Method and kit for template-independent nucleic acid synthesis

Abstract

A method for synthesizing a nucleic acid includes providing an initiator having a 3′ end having an unprotected hydroxyl group, providing a nucleic acid polymerase having at least a conservative catalytic polymerase domain of a family-B DNA polymerase, providing a nucleotide monomer, and exposing the initiator to the nucleotide monomer in the presence of the nucleic acid polymerase and a metal cofactor which is a bivalent cation, and in the absence of a template, such that the nucleotide monomer is incorporated to the initiator. The kit includes the initiator, the nucleic acid polymerase, and the nucleotide monomer, and is used according to the method.

1 - 20 . (canceled) 
     
     
         21 . A kit for nucleic acid synthesis, comprising:
 an initiator having a 3′ end having an unprotected hydroxyl group;   a nucleotide monomer;   a nucleic acid polymerase having at least a conservative catalytic polymerase domain of a family-B DNA polymerase for incorporation of the nucleotide monomer to the initiator.   
     
     
         22 . The kit of  claim 21 , wherein the nucleic acid synthesis is template-independent, and the incorporation is performed in absence of a template. 
     
     
         23 . The kit of  claim 21 , wherein the initiator has a non-self complementary sequence or a non-self complementarity forming sequence. 
     
     
         24 . The kit of  claim 21 , wherein the initiator is in a single-stranded form and comprises at least five nucleotides. 
     
     
         25 . The kit of  claim 21 , further comprises a solid support linked to a 5′ end of the initiator. 
     
     
         26 . The kit of  claim 25 , wherein the solid support is selected from the group consisting of a microarray, a bead, a column, an optical fiber, a wipe, nitrocellulose, nylon, glass, quartz, a diazotized membrane, a silicone, polyformaldehyde, cellulose, cellulose acetate, paper, a ceramic, a metal, a metalloid, a semiconductor material, a magnetic particle, a plastic, a gel-forming material, a gel, a nanostructured surface, a nanotube, a nanoparticle, and any combination thereof. 
     
     
         27 . The kit of  claim 21 , wherein the nucleotide monomer has a phosphate group selected from the group consisting of a monophosphate, a diphosphate, a triphosphate, a tetraphosphate, a pentaphosphate, a hexaphosphate, and any combination thereof. 
     
     
         28 . The kit of  claim 21 , wherein the nucleotide monomer has a removable blocking moiety selected from the group consisting of a 3′-O-blocking moiety, a base blocking moiety, and any combination thereof 
     
     
         29 . The kit of  claim 21 , the family-B DNA polymerase is selected from the group consisting of a bacterial family-B DNA polymerase, a eukaryotic family-B DNA polymerase, an archaeal family-B DNA polymerase, and a viral family-B DNA polymerase. 
     
     
         30 . The kit of  claim 21 , wherein the family-B polymerase is a Thermococcaceae DNA polymerase. 
     
     
         31 . The kit of  claim 21 , wherein the family-B polymerase is a  Thermococcus  DNA polymerase or a  Pyrococus  DNA polymerase. 
     
     
         32 . The kit of  claim 21 , wherein the family-B polymerase is selected from the group consisting of a family-B DNA polymerase of Thermococcus kodakaraensis KOD1, a family-B DNA polymerase of  Pyrococcus furious  (Pfu), and a family-B DNA polymerase of  Thermococcus litoralis  (Vent). 
     
     
         33 . The kit of  claim 21 , wherein the nucleic acid polymerase has a 3′ to 5′ exonuclease domain and the 3′ to 5′ exonuclease domain of the family-B DNA polymerase is inactivated. 
     
     
         34 . The kit of  claim 21 , wherein the incorporation is performed in the presence of a metal cofactor which is a bivalent cation. 
     
     
         35 . The kit of  claim 34 , the bivalent cation is selected from the group consisting of Mg 2+ , Ca 2+ , Sr 2+ , Ba 2+ , Mn 2+ , Co 2+ , Fe 2+ , Ni 2+ , Cu 2+ , Zn 2+ , and any combination thereof. 
     
     
         36 . The kit of  claim 34 , wherein the incorporation is performed in the presence of different bivalent cations. 
     
     
         37 . A method for synthesizing a nucleic acid, comprising:
 providing a kit of  claim 21 ; and   incorporating the nucleotide monomer to the initiator by the nucleic acid polymerase.   
     
     
         38 . The method of  claim 37 , wherein the nucleotide monomer is incorporated to the initiator by the nucleic acid polymerase at a temperature ranging from 10° C. to 90° C. 
     
     
         39 . The method of  claim 37 , wherein the nucleotide monomer is incorporated to the initiator by the nucleic acid polymerase at a pH of not less than 8.0. 
     
     
         40 . The method of  claim 37 , wherein the nucleotide monomer is incorporated to the initiator by the nucleic acid polymerase in the absence of a template and in the presence of a metal cofactor.