Methods and apparatus for synthesizing nucleic acids

Abstract

The invention provides improved methods for synthesizing polynucleotides, such as DNA and RNA, using enzymes and specially designed nucleotide analogs. Using the methods of the invention, specific sequences of polynucleotides can be synthesized de novo, base by base, in an aqueous environment, without the use of a nucleic acid template. Because the nucleotide analogs have an unmodified 3′ OH, i.e., as found in “natural” deoxyribose and ribose molecules, the analogs result in natural polynucleotides suitable for incorporation into biological systems.

1 . A method for non-template dependent oligonucleotide synthesis, the method comprising:
 exposing a nucleic acid strand to a terminal transferase enzyme capable of incorporating a single nucleotide and remaining bound to the strand and preventing further nucleotide incorporation until exposed to a releasing agent or releasing condition.   
     
     
         2 . The method of  claim 1 , wherein said single nucleotide is a nucleotide analog. 
     
     
         3 . The method of  claim 1 , wherein the terminal transferase enzyme is a modified terminal deoxynucleotidyl transferase (TdT) enzyme. 
     
     
         4 . The method of  claim 3 , wherein the modification comprises a mutation allowing the covalent attachment of a nucleotide analog to the TdT enzyme. 
     
     
         5 . The method of  claim 1 , wherein the releasing reagent comprises a salt buffer or a denaturant or a reducing agent or elevated pH. 
     
     
         6 . The method of  claim 1 , wherein the releasing condition is a temperature increase or agitation. 
     
     
         7 . A nucleotidyl transferase enzyme modified to remain bound to a nucleic acid strand after incorporating a nucleotide into the nucleic acid strand and to prevent subsequent incorporation of nucleotide analogs until released by a releasing agent or releasing condition. 
     
     
         8 . The nucleotidyl transferase enzyme of  claim 7 , wherein the nucleotidyl transferase enzyme incorporates the nucleotide analog into the nucleic acid strand in an absence of a nucleic acid template. 
     
     
         9 . The nucleotidyl transferase enzyme of  claim 7 , wherein the nucleotidyl transferase enzyme is a modified terminal deoxynucleotidyl transferase (TdT) enzyme. 
     
     
         10 . The nucleotidyl transferase enzyme of  claim 9 , wherein the modification comprises a mutation to allowing the covalent attachment of a nucleotide analog 
     
     
         11 . The nucleotidyl transferase enzyme of  claim 7 , wherein the releasing reagent comprises a salt buffer or a denaturant or a reducing agent or elevated pH. 
     
     
         12 . The nucleotidyl transferase enzyme of  claim 7 , wherein the releasing condition is a temperature increase or agitation. 
     
     
         13 . A method for non-template dependent oligonucleotide synthesis, the method comprising:
 exposing a covalent blocker-nucleic acid strand complex to an exonuclease to remove un complexed, unmodified nucleic acid strands;   removing the exonuclease; and   exposing the covalent blocker-nucleic acid strand complex to a releasing agent or a releasing condition.