Method for amplifying target nucleic acid sequence using nucleic acid introduced with modified oligonucleotide

Abstract

To provide a method for amplifying a target nucleic acid sequence using a nucleic acid introduced with a modified oligonucleotide.SOLUTION: The present invention provides a method for amplifying a target nucleic acid sequence using a nucleic acid introduced with a modified oligonucleotide, wherein the amplification reaction is performed in the presence of an enzyme which has at least 90% homology with a polymerase derived from Thermus aquaticus with deletion of amino acids 1 to 289, and also has DNA polymerase activity.SELECTED DRAWING: None

Description

[Technical Field]

[0001] The present invention broadly relates to a method for amplifying a target nucleic acid sequence using a nucleic acid into which a modified oligonucleotide has been introduced. [Background technology]

[0002] Nucleic acid modifications can be classified as modifications of the phosphate moiety, sugar moiety, or base moiety. Among these, modifications of the sugar moiety stabilize the unstable sugar moiety structure, resulting in stronger binding of the modified nucleic acid to a complementary strand. Stronger binding to a complementary strand increases the Tm value and improves specificity. Examples of such modified nucleic acids that have been developed include locked nucleic acids (LNA), bridged nucleic acids (BNA), peptide nucleic acids (PNA), and serinol nucleic acids (SNA).

[0003] The above-mentioned modified nucleic acids, which can form stable double-stranded nucleic acids, can be used for applications such as primers, labeled probes, and clamps for inhibiting amplification due to their sequence specificity and high stability.

[0004] Modified nucleic acids used to inhibit amplification are known to be particularly useful for detecting mutations. A sequence having a modified nucleic acid complementary to a wild-type sequence forms a strong double-stranded chain with the wild-type sequence, and maintains this double-stranded structure even during an extension reaction using a polymerase, thereby inhibiting the amplification of the wild-type sequence. As a result, it becomes possible to specifically amplify a mutant sequence and detect mutations.

[0005] For example, polymerases that have proofreading activity (3'→5' exonuclease activity), which is an indicator of fidelity, have been used in amplification reactions such as PCR, which uses locked nucleic acids (LNAs) as modified nucleic acids to inhibit the amplification of specific sequences (Non-Patent Document 1). [Prior art documents] [Non-patent literature]

[0006] [Non-Patent Document 1] Lone Hummelshoj et al. Locked nucleic acid inhibits amplification of contaminating DNA in real-time PCR. BioTechniques. 2005, 38(4), 605-610. Summary of the Invention [Problem to be solved by the invention]

[0007] An object of the present invention is to provide a novel polymerase that has never been used before in an amplification reaction of a target nucleic acid sequence using a nucleic acid into which a modified oligonucleotide has been introduced.Another object is to provide a novel polymerase that has high activity and is capable of inhibiting the amplification of sequences other than the target nucleic acid sequence in an amplification reaction of a target nucleic acid sequence using a nucleic acid into which a modified oligonucleotide has been introduced.

[0008] A further object of the present invention is to provide a method for amplifying a target nucleic acid sequence using these polymerases. [Means for solving the problem]

[0009] As a result of extensive research, the present inventors have found that an enzyme highly homologous to a polymerase derived from Thermus aquaticus, which lacks proofreading activity (3'→5' exonuclease activity) and lacks a specific region of amino acids, can be used in amplification reactions of target nucleic acid sequences using nucleic acids incorporating modified oligonucleotides. Furthermore, when the polymerase is produced by expression in a host, it has been found that by using a polymerase produced by a method including a step of heating a host-derived protein at a predetermined temperature in an amplification reaction, it is possible to inhibit the amplification of sequences other than the target nucleic acid sequence with higher activity.

[0010] That is, the present application includes the following inventions. (1) A method for amplifying a target nucleic acid sequence using a nucleic acid incorporating a modified oligonucleotide, wherein the amplification reaction is carried out in the presence of an enzyme having at least 90% homology to a polymerase derived from Thermus aquaticus in which the first to 289th amino acids are deleted and having DNA polymerase activity. (2) The method according to (1), wherein the target nucleic acid sequence is a sequence having a mutation. (3) The method described in (2), which includes a step in which a nucleic acid into which a modified oligonucleotide has been introduced forms a double strand with a wild-type target nucleic acid sequence, thereby inhibiting the amplification of the wild-type target nucleic acid sequence. (4) The method according to any one of (1) to (3), wherein the modified oligonucleotide is a locked nucleic acid (LNA). (5) The method according to any one of (1) to (4), wherein the enzyme is produced by expression in a host. (6) The method according to (5), wherein the enzyme is produced by a method including a step of heating a host whose cell wall has been destroyed at a temperature of less than 64°C, at which proteins derived from the host cell are denatured. (7) The method according to (6), wherein the amplification of a wild-type target nucleic acid sequence is further inhibited than when the amplification reaction is carried out in the presence of an enzyme, produced by a method including a step of heating a host whose cell wall has been destroyed at a temperature of 65°C or higher. (8) The method according to any one of (1) to (7), wherein the concentration of the modified oligonucleotide in the reaction solution for the amplification reaction is less than 100 nM. (9) The method according to any one of (1) to (8), wherein the enzyme does not have 5'→3' exonuclease activity and 3'→5' exonuclease activity. (10) The method according to any one of (1) to (9), wherein the amplification reaction is carried out by PCR. (11) A kit for amplifying a target nucleic acid sequence, comprising: 1) a nucleic acid into which a modified oligonucleotide has been introduced; 2) an enzyme having at least 90% homology to a polymerase derived from Thermus aquaticus in which the 1st to 289th amino acids are deleted and having DNA polymerase activity; Kit including: (12) The kit according to (11), wherein the target nucleic acid sequence is a sequence having a mutation. (13) The kit according to (11) or (12), wherein the amplification reaction is carried out in the presence of the enzyme of (2). (14) The kit according to any one of (11) to (13), wherein the modified oligonucleotide is a locked nucleic acid (LNA). [Effects of the Invention]

[0011] The present invention provides novel polymerases that can be used in amplification reactions of target nucleic acid sequences using nucleic acids incorporating modified oligonucleotides. Furthermore, when the polymerases are produced by expression in a host, by heating host-derived proteins at a temperature below 64°C, at which the host cell-derived proteins denature, novel polymerases can be provided that have higher activity and can inhibit the amplification of sequences other than the target nucleic acid sequence. Furthermore, methods for amplifying target nucleic acid sequences using these polymerases can be provided. [Brief explanation of the drawings]

[0012] [Figure 1] Shown are the results of LNA-blocked PCR carried out in the presence of mutant KlenTaq, commercially available AccuTaq LA DNA polymerase, and HiDi DNA polymerase. [Figure 2] The results of LNA block PCR performed in the presence of mutant KlenTaqs with different heat treatment temperatures during enzyme purification and commercially available HiDi DNA polymerase are shown. [Figure 3]The results of LNA block PCR carried out using primer / LNA block set numbers 2 to 10 in the presence of mutant KlenTaq and commercially available HiDi DNA polymerase are shown. [Figure 4] The results of LNA block PCR carried out using primer / LNA block set numbers 11 to 19 in the presence of mutant KlenTaq and commercially available HiDi DNA polymerase are shown. [Figure 5] 1 shows the results of LNA block PCR performed using primer / LNA block set number 11, which has a lower concentration of LNA block, in the presence of mutant KlenTaq and commercially available HiDi DNA polymerase.

[0013] Hereinafter, an embodiment of the present invention (hereinafter referred to as "the present embodiment") will be described, but the scope of the present invention should not be interpreted as being limited to the following embodiment.

[0014] (Method for amplifying a target nucleic acid sequence using a nucleic acid incorporating a modified oligonucleotide) In a first embodiment, a method for amplifying a target nucleic acid sequence using a nucleic acid incorporating a modified oligonucleotide is provided, wherein the amplification reaction is carried out in the presence of an enzyme having at least 90% homology to a polymerase derived from Thermus aquaticus in which amino acids 1 to 289 are deleted and having DNA polymerase activity.

[0015] Method for amplifying a target nucleic acid sequence using a nucleic acid incorporating a modified oligonucleotide As used herein, "nucleic acid" refers to deoxyribonucleic acid (DNA), ribonucleic acid (RNA), or an oligonucleotide consisting of one or more deoxyribonucleic acids (DNA) and / or ribonucleic acids (RNA), and may be an artificially synthesized nucleic acid, and may be a single-stranded nucleic acid or a double-stranded nucleic acid. The term "nucleic acid" can be used interchangeably with "nucleotide," and the term "oligonucleotide" can be used interchangeably with "polynucleotide." In this embodiment, the nucleic acid into which the modified oligonucleotide has been introduced is preferably a single-stranded nucleic acid.

[0016] As used herein, "nucleic acid sequence" refers to an oligonucleotide or polynucleotide comprising multiple deoxyribonucleic acid (DNA) and / or ribonucleic acid (RNA).

[0017] As used herein, the term "amplification reaction" refers broadly to a method for replicating or amplifying a specific sequence in a nucleic acid. The amplification reaction may be, for example, a polymerase chain reaction (PCR) or a PCR-based amplification reaction such as multiplex PCR, quantitative PCR, or droplet digital PCR.

[0018] As used herein, "modified oligonucleotide" refers to a nucleic acid in which the phosphate moiety, sugar moiety, and / or base moiety has been chemically modified or altered.

[0019] Examples of nucleic acids in which the phosphate moiety is modified include phosphorothioate-modified nucleic acids in which an oxygen atom is substituted with a sulfur atom, and boranophosphate-modified nucleic acids.

[0020] Examples of nucleic acids with modified sugar moieties include nucleic acids with modified hydroxyl groups at position 2, nucleic acids in which oxygen at position 2 and carbon at position 4 are bridged, and nucleic acids modified to acyclic forms, and specific examples include 2-F modified nucleic acids, 2-OMe modified nucleic acids, 2-MOE modified nucleic acids, locked nucleic acids (LNA), bridged nucleic acids (BNA), peptide nucleic acids (PNA), and serinol nucleic acids (SNA).

[0021] Examples of nucleic acids with modified base moieties include pseudouridine and methyladenosine.

[0022] In this embodiment, the modified oligonucleotide is preferably a nucleic acid whose sugar moiety is modified, and among these, LNA is more preferred.

[0023] The structure of the sugar moiety of the nucleic acid, which has a fluctuating structure, may be stabilized by modifying the hydroxyl group at position 2, bridging the oxygen at position 2 with the carbon at position 4, or substituting a peptide-derived backbone for the sugar backbone to make it non-cyclizable. Introduction of such modified oligonucleotides into nucleic acids strengthens the binding of the nucleic acid to the complementary strand, increasing the Tm value and improving specificity.

[0024] Nucleic acids incorporating modified oligonucleotides that have stronger binding to complementary strands and improved specificity can be used in amplification reactions, for example, as primers, labeled probes, clamps for inhibiting amplification, etc., but are preferably used to inhibit the amplification of specific sequences.

[0025] Therefore, in this embodiment, the nucleic acid into which the modified oligonucleotide has been introduced is preferably designed to have a sequence complementary to the sequence whose amplification needs to be inhibited, in order to inhibit the amplification of a specific sequence.

[0026] A nucleic acid into which a modified oligonucleotide has been introduced contains a sequence complementary to a sequence whose amplification needs to be inhibited, and thus forms a double strand with the sequence, thereby inhibiting amplification. That is, a nucleic acid that has been strongly bound to a complementary strand by the introduction of a modified oligonucleotide can maintain a double-stranded structure with the complementary strand even in the extension reaction step by a polymerase in the amplification reaction, thereby inhibiting the extension of the sequence by the polymerase and inhibiting the amplification of the sequence to which it is bound.

[0027] As used herein, the term "target nucleic acid sequence" refers to a nucleic acid sequence intended to be amplified. In this embodiment, when a nucleic acid into which a modified oligonucleotide has been introduced is used to inhibit a specific sequence, it is preferable that the nucleic acid into which the modified oligonucleotide has been introduced inhibits the amplification of nucleic acid sequences other than the target nucleic acid sequence. Therefore, in this specification, the target nucleic acid sequence may also mean a nucleic acid sequence whose amplification is not inhibited by a nucleic acid into which a modified oligonucleotide has been introduced. In other words, the target nucleic acid sequence may be a sequence for which inhibition of amplification is not required.

[0028] The nucleic acid into which the modified oligonucleotide has been introduced inhibits the amplification of nucleic acid sequences other than the target nucleic acid sequence, thereby enabling specific amplification of the target nucleic acid sequence. The method for amplifying a target nucleic acid sequence of this embodiment may be a method for specifically amplifying the target nucleic acid sequence, or a method for inhibiting the amplification of sequences other than the target nucleic acid sequence.

[0029] The Tm value of the nucleic acid into which the modified oligonucleotide has been introduced is preferably higher than the reaction temperature in the extension reaction step with polymerase, and is preferably, for example, 3 to 5° C. or more higher than the reaction temperature in the extension reaction step with polymerase, and more preferably, for example, 6 to 10° C. or more higher. This is because the binding with the complementary sequence is maintained even in the extension reaction step with polymerase in the amplification reaction.

[0030] In this embodiment, the target nucleic acid sequence may be wild-type, or may have one or more mutations in its sequence. When the target nucleic acid sequence has a mutation, it is preferable that the nucleic acid into which the modified oligonucleotide has been introduced has a sequence complementary to the wild-type target nucleic acid sequence, and that it maintains its binding to the wild-type target nucleic acid sequence even in the extension reaction step by polymerase in the amplification reaction, thereby inhibiting the amplification of the wild-type target nucleic acid sequence. It is preferable that the target nucleic acid sequence having a mutation is specifically amplified by inhibiting the amplification of the wild-type target nucleic acid sequence. By specifically amplifying the target nucleic acid sequence having a mutation, it is possible to detect whether the template nucleic acid has a mutation.

[0031] Therefore, the method for amplifying a target nucleic acid sequence according to this embodiment can be carried out to detect mutations, and therefore this embodiment also provides a method for detecting mutations using a nucleic acid into which a modified oligonucleotide has been introduced.

[0032] As used herein, "mutation" refers to one or more bases in which a base substitution, deletion, or insertion has occurred, or the state in which such a base substitution or deletion has occurred. "Mutation" can be used interchangeably with "polymorphism." When a target nucleic acid sequence has a mutation, the position of the base in the target nucleic acid sequence where the base substitution, deletion, or insertion has occurred, the number of bases substituted or deleted, and the number of bases inserted can be appropriately set depending on the purpose. The type of mutation is not particularly limited, but includes missense mutation, nonsense mutation, frameshift mutation, silent mutation, etc. In this embodiment, the mutation may be a single nucleotide polymorphism.

[0033] The nucleic acid into which a modified oligonucleotide has been introduced may be a nucleic acid sequence containing a sequence complementary to only a portion of a sequence whose amplification needs to be inhibited, as long as it maintains binding to the complementary sequence in the polymerase-mediated extension reaction step in the amplification reaction. Thus, the nucleic acid into which a modified oligonucleotide has been introduced may be a nucleic acid sequence containing a sequence complementary to a portion of a sequence whose amplification needs to be inhibited, or may be a nucleic acid sequence containing a sequence complementary to a portion of a sequence whose amplification needs to be inhibited and an arbitrary sequence. Furthermore, the nucleic acid into which a modified oligonucleotide has been introduced may be a nucleic acid sequence containing a sequence complementary to the entire sequence whose amplification needs to be inhibited, or may be a nucleic acid sequence containing a sequence complementary to the entire sequence whose amplification needs to be inhibited and an arbitrary sequence. The sequence complementary to a portion or all of a sequence whose amplification needs to be inhibited in the nucleic acid into which a modified oligonucleotide has been introduced preferably has a Tm value higher than the reaction temperature of the polymerase-mediated extension reaction in the amplification reaction.

[0034] The length of the nucleic acid into which the modified oligonucleotide has been introduced and the length of the sequence complementary to part or all of the sequence in the nucleic acid into which the modified oligonucleotide has been introduced, the amplification of which requires inhibition, can be determined appropriately by those skilled in the art, and may be determined, for example, based on the Tm value of the complementary portion, the reaction temperature in the amplification reaction, and, among reaction temperatures, the reaction temperature in the extension reaction by polymerase.

[0035] The number and site of modified oligonucleotides introduced into a nucleic acid may be appropriately determined by those skilled in the art, as long as the nucleic acid into which the modified oligonucleotides have been introduced maintains binding with a complementary sequence in the polymerase-mediated extension reaction step of the amplification reaction. Factors to be considered in this determination include, for example, the Tm value of the nucleic acid after the modified oligonucleotides have been introduced and the reaction temperature in the amplification reaction, and among the reaction temperatures, the reaction temperature in the polymerase-mediated extension reaction is important. The nucleic acid into which the modified oligonucleotides have been introduced may be a nucleic acid consisting solely of modified oligonucleotides.

[0036] The reaction solution for the amplification reaction may contain, in addition to the nucleic acid into which the modified oligonucleotide has been introduced and the template containing the target nucleic acid sequence, any buffer, nucleic acid, and other necessary reagents. The reaction solution may contain, for example, primers for amplifying the target nucleic acid sequence, deoxynucleoside triphosphates (dNTPs), (NH4)2SO4, Tween-20, MgCl2, Tris-HCl, BSA, DMSO, glycerol, water, DNA or RNA enzyme inhibitors, etc.

[0037] The number of types of nucleic acids to which modified oligonucleotides have been introduced, used in the amplification reaction, may be any number, but may be determined based on the number of types of sequences for which inhibition of amplification is required.

[0038] The nucleic acid serving as a template in the amplification reaction may contain any nucleic acid sequence in addition to the target nucleic acid sequence, but preferably contains a nucleic acid sequence similar to the target nucleic acid sequence. For example, when the target nucleic acid sequence has a mutation, the template nucleic acid preferably contains a wild-type target nucleic acid sequence as a template in addition to the target nucleic acid sequence.

[0039] When a normal amplification reaction is performed using a nucleic acid containing multiple sequences similar to each other as a template, such as the relationship between a mutant nucleic acid sequence and a wild-type nucleic acid sequence, and without using a nucleic acid into which a modified oligonucleotide has been introduced, not only the target nucleic acid sequence but also sequences similar to it will be amplified. However, by carrying out the method of this embodiment, which uses a nucleic acid into which a modified oligonucleotide has been introduced, it is possible to specifically amplify the target nucleic acid sequence.

[0040] Therefore, when the method of this embodiment is carried out using multiple nucleic acid sequences as templates that are similar to each other and would be amplified indiscriminately in a normal amplification reaction, the effect of specifically amplifying the target nucleic acid sequence can be more effectively achieved.

[0041] Any number of different templates may be used in the amplification reaction, in which case the number of different target nucleic acid sequences and the number of different sequences for which amplification inhibition is required may be any number.

[0042] The template nucleic acid may be in any form, but when the target nucleic acid sequence has a mutation, for example, genomic DNA derived from a cell line or tissue having the mutation is preferred. Furthermore, when amplifying by colony direct PCR or the like, cells or tissues may be used directly as templates instead of nucleic acids.

[0043] Those skilled in the art can appropriately determine which reaction steps to include in an amplification reaction, but it is preferable to include, for example, an annealing step, an extension reaction step using a polymerase, and a thermal denaturation step. A thermal denaturation step may be included before the reaction cycle. In the annealing step, the nucleic acid into which the modified oligonucleotide has been introduced or the primer binds to the template nucleic acid.

[0044] The number of cycles of the amplification reaction can also be appropriately determined by those skilled in the art, but is preferably 20 to 50 times, for example.

[0045] Various reaction conditions, such as the reaction temperature and reaction time for each step in the amplification reaction, can be appropriately determined by those skilled in the art. For example, the annealing temperature is preferably 45 to 65°C, the polymerase extension reaction temperature is preferably 65 to 75°C, and the thermal denaturation temperature is preferably 90°C or higher. The polymerase extension reaction temperature may be determined based on the optimal temperature of the polymerase used.

[0046] The nucleic acid into which the modified nucleotide has been introduced and the enzyme may be added to the reaction solution at any timing, and for example, may be contained in the reaction solution before the start of the reaction.

[0047] The amplification of a target nucleic acid sequence and the inhibition of amplification of a specific sequence other than the target nucleic acid sequence can be evaluated, for example, by analyzing the concentration of the amplification reaction product or qualitatively analyzing the amplification product by electrophoresis, etc. Specifically, such evaluation can involve, for example, using primers for amplifying the target nucleic acid, performing an amplification reaction using a nucleic acid containing the target nucleic acid sequence and / or a sequence whose amplification needs to be inhibited as a template, and using a nucleic acid into which a modified oligonucleotide has been introduced, and analyzing the amplification product of the target nucleic acid sequence and the amplification product of the sequence whose amplification needs to be inhibited. If the concentration of the amplification product of the sequence whose amplification needs to be inhibited is lower than the concentration of the amplification product of the sequence in the absence of a nucleic acid into which a modified oligonucleotide has been introduced, it can be evaluated that the amplification of the sequence has been inhibited by the nucleic acid into which a modified oligonucleotide has been introduced. Furthermore, for example, if the amplification of a specific sequence other than the target nucleic acid sequence is inhibited and the target nucleic acid sequence is amplified, it can be evaluated that the target nucleic acid sequence has been specifically amplified.

[0048] Enzymes in amplification reactions In this embodiment, the amplification reaction is carried out in the presence of an enzyme having DNA polymerase activity and at least 90% homology to the polymerase derived from Thermus aquaticus lacking amino acids 1 to 289. That is, the enzyme having DNA polymerase activity and at least 90% homology to the polymerase derived from Thermus aquaticus lacking amino acids 1 to 289 catalyzes the amplification reaction, specifically, the extension reaction of the complementary sequence of the template nucleic acid.

[0049] As used herein, "a Thermus aquaticus polymerase lacking amino acids 1 to 289" (also known as a Klenow fragment) refers to a DNA polymerase lacking 5' to 3' exonuclease activity and 3' to 5' exonuclease activity, which is a Thermus aquaticus polymerase lacking 5' to 3' exonuclease activity but lacking 3' to 5' exonuclease activity. Furthermore, a "Thermus aquaticus polymerase lacking amino acids 1 to 289" may be a polymerase capable of accurately recognizing a sequence containing a mutation, such as a polymerase that can be used for allele-specific PCR. The "polymerase derived from Thermus aquaticus in which the 1st to 289th amino acids are deleted" may be a "polymerase derived from Thermus aquaticus in which the 1st to 289th amino acids are deleted." In the following explanation, although not particularly limited thereto, a "polymerase derived from Thermus aquaticus in which the 1st to 289th amino acids are deleted" will be exemplified. When the polymerase of this embodiment is a "polymerase derived from Thermus aquaticus in which the 1st to 292nd amino acids are deleted," in the following description, "a polymerase derived from Thermus aquaticus in which the 1st to 289th amino acids are deleted" should be read as "a polymerase derived from Thermus aquaticus in which the 1st to 292nd amino acids are deleted."

[0050] The polymerase derived from Thermus aquaticus in which the 1st to 289th amino acids are deleted may be, for example, a polymerase consisting of the amino acid sequence of SEQ ID NO: 1. However, the amino acid sequence of the polymerase is not limited to that set forth in SEQ ID NO: 1, and any DNA polymerase derived from Thermus aquaticus that does not have 5'→3' exonuclease activity or 3'→5' exonuclease activity may be used.

[0051] As used herein, the term "DNA polymerase" broadly refers to an enzyme that catalyzes the synthesis of a DNA sequence complementary to a template nucleic acid, but it may be a polymerase that synthesizes DNA using DNA as a template, or a polymerase that synthesizes DNA using RNA as a template.

[0052] Here, an enzyme having at least 90% sequence identity with the polymerase derived from Thermus aquaticus from which the 1st to 289th amino acids have been deleted is an enzyme having at least 90% sequence identity with the amino acid sequence of the polymerase derived from Thermus aquaticus from which the 1st to 289th amino acids have been deleted.

[0053] As used herein, "homology" or "sequence homology" refers to the percentage (%) of identical or similar amino acids in the same position or line when two amino acid sequences are aligned. When two amino acid sequences are aligned, the percentage (%) of identical amino acids in the same position or line is called identity. In this specification, homology may also mean identity. For example, an enzyme having 90% homology to a specific enzyme consisting of 100 amino acids may be an enzyme that, when aligned with the amino acid sequence of the specific enzyme consisting of 100 amino acids, has 90 positions where identical or similar amino acids are lined up in the same position or row.

[0054] Furthermore, as used herein, "similar amino acids" refers to two or more amino acids that have similar chemical or physical properties. For example, two amino acids having similar chemical or physical properties means that the two amino acids are classified into the same amino acid group, such as acidic amino acids, basic amino acids, aromatic amino acids, aliphatic amino acids, amino acids with a hydroxyl group, hydrophilic amino acids, or hydrophobic amino acids.

[0055] The homology of the enzyme that catalyzes the amplification reaction to the polymerase derived from Thermus aquaticus, which lacks amino acids 1 to 289, is at least 90% or more, and may be determined appropriately by one skilled in the art, as long as the enzyme has DNA polymerase activity.

[0056] In one embodiment, the amplification reaction is carried out in the presence of an enzyme that has 90% or more, e.g., 91% or more, 92% or more, 93% or more, 94% or more, 95% or more, 96% or more, 97% or more, 98% or more, 99% or more, or 100% homology to the polymerase from Thermus aquaticus in which amino acids 1 to 289 are deleted, and that has DNA polymerase activity.

[0057] In one embodiment, the amplification reaction is carried out in the presence of an enzyme that has 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, or 100% homology to a polymerase derived from Thermus aquaticus lacking amino acids 1 through 289 and that has DNA polymerase activity.

[0058] In one embodiment, the amplification reaction is carried out in the presence of an enzyme consisting of the amino acid sequence set forth in SEQ ID NO:1.

[0059] In one embodiment, the amplification reaction is carried out in the presence of an enzyme consisting of the amino acid sequence set forth in SEQ ID NO: 1, in which arginine at position 368 has been mutated to valine.

[0060] A polymerase may be modified. For example, when the polymerase has at least 90% homology with a polymerase derived from Thermus aquaticus from which the amino acids 1 to 289 are deleted, one or several, or even multiple, amino acids may be added, substituted, or deleted in the amino acid sequence of the polymerase. The number of added, substituted, or deleted amino acids can be determined appropriately by those skilled in the art to be 10% or less of the total number of amino acids in a polymerase derived from Thermus aquaticus from which the amino acids 1 to 289 are deleted. As long as the polymerase has polymerase activity, any number of amino acids, for example, 10% or less of the total number of amino acids, may be added, substituted, or deleted.

[0061] In one embodiment, the amplification reaction is carried out in the presence of an enzyme having DNA polymerase activity, wherein 1 to 54, 1 to 50, 1 to 40, 1 to 30, 1 to 20, 1 to 10, or 1 to 5 amino acids have been added, substituted, or deleted from the amino acid sequence of a polymerase derived from Thermus aquaticus, in which amino acids 1 to 289 have been deleted.

[0062] In one embodiment, the amplification reaction is carried out in the presence of an enzyme derived from Thermus aquaticus in which amino acids 1 to 289 have been deleted and in which arginine 368 has been mutated to valine. When the polymerase of this embodiment is an enzyme in which arginine at position 368 has been mutated to valine, the polymerase has the amino acid sequence of SEQ ID NO: 1 in which arginine at position 368 has been mutated to valine. When the polymerase in this embodiment is an enzyme in which arginine at position 368 is mutated to valine, the polymerase may be selected from the group consisting of: 1) a polymerase consisting of an amino acid sequence in which arginine at position 368 is mutated to valine in SEQ ID NO: 1; 2) a polymerase consisting of an amino acid sequence that is at least 90% homologous to the amino acid sequence in which arginine at position 368 is mutated to valine in SEQ ID NO: 1; 3) a polymerase consisting of an amino acid sequence in which one or more amino acids are deleted, substituted, and / or added in the amino acid sequence in which arginine at position 368 is mutated to valine in SEQ ID NO: 1; and 4) a polymerase encoded by a nucleotide sequence that hybridizes under stringent conditions to a complementary sequence of a nucleotide sequence encoding the amino acid sequence in which arginine at position 368 is mutated to valine in SEQ ID NO: 1.

[0063] As used herein, the term "a base sequence that hybridizes under stringent conditions" refers to a DNA base sequence that can be obtained by using, as a probe, the complementary sequence of a wild-type gene encoding the amino acid sequence in which arginine at position 368 in SEQ ID NO: 1 is mutated to valine, and employing colony hybridization, plaque hybridization, Southern blot hybridization, or the like.

[0064] As used herein, "stringent conditions" refer to conditions under which signals from specific hybrids are clearly distinguished from signals from nonspecific hybrids, and vary depending on the hybridization system used and the type, sequence, and length of the probe. Such conditions can be determined by changing the hybridization temperature, washing temperature, and salt concentration.

[0065] For example, if a strong signal from a nonspecific hybrid is detected, specificity can be improved by increasing the hybridization and washing temperature and, if necessary, decreasing the salt concentration in the washing. If a signal from a specific hybrid is not detected, hybrids can be stabilized by decreasing the hybridization and washing temperature and, if necessary, increasing the salt concentration in the washing.

[0066] In some embodiments, specific examples of stringent conditions include the following: For example, a DNA probe is used as the probe, and hybridization is performed overnight (approximately 8 to 16 hours) using 5x SSC, 1.0% (w / v) blocking reagent for nucleic acid hybridization (Boehringer-Mannheim), 0.1% (w / v) N-lauroyl sarcosine, and 0.02% (w / v) SDS. Washing is performed twice for 15 minutes using 0.1 to 0.5x SSC and 0.1% (w / v) SDS, preferably 0.1x SSC and 0.1% (w / v) SDS. The temperature for hybridization and washing is 65°C or higher, preferably 68°C or higher.

[0067] Examples of base sequences that hybridize under stringent conditions include DNA obtained by hybridizing under the above-mentioned stringent conditions using a filter onto which DNA having a complementary sequence of a wild-type gene derived from a colony or plaque or a fragment of said DNA is immobilized; and DNA that can be identified by hybridizing in the presence of 0.5 to 2.0 M NaCl at 40 to 75°C, preferably in the presence of 0.7 to 1.0 M NaCl at 65°C, and then washing the filter at 65°C using 0.1 to 1×SSC solution (1×SSC solution is 150 mM sodium chloride, 15 mM sodium citrate). Probe preparation and hybridization methods can be carried out in accordance with the methods described in Molecular Cloning: A Laboratory Manual, 2nd Ed., Cold Spring Harbor Laboratory, Cold Spring Harbor, NY., 1989, Current Protocols in Molecular Biology, Supplement 1-38, John Wiley & Sons, 1987-1997, etc.

[0068] Those skilled in the art can appropriately set conditions for obtaining a base sequence that hybridizes under stringent conditions with the complementary sequence of a wild-type gene, taking into account not only the salt concentration of the buffer and temperature, but also other conditions such as probe concentration, probe length, and reaction time.

[0069] In this embodiment, the polymerase encoded by a base sequence that hybridizes under stringent conditions to the complementary sequence of a base sequence encoding an amino acid sequence in which arginine at position 368 in SEQ ID NO: 1 is mutated to valine is likely to be a polymerase having an amino acid sequence that has one to multiple, preferably several, amino acid deletions, substitutions, additions, etc. in the amino acid sequence of the polymerase encoded by the base sequence of the wild-type gene, but has the same polymerase activity as the polymerase encoded by the base sequence of the wild-type gene.

[0070] Furthermore, it is preferable that the enzyme has at least 90% homology with the polymerase derived from Thermus aquaticus, which has the amino acids 1 to 289 deleted, and has DNA polymerase activity but does not have 5'→3' exonuclease activity or 3'→5' exonuclease activity.

[0071] Specific amplification of a target nucleic acid sequence can be achieved by carrying out an amplification reaction using a nucleic acid into which a modified oligonucleotide has been introduced in the presence of an enzyme having DNA polymerase activity and at least 90% homology to the polymerase derived from Thermus aquaticus in which the amino acids 1 to 289 have been deleted. That is, the enzyme having DNA polymerase activity and at least 90% homology to the polymerase derived from Thermus aquaticus in which the amino acids 1 to 289 have been deleted can catalyze an amplification reaction using a nucleic acid into which a modified oligonucleotide has been introduced, specifically an extension reaction of a complementary sequence of a template nucleic acid, which inhibits the amplification of a specific sequence other than the target nucleic acid sequence and specifically amplifies the target nucleic acid sequence.

[0072] When the target nucleic acid sequence has a mutation, an enzyme that has at least 90% homology with the polymerase derived from Thermus aquaticus in which the 1st to 289th amino acids are deleted and has DNA polymerase activity can catalyze an amplification reaction using a nucleic acid containing a modified oligonucleotide that inhibits the amplification of the wild-type target nucleic acid sequence and specifically amplifies the target nucleic acid sequence having a mutation, specifically, an extension reaction of the complementary sequence of the target nucleic acid sequence that serves as a template.

[0073] How enzymes are produced An enzyme having at least 90% homology to the polymerase derived from Thermus aquaticus in which the 1st to 289th amino acids are deleted and having DNA polymerase activity may be produced by any method known to those skilled in the art.

[0074] For example, the target enzyme may be expressed by directly editing a gene related to a polymerase in cells derived from Bacillus aquaticus, or the target enzyme may be expressed in vitro rather than within a cell. However, it is preferable to produce the target enzyme by introducing a vector containing a gene that expresses the target enzyme into a host, transforming it, and allowing the host to express the enzyme.

[0075] As used herein, the term "host" refers to an organism, such as a bacterium, fungus, or plant cell, that has been transformed to express a desired protein derived from a thermophilic bacterium. The host bacterium may be Escherichia coli, Bacillus subtilis, actinomycete, or the like, and the host fungus may be a yeast or filamentous fungus, or the like.

[0076] In one embodiment, the host is E. coli.

[0077] When the host is a prokaryote, the protein expressed in the host may be a protein derived from a thermophilic prokaryote or a protein derived from a thermophilic eukaryote. Furthermore, when the host is a eukaryote, the protein expressed in the host may be a protein derived from a thermophilic eukaryote or a protein derived from a thermophilic prokaryote. For example, when the host is Escherichia coli, the protein expressed in the host may be a protein derived from any thermophilic bacterium, thermophilic fungus, or thermophilic archaea.

[0078] Transformation of host cells may be carried out by any method known to those skilled in the art, such as electroporation or heat shock.

[0079] To transform a host, an exogenous expression vector encoding a protein of interest is prepared. The expression vector may encode multiple proteins of interest. The expression vector preferably encodes a selectable marker, such as an antibiotic resistance gene, used to select transformants.

[0080] To increase the transformation efficiency, the host is preferably subjected to heat treatment or electrical treatment before transformation, which increases the efficiency of expression vector uptake in the resulting competent cells.

[0081] The transformed host is also referred to as a transformant. Selection of the transformant, growth of the transformant, induction of expression of the target protein, and recovery of the host expressing the target protein may be carried out by any method known to those skilled in the art.

[0082] Transformants can be obtained by culturing a mixture of an expression vector and host competent cells in a medium containing an antibiotic corresponding to the selection marker encoded by the expression vector, and selecting the colonies that form. While the type of medium varies depending on the host cell, LB medium is an example of a medium for E. coli. Plate media are preferred for selecting transformants. Culture conditions are appropriately adjusted, for example, at a temperature of approximately 30°C to 37°C and for a culture time of approximately 12 hours to overnight.

[0083] The selected transformant is grown by culturing. Pre-culturing may be performed before the main culturing. The growth of the transformant can also be carried out under the culture conditions used for selecting the transformant, but the culture medium is preferably a liquid medium. The culture temperature and culture time are appropriately adjusted depending on the desired level of growth of the transformant, and are not intended to be limiting, but are appropriately adjusted between about 16°C and 37°C and about 12 hours to overnight.

[0084] The conditions for inducing expression of a target protein vary depending on the expression system used. While not intended to be limiting, the pET system is a preferred expression system when E. coli is used as a host. The pET system is a protein expression system in which T7 RNA polymerase transcribes the target gene under the control of the lacUV5 promoter and is induced in the presence of allolactose or the lactose analog IPTG (isopropyl-β-thiogalactopyranoside).

[0085] In the case of an expression system using the pET system, expression of the target protein can be induced by adding IPTG to the medium and culturing under specified conditions. The culture temperature and culture time are adjusted appropriately depending on the desired level of expression, and are not intended to be limiting, but are appropriately adjusted between 1 hour and overnight, and between 16°C and 37°C.

[0086] To recover expressed proteins from the host, host cells are subjected to a disruption process. As used herein, cell disruption refers to the destruction of cell walls. The disruption method is not particularly limited, but can be broadly divided into mechanical and non-mechanical methods. Examples of mechanical methods include ultrasonic disruption, disruption using known devices such as homogenizers and blenders. Non-mechanical methods are methods that do not fall under the mechanical method category, and examples include chemical methods using chemical agents such as surfactants or enzymes such as lysozyme. When disrupting host cells using chemical agents such as surfactants or enzymes such as lysozyme, protease inhibitors, DNase, etc. may be added as needed in addition to the surfactants and lysozyme. Non-mechanical methods are not limited to chemical methods and may also utilize osmotic pressure differences.

[0087] Various conditions, such as temperature conditions, used in the process of disrupting host cells can be appropriately determined by those skilled in the art. For example, mechanical methods are generally performed on ice because heat is generated during the disruption process. Non-mechanical chemical methods include the lysis reagent method, in which the host cell wall is disrupted by suspending it in a buffer containing a surfactant or lysozyme and treating it at room temperature. However, when a non-mechanical method is used to disrupt host cells in the present invention, it is preferable that the treatment temperature is not room temperature. Room temperature may be, for example, 10 to 30°C.

[0088] The conditions for non-mechanical disruption of host cells at temperatures other than room temperature can be determined appropriately by those skilled in the art. For example, when disrupting host cells using a lysis reagent, among other non-mechanical methods, the treatment temperature is adjusted by mixing the host in a buffer containing necessary reagents, such as a surfactant or lysozyme, on ice. When the mixture of the host and buffer containing the reagent is removed from the ice and suspended using a known device such as a vortex mixer, it is preferable to suspend the mixture quickly, for example, within 10 seconds, so that the temperature of the mixture does not rise to room temperature. The procedure of removing the cells from the ice, suspending them using a vortex mixer, returning them to ice, and then removing them from the ice and suspending them using a vortex mixer may be repeated. When suspending without using a known device such as a vortex mixer, it is preferable to always perform the suspension procedure on ice. Avoiding incubation at room temperature after mixing and suspension allows for rapid transition to the heating step. If the heating step is not to be transitioned to immediately after mixing and suspension, it is preferable to store the mixture on ice.

[0089] After the disruption step, the host cells are subjected to a heating step. The heating step is carried out to denature the host cell-derived proteins, and is carried out at a temperature below 64°C at which the host cell-derived proteins denature. The temperature used in the heating step is also referred to as the "heating temperature" or the "treatment temperature," and these terms are used interchangeably. Denaturation occurs when the helical or sheet structure of the protein is disrupted, and the temperature at which the protein denatures can be determined by known methods for analyzing the secondary structure of proteins, such as circular dichroism spectroscopy or fluorescence spectroscopy.

[0090] As used herein, "a temperature below 64°C at which a host cell-derived protein denatures" refers to a temperature equal to or higher than a specific temperature at which the structure of a host cell-derived protein changes and the function of the protein is lost, but lower than 64°C. It also refers to a temperature at which a host cell-derived protein denatures but a thermophilic bacterium-derived protein does not denature.

[0091] The heating temperature can be appropriately determined by those skilled in the art depending on the growth temperature of the thermophilic bacterium. For example, when a thermophilic bacterium with a low growth temperature, such as a moderate thermophilic bacterium, is used, the treatment may be carried out at a lower temperature than that used in the heating step for an extreme thermophilic bacterium.

[0092] In one embodiment, the heating temperature is 50°C or higher.

[0093] The heating temperature is 50°C, 51°C, 52°C, 53°C, 54°C, 55°C, 56°C, 57°C, 58°C, 59°C, 60°C, 61°C, 62°C, or 63°C. The heating temperature is preferably 50°C to 60°C, more preferably 55°C to 60°C. The heating step time varies depending on the heating temperature, but may be any time as long as the host cell-derived protein is denatured.

[0094] In one embodiment, the heating temperature is about 50 to 62°C, preferably about 54 to 60°C.

[0095] In one embodiment, when the heating temperature is within the range of 50 to 60°C, the heating step time is, for example, 5 minutes, 6 minutes, 7 minutes, 8 minutes, 9 minutes, 10 minutes, 11 minutes, 12 minutes, 13 minutes, 14 minutes, 15 minutes, 16 minutes, 17 minutes, 18 minutes, or 19 minutes.

[0096] Heating may be performed using any device known to those skilled in the art, for example, an incubator such as a heat block, or, in the case of industrial production, a heating tank.

[0097] The host and / or the protein produced by the host may be cooled after the heating step. Proteins produced by the host are usually separated from other components by a step such as centrifugation, but by cooling the host and / or the protein produced by the host, the insoluble fraction and the soluble fraction can be separated more clearly than when not cooled, thereby increasing the recovery amount of the target protein contained in either fraction. Cooling is preferably performed on ice, but may be performed at a temperature lower than room temperature, for example, at 0 to 10°C. The cooling time may be any time as long as the insoluble fraction and the soluble fraction can be clearly separated, for example, 1 to 30 minutes. The cooling step is preferably performed promptly after the heat treatment.

[0098] After cooling, the protein produced from the host is collected by centrifugation or the like.

[0099] The produced protein may be purified by any method known to those skilled in the art, for example, affinity chromatography, ion exchange chromatography, or gel filtration chromatography.

[0100] When an amplification reaction is carried out in the presence of an enzyme produced by a method including a step of heating a host whose cell wall has been destroyed at a temperature below 64° C. at which host cell-derived proteins denature, inhibition of amplification of a specific sequence by nucleic acid into which a modified oligonucleotide has been introduced is further enhanced than when an amplification reaction is carried out in the presence of an enzyme produced by a method including a step of heating at a temperature of 65° C. or higher. In other words, when an amplification reaction is carried out in the presence of an enzyme produced by a method including a step of heating at a temperature below 64° C. at which host cell-derived proteins denature, the amount of amplification product of a specific sequence whose amplification is not inhibited by nucleic acid into which a modified oligonucleotide has been introduced is smaller.

[0101] Furthermore, by carrying out an amplification reaction in the presence of an enzyme produced by a method including a step of heating at a temperature below 64°C at which host cell-derived proteins denature, a smaller amount of nucleic acid into which a modified oligonucleotide has been introduced is required to inhibit the amplification of a specific sequence than when carrying out an amplification reaction in the presence of an enzyme produced by a method including a step of heating at a temperature of 65°C or higher. In other words, a sufficient amplification inhibitory effect can be achieved with a lower concentration of modified oligonucleotide. For example, carrying out an amplification reaction in the presence of an enzyme produced by a method including a step of heating at a temperature below 64°C allows a smaller amount of nucleic acid into which a modified oligonucleotide has been introduced to inhibit a certain amount of a specific sequence than when carrying out an amplification reaction in the presence of an enzyme produced by a method including a step of heating at a temperature of 65°C or higher.

[0102] When an enzyme having at least 90% homology to a polymerase derived from Thermus aquaticus in which the 1st to 289th amino acids are deleted and having DNA polymerase activity is produced by a method including a step of heating a host whose cell wall has been destroyed to a temperature of less than 64°C at which proteins derived from the host cell are denatured, the concentration of nucleic acid containing modified oligonucleotides used in the amplification reaction is preferably less than 100 nM, more preferably less than 50 nM. In one embodiment, when an enzyme having at least 90% homology to a polymerase derived from Thermus aquaticus in which amino acids 1 to 289 are deleted and having DNA polymerase activity is produced by a method comprising a step of heating a host whose cell wall has been destroyed to a temperature of less than 64°C at which proteins derived from the host cell are denatured, the concentration of nucleic acid containing a modified oligonucleotide used in the amplification reaction is 0.1 nM, 0.2 nM, 0.3 nM, 0.4 nM, 0.5 nM, 0.6 nM, 0.7 nM, 0.8 nM, 0.9 nM, 1 nM, 2 nM, 3 nM, 4 nM, 5 nM, 6 nM, 7 nM, 8 nM, 9 nM, or 10 nM.

[0103] Similar to an enzyme having DNA polymerase activity and at least 90% homology to the polymerase derived from Thermus aquaticus lacking the 1st to 289th amino acids, any polymerase lacking 5'→3' exonuclease activity and 3'→5' exonuclease activity can amplify a target nucleic acid sequence more specifically by changing the temperature conditions of the heating step in the production method. That is, when any polymerase lacking 5'→3' exonuclease activity and 3'→5' exonuclease activity is produced by a method including a step of heating at a temperature below 64°C at which host cell-derived proteins denature, inhibition of amplification of a specific sequence by a nucleic acid into which a modified oligonucleotide has been introduced is further enhanced in an amplification reaction catalyzed by the any polymerase, compared to when the polymerase is produced by a method including a step of heating at a temperature of 65°C or higher.

[0104] This embodiment also provides an enzyme having at least 90% homology to a polymerase derived from Thermus aquaticus lacking amino acids 1 to 289 and having DNA polymerase activity, for amplifying a target nucleic acid sequence using a nucleic acid containing a modified oligonucleotide. The present embodiment also provides a method for producing an enzyme having at least 90% homology to a polymerase derived from Thermus aquaticus lacking the 1st to 289th amino acids and having DNA polymerase activity, for amplifying a target nucleic acid sequence using a nucleic acid into which a modified oligonucleotide has been introduced, the method comprising the step of heating a host expressing the enzyme, the host having a destroyed cell wall, at a temperature below 64°C at which proteins derived from the host cell are denatured.

[0105] Furthermore, an enzyme that is at least 90% homologous to the polymerase derived from Thermus aquaticus lacking the 1st to 289th amino acids and that has DNA polymerase activity can inhibit the amplification of a specific sequence with even greater activity than conventional polymerases that have been used in amplification reactions such as PCR that inhibit the amplification of specific sequences, such as AccuTaq LA DNA polymerase (Lone Hummelshoj et al. Locked nucleic acid inhibits amplification of contaminating DNA in real-time PCR. BioTechniques. 2005, 38(4), 605-610.). Furthermore, an enzyme that has at least 90% homology to the polymerase derived from Thermus aquaticus in which the 1st to 289th amino acids are deleted and that has DNA polymerase activity can inhibit the amplification of the specific sequence with higher activity than commercially available enzymes that exhibit properties similar to the enzyme, such as polymerases that do not have 5'→3' exonuclease activity and 3'→5' exonuclease activity, specifically, HiDi DNA polymerase.

[0106] (Kit for amplifying a target nucleic acid sequence) In a second embodiment, a kit for amplifying a target nucleic acid sequence is provided, comprising: 1) a nucleic acid having a modified oligonucleotide introduced therein; and 2) an enzyme having at least 90% homology to a polymerase derived from Thermus aquaticus, which has a deletion of amino acids 1 to 289, and having DNA polymerase activity.

[0107] The nucleic acid into which the modified oligonucleotide of 1) has been introduced and the enzyme of 2) provided as the kit of this embodiment may be provided together with any buffer, nucleic acid, other necessary reagents, etc. The nucleic acid into which the modified oligonucleotide of 1) has been introduced and the enzyme of 2) may be provided together with, for example, a primer, deoxynucleoside triphosphate (dNTP), (NH4)2SO4, Tween-20, MgCl2, water, Tris-HCl, BSA, DMSO, glycerol, a DNA or RNA degrading enzyme inhibitor, etc.

[0108] In the kit of this embodiment, 1) the nucleic acid into which a modified oligonucleotide has been introduced and 2) the first nucleic acid sequence may be provided in the same container, or each may be provided in a separate container. Furthermore, when 1) the nucleic acid into which a modified oligonucleotide has been introduced and / or 2) the first nucleic acid sequence are provided in separate containers, these containers may be provided together in a single box or the like.

[0109] The kit of this embodiment uses a nucleic acid containing a target nucleic acid sequence as a template, provides a nucleic acid to which a modified oligonucleotide (1) has been introduced to a reaction solution for an amplification reaction, and performs the amplification reaction in the presence of an enzyme (2), thereby enabling specific amplification of the target nucleic acid sequence. When the target nucleic acid sequence has a mutation, the template nucleic acid also contains a wild-type target nucleic acid sequence, and amplification of the wild-type target nucleic acid sequence is inhibited by the nucleic acid into which the modified oligonucleotide of 1) has been introduced. As a result, the target nucleic acid sequence having the mutation is specifically amplified, making it possible to detect whether the template nucleic acid has a mutation.

[0110] The kit of this embodiment may be a kit for specifically amplifying a target nucleic acid sequence, a kit for inhibiting the amplification of a sequence other than the target nucleic acid sequence, or a kit for detecting a mutation.

[0111] This embodiment also provides a method for amplifying a target nucleic acid sequence using a kit comprising: 1) a nucleic acid incorporating a modified oligonucleotide; and 2) an enzyme having at least 90% homology to a polymerase derived from Thermus aquaticus lacking amino acids 1 to 289 and having DNA polymerase activity.

[0112] The present invention will be specifically explained below by showing examples, but the present invention is not limited to these examples. [Example]

[0113] Example 1 <Test Method> 1. Purification of mutant Taq KlenTaq (Taq KlenTaq(R660V)) <Transformation> BL21(DE3) competent cells (Thermo Fisher, Cat: EC0114) were transformed with a pET vector containing KlenTaq (R660V), a polymerase derived from Thermus aquaticus that lacks amino acids 1 to 292. The arginine at position 368 in KlenTaq (SEQ ID NO: 1) was mutated to valine. The transformed E. coli was plated on an ampicillin-containing LB plate and cultured overnight at 37°C to allow colony formation.

[0114] [Table 1]

[0115] <Induction of enzyme protein expression> A single colony was inoculated into a small amount of liquid LB medium containing carbenicillin and cultured with shaking overnight at 37 °C (225 rpm, pre-culture). The next day, 1 / 40 volume of the pre-culture solution was added to a new liquid LB medium containing carbenicillin and cultured for 4 hours at 37 °C (225 rpm, main culture). It was confirmed that the OD600 value of the main culture solution was between 0.4 and 0.6, IPTG (FUJIFILM Wako, Cat: 099-05013) with a final concentration of 0.1 mM was added, and cultured for 3 hours at 30 °C (225 rpm, IPTG induction). The IPTG-induced E. coli solution was collected, and the E. coli pellet was collected by centrifugation (10,000 xg, 10 minutes, 4 °C). After measuring the wet weight, it was frozen in a -80 °C freezer overnight or longer.

[0116] <Lysis of Escherichia coli and collection of soluble fraction> The frozen E. coli was transferred onto ice, and BugBuster (Novagen, Cat: 70921-4) containing 1,000 U / mL rLysozyme (Novagen, Cat: 71110-4) + 1 / 200 Volume Protease inhibitor cocktail (Millipore, Cat: 539134-1MLCN) was added at 5 mL per 1 g of the wet weight of E. coli, and immediately vortexed thoroughly to suspend without warming. The E. coli suspension was dispensed into 1.5 mL microtubes, transferred to each heat block set at 60 °C or 72 °C, and subjected to a shaking reaction for 15 minutes (600 rpm). After the heat treatment, it was immediately transferred onto an aluminum block placed on ice and cooled for 15 minutes to age the precipitate. After centrifugation (16,000 xg, 20 minutes, 4 °C), the soluble fraction was collected and transferred to a new 1.5 mL microtube.

[0117] <His affinity purification> 250 μL of His-Affinity Gel (included in the His-Spin Protein Miniprep kit (ZymoResearch, Cat. P2002)) was transferred to a His-Spin collection tube and centrifuged (15,000 × g, 20 seconds, 4°C) to remove the gel dispersion. 300 μL of the soluble fraction was added, and the mixture was shaken (1,500 rpm) at 25°C for 5 minutes. After centrifugation (as above), the flow-through liquid was decanted and discarded. 250 μL of wash buffer (10 mM imidazole (Nacalai Tesque, Cat. No. 08787-22), 0.03% Triton® X-100 (Fujifilm Wako, Cat. No. 160-24751) was added, the mixture was mixed gently by vortexing, and centrifuged (as above). The flow-through was discarded, and the same washing procedure was repeated two more times. The column was placed in a new 1.5 mL microtube, and 250 μL of His-Elution Buffer (included in the His-Spin Protein Miniprep kit) was added. The column was mixed by vortexing for approximately 10 seconds and then allowed to stand on ice for at least 1 minute. The eluate was collected by centrifugation (as above). The volume of the eluate was accurately recorded.

[0118] <Measurement of protein concentration in eluate> Protein concentration was measured using an absorbance spectrometer by measuring the absorbance at 280 nm derived from the side chains of aromatic amino acids (tyrosine, tryptophan) that make up the protein. His-Elution Buffer was used as a blank solution.

[0119] 2. LNA Block PCR Reaction <LNA-blocked PCR reaction using mutant KlenTaq> 200 nM mutant KlenTaq, 200 nM forward primer (DNA oligos of SEQ ID NO: 2-4), 200 nM reverse primer (DNA oligos of SEQ ID NO: 5-11), 0-100 nM LNA block (DNA oligos of SEQ ID NO: 12-18), 10 ng of genomic DNA purified from colorectal cancer-derived cancer cell line HCT-116 (ATCC, Cat: CCL-247) or melanoma-derived cancer cell line A375 (ATCC, Cat: CRL-1619), 200 μM dNTP (TOYOBO, Ref: NTP-201), 50 mM Tris-HCl, pH 9.2, 16 mM (NH4)2SO4 (Nacalai Tesque, Cat: 02633-15), 0.1% Tween-20 (Karyo Tech, Cat: 28353-14), 2.5 mM MgCl2 (Nacalai Tesque, Cat: 20937-72) reaction solution (total volume of the reaction solution is 10 μL or 15 μL) was prepared. In addition, primers and LNA blocks with matching set numbers were used (see Table 3). After the first heat treatment reaction at 95°C for 2 minutes, a PCR reaction with cycles of 95°C for 15 seconds, 55°C or 60°C for 30 seconds, and 72°C for 30 seconds was performed for 35 cycles. The purified product was electrophoretically separated on a 2.0% or 3.0% agarose / TAE or TBE gel, stained with SYBR Gold staining reagent for 10 minutes, and the PCR amplicon of the target length was detected.

[0120]

Table 2

[0121]

Table 3

[0122] <LNA Block PCR Reaction Using AccuTaq LA DNA Polymerase> 0.5 U AccuTaq LA DNA polymerase (Sigma Aldrich, Ref: D8045-125UN), 200 nM forward primer (DNA oligo of SEQ ID NO: 2), 200 nM reverse primer (DNA oligo of SEQ ID NO: 5), 0 - 100 nM LNA block (DNA oligo of SEQ ID NO: 12), genomic DNA purified from 10 ng of colorectal cancer-derived cancer cell line HCT-116 (ATCC, Cat: CCL-247) or melanoma-derived cancer cell line A375 (ATCC, Cat: CRL-1619), 500 μM dNTP (TOYOBO, Ref: NTP-201), 50 mM Tris-HCl, pH 9.3, 15 mM (NH4)2SO4 (Nacalai Tesque, Cat: 02633-15), 0.02% DMSO (attached to AccuTaq LA DNA polymerase), 5 mM MgCl2 (attached to AccuTaq LA DNA polymerase) reaction solution (total volume of the reaction solution is 10 μL) was prepared. After the first heat treatment reaction at 95°C for 2 minutes, PCR reaction with one cycle of 95°C for 15 seconds, 55°C for 30 seconds, and 72°C for 30 seconds was performed for 35 cycles, and the purified product was electrophoretically separated on a 3.0% agarose / TBE gel, stained with SYBR Gold staining reagent for 10 minutes, and the PCR amplicon of the target length was detected.

[0123] <LNA Block PCR Reaction Using HiDi DNA Polymerase> The kit contained 0.5U HiDi DNA polymerase (myPOLS Biotec, Ref: 9001M), 200 nM forward primer (DNA oligo of SEQ ID NO: 2), 200 nM reverse primer (DNA oligo of SEQ ID NO: 5), 0-100 nM LNA block (DNA oligo of SEQ ID NO: 12), 10 ng of purified genomic DNA from the colon cancer cell line HCT-116 (ATCC, Cat: CCL-247) or the melanoma cell line A375 (ATCC, Cat: CRL-1619), 200 μM dNTP (TOYOBO, Ref: NTP-201), 50 mM Tris-HCl, pH 9.2, 16 mM (NH4)2SO4 (Nacalai Tesque, Cat: 02633-15), 0.1% A 10 μL reaction mixture containing Tween-20 (Calai Tesque, Cat. 28353-14) and 2.5 mM MgCl2 (Nacalai Tesque, Cat. 20937-72) was prepared. The primers and LNA blocks used were identical in set number (see Table 3). After an initial heat treatment at 95°C for 2 minutes, 35 PCR cycles were performed, each cycle consisting of 95°C for 15 seconds, 55°C or 60°C for 30 seconds, and 72°C for 30 seconds. The purified products were electrophoretically separated on a 2.0% or 3.0% agarose / TAE or TBE gel and stained with SYBR Gold staining reagent for 10 minutes to detect PCR amplicons of the desired length.

[0124] <Result 1> LNA block PCR was performed using LNA blocks targeting the wild-type sequence of the BRAF gene and mutant KlenTaq to evaluate their ability to suppress PCR amplification of the wild-type sequence. HCT-116 cells, from which the gDNA template was derived, harbor the BRAF wild-type allele, while A375 cells harbor the BRAF V600E1 mutant allele. For comparison, two commercially available enzymes were used: AccuTaq, which has been shown to have relatively high LNA blocking capacity in a previous publication (Reference 1, Christian Gloechner et al., doi:10.1002 / anie.200603987), and HiDi, a KlenTaq variant of Taq, similar to mutant KlenTaq. As shown in Figure 1, mutant KlenTaq exhibited at least 10-fold higher LNA blocking activity than the other two enzymes. On the other hand, for both enzymes, the LNA block did not have any inhibitory effect on PCR amplification of the mutant gene, suggesting that the LNA block acted specifically on the wild-type BRAF gene. Taq polymerase, including its mutants, is an enzyme that has been widely used in genetic engineering experiments, and many studies on recombinant protein expression and purification methods have been reported (Reference 2, Allison Laws et al., published at: https: / / our.oakland.edu / bitstream / handle / 10323 / 11462 / HonorsThesis_AllisonLaws.pdf?sequence=1&isAllowed=y; Reference 3, Sique Chen et al., doi: 10.1016 / j.ejbt.2015.08.001; Reference 4, Nosaibah Samman et al., doi: 10.1016 / j.jksus.2023.102565; Reference 5, Samaneh Golayj et al., doi: 10.12988 / asb.2014.4627; Reference 6, Maria J. (Manzur et al., doi: 10.2225 / vol9-issue3-fulltext-16). As reported in these reports, in the purification process of Taq polymerase and its analogs from E. coli, the E. coli cells in which the proteins were induced are lysed, followed by heat treatment at 72–75°C for 30 minutes to 2 hours to denature and precipitate the E. coli-derived proteins, thereby obtaining the target protein, Taq, in the soluble fraction. The mutant KlenTaq used in our study had a thermal denaturation temperature of 60°C and a treatment time of 15 minutes. We attributed the high LNA blocking capacity of the mutant KlenTaq observed in Figure 1 to differences in the treatment temperature during purification. Therefore, we purified the mutant KlenTaq from the soluble fraction by heat treatment at 72°C, the lower limit of the temperatures reported in previous literature, for a treatment time of 15 minutes, consistent with our own treatment time (hereafter referred to as 72°C-mutant KlenTaq).

[0125] <Result 2> We evaluated the PCR inhibition ability of two types of mutant KlenTaq purified at different temperatures and a commercially available product (HiDi DNA polymerase) by LNA blocking PCR against the wild-type BRAF sequence. As shown in Figure 2, the mutant KlenTaq purified under heat denaturation conditions at 60°C exhibited LNA blocking ability nearly 10 times that of the commercial product, while the mutant KlenTaq purified under heat denaturation conditions at 72°C (72°C-mutant KlenTaq) exhibited activity similar to that of the commercial product. This indicates that LNA blocking ability varies depending on the heat denaturation temperature, and an enzyme with high LNA blocking ability was obtained by denaturing at 60°C.

[0126] <Result 3> To confirm the generality of the high LNA blocking ability of mutant KlenTaq, 18 primer / LNA block sets designed for the wild-type gene in exon numbers 3 or 4 of the KRAS gene were evaluated. HCT-116 cells, from which the gDNA used as template was derived, contain the wild-type KRAS allele. The results for sets 2 to 10 are shown in Figure 3. With the exception of set 5, the use of mutant KlenTaq clearly improved blocking ability.

[0127] The results for sets 11 to 19 are shown in Figure 4. For sets 16 and 19, no PCR amplification suppression ability due to the LNA block was observed. For sets 11, 14, and 17, sufficient suppression was observed at an LNA block concentration of 10 nM regardless of the enzyme used, so no differences between the enzymes could be confirmed. For the other sets, it was confirmed that the use of mutant KlenTaq improved blocking performance. <Result 4> Figure 5 shows the results of a comparative study using Set 11 under conditions with a lower LNA block concentration. While the mutant KlenTaq completely inhibited PCR amplification with a 1 nM LNA block, the commercial product required 10 nM. Furthermore, the band intensity with a 0.1 nM LNA block when using the mutant KlenTaq was nearly identical to that with a 3 nM LNA block when using the commercial product, demonstrating that the mutant KlenTaq has 10 to 30 times the LNA blocking ability of the commercial product.

Claims

1. A method for amplifying a target nucleic acid sequence using a nucleic acid containing a modified oligonucleotide, wherein the amplification reaction is carried out in the presence of an enzyme having at least 90% homology to a polymerase derived from Thermus aquaticus in which the first to 289th amino acids are deleted, and having DNA polymerase activity.

2. The method of claim 1 , wherein the target nucleic acid sequence is a sequence having a mutation.

3. The method according to claim 2, comprising a step of forming a duplex with a wild-type target nucleic acid sequence using a nucleic acid to which a modified oligonucleotide has been introduced, thereby inhibiting amplification of the wild-type target nucleic acid sequence.

4. The method of any one of claims 1 to 3, wherein the modified oligonucleotide is a locked nucleic acid (LNA).

5. The method according to any one of claims 1 to 3, wherein the enzyme is produced by expression in a host.

6. The method according to claim 5, wherein the enzyme is produced by a method including a step of heating a host whose cell wall has been destroyed at a temperature of less than 64°C, at which proteins derived from the host cell are denatured.

7. The method of claim 6, wherein the amplification of a wild-type target nucleic acid sequence is further inhibited than when the amplification reaction is carried out in the presence of an enzyme produced by a method including a step of heating a host whose cell wall has been destroyed at a temperature of 65°C or higher.

8. The method of claim 6, wherein the concentration of the modified oligonucleotide in the reaction solution for the amplification reaction is less than 100 nM.

9. The method according to any one of claims 1 to 3, wherein the enzyme does not have 5' to 3' exonuclease activity and 3' to 5' exonuclease activity.

10. The method according to any one of claims 1 to 3, wherein the amplification reaction is carried out by PCR.

11. 1. A kit for amplifying a target nucleic acid sequence, comprising: 1) a nucleic acid into which a modified oligonucleotide has been introduced; 2) an enzyme having at least 90% homology to the polymerase derived from Thermus aquaticus, which lacks the 1st to 289th amino acids, and having DNA polymerase activity; Kit including:

12. The kit of claim 11 , wherein the target nucleic acid sequence is a sequence having a mutation.

13. 12. The kit according to claim 11, wherein the amplification reaction is carried out in the presence of the enzyme of 2).

14. The kit according to any one of claims 11 to 13, wherein the modified oligonucleotide is a locked nucleic acid (LNA).

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