Enzymatic Nucleic Acid Synthesis

Abstract

The disclosure provides methods for making a polynucleotide wherein the addition of nucleotides can be physically, chemically and/or enzymatically controlled. The methods include combining a selected nucleotide, cations, an error prone or template independent DNA polymerase at a reaction site including an initiator sequence attached thereto and having a 3′ terminal nucleotide, wherein the reaction reagents can be modulated and under conditions that allow covalent addition of one or more of a selected nucleotide to the 3′ terminal nucleotide such that the selected nucleotide becomes a 3′ terminal nucleotide, and repeating the addition step until the polynucleotide is formed.

1 . A method for making a polynucleotide comprising
 (a) delivering a reaction reagent mobile phase including at least an error prone template independent DNA polymerase, a selected nucleotide triphosphate and cations along a fluidic channel to a reaction site, wherein the reaction site includes an initiator attached thereto and having a 3′ terminal nucleotide, wherein reaction reagents are present in the reaction reagent mobile phase at selected concentrations, wherein the reaction reagent mobile phase has a selected volume and a selected flow rate to achieve a selected residence time at the reaction site under conditions which covalently add one or more of the selected nucleotide to the 3′ terminal nucleotide such that the selected nucleotide becomes a 3′ terminal nucleotide,   (b) delivering an organic wash mobile phase to the reaction site at a fluid flow rate to remove the reaction reagents from the reaction site, and   (c) repeating steps (a) and (b) until the polynucleotide is formed, with the proviso that step (b) is not required to be performed after the polynucleotide is formed.   
     
     
         2 . The method of  claim 1  wherein the selected volume and selected flow rate for the reaction reagent mobile phase is determined based on reactivity of the selected nucleotide triphosphate present in the reaction reagent mobile phase. 
     
     
         3 . The method of  claim 1  wherein the selected volume and selected flow rate for the reaction reagent mobile phase differ based on the selected nucleotide triphosphate present in the reaction reagent mobile phase. 
     
     
         4 . The method of  claim 1  wherein the selected flow rate for the reaction reagent mobile phase is constant and the selected volume differs based on the selected nucleotide triphosphate present in the reaction reagent mobile phase. 
     
     
         5 . The method of  claim 1  wherein the selected volume of the reaction reagent mobile phase is constant and the selected flow rate differs based on the selected nucleotide triphosphate present in the reaction reagent mobile phase. 
     
     
         6 . The method of  claim 1  wherein the selected flow rate for the reaction reagent mobile phase is constant and the selected volume differs based on the selected nucleotide triphosphate present in the reaction reagent mobile phase and the desired number of the selected nucleotides to be added to the 3′ end of the polynucleotide. 
     
     
         7 . The method of  claim 1  wherein the selected volume of the reaction reagent mobile phase is constant and the selected flow rate differs based on the selected nucleotide triphosphate present in the reaction reagent mobile phase and the desired number of the selected nucleotides to be added to the 3′ end of the polynucleotide. 
     
     
         8 . The method of  claim 1  wherein the reaction site is a surface area on the surface of the fluidic channel. 
     
     
         9 . The method of  claim 1  wherein the selected concentration of reaction reagents in the reaction reagent mobile phase is determined by the selected nucleotide triphosphate present in the reaction reagent mobile phase. 
     
     
         10 . The method of  claim 1  wherein the reaction site is within the fluidic channel. 
     
     
         11 . The method of  claim 1  wherein the reaction site is a structure within the fluidic channel. 
     
     
         12 . The method of  claim 1  wherein the reaction site is a collection of beads within the fluidic channel. 
     
     
         13 . The method of  claim 1  wherein the reaction site is an electrode on the surface of the fluidic channel. 
     
     
         14 . The method of  claim 1  wherein the reaction site is an electrode within the fluidic channel. 
     
     
         15 . The method of  claim 1  wherein the initiator includes one or more nucleotides. 
     
     
         16 . The method of  claim 1  wherein the residence time is sufficient to limit the number of covalent additions of the selected nucleotide. 
     
     
         17 . The method of  claim 1  wherein the organic wash mobile phase is immiscible with the reaction reagent mobile phase. 
     
     
         18 . The method of  claim 1  wherein the reaction reagent mobile phase is bounded on either end by an organic wash mobile phase. 
     
     
         19 . The method of  claim 1  wherein the organic wash mobile phase inactivates the reaction reagent mobile phase at the reaction site. 
     
     
         20 . The method of  claim 1  wherein an air plug is used instead of or in addition to the organic wash mobile phase. 
     
     
         21 . The method of  claim 1  wherein an aqueous wash mobile phase is used instead of or in addition to the organic wash mobile phase. 
     
     
         22 . The method of  claim 20  wherein an aqueous wash mobile phase is used instead of or in addition to an air plug. 
     
     
         23 . The method of  claim 1  wherein a plurality of reaction reagent mobile phases bounded on either end by an organic wash mobile phase flow to the reaction site. 
     
     
         24 . The method of  claim 1  further including the step of monitoring covalent addition of the selected nucleotide. 
     
     
         25 . The method of  claim 1  wherein the error prone template independent DNA polymerase is terminal deoxynucleotide transferase. 
     
     
         26 . The method of  claim 1  wherein the cations are one or more of Zn +2 , Co +2 , Mg +2  or Mn +2 . 
     
     
         27 . The method of  claim 1  wherein the selected nucleotide is a natural nucleotide or a nucleotide analog. 
     
     
         28 . The method of  claim 1  wherein the selected nucleotide is a member selected from the group consisting of 1-borano-dATP, 2-amino-dATP, 7-deaza-7-bromo-dATP, 7-deaza-7-iodo-dATP, 7-deaza-dATP, 5-aminoallyl-dUTP, 5-bromo-dUTP, 5-iodo-dUTP, 5-propargylamino-dUTP, 5-propynyl-dUTP, 5-hydroxy-dCTP, 5-hydroxymethyl-dCTP, 5-bromo-dCTP, 5-iodo-dCTP and 5-methyl-sCTP. 
     
     
         29 . The method of  claim 1  wherein the reaction reagent mobile phase includes a buffer comprising a monovalent salt, a divalent salt, a buffering agent, and a reducing agent at a suitable pH and temperature. 
     
     
         30 . The method of  claim 1  wherein the reaction reagent mobile phase includes a buffer comprising 10 to 20 mM tris-acetate, 20 to 50 mM potassium acetate, 5 to 8 mM magnesium acetate, 0.5 to 1.0 mM DTT and with a pH of about 2 to 12 and at a temperature of about 10 and 80° C. 
     
     
         31 . The method of  claim 1  wherein the reaction reagent mobile phase includes a buffer comprising 14 mM tris-acetate, 35 mM potassium acetate, 7 mM magnesium acetate, 0.7 mM DTT and with a pH of about 7.9 and at a temperature of about 25° C. 
     
     
         32 . The method of  claim 1  wherein the initiator is attached by a cleavable moiety. 
     
     
         33 . The method of  claim 1  further comprising releasing the polynucleotide from the reaction site after the desired sequence of nucleotides has been added to the 3′ end of the polynucleotide. 
     
     
         34 . The method of  claim 1  further comprising releasing the polynucleotide from the reaction site using an enzyme, a chemical, light, heat or other suitable method or reagent. 
     
     
         35 . The method of  claim 1  further comprising releasing the polynucleotide from the reaction site, collecting the polynucleotide, amplifying the polynucleotide and sequencing the polynucleotide. 
     
     
         36 . A method for making a polynucleotide comprising
 (a) combining a selected nucleotide triphosphate, cations, an error prone or template independent DNA polymerase, and a nucleotide triphosphate inactivating enzyme at a reaction site including an initiator sequence attached thereto and having a 3′ terminal nucleotide, wherein reaction reagents are present at selected concentrations and under conditions which covalently add one or more of the selected nucleotide to the 3′ terminal nucleotide such that the selected nucleotide becomes a 3′ terminal nucleotide and under conditions which inactivate free nucleotide triphosphates until free nucleotide triphosphates are substantially inactivated, wherein a desired number of the selected nucleotide is added to the initiator sequence, and   (b) repeating step (a) until the polynucleotide is formed.   
     
     
         37 . The method of  claim 36  wherein the nucleotide inactivating enzyme is a nucleotide triphosphate degrading enzyme. 
     
     
         38 . The method of  claim 36  wherein the nucleotide triphosphate inactivating enzyme is a nucleotide triphosphate degrading enzyme that degrades nucleotide triphosphates at a rate slower than rate of addition of nucleotides by the error prone or template independent DNA polymerase. 
     
     
         39 . The method of  claim 36  wherein the nucleotide triphosphate inactivating enzyme is a nucleotide triphosphate degrading enzyme present at a concentration that degrades nucleotide triphosphates at a rate slower than rate of addition of nucleotides by the present concentration of the error prone or template independent DNA polymerase. 
     
     
         40 . The method of  claim 36  wherein the nucleotide triphosphate inactivating enzyme comprises ATP diphosphohydrolase, dNTP pyrophosphatases, dNTPases, and phosphatases. 
     
     
         41 . The method of  claim 36  wherein the concentration of nucleotide triphosphate inactivating enzyme is modulated to control addition of one or more nucleotides. 
     
     
         42 . The method of  claim 36  wherein the nucleotide triphosphate inactivating enzyme renders free nucleotide triphosphates inactive. 
     
     
         43 . The method of  claim 36  wherein the nucleotide inactivating enzyme renders free nucleotide triphosphates inactive by degradation. 
     
     
         44 . The method of  claim 36  wherein the nucleotide inactivating enzyme renders free nucleotide triphosphates inactive by polymerizing them with each other. 
     
     
         45 . The method of  claim 36  wherein the reaction conditions present a competing reaction between addition of free nucleotide triphosphates to the initiator sequence and degradation of free nucleotide triphosphates. 
     
     
         46 . The method of  claim 36  wherein the selected nucleotide is added to the reaction site including the initiator sequence having the terminal nucleotide, the error prone or template independent DNA polymerase and the nucleotide inactivating enzyme. 
     
     
         47 . The method of  claim 36  wherein the error prone or template independent DNA polymerase and the nucleotide inactivating enzyme are added to the reaction site including the initiator sequence having the terminal nucleotide, and the selected nucleotide. 
     
     
         48 . The method of  claim 36  wherein the nucleotide inactivating enzyme is added to the reaction site including the initiator sequence having the terminal nucleotide, the error prone or template independent DNA polymerase and the selected nucleotide under conditions where the polymerase is inactive, and wherein the polymerase is activated upon addition of the nucleotide inactivating enzyme. 
     
     
         49 . The method of  claim 36  wherein step (b) is repeated a plurality of times after which the reaction reagents are removed from the reaction site and additional reaction reagents are provided to the reaction site. 
     
     
         50 . The method of  claim 47  wherein the reaction reagents are removed from the reaction site and additional reaction reagents are provided to the reaction site after each round of addition. 
     
     
         51 . The method of  claim 48  wherein the reaction reagents are removed from the reaction site and additional reaction reagents are provided to the reaction site after each round of addition. 
     
     
         52 . A method for making a polynucleotide comprising
 (a) combining a selected inactive nucleotide, cations, an error prone or template independent DNA polymerase at a reaction site including an initiator sequence attached thereto and having a 3′ terminal nucleotide, activating the selected inactive nucleotide, wherein reaction reagents are present at selected concentrations and under conditions which covalently add one or more of a selected activated nucleotide to the 3′ terminal nucleotide such that the selected activated nucleotide becomes a 3′ terminal nucleotide and under conditions wherein a desired number of the selected activated nucleotide is added to the initiator sequence, and   (b) repeating step (a) until the polynucleotide is formed.   
     
     
         53 . The method of  claim 52  wherein the inactive nucleotide is rendered active by a chemical reaction, an enzyme, heat, light or pH. 
     
     
         54 . The method of  claim 52  wherein the inactive nucleotide includes a protecting group and the protecting group is removed. 
     
     
         55 . The method of  claim 54  wherein the inactive nucleotide comprises NPE-caged nucleotides or similar caged nucleotides that are removed by light, heat, an enzyme, a chemical reaction, or pH. 
     
     
         56 . The method of  claim 55  wherein the NPE-caged nucleotides comprise deoxynucleotide 5′-Triphosphate, P3-(1-(2-Nitrophenyl)Ethyl) esters. 
     
     
         57 . The method of  claim 52  wherein the selected inactive nucleotide in a nucleoside, nucleotide monophosphate, or nucleotide diphosphate form that is rendered into the active nucleotide triphosphate form by an activating enzyme such as nucleotide diphosphate kinase. 
     
     
         58 . The method of  claim 52  wherein the inactive nucleotide is rendered active at a rate which allows addition of one or more activated nucleotides. 
     
     
         59 . The method of  claim 52  wherein the inactive nucleotide is rendered active at a rate which allows addition of one or more activated nucleotides after which either the activated nucleotides or the polymerase is rendered inactive. 
     
     
         60 . The method of  claim 52  wherein the inactive nucleotide is rendered active allowing addition of one or more activated nucleotides after which the polymerase is rendered inactive. 
     
     
         61 . The method of  claim 52  wherein the polymerase is rendered inactive by a chemical reaction, divalent cations, an enzyme, heat, light or pH. 
     
     
         62 . The method of  claim 52  wherein the selected inactive nucleotide is added to the reaction site including the initiator sequence having the terminal nucleotide, and the error prone or template independent DNA polymerase and the inactive nucleotide is activated. 
     
     
         63 . The method of  claim 52  wherein step (b) is repeated a plurality of times after which the reaction reagents are removed from the reaction site and additional reaction reagents are provided to the reaction site. 
     
     
         64 . A method for making a polynucleotide comprising
 (a) combining a selected nucleotide triphosphate, cations, an inactive error prone or template independent DNA polymerase at a reaction site including an initiator sequence attached thereto and having a 3′ terminal nucleotide, activating the inactive error prone or template independent DNA polymerase, wherein reaction reagents are present at selected concentrations and under conditions which covalently add one or more of a selected nucleotide to the 3′ terminal nucleotide such that the selected nucleotide becomes a 3′ terminal nucleotide and under conditions wherein a desired number of the selected nucleotide is added to the initiator sequence, and   (b) repeating step (a) until the polynucleotide is formed.   
     
     
         65 . The method of  claim 64  wherein the inactive error prone or template independent DNA polymerase is rendered active by a chemical reaction, divalent cations, an enzyme, heat, light or pH. 
     
     
         66 . The method of  claim 64  wherein the inactive error prone or template independent DNA polymerase is rendered active by a chemical reaction, an enzyme, heat, light or pH and rendered inactive again by a chemical reaction, an enzyme, heat, light or pH after addition of the desired number of the selected nucleotide onto the initiator. 
     
     
         67 . The method of  claim 64  wherein the inactive error prone or template independent DNA polymerase includes a protecting group and the protecting group is removed. 
     
     
         68 . The method of  claim 67  wherein the protecting group comprises a chemical group that is incorporated into the polymerase and is removable by light, heat, pH, or enzymes to control the polymerase activity. 
     
     
         69 . The method of  claim 64  wherein the inactive error prone or template independent DNA polymerase is rendered active at a rate which allows addition of one or more nucleotides. 
     
     
         70 . The method of  claim 64  wherein the inactive error prone or template independent DNA polymerase is rendered active at a rate which allows addition of one or more nucleotides after which either the nucleotides or the polymerase is rendered inactive. 
     
     
         71 . The method of  claim 64  wherein the inactive error prone or template independent DNA polymerase is rendered active allowing addition of one or more nucleotides after which the polymerase is rendered inactive. 
     
     
         72 . The method of  claim 64  wherein the polymerase is rendered inactive by a chemical reaction, an enzyme, heat, light or pH. 
     
     
         73 . The method of  claim 64  wherein the inactive error prone or template independent DNA polymerase is added to the reaction site including the initiator sequence having the terminal nucleotide, and the selected nucleotide triphosphate and wherein the inactive error prone or template independent DNA polymerase is activated. 
     
     
         74 . The method of  claim 64  wherein step (b) is repeated a plurality of times after which the reaction reagents are removed from the reaction site and additional reaction reagents are provided to the reaction site. 
     
     
         75 . A method for making a polynucleotide comprising
 (a) combining a selected nucleotide triphosphate, cations, an error prone or template independent DNA polymerase at a reaction site including an initiator sequence attached thereto and having a 3′ terminal nucleotide, wherein reaction reagents are present at selected concentrations and under conditions which covalently add one or more of a selected nucleotide to the 3′ terminal nucleotide such that the selected nucleotide becomes a 3′ terminal nucleotide and wherein the error prone or template independent DNA polymerase is inactivated to terminate addition of the selected nucleotide, and   (b) repeating step (a) until the polynucleotide is formed.   
     
     
         76 . The method of  claim 75  wherein the inactive error prone or template independent DNA polymerase is rendered active by a chemical reaction, an enzyme, heat, light or pH. 
     
     
         77 . The method of  claim 75  wherein the active error prone or template independent DNA polymerase is rendered inactive by a chemical reaction, an enzyme, heat, light or pH after the addition of a desired number of the selected nucleotide and rendered active again by a chemical reaction, an enzyme, heat, light or pH for the addition of the next selected nucleotide to the 3′ terminal nucleotide of the polynucleotide. 
     
     
         78 . The method of  claim 75  wherein the inactive error prone or template independent DNA polymerase includes a protecting group and the protecting group is removed. 
     
     
         79 . The method of  claim 78  wherein the protecting group comprises a chemical group that is incorporated into the polymerase and is removable by light, heat, pH, or enzymes to control the polymerase activity. 
     
     
         80 . The method of  claim 75  wherein the error prone or template independent DNA polymerase is rendered inactive at a rate which allows addition of one or more nucleotides. 
     
     
         81 . The method of  claim 75  wherein the error prone or template independent DNA polymerase is rendered inactive at a rate which allows addition of one or more nucleotides. 
     
     
         82 . The method of  claim 75  wherein the error prone or template independent DNA polymerase is added to the reaction site including the initiator sequence having the terminal nucleotide, and the selected nucleotide triphosphate and wherein the error prone or template independent DNA polymerase is rendered inactive. 
     
     
         83 . The method of  claim 75  wherein step (b) is repeated a plurality of times after which the reaction reagents are removed from the reaction site and additional reaction reagents are provided to the reaction site. 
     
     
         84 . A method for making a polynucleotide comprising
 (a) combining a selected inactive nucleotide, cations, an inactive error prone or template independent DNA polymerase at a reaction site including an initiator sequence attached thereto and having a 3′ terminal nucleotide, activating the nucleotide and activating the error prone or template independent DNA polymerase, wherein reaction reagents are present at selected concentrations and under conditions which covalently add one or more of a selected nucleotide to the 3′ terminal nucleotide such that the selected nucleotide becomes a 3′ terminal nucleotide, and   (b) repeating step (a) until the polynucleotide is formed.   
     
     
         85 . The method of  claim 84  wherein either the active nucleotide or the active error prone or template independent DNA polymerase is rendered inactive to terminate addition of the selected nucleotide. 
     
     
         86 . The method of  claim 84  wherein the active error prone or template independent DNA polymerase is rendered inactive by a chemical reaction, an enzyme, heat, light or pH after the addition of a desired number of the selected nucleotide and rendered active again by a chemical reaction, an enzyme, heat, light or pH for the addition of the next selected nucleotide to the 3′ terminal nucleotide of the polynucleotide. 
     
     
         87 . The method of  claim 84  wherein the inactive error prone or template independent DNA polymerase is rendered active by a chemical reaction, an enzyme, heat, light or pH. 
     
     
         88 . The method of  claim 84  wherein the inactive error prone or template independent DNA polymerase includes a protecting group and the protecting group is removed. 
     
     
         89 . The method of  claim 88  wherein the protecting group comprises a chemical group that is incorporated into the polymerase and is removable by light, heat, pH, or enzymes to control the polymerase activity. 
     
     
         90 . The method of  claim 84  wherein the inactive nucleotide is rendered active by a chemical reaction, an enzyme, heat, light or pH. 
     
     
         91 . The method of  claim 90  wherein the inactive nucleotide includes a protecting group and the protecting group is removed. 
     
     
         92 . The method of  claim 91  wherein the inactive nucleotide comprises NPE-caged nucleotides or similar caged nucleotides that are removed by light, heat, an enzyme, a chemical reaction, or pH. 
     
     
         93 . The method of  claim 92  wherein the NPE-caged nucleotides comprise deoxynucleotide 5′-Triphosphate, P3-(1-(2-Nitrophenyl)Ethyl) esters. 
     
     
         94 . The method of  claim 84  wherein the selected inactive nucleotide in a nucleoside, nucleotide monophosphate, or nucleotide diphosphate form that is rendered into the active nucleotide triphosphate form by an activating enzyme such as nucleotide diphosphate kinase. 
     
     
         95 . The method of  claim 84  wherein either the inactive nucleotide or inactive error prone or template independent DNA polymerase is rendered active at a rate which allows addition of one or more nucleotides. 
     
     
         96 . The method of  claim 84  wherein step (b) is repeated a plurality of times after which the reaction reagents are removed from the reaction site and additional reaction reagents are provided to the reaction site.