Methods and kits for template-independent nucleic acid synthesis

Abstract

The present disclosure provides a method for synthesizing nucleic acids, which comprises: providing a starter having an unprotected nucleobase at the 3'-end and a 3'-hydroxyl group; providing a nucleic acid polymerase having at least one conserved catalytic polymerase domain of a family B DNA polymerase; providing nucleotide monomers; and exposing the starter to the nucleotide monomers in the presence of the nucleic acid polymerase and at least one metal cofactor belonging to divalent cations and in the absence of a template, such that the nucleotide monomers are incorporated into the starter. The present disclosure also provides a kit comprising the starter, the nucleic acid polymerase, the nucleotide monomers, and at least one metal cofactor, and used according to the method.

Description

[0001] Cross - reference to related applications

[0002] This application claims priority to U.S. Provisional Application No. 16 / 725,420, filed on December 23, 2019, which is incorporated herein by reference in its entirety. Technical field

[0003] The present disclosure relates to a method and kit for nucleic acid synthesis, particularly a method and kit for template - independent nucleic acid synthesis. Background art

[0004] Over the past few decades, de novo DNA synthesis without a DNA template has been developed. Among the currently available template - independent DNA synthesis methods, phosphoramidite - based chemical DNA synthesis has been well - known since the early 1980s but has remained essentially unchanged since then. The phosphoramidite - based chemical DNA synthesis requires four consecutive reaction steps, including de - blocking, coupling, capping, and oxidation steps, to add a nucleoside to another nucleoside attached to a solid support. However, one of the major drawbacks of phosphoramidite - based chemical DNA synthesis is the unavoidable use of harmful chemicals in the above - mentioned reaction steps.

[0005] Due to the increasing demand for environmental protection, green technologies applicable to DNA synthesis have attracted the attention of researchers. Therefore, enzymatic DNA synthesis, which can significantly reduce the use of harmful chemicals, seems promising due to advantages such as longer chain generation, lower error rate, faster cycle time, and lower production cost.

[0006] In terms of template-independent enzymatic DNA synthesis, terminal deoxynucleotidyltransferase (TdT) has been found to be a template-independent DNA polymerase that can attach all four deoxynucleoside triphosphates (dNTPs) to the 3'-end of a DNA strand. TdT belongs to the X Family of low-fidelity DNA polymerases. TdT-based DNA synthesis only requires two reaction steps, namely single nucleotide addition via TdT to the 3'-end of the extending single-stranded DNA strand being synthesized and subsequent removal of the 3'-protecting group. Although TdT and its homologs have been applied to many DNA synthesis platforms, template-independent enzymatic DNA synthesis based on TdT has been difficult to commercialize due to unsatisfactory product length, reagent recyclability, cycle time, etc. Summary of the Invention

[0007] Accordingly, an object of the present disclosure is to provide a method and a kit for synthesizing nucleic acids, which can alleviate at least one drawback of the prior art.

[0008] The method includes providing an initiator having an unprotected nucleobase and a 3'-hydroxyl group at the 3'-end; providing a nucleic acid polymerase having at least one conserved catalytic polymerase domain of a family-B DNA polymerase; providing nucleotide monomers; and exposing the initiator to the nucleotide monomers in the presence of the nucleic acid polymerase and at least one metal cofactor belonging to divalent cations and in the absence of a template such that the nucleotide monomers are incorporated into the initiator.

[0009] The kit includes the initiator as described above, the nucleic acid polymerase as described above, at least one metal cofactor as described above, and the nucleotide monomers as described above. The kit is used according to the method as described above. Brief Description of the Drawings

[0010] Other features and advantages of the present invention will become apparent in the following detailed description of embodiments with reference to the accompanying drawings, in which:

[0011] Figure 1 is de novo nucleic acid synthesis using a family-B DNA polymerase;

[0012] Figure 2 is an image of a denaturing urea-polyacrylamide gel showing the products of template-independent nucleic acid synthesis obtained using KOD1 exo- DNA polymerase at different reaction temperatures, where the symbol "S" indicates the position of the initiator DNA;

[0013] Figure 3 is an image of a denaturing urea-polyacrylamide gel showing the products of template-independent nucleic acid synthesis obtained using Vent exo- DNA polymerase at different reaction temperatures, where the symbol "S" indicates the position of the initiator DNA;

[0014] Figure 4 is an image of a denaturing urea-polyacrylamide gel showing the products of template-independent nucleic acid synthesis obtained using Pfu exo- DNA polymerase at different reaction temperatures, where the symbol "S" indicates the position of the initiator DNA; and

[0015] Figure 5 is an image of a denaturing urea-polyacrylamide gel showing the products of template-independent nucleic acid synthesis obtained using Vent 2+ DNA polymerase, KOD1 2+ DNA polymerase or Pfu exo- DNA polymerase in the presence of only Mg exo- or in combination with Mn exo- DNA polymerase, where the symbol "S" indicates the position of the initiator DNA, and the symbols "V", "K", "P" represent Vent exo- DNA polymerase, KOD1 exo- DNA polymerase and Pfu exo- DNA polymerase. Detailed Description

[0016] It should be understood that if any prior art publication is cited herein, such prior art publication does not constitute an admission that in any country or region, such prior art publication forms part of the common general knowledge in the art.

[0017] For the purposes of this specification, it will be clearly understood that the term "comprising" means "including but not limited to", and the term "comprises" has a corresponding meaning.

[0018] Unless otherwise defined, all technical and scientific terms used herein have the meaning commonly understood by those of ordinary skill in the art to which this invention belongs. Those of ordinary skill in the art will recognize many methods and materials similar or equivalent to those described herein, which can be used to practice the present invention. Of course, the present invention is in no way limited to the methods and materials described.

[0019] The Applicant has unexpectedly discovered that a DNA polymerase of Family B, which is well-known as a template-dependent DNA polymerase, can be used for template-independent nucleic acid synthesis (i.e., de novo nucleic acid synthesis). See Figure 1 , which illustrates a flowchart of de novo nucleic acid synthesis using a DNA polymerase of Family B.

[0020] DNA polymerases of Family B (also known as DNA polymerases of Type B) are replicative and repair polymerases that inherently 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" means a structural part or region of the amino acid sequence of a protein that has the catalytic DNA / RNA polymerase activity of the protein, and that does not contain other catalytic activities, such as editing activities (e.g., the proofreading activity of the 3'-to-5' exonuclease domain), the activity for excising Okazaki primers during replication, and the activity for interacting with other proteins. The catalytic polymerase domain of DNA polymerases of Family B has a common overall structure that resembles a right hand and is composed of a thumb, a palm, and a finger domain. The most conserved region is the palm domain that contains the catalytic site.

[0021] Examples of DNA polymerases of Family B include, but are not limited to, DNA polymerases of Family B from bacteria (e.g., Pol II), DNA polymerases of Family B from eukaryotes (e.g., Polα, Polδ, Polε, and Polζ), DNA polymerases of Family B from archaea (e.g., Pol B, Pol BI, Pol BII, Pol BIII, 9°N, Kod1, Pfu, Tgo, and Vent), and DNA polymerases of Family B from viruses (e.g., HSV-1, RB69, T4, B103, and Φ29).

[0022] Accordingly, the present disclosure provides a method for synthesizing a nucleic acid, which comprises:

[0023] providing a starter having an unprotected nucleobase at the 3'-end and a 3'-hydroxyl group;

[0024] Provided is a nucleic acid polymerase having at least one conserved catalytic polymerase domain of a family B DNA polymerase;

[0025] Provided are nucleotide monomers; and

[0026] In the presence of the nucleic acid polymerase and at least one metal cofactor belonging to divalent cations, and in the absence of a template, exposing the initiator to the nucleotide monomers such that the nucleotide monomers are incorporated into the initiator.

[0027] As used herein, terms such as "nucleic acid", "nucleic acid sequence", or "nucleic acid fragment" refer to a deoxyribonucleotide sequence or ribonucleotide sequence in single-stranded or double-stranded form, and include known naturally occurring nucleotides or artificial chemical mimics. As used herein, the term "nucleic acid" may be used interchangeably with "oligonucleotide", "polynucleotide", "DNA", "RNA", "gene", "cDNA", and "mRNA".

[0028] Generally, a "template" is a polynucleotide containing a target nucleotide sequence. In some cases, the terms "target sequence", "template polynucleotide", "target nucleic acid", "target polynucleotide", "nucleic acid template", "template sequence", and variations thereof may be used interchangeably. Specifically, the term "template" means a nucleic acid strand on which a complementary copy is synthesized from nucleotides or nucleotide analogs by the activity of a template-dependent nucleic acid polymerase. In a duplex, by convention, the template strand is depicted and described as the "bottom" strand. Similarly, the non-template strand is typically depicted and described as the "top" strand. The template strand may also be referred to as the "sense" strand, while the non-template strand is referred to as the "antisense" strand.

[0029] The terms "incorporated" or "incorporation" mean becoming part of a nucleic acid. There is known flexibility in the terminology regarding the incorporation of nucleic acid precursors. For example, the nucleotide dGTP is deoxyribonucleoside triphosphate. When incorporated into DNA, dGTP becomes dGMP, which is the deoxyguanosine monophosphate moiety. Although DNA does not include dGTP molecules, one can say that dGTP is incorporated into DNA.

[0030] The term "initiator" refers to a mononucleoside, mononucleotide, oligonucleotide, polynucleotide, or a modified analogue thereof from which a nucleic acid is to be synthesized de novo. The term "initiator" can also refer to xeno nucleic acids (XNA) or peptide nucleic acids (PNA) having a 3'-hydroxy group.

[0031] According to the present disclosure, the initiator can have a sequence selected from the following: a non-self-complementary sequence and a sequence forming non-self-complementarity. The term "self-complementary" means that a sequence (e.g., a nucleotide sequence or a PNA sequence) self-folds (e.g., a region of the sequence binds or hybridizes to another region of the sequence), thereby generating a duplex, duplex-like structure that can serve as a template for nucleic acid synthesis. Depending on how the complementary regions of the sequence are brought together, the strands can form, for example, 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 PNA sequence) that forms a complementary extension from the sequence when the sequence serves as a template (i.e., forms a self-complementary sequence based on the sequence serving as a template). For example, a sequence forming self-complementarity can be "ATCC". When the "ATCC" sequence serves as a template, a complementary extension "GGAT" is formed with the sequence (i.e., the self-complementary sequence "ATCCGGAT" is formed).

[0032] The terms "conservative" or "conserved" are used to describe a domain containing the same amino acid residues in multiple proteins having the same structure and / or function. Regions of conserved amino acid residues can be important for protein structure or function. Thus, a continuous stretch of conserved amino acid residues identified in a three-dimensional protein can be important for protein structure or function.

[0033] For example, as reported in Albà (2001), Genome Biology, 2(1): reviews3002.1 to review3002.4, family B DNA polymerases have Regions I and II, which form part of the active site of the catalytic polymerase domain and may contain the conserved amino acid residues "DT" and "SLYPS", respectively. 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.

[0034] 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: family B DNA polymerases of bacteria, family B DNA polymerases of eukaryotes, family B DNA polymerases of archaea, and family B DNA polymerases of viruses. In some embodiments, the family B DNA polymerase is selected from the group consisting of: the family B DNA polymerase of Thermococcus kodakaraensis KOD1 (Thermococcus kodakaraensis KOD1), the family B DNA polymerase of Pyrococcus furious (Pfu), and the family B DNA polymerase of Thermococcus litoralis (Vent).

[0035] According to the present disclosure, alternatively, the 3' to 5' exonuclease domain of the family B DNA polymerase may be inactive. Alternatively, the activity of the 3' to 5' exonuclease of the family B DNA polymerase may be reduced. Yet alternatively, the 3' to 5' exonuclease domain of the family B DNA polymerase may remain unchanged, and in the methods of the present invention, an inhibitor may be used to inhibit the 3' to 5' exonuclease domain of the family B DNA polymerase.

[0036] According to the present disclosure, alternatively, the nucleic acid polymerase may only have the aforementioned conserved catalytic polymerase domain. In some embodiments, the nucleic acid polymerase was initially designed to only have the aforementioned original conserved catalytic polymerase domain. In other embodiments, the nucleic acid polymerase is an original family B DNA polymerase having a 3' to 5' exonuclease domain, and the domain has been removed from the nucleic acid polymerase.

[0037] In some embodiments, the initiator is in single-stranded form.

[0038] In some embodiments, the initiator has at least five nucleotides. In an exemplary embodiment, the initiator has forty-five nucleotides.

[0039] In some embodiments, the initiator is exposed to the nucleotide monomers at a temperature in the range of 10 °C to 90 °C, and / or the initiator is exposed to the nucleotide monomers at a pH of not less than 8.0 (e.g., 8.8).

[0040] According to the present disclosure, the nucleotide monomers may be natural nucleic acid nucleotides, which consist of a sugar, a phosphate group, and a nitrogenous 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 nitrogenous bases are selected from adenine, guanine, uracil, cytosine, and thymine. Alternatively, the nucleotide monomers may be nucleotides modified on at least one of the three components. For example, the modification may occur on the base moiety, resulting in modified products (such as inosine, methyl-5-deoxycytidine, deoxyuridine, dimethylamino-5-deoxyuridine, diaminopurine, or bromo-5-deoxyuridine, and any other modified bases that allow hybridization), on the sugar moiety (e.g., by replacing deoxyribose with an analogue), or on the phosphate group moiety (e.g., borate esters, alkyl phosphonates, or thiophosphate derivatives).

[0041] According to the present disclosure, the nucleotide monomers have phosphate groups selected from the group consisting of monophosphate, diphosphate, triphosphate, tetraphosphate, pentaphosphate, and hexaphosphate.

[0042] According to the present disclosure, the metal cofactors are 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 combinations thereof. In an exemplary embodiment, the metal cofactor is Mg 2+ . In another embodiment, the metal cofactor is a combination of Mg 2+ and Mn 2+ .

[0043] According to the present disclosure, the nucleotide monomers have removable blocking moieties. Examples of the removable blocking moieties include, but are not limited to, 3'-O-blocking moieties, base blocking moieties, and combinations thereof.

[0044] The nucleotide monomer having a removable blocking moiety is also referred to as a reversible terminator. Thus, the nucleotide monomer having a 3'-O-blocking moiety is also referred to as a 3'-blocked reversible terminator or a 3'-O-modified reversible terminator, and the nucleotide monomer having a base blocking moiety is also referred to as a 3'-unblocked reversible terminator or a 3'-OH unblocked reversible terminator.

[0045] As used herein, the term "reversible terminator" refers to a chemically modified nucleotide monomer. When the reversible terminator is incorporated into a growing nucleic acid by a polymerase, it blocks further incorporation of nucleotide monomers by the polymerase. The "reversible terminator" base and nucleic acid can be deprotected by chemical or physical treatment, and after deprotection, the nucleic acid can be extended in one step by a polymerase.

[0046] Examples of 3'-O-blocking moieties include, but are not limited to, O-azidomethyl, O-amino, O-allyl, O-phenoxyacetyl, O-methoxyacetyl, O-acetyl, O-(p-toluene)sulfonate, O-phosphate, O-nitrate, O-[4-methoxy]-tetrahydropyranyl, O-tetrahydropyranyl, O-[5-methyl]-tetrahydrofuranyl, O-[2-methyl,4-methoxy]-tetrahydropyranyl, O-[5-methyl]-tetrahydropyranyl, and O-tetrahydrothiopyranyl, O-2-nitrobenzyl, O-methyl, and O-acyl.

[0047] Examples of 3'-unblocked reversible terminators include, but are not limited to, 7-[(S)-1-(5-methoxy-2-nitrophenyl)-2,2-dimethyl-propoxy]methyl-7-deazaATP, 5-[(S)-1-(5-methoxy-2-nitrophenyl)-2,2-dimethyl-propoxy]methyl-dCTP, 1-[(5-methoxy-2-nitrophenyl)-2,2-dimethyl-propoxy]methyl-7-deaza-dGTP, 5-[(S)-1-(5-methoxy-2-nitrophenyl)-2,2-dimethyl-propoxy]methyl-dUTP, and 5-[(S)-1-(2-nitrophenyl)-2,2-dimethyl-propoxy]methyl-dUTP.

[0048] According to the present disclosure, the base-blocking moiety is a reversible dye-terminator. Examples of reversible dye-terminators include, but are not limited to, the reversible dye-terminator of Illumina NovaSeq, the reversible dye-terminator of Illumina NextSeq, the reversible dye-terminator of Illumina MiSeq, the reversible dye-terminator of Illumina HiSeq, the reversible dye-terminator of Illumina Genome Analyzer IIX, the lightning terminator of LaserGen, and the reversible dye-terminator of Helicos Biosciences Heliscope.

[0049] Since reversible terminators are well-known and commonly used by those skilled in the art, more details thereof 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 removing the removable blocking moiety) can be found, for example, in 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.

[0050] According to the present disclosure, the initiator can be linked to a solid support and has a 5'-end linked to the solid support. The initiator can be directly attached to the support or can be attached to the support via a linker.

[0051] According to the present disclosure, examples of solid supports include, but are not limited to, microarrays, beads (coated or uncoated), columns, optical fibers, wipes, nitrocellulose, nylon, glass, quartz, diazotized membranes (paper or nylon), silicones, polyoxymethylene, cellulose, cellulose acetate, paper, ceramics, metals, metalloids, semiconductor materials, magnetic particles, plastics (such as polyethylene, polypropylene, and polystyrene), gel-forming materials (such as proteins, e.g., gelatin), lipopolysaccharides, silicates, agarose, polyacrylamide, polymethyl methacrylate polymers, sol-gels, porous polymer hydrogels, nanostructured surfaces, nanotubes (such as carbon nanotubes), and nanoparticles (such as gold nanoparticles or quantum dots).

[0052] In addition, the present invention provides a kit for synthesizing nucleic acids, which includes the above-mentioned initiator, the above-mentioned nucleic acid polymerase, the above-mentioned nucleotide monomers and then the above-mentioned at least one divalent cation. The kit is used according to the method of the present invention.

[0053] The present disclosure will be further described with reference to the following examples, but it should be understood that these examples are for illustrative purposes only and should not be construed as limitations on the implementation of the present invention.

[0054] Examples

[0055] Example 1. Template-independent nucleic acid synthesis using a family B DNA polymerase from the hyperthermophilic archaeon Thermococcus kodakaraensis KOD1

[0056] A synthesis reaction mixture was prepared using appropriate amounts of the following components: a single-stranded initiator having the nucleotide sequence of SEQ ID NO: 1, and having an unprotected hydroxyl group at the 3'-end and a 5'-end labeled with fluoresceinamidite (FAM); deoxynucleoside triphosphates, which serve as nucleotide monomers and include dATP, dGTP, dCTP, and dTTP; a family B DNA polymerase from the hyperthermophilic archaeon Thermococcus kodakaraensis KOD1, which has an inactive 3'-to-5' exonuclease domain and is referred to as KOD1 exo- DNA polymerase; and Tris-HCl buffer (pH 8.8). Specifically, the synthesis reaction mixture contained 100 nM of the initiator, 100 μM of dNTPs, and 200 nM of KOD1 exo- DNA polymerase.

[0057] KOD1 exo- The KOD1 exo- DNA polymerase was prepared as follows. A gene construct encoding a family B DNA polymerase from the hyperthermophilic archaeon Thermococcus kodakaraensis KOD1 (without an intein but having a normal 3'-to-5' exonuclease domain) was synthesized by Genomics BioSci&Tech Co. To obtain KOD1 exo- DNA polymerase, by substituting Asp 141 with Ala (D141A) and Glu 143Replacement with Ala (E143A) was used to inactivate the conserved 3' to 5' exonuclease domain, that is, to modify the conserved amino acid residues "DIE" of the conserved 3' to 5' exonuclease domain. Specifically, to complete the amino acid modifications of "D141A" and "E143A", site-directed mutagenesis was performed on the corresponding nucleotide residues of the above gene construct using the Q5 Site-Directed Mutagenesis Kit (New England Biolabs, Ipswich, MA, USA). The obtained mutant gene construct was expressed in BL21(DE3) cells, and the expressed protein was purified using an Akta Pure FPLC system (GE Healthcare Life Sciences, Marlborough, MA, USA) and sequentially through HisTrap Q and Heparin columns. The resulting KOD1 exo- DNA polymerase has the amino acid sequence of SEQ ID NO:2.

[0058] A 10-μL nucleic acid synthesis reaction mixture was pre-incubated 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, an appropriate amount of Mg 2+ (serving as a metal cofactor) was added to each reaction mixture to initiate template-independent nucleic acid synthesis, which was allowed to proceed for 5 minutes. The synthesis was terminated by adding 10 μL of 2X quench solution (containing 95% deionized formamide and 25 mM ethylenediaminetetraacetic acid).

[0059] The resulting synthesis reaction product was denatured at 98°C for 10 minutes. Subsequently, the synthesis reaction product was analyzed by 15% denaturing urea-polyacrylamide gel. The synthesis reaction product on the gel was observed by an Amersham Typhoon Imager (GE Healthcare Life Sciences, Marlborough, MA, USA).

[0060] Results:

[0061] As Figure 2 shown, KOD1 exo- DNA polymerase can perform template-independent nucleic acid synthesis at each of the tested temperatures, indicating that family B DNA polymerases can be used to synthesize nucleic acids in the absence of a template.

[0062] Example 2. Template-independent nucleic acid synthesis using the family B DNA polymerase of Thermococcus litoralis (Vent)

[0063] Template-independent nucleic acid synthesis and analysis of the reaction products were carried out generally according to the operating procedures described in Example 1, but using the family B DNA polymerase of Thermococcus litoralis (Vent TM ), which has an inactive 3'-to-5' exonuclease domain and is thus called Vent exo- DNA polymerase). Vent exo- DNA polymerase was prepared by the same operating procedures as those used for preparing KOD1 exo- DNA polymerase (see Example 1), but using a gene construct encoding the family B DNA polymerase of the hyperthermophilic archaeon KOD1 (without intein but with a normal 3'-to-5' exonuclease domain). Vent exo- DNA polymerase has the amino acid sequence of SEQ ID NO:3.

[0064] Results:

[0065] As Figure 3 shown, Vent exo- DNA polymerase can perform template-independent nucleic acid synthesis at each of the tested temperatures, indicating that family B DNA polymerases can be used for synthesizing nucleic acids in the absence of a template.

[0066] Example 3. Template-independent nucleic acid synthesis using the family B DNA polymerase of Pyrococcus furiosus (Pfu)

[0067] Template-independent nucleic acid synthesis and analysis of the reaction products were carried out generally according to the operating procedures described in Example 1, but using the family B DNA polymerase of Pfu (which has an inactive 3'-to-5' exonuclease domain and is thus called Pfu exo- DNA polymerase). Pfu exo- DNA polymerase was prepared by the same operating procedures as those used for preparing KOD1 exo- DNA polymerase (see Example 1), but using a gene construct encoding the family B DNA polymerase of Pfu (without intein but with a normal 3'-to-5' exonuclease domain). Pfu exo- DNA polymerase has the amino acid sequence of SEQ ID NO:4.

[0068] Results:

[0069] As Figure 4 shown, Pfuexo- DNA polymerases can perform template - independent nucleic acid synthesis at each of the tested temperatures, indicating that family B DNA polymerases can be used for synthesizing nucleic acids in the absence of a template.

[0070] Example 4. Template - independent nucleic acid synthesis by family B DNA polymerases in combination with a single type of divalent cation or different divalent cations

[0071] To evaluate whether different types of divalent cations would affect the efficiency of template - independent nucleic acid synthesis by family B DNA polymerases, the following experiment was conducted.

[0072] Template - independent nucleic acid synthesis and analysis of the reaction products were generally carried out according to the operating procedures described in Example 1, but using KOD1 exo- DNA polymerase (described in Example 1), Vent exo- DNA polymerase (described in Example 2), and Pfu exo- DNA polymerase (described in Example 3) respectively; pre - incubating each synthesis reaction mixture at 70 °C; and adding only Mg 2+ or a combination of Mg 2+ and Mn 2+ to each reaction mixture.

[0073] Results:

[0074] As Figure 5 shown, in the presence of two different types of divalent cations, template - independent nucleic acid synthesis using any one of the three family B DNA polymerases was more efficient (more newly synthesized nucleic acids were found), demonstrating that the use of a combination of different types of divalent cations can enhance the efficiency of template - independent nucleic acid synthesis using family B DNA polymerases.

[0075] All patents and literature cited in this specification are incorporated herein by reference in their entirety. In case of conflict, the detailed description (including definitions) in this case shall prevail.

[0076] Although the present invention has been described with reference to what are considered to be exemplary embodiments, it is to be understood that the disclosure is not limited to the described embodiments, but is intended to cover various different configurations included within the spirit and scope of the broadest interpretation, including all such modifications and equivalent configurations.

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Claims

1. A method for synthesizing nucleic acid, which comprises: providing a starter, which is in single-stranded form and has an unprotected nucleobase and a 3'-hydroxyl group at the 3'-end; providing a nucleic acid polymerase having at least one conserved catalytic polymerase domain of a family B DNA polymerase; providing nucleotide monomers; and exposing the starter to the nucleotide monomers in the presence of the nucleic acid polymerase and at least one metal cofactor belonging to divalent cations and in the absence of a template, such that the nucleotide monomers are incorporated into the starter, wherein the nucleic acid polymerase further has a 3'-to-5' exonuclease domain, and the family B DNA polymerase is selected from the group consisting of: the family B DNA polymerase of Thermococcus kodakaraensis (KOD1), the family B DNA polymerase of Pyrococcus furious (Pfu), the family B DNA polymerase of Thermococcus sp. (9°N), the family B DNA polymerase of Thermococcus gorgonarius (Tgo), and the family B DNA polymerase of Thermococcus litoralis (Vent).

2. The method according to claim 1, wherein the starter has a sequence selected from the group consisting of: a non-self-complementary sequence and a sequence forming non-self-complementarity.

3. The method according to claim 1, wherein the starter is linked to a solid support and has a 5'-end linked to the solid support.

4. The method according to claim 3, wherein the solid support is selected from the group consisting of: microarray, bead, column, optical fiber, swab, nitrocellulose, nylon, glass, quartz, diazotized membrane, silicone resin, polyoxymethylene, cellulose, cellulose acetate, paper, ceramic, metal, metalloid, semiconductor material, magnetic particle, plastic, gel-forming material, gel, nanostructured surface, nanotube, and nanoparticle.

5. The method according to claim 1, wherein the starter is exposed to the nucleotide monomers at a temperature in the range of 10°C to 90°C.

6. The method according to claim 1, wherein the starter is exposed to the nucleotide monomers at a pH of not less than 8.

0.

7. The method according to claim 1, wherein the metal cofactor 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 combinations thereof.

8. The method according to claim 1, wherein the 3'-to-5' exonuclease domain of the family B DNA polymerase is modified in a manner selected from the group consisting of: deactivated, attenuated, and deleted.

9. The method according to claim 1, wherein the starter has at least five nucleotide monomers.

10. The method according to claim 1, wherein the nucleotide monomers have a phosphate group selected from the group consisting of: monophosphate, diphosphate, triphosphate, tetraphosphate, pentaphosphate, and hexaphosphate.

11. The method according to 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 combinations thereof.

12. A kit for synthesizing nucleic acids, comprising: an initiator, which is in single-stranded form and has an unprotected nucleobase and a 3'-hydroxyl group at the 3'-end; a nucleic acid polymerase having at least one conserved catalytic polymerase domain of a family B DNA polymerase, and nucleotide monomers; wherein the kit is used according to the method of claim 1, wherein the nucleic acid polymerase further has a 3'-to-5' exonuclease domain, and the family B DNA polymerase is selected from the group consisting of: the family B DNA polymerase of Thermococcus kodakaraensis (KOD1), the family B DNA polymerase of Pyrococcus furious (Pfu), the family B DNA polymerase of Thermococcus sp. (9°N), the family B DNA polymerase of Thermococcus gorgonarius (Tgo), and the family B DNA polymerase of Thermococcus litoralis (Vent).

13. The kit according to claim 12, wherein the initiator has a sequence selected from the group consisting of: a non-self-complementary sequence and a sequence that forms non-self-complementarity.

14. The kit according to claim 12, wherein the family B DNA polymerase is selected from the group consisting of: the family B DNA polymerase of Thermococcus kodakaraensis KOD1, the family B DNA polymerase of Pyrococcus furious (Pfu), the family B DNA polymerase of Thermococcus sp. (9°N), the family B DNA polymerase of Thermococcus gorgonarius (Tgo), and the family B DNA polymerase of Thermococcus litoralis (Vent).

15. The kit according to claim 12, wherein the 3'-to-5' exonuclease domain of the family B DNA polymerase is modified in a manner selected from the group consisting of: deactivated, attenuated, and deleted.

16. The kit according to claim 12, 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 combinations thereof.

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