Array-based enzymatic oligonucleotide synthesis

Abstract

Array-based enzymatic oligonucleotide synthesis creates a large number of polynucleotides using an uncontrolled and template independent polymerase such as terminal deoxynucleotidyl transferase (TdT). Spatial control of reaction conditions on the surface of the array allows creation of polynucleotides with a variety of arbitrary sequences. Spatial control may be implemented by removing protecting groups attached to nucleotides only at a selected location on the array or by other techniques such as location-specific regulation of enzymatic activity. The ratio of polynucleotides with protecting groups to unprotected polynucleotides used during a cycle of synthesis is adjusted to control the length of homopolymers created by the polymerase. Digital information may be encoded in the enzymatically synthesized polynucleotides. An encoding scheme for representing digital information in a nucleotide sequence accounts for homopolymers in the polynucleotides by collapsing homopolymer strings in the sequence data to a single nucleotide or to a shorter homopolymer.

1 . A method of synthesis of multiple polynucleotides having different sequences using a template independent polymerase, the method comprising:
 (a) incubating an array covered with a plurality of initiators for a reaction time at a reaction temperature with a reaction reagent solution comprising the template independent polymerase and a selected nucleotide such that the template independent polymerase adds a variable number of nucleotides to 3′ ends of the initiators, the variable number of nucleotides based on a ratio of the template independent polymerase to the initiators to the selected nucleotide;   (b) activating polymerization at a selected location on the array;   (c) after the reaction time, stopping activity of the template independent polymerase;   (d) delivering a wash solution to the array to remove the reaction reagent solution; and   (e) iteratively repeating steps (a), (b), (c), and (d) until the multiple polynucleotides are formed, wherein during iterations of repeating steps (a), (b), (c), and (d) the selected nucleotide and the selected location both change at least once.   
     
     
         2 . The method of  claim 1 , wherein a target value for the variable number of nucleotides is predetermined prior to the incubating. 
     
     
         3 . The method of  claim 1 , wherein the activating the polymerization at the selected location comprises changing an oxidation state of a metal cofactor of the template independent polymerase at the selected location, removing a protecting group from the template independent polymerase at the selected location, or adding the selected nucleotide at the selected location. 
     
     
         4 . The method of  claim 1 , wherein the activating the polymerization at the selected location comprises inhibiting the polymerization at locations on the array other than the selected location by physically blocking the template independent polymerase, the selected nucleotide, or both from accessing the locations on the array other than the selected location during the incubating. 
     
     
         5 . The method of  claim 1 , wherein stopping activity of the template independent polymerase comprises heating the reaction reagent solution to at least 60° C., denaturing the template independent polymerase with a surfactant, adding a chelator to the reaction reagent solution, or changing an oxidation state of a metal cofactor for the template independent polymerase to an oxidation state other than +2. 
     
     
         6 . The method of  claim 1 , wherein the activating the polymerization at the selected location comprises changing an oxidation state of a metal cofactor of the template independent polymerase at the selected location to +2 and stopping activity of the template independent polymerase comprises changing the oxidation state of the metal cofactor to an oxidation state other than +2. 
     
     
         7 . The method of  claim 1 , wherein one or more of the initiators has a random sequence of nucleotides, has a sequence of nucleotides that is cleaved by a restriction endonuclease, or has a sequence of nucleotides that includes a primer binding site. 
     
     
         8 . The method of  claim 1 , wherein the ratio of the template independent polymerase to the initiators to the selected nucleotide is 10 units of template independent polymerase for each 10 pmol of initiators and 0.5 mmol of the selected nucleotide. 
     
     
         9 . The method of  claim 7 , wherein the variable number of nucleotides is about 5. 
     
     
         10 . The method of  claim 1 , wherein the reaction reagent solution comprises the selected nucleotide only in unprotected form. 
     
     
         11 . A method of decoding digital information in a polynucleotide synthesized by a template independent polymerase comprising:
 receiving an output string from sequencing of the polynucleotide that encodes the digital information according to a nucleotide sequence string generated by an encoding scheme, wherein synthesis with the template independent polymerase causes the polynucleotide to include a homopolymer that is not present in the nucleotide sequence string;   converting the output string to a collapsed output string by replacing the homopolymer in the output string with fewer nucleotides; and   decoding the collapsed output string using the encoding scheme to recover the digital information.   
     
     
         12 . The method of  claim 11 , wherein the nucleotide sequence string generated by the encoding scheme excludes homopolymers and the fewer nucleotides is a single nucleotide. 
     
     
         13 . The method of  claim 11 , wherein the nucleotide sequence string generated by the encoding scheme includes homopolymers and a number of the fewer nucleotides is based on a length of the homopolymer. 
     
     
         14 . The method of  claim 13 , wherein the number of the fewer nucleotides is further based on a variable number of nucleotides added by the template independent polymerase during a single synthesis cycle. 
     
     
         15 . The method of  claim 13 , wherein a cutoff used for determining the number of the fewer nucleotides is a midpoint between one extension length and a next extension length. 
     
     
         16 . The method of  claim 11 , further comprising identifying a length of the homopolymer in the output string, wherein decoding the collapsed output string is based on a number of nucleotides in the homopolymer. 
     
     
         17 . The method of  claim 16 , further comprising recording a ratio of the number of nucleotides in the output string that comprise the homopolymer and a resulting number of nucleotides in the collapsed output string. 
     
     
         18 . The method of  claim 11 , further comprising sequencing the polynucleotide encoding the digital information thereby generating the output string. 
     
     
         19 . The method of  claim 11 , further comprising:
 generating the nucleotide sequence string from the digital information using the encoding scheme;   synthesizing the polynucleotide encoding the digital information according to the nucleotide sequence string with the template independent polymerase; and   sequencing the polynucleotide encoding the digital information thereby generating the output string.   
     
     
         20 . The method of  claim 19 , wherein the encoding scheme includes homopolymers and synthesizing the polynucleotide comprises controlling extension length of a selected nucleotide to create a nucleotide sequence that encodes a homopolymer.