METHODS AND APPARATUS FOR SYNTHESIZING NUCLEIC ACIDS
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 nova, 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 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.