WO2021133713A1

WO2021133713A1 - Method and kit for template-independent nucleic acid synthesis

Info

Publication number: WO2021133713A1
Application number: PCT/US2020/066336
Authority: WO
Inventors: Cheng-Yao Chen
Original assignee: Individual
Current assignee: Individual
Priority date: 2019-12-23
Filing date: 2020-12-21
Publication date: 2021-07-01
Anticipated expiration: 2022-06-23
Also published as: CA3162466C; US20230220436A1; KR102478977B1; CN114846149B; TW202126816A; IL294266A; JP7511100B2; TWI766485B; EP4081648A1; US11591629B2; AU2020412607A1; CA3162466A1; EP4081648A4; AU2020412607C1; JP2022554037A; US20210189447A1; KR20220113522A; CN114846149A; AU2020412607B2

Abstract

A method for synthesizing a nucleic acid includes providing an initiator having an unprotected nucleoside base and a 3' hydroxyl group at a 3' terminus thereof, 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 at least one type of metal cofactors which are divalent cations, and in the absence of a template, such that the nucleotide monomer is incorporated to the initiator. A kit includes the initiator, the nucleic acid polymerase, the nucleotide monomer, and the at least one type of metal cofactors, and is used according to the method.

Description

METHOD AND KIT FOR TEMPLATE-INDEPENDENT NUCLEIC ACID SYNTHESIS

CROSS-REFERENCE TO RELATED APPLICATION

This application claims priority of U.S. Patent Application No.16/725,420, filed on December 23, 2019, the entire content of which is incorporated herein by reference .

FIELD

The disclosure relates to a method and a kit for nucleic acid synthesis, more particularly to a method and a kit for template-independent nucleic acid synthesis .

BACKGROUND

De novo DNA synthesis dispensing with a DNA template has been developed during past decades. Among the currently available template-independent DNA synthesis methods, the phosphoramidite-based chemical DNA synthesis has been well-known since early 1980's, but basically has remained unchanged since then. The phosphoramidite-based chemical DNA Synthesis requires four consecutive reaction steps, including de-blocking, coupling, capping, and oxidation steps, to add one nucleoside to another nucleoside tethered to a solid support. However, one of the major drawbacks of the phosphoramidite-based chemical DNA synthesis is inevitable use of hazardous chemicals in the aforesaid reaction steps.

Due to growing demand for environmental protection, green technology applicable to DNA synthesis has drawn attention of researchers. Therefore, enzymatic DNA synthesis, which can greatly reduce use of hazardous chemicals, seems promising since such synthesis has merits such as longer strand generation, a lower error rate, a faster cycle time, a lower production cost,etc.

Speaking of template-independent enzymatic DNA synthesis, terminal deoxynucleotidyl transferase (TdT) has been found to be a template-independent DNA polymerase that adds all four deoxynucleoside triphosphates (dNTPs) to the 3^' termini of DNA strands. TdT belongs to the X Family of low-fidelity DNA polymerases. The TdT-based DNA synthesis requires only two reaction steps, namely, a single-nucleotide addition by TdT and subsequent removal of the 3'-protective group from the extended 3'-end of the single-stranded DNA strand being synthesized. Even though TdT and its homologs have been applied to numerous DNA synthesis platforms, template-independent enzymatic DNA synthesis based on TdT can be hardly commercialized due to unsatisfactory product length, reagent reusability, cycle time, and so forth.

SUMMARY

Therefore, an object of the disclosure is to provide amethod and a kit for synthesizing anucleic acid,which can alleviate at least one of the drawbacks of the prior art.

The method includes providing an initiator having an unprotected nucleoside base and a 3' hydroxyl group at a 3' terminus thereof, 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 at least one type of metal cofactors,which are divalent cations, and in the absence of a template, such that the nucleotide monomer is incorporated to the initiator. The kit includes an initiator as described above, a nucleic acid polymerase as described above, at least one type of metal cofactors as described above, and a nucleotide monomer as described above. The kit is used according to a method as described above. BRIEF DESCRIPTION OF THE DRAWINGS

Other features and advantages of the disclosure will become apparent in the following detailed description of the embodiments with reference to the accompanying drawings, of which: FIG. 1 is a de novo nucleic acid synthesis scheme using family-B DNA polymerases;

FIG. 2 is an image ofdenaturing urea-polyacrylamide gel showing products of template-independent nucleic acid synthesis obtained at different reaction temperatures using KOD1^exo_ DNA polymerase, in which the symbol "S" indicates the position of initiator DNA; FIG. 3 is an image of denaturing urea-polyacrylamide gel showing products of template-independent nucleic acid synthesis obtained at different reaction temperatures using Vent^exo_ DNA polymerase, in which the symbol "S" indicates the position of initiator DNA; FIG. 4 is an image of denaturing urea-polyacrylamide gel showing products of template-independent nucleic acid synthesis obtained at different reaction temperatures using Pfu^exo_ DNA polymerase, in which the symbol "S" indicates the position of initiator DNA; and FIG. 5 is an image of denaturing urea-polyacrylamide gel showing products of template-independent nucleic acid synthesis obtained, in the presence of Mg²⁺ only or in combination with Mn²⁺, using Vent^exo_ DNA polymerase, KOD1^exo_ DNA polymerase, or Pfu^exo_ DNA polymerase, in which the symbol "S" indicates the position of initiator DNA, and the symbols "V", "K" and "P" stand for Vent^exo“ DNA polymerase, KOD1^exo_ DNA polymerase, and Pfu^exo_ DNA polymerase, respectively.

DETAILED DESCRIPTION It is to be understood that, if any prior art publication is referred to herein, such reference does not constitute an admission that the publication forms a part of the common general knowledge in the art, in Taiwan or any other country.

For the purpose of this specification, it will be clearly understood that the word "comprising" means "including but not 1imited to", and that the word "comprises" has a corresponding meaning.

Unless defined otherwise, all technical and scientific terms used herein have the meaning commonly understood by a person skilled in the art to which the present disclosure belongs. One skilled in the art will recognize many methods and materials similar or equivalent to those described herein, which could be used in the practice of the present disclosure. Indeed, the present disclosure is in noway limited to themethodsandmaterials described.

The applicant surprisingly found that family-B DNA polymerases, which are well-known as template-dependent DNA polymerases, can be used to conduct template-independent nucleic acid synthesis (i.e. denovo nucleic acid synthesis).Referring to FIG.

1, a de novo nucleic acid synthesis scheme using family-B DNA polymerases is illustrated.

Family-B DNA polymerases (also known as type-B DNA polymerases) are replicative and repair polymerases that intrinsically have a catalytic polymerase domain and a 3' to 5' exonuclease domain, and can be found in bacteria, archaea, eukaryotes, and viruses. The term "catalytic polymerase domain" refers to a structural portion or region ofthe amino acid sequence ofa protein which possesses the catalytic DNA/RNA polymerase activity of the protein, and which does not contain other catalytic activity, such asediting activity (e.g. proofreading activity ofa 3'to 5'exonuclease domain), activity for excision of Okazaki primers during replication, and activity for interaction with other proteins. The catalytic polymerase domains of family-B DNA polymerases have a common overall architecture, which resembles a righthand and consists of thumb,palm, and fingers domains. The most conserved region is the palm domain, which contains the catalytic site.

Examples of family-B DNA polymerases include, but are not limited to, bacterial family-B DNA polymerases (e.g. Pol II),eukaryotic family-B DNA polymerases (e.g. Pol α, Pol d, and Pol e, and Pol z), archaeal family-B DNA polymerases (e.g. Pol B, Pol BI, Pol BII, Pol Bill, 9°N, Kodl, Pfu, Tgo, and Vent), and viral family-B DNA polymerases (e.g. HSV-1, RB69, T4, B103 and F29).

Therefore, the present disclosure provides a method for synthesizing a nucleic acid, which includes: providing an initiator having an unprotected nucleoside base and a 3' hydroxyl group at a 3' terminus thereof; 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 at least one type of metal cofactors, which are divalent cations, and in the absence of a template, such that the nucleotide monomer is incorporated to the initiator.

The terms "nucleic acid", "nucleic acid sequence", and "nucleic acid fragment" as used herein refer to a deoxyribonucleotide or ribonucleotide sequence in single-stranded or double-stranded form, and comprise naturally occurring nucleotides or artificial chemical mimics. The term "nucleic acid" as used herein is interchangeable with the terms "oligonucleotide", "polynucleotide", "DNA", "RNA", "gene", "cDNA", and "mRNA" in use. Generally, a "template" is a polynucleotide that contains the target nucleotide sequence. In some instances, the terms "target sequence", "template polynucleotide", "target nucleic acid", "target polynucleotide", "nucleic acid template", "template sequence", and variations thereof, are used interchangeably. Specifically, the term "template" refers to a strand of nucleic acid on which a complementary copy is synthesized from nucleotides or nucleotide analogs through the activity of a template-dependent nucleic acid polymerase. Within a duplex, the template strand is,by convention,depicted and described as the "bottom" strand. Similarly, the non-template strand is often depicted and described as the "top" strand. The "template" strand may also be referred to as the "sense" strand, and the non-template strand as the "antisense" strand. The term "incorporated" or "incorporation" refers to becoming a part of a nucleic acid. There is a known flexibility in the terminology regarding incorporation of nucleic acid precursors.For example, the nucleotide dGTP is a deoxyribonucleoside triphosphate. Upon incorporation into DNA, dGTP becomes dGMP, that is, a deoxyguanosine monophosphate moiety.Although DNA does not include dGTP molecules, one may say that one incorporates dGTP into DNA.

The term "initiator" refers to a mononucleoside, a mononucleotide, an oligonucleotide, a polynucleotide, or modified analogs thereof, from which a nucleic acid is to be synthesized de novo. The term "initiator" may also refer to a Xeno nucleic acids (XNA) or a peptide nucleic acid (PNA) having a 3'-hydroxyl group. According to the present disclosure, the initiator may have a sequence selected from a non-self complementary sequence and a non-self complementarity forming sequence. The term "self complementary" means that a sequence (e.g. a nucleotide sequence or a PNA sequence) folds back on itself (i.e. a region of the sequence binds or hybridizes to another region of the sequence), creating a duplex, double-strand like structure which can serve as a template for nucleic acid synthesis. Depending on how close together the complementary regions of the sequence are, the strand may form, for instance, hairpin loops, junctions, bulges or internal loops . The term "self complementarity forming" is used to describe a sequence (e.g. a nucleotide sequence, XNA, or a PNA sequence) from which a complementary extended portion is formed when such sequence serves as a template (namely, a self-complementary sequence is formed based on such sequence serving as a template).For instance, the self complementarity forming sequence may be "ATCC". When the "ATCC" sequence serves as a template, an extended portion "GGAT"complementary to such sequence is formed from such sequence (i.e.a self-complementary sequence "ATCCGGAT" is formed).

The term "conservative" or "conserved" is used to describe domains containing amino acid residues that are the same among a plurality of proteins having the same structure and/or function. A region of conserved amino acid residues may be important for protein structure or function. Thus, contiguous conserved amino acid residues as identified in a three-dimensional protein may be important for protein structure or function.

For instance, as reported in Alba (2001), Genome Biology, 2 (1):reviews3002.1 to reviews3002.4, family-B DNA polymerases have Regions I and II that form part of the active sites of the catalytic polymerase domain, and that may respectively contain conserved amino acid residues "DT" and "SLYPS".Region I may span amino acid residues 512 to 582, amino acid residues 513 to 582 or 583, or amino acid residues 535 to 604. Region II may span amino acid residues 375 to 441 or 442, or amino acid residues 397 to 464.

According to the present disclosure, the nucleic acid polymerase may further have a 3' to 5' exonuclease domain and may be a family-B DNA polymerase 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. In some embodiments, the family-B DNA polymerase is selected from the group consisting of a family-B DNA polymerase of Thermococcus kodakaraensis KOD1, a family-B DNA polymerase of Pyrococus furious (Pfu), and a family-B DNA polymerase of Thermococcus litoralis (Vent).

According to the present disclosure, the 3' to 5' exonuclease domain of the family-B DNA polymerase may be inactivated.Alternatively, the 3' to 5'exonuclease activity of the family-B DNA polymerase may be reduced. Still alternatively, the 3' to 5' exonuclease domain of the family-B DNA polymerase may remain unchanged, and an inhibitor may be used to inhibit the 3' to 5' exonuclease domain of the family-B DNA polymerase during the method of the present disclosure.

According to the present disclosure, alternatively, the nucleic acid polymerase may only have the aforesaid conservative catalytic polymerase domain. In some embodiments, the nucleic acid polymerase is designed to only have the aforesaid conservative catalytic polymerase domain originally. In other embodiments, the nucleic acid polymerase was originally a family-B DNA polymerase having a 3' to 5' exonuclease domain, and such domain has been removed from the nucleic acid polymerase.

In some embodiments, the initiator is in single-stranded form. In some embodiments, the initiator has at least five nucleotides. In an exemplary embodiment, the initiator has forty-five nucleotides.

In some embodiments, the initiator is exposed to the nucleotide monomer at a temperature ranging from 10°C to 90°C, and/or the initiator is exposed to the nucleotide monomer at a pH of not less than 8.0 (for instance, 8.8). According to the present disclosure, the nucleotide monomer may be a natural nucleic acid nucleotide whose constituent elements are a sugar, a phosphate group and a nitrogen base. The sugar may be ribose in RNA or 2'-deoxyribose in DNA. Depending on whether the nucleic acid to be synthesized is DNA or RNA, the nitrogenbase is selected from adenine,guanine, uracil, cytosine and thymine. Alternatively, the nucleotide monomermay be a nucleotidewhich ismodified in at least one of the three constituent elements. By way of example, the modification can take place at the level of the base, generating a modified product (such as inosine, methyl-5-deoxycytidine, deoxyuridine, dimethylamino-5-deoxyuridine, diamino-2,6-purine or bromo-5-deoxyuridine, and any other modified base which permits hybridization), at the level of the sugar (for example,replacement ofa deoxyriboseby an analog), or at the level of the phosphate group (for example, boronate, alkylphosphonate, or phosphorothioate derivatives).

According to the present disclosure, the nucleotide monomer may have a phosphate group selected from a monophosphate, a diphosphate, a triphosphate, a tetraphosphate , a pentaphosphate, and a hexaphosphate .

According to the present disclosure, the metal cofactor may be selected from the group consisting of Mg²⁺, Ca²⁺, Sr²⁺, Ba²⁺, Mn²⁺, Co²⁺, Fe²⁺, Ni²⁺, Cu²⁺, Zn²⁺, and their combinations thereof. In an exemplary embodiment, the cofactor isMg²⁺.In another embodiment, the cofactor is a combination of Mg²⁺ and Mn²⁺. According to the present disclosure, the nucleotide monomer may have a removable blocking moiety. Examples of the removable blocking moiety include, but are not limited to, a 3'-O-blocking moiety, a base blocking moiety, and a combination thereof. The nucleotide monomer having a removable blocking moiety is also referred to as a reversible terminator. Therefore, the nucleotide monomer having the 3'-O-blocking moiety is also referred to as 3'-blocked reversible terminator or a 3 -O-modified reversible terminator, and the nucleotide monomer having the base blocking moiety is also referred to as a 3'-unblocked reversible terminator or a 3'-OH unblocked reversible terminator .

As used herein, the term "reversible terminator" refers to a chemically modified nucleotide monomer. When such a reversible terminator is incorporated into a growing nucleic acid by a polymerase, it blocks the further incorporation of a nucleotide monomer by the polymerase. Such "reversible terminator" base and a nucleic acid can be deprotected by chemical or physical treatment, and following such deprotection, the nucleic acid can be further extended by a polymerase. Examples of the 3'-0-blocking moiety include, but are not limited to, O-azidomethyl, O-amino, O-allyl, O-phenoxyacetyl, O-methoxyacetyl, O-acetyl,

0-(p-toluene)sulfonate, O-phosphate, 0-nitrate, 0-[4-methoxy]-tetrahydrothiopyranyl , 0- tetrahydrothiopyranyl , 0- [5-methyl]- tetrahydrofuranyl , 0-[2-methyl,4-methoxy]- tetrahydropyranyl , 0-[5-methyl]-tetrahydropyranyl, and 0-tetrahydrothiof uranyl, 0-2-nitrobenzyl, 0-methyl, and 0-acyl.

Examples of the 3'-unblocked reversible terminators include, but are not limited to, 7-[(S)-1- (5-methoxy-2-nitrophenyl)-2,2-dimethyl- propyloxy]methyl-7-deaza-dATP, 5-[(S)-1- (5-methoxy- 2-nitrophenyl)-2 ,2-dimethyl-propyloxy]methyl-dCTP,

1-(5-methoxy-2-nitrophenyl) -2,2-dimethyl-propyloxy] methyl-7-deaza-dGTP, 5-[(S)-1- (5-methoxy-2- nitrophen-yl)-2,2-dimethyl-propyloxy] methyl-dUTP, and 5-[(S)-1-(2-nitrophenyl)-2,2-dimethyl- propyloxy]methyl-dUTP .

According to the present disclosure, the base blocking moiety may be a reversible dye-terminator. Examples of the reversible dye-terminator include, but are not limited to, a reversible dye-terminator of Illumina NovaSeq, a reversible dye-terminator of

Illumina NextSeq, a reversible dye-terminator of Illumina MiSeq, a reversible dye-terminator of Illumina HiSeq, a reversible dye-terminator of Illumina Genome Analyzer IIX, a lightning terminator of LaserGen, and a reversible dye-terminator of Helicos Biosciences Heliscope. Since the reversible terminators are well-known to and commonly used by those skilled in the art, further details of the same are omitted herein for the sake of brevity. Nevertheless, applicable 3'-blocked reversible terminators, applicable 3'-unblocked reversible terminators, and applicable conditions for protection and deprotection (i.e. conditions for adding and eliminating the removable blocking moiety) can be found in, for example, Gardner et al. (2012), Nucleic Acids Research, 40(15):7404-7415, Litosh et al (2011), Nucleic Acids Research, 39(6):e39, and Chen et al. (2013), Genomics Proteomics Bioinformatics, 11:34-40.

According to the present disclosure, the initiator may be linked to a solid support and have a 5' end linked to the solid support. The initiator may be directly attached to the support, or may be attached to the support via a linker.

According to the present disclosure, examples of the solid support include, but are not limited to, microarrays, beads (coated or non-coated), columns, optical fibers, wipes, nitrocellulose, nylon, glass, quartz, diazotized membranes (paper or nylon), silicones, polyformaldehyde, cellulose, cellulose acetate, paper, ceramics, metals, metalloids, semiconductive materials, magnetic particles, plastics (such as polyethylene, polypropylene, and polystyrene, gel-forming materials [such as proteins (e.g., gelatins), lipopolysaccharides, silicates, agarose, polyacrylamides, methylmethracrylate polymers], sol gels, porous polymer hydrogels, nanostructured surfaces, nanotubes (such as carbon nanotubes), and nanoparticles (such as gold nanoparticles or quantum dots).

In addition, the present disclosure provides a kit for synthesizing a nucleic acid, which includes the aforesaid initiator, the aforesaid nucleic acid polymerase, the aforesaid nucleotide monomer, and the aforesaid at least one type of divalent cations. The kit is used according to the method of the present disclosure .

The disclosure will be further described by way of the following examples. However, it should be understood that the following examples are solely intended for the purpose of illustration and should not be construed as limiting the disclosure in practice. EXAMPLES Example 1.Template-independent nucleic acid synthesis using family-B DNA polymerase of Thermococcus kodakaraensisKOO1 A synthesis reaction mixture was prepared using suitable amounts of the following ingredients: a single-stranded initiator that has a nucleotide sequence of SEQ ID NO: 1 and a 3' end possessing an unprotected hydroxyl group and a 5' end labeled with fluorescein amidite (FAM); deoxynucleoside triphosphates (dNTPs) serving as nucleotide monomers, including dATP, dGTP, dCTP, and dTTP; a family-B DNA polymerase of Thermococcus kodakaraensisKOD1 that has an inactivated 3' to 5' exonuclease domain and that is referred to as KOD1^exo_ DNA polymerase; and a Tris-HCl buffer (pH 8.8). Specifically, the synthesis reaction mixture contained 100 nM of the initiator, 100 mM of the dNTPs, and 200 nM of KOD1^exo_ DNA polymerase. KOD1^exo_ DNA polymerase was prepared as follows. A gene construct encoding a family-B DNA polymerase of Thermococcus kodakaraensis KOD1 (intein-free and having a normal 3' to 5' exonuclease domain) was synthesized by Genomics BioSci & Tech Co. (New Taipei City, Taiwan). To obtain KOD1^exo_ DNA polymerase, the inactivation of the conservative 3' to 5' exonuclease domain was achieved by changing Asp¹⁴¹ to Ala (D141A) and Glu¹⁴³ to Ala (E143A), i.e. modifying the conserved amino residues "DIE" of the conservative 3' to 5' exonuclease domain. Specifically, to accomplish the amino acid modifications "D141A" and "E143A", the corresponding nucleotide residues on the aforesaid gene construct were subjected to site-directed mutagenesis using Q5 Site-directed Mutagenesis Kit (New England Biolabs, Ipswich, MA, USA). The resulting mutagenized gene construct was expressed in BL21(DE3) cells, and the expressed protein was purified using Akta Pure FPLC system (GE Healthcare Life Sciences, Marlborough, MA, USA) through HisTrap Q and Heparin columns sequentially. KOD1^exo_ DNA polymerase thus obtained has an amino acid sequence of SEQ ID NO: 2. 10 pL of the nucleic acid synthesis reaction mixture was preincubated for 2 minutes at one of the following temperatures: 10°C, 20°C, 30°C, 35°C, 40°C, 45°C, 50°C, 55°C, 60°C, 70°C, 80°C, and 90°C.

Subsequently, a suitable amount of Mg²⁺ serving as cofactors were added into the respective reaction mixture to initiate the template-independent nucleic acid synthesis, which was allowed to proceed for 5 minutes. The synthesis was terminated by adding 10 pL of 2X quench solution (containing 95% de-ionized formamide and 25 mM ethylenediaminetetraacetic acid (EDTA)).

The resulting synthesis reaction products were subjected to denaturation at 98°C for 10 minutes. Subsequently, the synthesis reaction products were analyzed by a 15% denaturing urea-polyacrylamide gel. The synthesis reaction products on the gel were visualized by Amersham Typhoon Imager (GE Healthcare Life Sciences, Marlborough, MA, USA).

Results:

As shown in FIG. 2, KOD1^exo_ DNA polymerase is able to perform the template-independent nucleic acid synthesis at each of the temperatures tested, thereby indicating that a family-B DNA polymerase can be used to synthesize a nucleic acid in the absence of a template. Example 2.Template-independent nucleic acid synthesis using family-B DNA polymerase of Thermococcus litoralis (Vent)

Template-independent nucleic acid synthesis and analysis of the reaction products were conducted generally according to the procedures set forth in Example 1, except for use of a family-B DNA polymerase of Thermococcus litoralis (Vent) which has an inactivated 3' to 5' exonuclease domain and is referred to as Vent^exo" DNA polymerase accordingly. Vent^exo_ DNA polymerase was prepared following the same procedure as that for preparing KOD1^exo_ DNA polymerase (see Example 1), except that a gene construct encoding a family-B DNA polymerase of Thermococcus litoralis (intein-free and having a normal 3' to 5' exonuclease domain) was used. Vent^ex°^" DNA polymerase has an amino acid sequence of SEQ ID NO: 3.

Results: As shown in FIG. 3, Vent^exo_ DNA polymerase is able to perform the template-independent nucleic acid synthesis at each of the temperatures tested, thereby indicating that a family-B DNA polymerase can be used to synthesize a nucleic acid in the absence of a template.

Example 3.Template-independent nucleic acid synthesis using family-B DNA polymerase of Pyrococus furious (Pfu) Template-independent nucleic acid synthesis and analysis of the reaction products were conducted generally according to the procedures set forth in Example 1, except for use of a family-B DNA polymerase of Pfu which has an inactivated 3' to 5' exonuclease domain and is referred to as Pfu^exo_ DNA polymerase accordingly. Pfu^exo_ DNA polymerase was prepared following the same procedure as that for preparing KOD1^exo_ DNA polymerase (see Example 1), except that a gene construct encoding a family-B DNA polymerase of Pfu (intein-free and having a normal 3' to 5' exonuclease domain)was used.Pfu^exo_ DNA polymerase has an amino acid sequence of SEQ ID NO: 4.

Results:

As shown in FIG. 4, Pfu^exo_ DNA polymerase is able to perform template-independent nucleic acid synthesis at each of the temperatures tested, thereby indicating that a family-B DNA polymerase can be used to synthesize a nucleic acid in the absence of a template.

Example 4.Template-independent nucleic acid synthesis by family-B DNA polymerase with a single type of divalent cation or a combination of different divalent cations

In order to evaluate whether different types of divalent cations may affect the efficiency of template-independent nucleic acid synthesis by a family-B polymerase, the following experiment was performed.

Template-independent nucleic acid synthesis and analysis of the reaction products were conducted generally according to the procedures set forth in Example 1, except that: a respective one of K0Dl^exo_ DNA polymerase (described in Example 1), Vent^exo_ DNA polymerase (described in Example 2), and Pfu^exo_ DNA polymerase (described in Example 3) was used; a respective synthesis reaction mixture was preincubated at 70°C; and Mg²⁺ only or Mg²⁺ in combination with Mn²⁺ were added into the respective reaction mixture. Results:

As shown in FIG. 5, template-independent nucleic acid synthesis with any of the three family-B DNA polymerases was more efficient (more newly synthesized nucleic acids were found) in the presence of two different types of divalent cations, thus manifesting that the efficiency of template-independent nucleic acid synthesis with a family-B polymerase can be enhanced using a combination of different types of divalent cations. All patents and references cited in this specification are incorporated herein in their entirety as reference. Where there is conflict, the descriptions in this case, including the definitions, shall prevail. While the disclosure has been described in connection with what are considered the exemplary embodiments, it is understood that this disclosure is not limited to the disclosed embodimentsbut is intended to cover various arrangements included within the spirit and scope of the broadest interpretation so as to encompass all such modifications and equivalent arrangements.

SEQUENCE LISTING

<110> Chen, Cheng-Yao

<120> METHOD AND KIT FOR TEMPLATE-INDEPENDENT NUCLEIC ACID SYNTHESIS

<130> PE-63427-W0

<160> 4

<170> Patent In version 3.5

<210> 1

<211> 45

<212> DNA

<213> Artificial Sequence

<220>

<223> Initiator for template-independent nucleic acid synthesis <400> 1 ctcggcctgg cacaggtccg ttcagtgctg cggcgaccac cgagg 45 <210> 2

<211> 774

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<213> Artificial Sequence

<220> <223> K0D1(exo-) DNA polymerase

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Met lie Leu Asp Thr Asp Tyr lie Thr Glu Asp Gly Lys Pro Val lie 1 5 10 15

Arg lie Phe Lys Lys Glu Asn Gly Glu Phe Lys lie Glu Tyr Asp Arg 20 25 30 Thr Phe Glu Pro Tyr Phe Tyr Ala Leu Leu Lys Asp Asp Ser Ala lie 35 40 45

Glu Glu Val Lys Lys lie Thr Ala Glu Arg His Gly Thr Val Val Thr 50 55 60 Val Lys Arg Val Glu Lys Val Gin Lys Lys Phe Leu Gly Arg Pro Val 65 70 75 80

Glu Val Trp Lys Leu Tyr Phe Thr His Pro Gin Asp Val Pro Ala lie 85 90 95

Arg Asp Lys lie Arg Glu His Pro Ala Val lie Asp lie Tyr Glu Tyr 100 105 110

Asp lie Pro Phe Ala Lys Arg Tyr Leu lie Asp Lys Gly Leu Val Pro 115 120 125

Met Glu Gly Asp Glu Glu Leu Lys Met Leu Ala Phe Ala lie Ala Thr 130 135 140 Leu Tyr His Glu Gly Glu Glu Phe Ala Glu Gly Pro lie Leu Met lie 145 150 155 160

Ser Tyr Ala Asp Glu Glu Gly Ala Arg Val lie Thr Trp Lys Asn Val 165 170 175

Asp Leu Pro Tyr Val Asp Val Val Ser Thr Glu Arg Glu Met lie Lys 180 185 190

Arg Phe Leu Arg Val Val Lys Glu Lys Asp Pro Asp Val Leu lie Thr 195 200 205

Tyr Asn Gly Asp Asn Phe Asp Phe Ala Tyr Leu Lys Lys Arg Cys Glu 210 215 220 Lys Leu Gly lie Asn Phe Ala Leu Gly Arg Asp Gly Ser Glu Pro Lys 225 230 235 240 lie Gin Arg Met Gly Asp Arg Phe Ala Val Glu Val Lys Gly Arg lie 245 250 255

His Phe Asp Leu Tyr Pro Val lie Arg Arg Thr lie Asn Leu Pro Thr 260 265 270 Tyr Thr Leu Glu Ala Val Tyr Glu Ala Val Phe Gly Gin Pro Lys Glu 275 280 285

Lys Val Tyr Ala Glu Glu lie Thr Thr Ala Trp Glu Thr Gly Glu Asn 290 295 300 Leu Glu Arg Val Ala Arg Tyr Ser Met Glu Asp Ala Lys Val Thr Tyr 305 310 315 320

Glu Leu Gly Lys Glu Phe Leu Pro Met Glu Ala Gin Leu Ser Arg Leu 325 330 335 lie Gly Gin Ser Leu Trp Asp Val Ser Arg Ser Ser Thr Gly Asn Leu 340 345 350

Val Glu Trp Phe Leu Leu Arg Lys Ala Tyr Glu Arg Asn Glu Leu Ala

355 360 365

Pro Asn Lys Pro Asp Glu Lys Glu Leu Ala Arg Arg Arg Gin Ser Tyr 370 375 380 Glu Gly Gly Tyr Val Lys Glu Pro Glu Arg Gly Leu Trp Glu Asn lie 385 390 395 400

Val Tyr Leu Asp Phe Arg Ser Leu Tyr Pro Ser lie lie lie Thr His 405 410 415

Asn Val Ser Pro Asp Thr Leu Asn Arg Glu Gly Cys Lys Glu Tyr Asp 420 425 430

Val Ala Pro Gin Val Gly His Arg Phe Cys Lys Asp Phe Pro Gly Phe

435 440 445 lie Pro Ser Leu Leu Gly Asp Leu Leu Glu Glu Arg Gin Lys lie Lys 450 455 460 Lys Lys Met Lys Ala Thr lie Asp Pro lie Glu Arg Lys Leu Leu Asp 465 470 475 480

Tyr Arg Gin Arg Ala lie Lys lie Leu Ala Asn Ser Tyr Tyr Gly Tyr 485 490 495

Tyr Gly Tyr Ala Arg Ala Arg Trp Tyr Cys Lys Glu Cys Ala Glu Ser 500 505 510 Val Thr Ala Trp Gly Arg Glu Tyr lie Thr Met Thr lie Lys Glu lie 515 520 525

Glu Glu Lys Tyr Gly Phe Lys Val lie Tyr Ser Asp Thr Asp Gly Phe 530 535 540 Phe Ala Thr lie Pro Gly Ala Asp Ala Glu Thr Val Lys Lys Lys Ala 545 550 555 560

Met Glu Phe Leu Lys Tyr lie Asn Ala Lys Leu Pro Gly Ala Leu Glu 565 570 575

Leu Glu Tyr Glu Gly Phe Tyr Lys Arg Gly Phe Phe Val Thr Lys Lys 580 585 590

Lys Tyr Ala Val lie Asp Glu Glu Gly Lys lie Thr Thr Arg Gly Leu

595 600 605

Glu lie Val Arg Arg Asp Trp Ser Glu lie Ala Lys Glu Thr Gin Ala 610 615 620 Arg Val Leu Glu Ala Leu Leu Lys Asp Gly Asp Val Glu Lys Ala Val 625 630 635 640

Arg lie Val Lys Glu Val Thr Glu Lys Leu Ser Lys Tyr Glu Val Pro 645 650 655

Pro Glu Lys Leu Val lie His Glu Gin lie Thr Arg Asp Leu Lys Asp 660 665 670

Tyr Lys Ala Thr Gly Pro His Val Ala Val Ala Lys Arg Leu Ala Ala

675 680 685

Arg Gly Val Lys lie Arg Pro Gly Thr Val lie Ser Tyr lie Val Leu 690 695 700 Lys Gly Ser Gly Arg lie Gly Asp Arg Ala lie Pro Phe Asp Glu Phe 705 710 715 720

Asp Pro Thr Lys His Lys Tyr Asp Ala Glu Tyr Tyr lie Glu Asn Gin 725 730 735

Val Leu Pro Ala Val Glu Arg lie Leu Arg Ala Phe Gly Tyr Arg Lys 740 745 750 Glu Asp Leu Arg Tyr Gin Lys Thr Arg Gin Val Gly Leu Ser Ala Trp 755 760 765

Leu Lys Pro Lys Gly Thr 770

<210> 3

<211> 774

<212> PRT

<213> Artificial Sequence <220>

<223> Vent(exo-) DNA polymerase <400> 3

Met lie Leu Asp Thr Asp Tyr lie Thr Lys Asp Gly Lys Pro lie lie 1 5 10 15 Arg lie Phe Lys Lys Glu Asn Gly Glu Phe Lys lie Glu Leu Asp Pro

20 25 30

His Phe Gin Pro Tyr lie Tyr Ala Leu Leu Lys Asp Asp Ser Ala lie 35 40 45

Glu Glu lie Lys Ala lie Lys Gly Glu Arg His Gly Lys Thr Val Arg 50 55 60

Val Leu Asp Ala Val Lys Val Arg Lys Lys Phe Leu Gly Arg Glu Val 65 70 75 80

Glu Val Trp Lys Leu lie Phe Glu His Pro Gin Asp Val Pro Ala Met 85 90 95 Arg Gly Lys lie Arg Glu His Pro Ala Val Val Asp lie Tyr Glu Tyr

100 105 110

Asp lie Pro Phe Ala Lys Arg Tyr Leu lie Asp Lys Gly Leu lie Pro 115 120 125

Met Glu Gly Asp Glu Glu Leu Lys Leu Leu Ala Phe Ala lie Ala Thr 130 135 140 Phe Tyr His Glu Gly Asp Glu Phe Gly Lys Gly Glu lie lie Met lie

145 150 155 160

Ser Tyr Ala Asp Glu Glu Glu Ala Arg Val lie Thr Trp Lys Asn lie 165 170 175

Asp Leu Pro Tyr Val Asp Val Val Ser Asn Glu Arg Glu Met lie Lys 180 185 190

Arg Phe Val Gin Val Val Lys Glu Lys Asp Pro Asp Val lie lie Thr 195 200 205

Tyr Asn Gly Asp Asn Phe Asp Leu Pro Tyr Leu lie Lys Arg Ala Glu

210 215 220

Lys Leu Gly Val Arg Leu Val Leu Gly Arg Asp Lys Glu His Pro Glu

225 230 235 240

Pro Lys lie Gin Arg Met Gly Asp Ser Phe Ala Val Glu lie Lys Gly 245 250 255

Arg lie His Phe Asp Leu Phe Pro Val Val Arg Arg Thr lie Asn Leu 260 265 270

Pro Thr Tyr Thr Leu Glu Ala Val Tyr Glu Ala Val Leu Gly Lys Thr 275 280 285

Lys Ser Lys Leu Gly Ala Glu Glu lie Ala Ala lie Trp Glu Thr Glu

290 295 300

Glu Ser Met Lys Lys Leu Ala Gin Tyr Ser Met Glu Asp Ala Arg Ala

305 310 315 320

Thr Tyr Glu Leu Gly Lys Glu Phe Phe Pro Met Glu Ala Glu Leu Ala 325 330 335

Lys Leu lie Gly Gin Ser Val Trp Asp Val Ser Arg Ser Ser Thr Gly 340 345 350

Asn Leu Val Glu Trp Tyr Leu Leu Arg Val Ala Tyr Ala Arg Asn Glu 355 360 365

Leu Ala Pro Asn Lys Pro Asp Glu Glu Glu Tyr Lys Arg Arg Leu Arg

370 375 380 Thr Thr Tyr Leu Gly Gly Tyr Val Lys Glu Pro Glu Lys Gly Leu Trp 385 390 395 400

Glu Asn lie lie Tyr Leu Asp Phe Arg Ser Leu Tyr Pro Ser lie lie 405 410 415

Val Thr His Asn Val Ser Pro Asp Thr Leu Glu Lys Glu Gly Cys Lys 420 425 430

Asn Tyr Asp Val Ala Pro lie Val Gly Tyr Arg Phe Cys Lys Asp Phe 435 440 445

Pro Gly Phe lie Pro Ser lie Leu Gly Asp Leu lie Ala Met Arg Gin 450 455 460

Asp lie Lys Lys Lys Met Lys Ser Thr lie Asp Pro lie Glu Lys Lys 465 470 475 480

Met Leu Asp Tyr Arg Gin Arg Ala lie Lys Leu Leu Ala Asn Ser Tyr 485 490 495

Tyr Gly Tyr Met Gly Tyr Pro Lys Ala Arg Trp Tyr Ser Lys Glu Cys 500 505 510

Ala Glu Ser Val Thr Ala Trp Gly Arg His Tyr lie Glu Met Thr lie 515 520 525

Arg Glu lie Glu Glu Lys Phe Gly Phe Lys Val Leu Tyr Ala Asp Thr 530 535 540

Asp Gly Phe Tyr Ala Thr lie Pro Gly Glu Lys Pro Glu Leu lie Lys 545 550 555 560

Lys Lys Ala Lys Glu Phe Leu Asn Tyr lie Asn Ser Lys Leu Pro Gly 565 570 575

Leu Leu Glu Leu Glu Tyr Glu Gly Phe Tyr Leu Arg Gly Phe Phe Val 580 585 590

Thr Lys Lys Arg Tyr Ala Val lie Asp Glu Glu Gly Arg lie Thr Thr 595 600 605

Arg Gly Leu Glu Val Val Arg Arg Asp Trp Ser Glu lie Ala Lys Glu 610 615 620 Thr Gin Ala Lys Val Leu Glu Ala lie Leu Lys Glu Gly Ser Val Glu 625 630 635 640

Lys Ala Val Glu Val Val Arg Asp Val Val Glu Lys lie Ala Lys Tyr 645 650 655 Arg Val Pro Leu Glu Lys Leu Val lie His Glu Gin lie Thr Arg Asp

660 665 670

Leu Lys Asp Tyr Lys Ala lie Gly Pro His Val Ala lie Ala Lys Arg 675 680 685

Leu Ala Ala Arg Gly lie Lys Val Lys Pro Gly Thr lie lie Ser Tyr 690 695 700 lie Val Leu Lys Gly Ser Gly Lys lie Ser Asp Arg Val lie Leu Leu 705 710 715 720

Thr Glu Tyr Asp Pro Arg Lys His Lys Tyr Asp Pro Asp Tyr Tyr lie 725 730 735 Glu Asn Gin Val Leu Pro Ala Val Leu Arg lie Leu Glu Ala Phe Gly

740 745 750

Tyr Arg Lys Glu Asp Leu Arg Tyr Gin Ser Ser Lys Gin Thr Gly Leu 755 760 765

Asp Ala Trp Leu Lys Arg 770

<210> 4

<211> 775

<212> PRT <213> Artificial Sequence

<220>

<223> Pfu(exo-) DNA polymerase <400> 4

Met lie Leu Asp Val Asp Tyr lie Thr Glu Glu Gly Lys Pro Val lie 1 5 10 15 Arg Leu Phe Lys Lys Glu Asn Gly Lys Phe Lys lie Glu His Asp Arg 20 25 30

Thr Phe Arg Pro Tyr lie Tyr Ala Leu Leu Arg Asp Asp Ser Lys lie 35 40 45

Glu Glu Val Lys Lys lie Thr Gly Glu Arg His Gly Lys lie Val Arg 50 55 60 lie Val Asp Val Glu Lys Val Glu Lys Lys Phe Leu Gly Lys Pro lie 65 70 75 80

Thr Val Trp Lys Leu Tyr Leu Glu His Pro Gin Asp Val Pro Thr lie 85 90 95

Arg Glu Lys Val Arg Glu His Pro Ala Val Val Asp lie Phe Glu Tyr 100 105 110

Asp lie Pro Phe Ala Lys Arg Tyr Leu lie Asp Lys Gly Leu lie Pro 115 120 125

Met Glu Gly Glu Glu Glu Leu Lys lie Leu Ala Phe Ala lie Ala Thr 130 135 140

Leu Tyr His Glu Gly Glu Glu Phe Gly Lys Gly Pro lie lie Met lie 145 150 155 160

Ser Tyr Ala Asp Glu Asn Glu Ala Lys Val lie Thr Trp Lys Asn lie 165 170 175

Asp Leu Pro Tyr Val Glu Val Val Ser Ser Glu Arg Glu Met lie Lys 180 185 190

Arg Phe Leu Arg lie lie Arg Glu Lys Asp Pro Asp lie lie Val Thr 195 200 205

Tyr Asn Gly Asp Ser Phe Asp Phe Pro Tyr Leu Ala Lys Arg Ala Glu 210 215 220

Lys Leu Gly lie Lys Leu Thr lie Gly Arg Asp Gly Ser Glu Pro Lys 225 230 235 240

Met Gin Arg lie Gly Asp Met Thr Ala Val Glu Val Lys Gly Arg lie 245 250 255 His Phe Asp Leu Tyr His Val lie Thr Arg Thr lie Asn Leu Pro Thr 260 265 270

Tyr Thr Leu Glu Ala Val Tyr Glu Ala lie Phe Gly Lys Pro Lys Glu 275 280 285

Lys Val Tyr Ala Asp Glu lie Ala Lys Ala Trp Glu Ser Gly Glu Asn 290 295 300

Leu Glu Arg Val Ala Lys Tyr Ser Met Glu Asp Ala Lys Ala Thr Tyr 305 310 315 320

Glu Leu Gly Lys Glu Phe Leu Pro Met Glu lie Gin Leu Ser Arg Leu 325 330 335

Val Gly Gin Pro Leu Trp Asp Val Ser Arg Ser Ser Thr Gly Asn Leu 340 345 350

Val Glu Trp Phe Leu Leu Arg Lys Ala Tyr Glu Arg Asn Glu Val Ala 355 360 365

Pro Asn Lys Pro Ser Glu Glu Glu Tyr Gin Arg Arg Leu Arg Glu Ser 370 375 380

Tyr Thr Gly Gly Phe Val Lys Glu Pro Glu Lys Gly Leu Trp Glu Asn 385 390 395 400 lie Val Tyr Leu Asp Phe Arg Ala Leu Tyr Pro Ser lie lie lie Thr 405 410 415

His Asn Val Ser Pro Asp Thr Leu Asn Leu Glu Gly Cys Lys Asn Tyr 420 425 430

Asp lie Ala Pro Gin Val Gly His Lys Phe Cys Lys Asp lie Pro Gly 435 440 445

Phe lie Pro Ser Leu Leu Gly His Leu Leu Glu Glu Arg Gin Lys lie 450 455 460

Lys Thr Lys Met Lys Glu Thr Gin Asp Pro lie Glu Lys lie Leu Leu 465 470 475 480

Asp Tyr Arg Gin Lys Ala lie Lys Leu Leu Ala Asn Ser Phe Tyr Gly 485 490 495 Tyr Tyr Gly Tyr Ala Lys Ala Arg Trp Tyr Cys Lys Glu Cys Ala Glu 500 505 510

Ser Val Thr Ala Trp Gly Arg Lys Tyr lie Glu Leu Val Trp Lys Glu 515 520 525 Leu Glu Glu Lys Phe Gly Phe Lys Val Leu Tyr lie Asp Thr Asp Gly 530 535 540

Leu Tyr Ala Thr lie Pro Gly Gly Glu Ser Glu Glu lie Lys Lys Lys 545 550 555 560

Ala Leu Glu Phe Val Lys Tyr lie Asn Ser Lys Leu Pro Gly Leu Leu 565 570 575

Glu Leu Glu Tyr Glu Gly Phe Tyr Lys Arg Gly Phe Phe Val Thr Lys

580 585 590

Lys Arg Tyr Ala Val lie Asp Glu Glu Gly Lys Val lie Thr Arg Gly 595 600 605 Leu Glu lie Val Arg Arg Asp Trp Ser Glu lie Ala Lys Glu Thr Gin 610 615 620

Ala Arg Val Leu Glu Thr lie Leu Lys His Gly Asp Val Glu Glu Ala 625 630 635 640

Val Arg lie Val Lys Glu Val lie Gin Lys Leu Ala Asn Tyr Glu lie 645 650 655

Pro Pro Glu Lys Leu Ala lie Tyr Glu Gin lie Thr Arg Pro Leu His 660 665 670

Glu Tyr Lys Ala lie Gly Pro His Val Ala Val Ala Lys Lys Leu Ala 675 680 685 Ala Lys Gly Val Lys lie Lys Pro Gly Met Val lie Gly Tyr lie Val 690 695 700

Leu Arg Gly Asp Gly Pro lie Ser Asn Arg Ala lie Leu Ala Glu Glu 705 710 715 720

Tyr Asp Pro Lys Lys His Lys Tyr Asp Ala Glu Tyr Tyr lie Glu Asn 725 730 735 Gin Val Leu Pro Ala Val Leu Arg lie Leu Glu Gly Phe Gly Tyr Arg 740 745 750

Lys Glu Asp Leu Arg Tyr Gin Lys Thr Arg Gin Val Gly Leu Thr Ser 755 760 765 Trp Leu Asn lie Lys Lys Ser 770 775

Claims

WHAT IS CLAIMED IS:

1. A method for synthesizing a nucleic acid, comprising : providing an initiator having an unprotected nucleoside base and a 3' hydroxyl group at a 3' terminus thereof; 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 at least one type of metal cofactors which are divalent cations, and in the absence of a template, such that the nucleotide monomer is incorporated to the initiator.

2. The method of Claim 1, wherein the initiator has a sequence selected from a non-self complementary sequence and a non-self complementarity forming sequence.

3. The method of Claim 1, wherein the initiator is linked to a solid support and has a 5' end linked to the solid support.

4. The method of Claim 3, wherein the solid support is selected from 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, and a nanoparticle.

5. The method of Claim 1, wherein the initiator is exposed to the nucleotide monomer at a temperature ranging from 10°C to 90°C.

6. The method of Claim 1, wherein the initiator is exposed to the nucleotide monomer at a pH of not less than 8.0.

7. The method of Claim 1, wherein the metal cofactor is selected from the group consisting of Mg²⁺, Ca²⁺, Sr²⁺, Ba²⁺,Mn²⁺,Co²⁺, Fe²⁺,Ni²⁺, Cu²⁺, Zn²⁺ and combinations thereof.

8. The method of Claim 1, wherein the nucleic acid polymerase further has a 3' to 5' exonuclease domain and is a family-B DNA polymerase 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.

9. The method of Claim 8, 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 Pyrococus furious (Pfu), a family-B DNA polymerase of Thermococcus sp. (9°N), a family-B DNA polymerase of Thermococcus gorgonarius (Tgo), and a family-B DNA polymerase of Thermococcus litoralis (Vent).

10.The method of Claim 8, wherein the 3' to 5' exonuclease domain of the family-B DNA polymerase is modified in a manner selected from the group consisting of inactivation, attenuation, and deletion.

11.The method of Claim 1, wherein the initiator is in a single-stranded form.

12.The method of Claim 1, wherein the initiator has at least five nucleotide monomers.

13.The method of Claim 1, wherein the nucleotide monomer has a phosphate group selected from a monophosphate, a diphosphate, a triphosphate, a tetraphosphate, a pentaphosphate, and a hexaphosphate.

14.The method of Claim 1, 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 a combination thereof.

15.A kit for synthesizing a nucleic acid, comprising: an initiator having an unprotected nucleoside base and a 3'hydroxyl group at a 3' terminus thereof; a nucleic acid polymerase having at least a conservative catalytic polymerase domain of a family-B DNA polymerase; a nucleotide monomer; and at least one type of metal cofactors, wherein the kit is used according to a method as described in Claim 1.

16.The kit of Claim 15, wherein the initiator has a sequence selected from a non-self complementary sequence and a non-self complementarity forming sequence.

17.The kit of Claim 15, wherein the nucleic acid polymerase further has a 3' to 5' exonuclease domain and is a family-B DNA polymerase 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.

18.The kit of Claim 17, 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 Pyrococus furious (Pfu), a family-B DNA polymerase of Thermococcus sp . (9°N), a family-B DNA polymerase of Thermococcus gorgonarius (Tgo), and a family-B DNA polymerase of Thermococcus litoralis (Vent).

19.The kit of Claim 17, wherein the 3' to 5' exonuclease domain of the family-B DNA polymerase is modified in a manner selected from the group consisting of inactivation, attenuation, and deletion.

20.The kit of Claim 15, 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 a combination thereof.