wherein multiple thermal cycling incubations steps are performed. 
     
     
         4 . The method of  claim 1 , wherein the at least two DNA fragments comprise one or more portions of a vector. 
     
     
         5 . The method of  claim 3 , wherein the one or more portions of a vector comprises an origin of replication, selectable marker, reporter gene, promoter, and/or ribosomal binding site. 
     
     
         6 . The method of  claim 1 , wherein at least three DNA fragments comprising ends that can selectively hybridize with one another are obtained in step (a). 
     
     
         7 . The method of  claim 1 , wherein at least five DNA fragments comprising ends that can selectively hybridize with one another are obtained in step (a). 
     
     
         8 . The method of  claim 1 , wherein the product produced comprises an assembled circular DNA comprising at least a portion of each of the at least two double-stranded DNA fragments. 
     
     
         9 . The method of  claim 1 , wherein the product produced comprises an assembled linear DNA comprising at least a portion of each of the at least two double-stranded DNA fragments. 
     
     
         10 . The method of  claim 1 , further comprising expressing the product produced in a host cell. 
     
     
         11 . The method of  claim 1   0 , wherein the host cell is a bacterial cell, a viral cell, a yeast cell, or a mammalian cell. 
     
     
         12 . The method of  claim 1 , wherein the ends of the at least two DNA fragments that can selectively hybridize are at least 10 nucleotides in length. 
     
     
         13 . The method of  claim 1 , wherein the flap endonuclease is FEN-1. 
     
     
         14 . The method of  claim 1 , wherein each double-stranded DNA fragment is flanked by a terminal adapter sequence. 
     
     
         15 . The method of  claim 1 , wherein the product produced encodes for a gene. 
     
     
         16 . The method of  claim 1 , wherein each of the least two double single-stranded DNA fragments comprise a cDNA sequence. 
     
     
         17 . A nucleic acid assembly method comprising:
 a) de novo synthesizing at least two single-stranded DNA fragments;   b) amplifying the at least two single-stranded DNA fragments to generate at least two double single-stranded DNA fragments, wherein the at least two DNA fragments comprise ends that can selectively hybridize with one another; and   c) contacting the at least two double-stranded DNA fragments with:
 (i) a DNA polymerase; 
 (ii) a flap endonuclease; and 
 (iii) a DNA ligase, 
   
       under reaction conditions that promote hybridization of the at least two DNA fragments and support the activities of components (i), (ii) and (iii), to produce a product comprising an assembled nucleic acid comprising at least a portion of each of the at least two DNA fragments.