Method for assembly of multiple DNA fragments

Abstract

A method for assembling two or more DNA fragments with high efficiency, comprises: a) providing, for each DNA fragment, at least one protruding terminus, or “overhang”, capable of hydrogen bonding to a complementary sequence on at least one strand of a second DNA fragment, said overhang having at least 15 bases; and b) mixing two or more said DNA fragments under conditions suitable to promote joining thereof.

1 . A method for assembling two or more DNA fragments with high efficiency, comprising: 
 a) providing, for each DNA fragment, at least one protruding terminus, or “overhang”, capable of hydrogen bonding to a complementary sequence on at least one strand of a second DNA fragment, said overhang and said complementary sequence having at least 15 bases; and    b) mixing two or more said DNA fragments under conditions suitable to promote joining thereof.    
     
     
         2 . A method according to  claim 1 , wherein the number of fragments joined is three or more.  
     
     
         3 . A method according to  claim 1  or  2 , wherein the overhang has at least 20 bases.  
     
     
         4 . A method according to  claim 1  or  2 , wherein the number of bases in the overhang is between about 20 and about 30.  
     
     
         5 . A method according to  claim 2 , wherein the molar ratio of each DNA pair is between about 1:1 and 1:50  
     
     
         6 . A method according to any one of  claims 1  to  5 , essentially as described and with particular reference to the examples.  
     
     
         7 . A DNA construct, whenever prepared by the method of any one of  claims 1  to  5 .  
     
     
         8 . A DNA fragment comprising an overhang of at least 15 nucleotides or an end portion suitable to be converted into such an overhang.  
     
     
         9 . A DNA fragment as claimed in  claim 8 , for use in the method of  claim 1.