Method for evaluating performance of hot start nucleic acid polymerase

Abstract

The present invention relates to a method for evaluating the performance of a hot start nucleic acid polymerase, a method for obtaining a QC-able hot start nucleic acid polymerase, or a method for providing a blending hot start nucleic acid polymerase that mimics the performance of a target hot start nucleic acid polymerase. In the present invention, the activity profile of a hot start nucleic acid polymerase is used with respect to heat activation time so that the performance of the hot start nucleic acid polymerase can be evaluated, a QC-able hot start nucleic acid polymerase can be provided, and a blending nucleic acid polymerase that mimics the performance of a target hot start nucleic acid polymerase can be provided.

Description

Method for evaluating the performance of hot-start nucleic acid polymerases

[0001] Cross-reference to related applications

[0002] This patent application claims priority to Republic of Korea Patent Application No. 2023-0129456, filed with the Korean Intellectual Property Office on September 26, 2023, the disclosures of which are incorporated herein by reference.

[0003] Technology field

[0004] The present invention relates to a method for evaluating the performance of a hot start nucleic acid polymerase, a method for obtaining a QC-capable hot start nucleic acid polymerase, or a method for providing a blended hot start nucleic acid polymerase that mimics the performance of a target hot start nucleic acid polymerase.

[0005] Molecular diagnostics is an in vitro diagnostic method that amplifies and tests DNA or RNA using techniques such as polymerase chain reaction (PCR). The importance of PCR continues to grow, even in recent molecular diagnostic tests for preemptive response to the COVID-19 pandemic. To facilitate PCR, it is crucial to utilize enzymes that are stable at the various temperatures used in the chain reaction and maintain activity. Currently, most polymerase chain reaction-based molecular diagnostic reagents utilize polymerases derived from high-temperature-stable microorganisms (extremophiles), such as Taq DNA polymerase derived from T. aquaticus (Chien A., Edgar DB, Trela ​​JMJ Bacteriol. 1976, 127, 1550-1557).

[0006] Among the polymerases used for molecular diagnostics, DNA polymerases perform a DNA polymerization reaction using DNA or RNA as a template. Essentially, polymerases consume nucleoside triphosphates as substrates, and since their primary purpose is polymerization, their activity is measured by how quickly they can consume substrate and polymerize. This is defined as units, a unique property of the enzyme being measured.

[0007] A unit of a polymerase is defined as the polymerization of one nucleoside triphosphate in a unit time at a unit temperature, rendering it insoluble in acid. For example, Taq DNA polymerase from New England Biolabs defines one unit as 15 nmol of nucleoside triphosphates becoming insoluble in acid (i.e., completely polymerized) at 75°C in 30 minutes. Similarly, Bst DNA polymerase, light fragment, defines one unit as 10 nmol of nucleoside triphosphates becoming insoluble in acid at 65°C in 30 minutes.

[0008] Although the definition of this unit may differ slightly depending on the company or product, such as temperature, amount of substrate, or type of material containing radioactive isotope, they generally have in common that the reaction rate is calculated by measuring the amount of substrate consumed using radioactive isotope or measuring the amount of polymer that has become insoluble, and the unit is defined through this (Charles C.Richardson, Carl L.Schildkraut, H. Vasken Aposhian, Arthur Kornberg J. Biol. Chem. 1964, 239(1), 222-232.).

[0009] The definition of a unit not only serves as a performance indicator for molecular diagnostic reagents but can also serve as a diagnostic assay in itself. Therefore, accurately analyzing the unit measurements of polymerases is becoming a critical issue for several reasons.

[0010] Among the methods for measuring enzyme units, the method for measuring enzyme units based on radioactive isotopes is a highly accurate method, but radioactive isotopes can cause environmental problems and, above all, can affect the health of the experimenter. Therefore, research has been conducted for the past several decades to find a method to define enzyme units without using isotopes. There have been attempts to measure units by measuring the change in the fluorescence value of single-stranded binding proteins (Mark A.Griep, Anal. Biochem., 1995, 232, 180-189.), using microplate reading of fluorescently labeled nucleotides, or using molecular beacons or electrochemical methods.

[0011] Molecular diagnostic products play a crucial role in determining a patient's symptoms, so their sensitivity and specificity are crucial. They must be able to diagnose actual viral or microbial infection, or clearly confirm the absence of infection. Therefore, it is crucial to avoid inaccurate diagnostic results due to enzyme errors before the actual PCR reaction is performed. Polymerase chain reaction (PCR) can sometimes produce inaccurate results due to several issues. For example, primers used in PCR can bind to unintended sites on the template DNA, or primers can bind nonspecifically to each other. When the polymerase, which is partially active at low temperatures, recognizes this and amplifies it, unintended results occur. Therefore, to prevent such unintended amplification reactions, hot-start polymerases, whose polymerase activity is suppressed before high-temperature treatment, have been developed.

[0012] These hot start methods are categorized as physical, chemical, and other methods. Physical methods use wax or beads to physically separate the enzyme from the reactants, inhibiting the reaction until the boundary collapses above a certain temperature. Chemical hot start methods chemically modify the enzyme itself to render it inactive without pretreatment at a specific temperature. Chemicals such as formaldehyde and citraconic anhydride are primarily used (US Pat. No. 5,677,152). In addition, methods that inhibit the reaction using antibodies or aptamers that can bind to polymerases, or control magnesium ions or primers that are important for the reaction, have also been developed and reported.

[0013] Most of these hot start methods remove or weaken inhibitors through heat pretreatment before performing the main PCR reaction, thereby allowing the enzyme to participate only in the main reaction.

[0014] Enzymes for use in diagnostic reagents are added to the product based on specific activity units according to the characteristics of the product when applied to the actual reagent after production. As mentioned above, hot-start enzymes are generally in a suppressed state, so existing methods for measuring the activity of polymerases for diagnostic reagents result in very low or no activity measured at all. Therefore, it is very important to develop a technology that can determine whether the performance of these enzymes satisfies a certain level or whether a certain concentration of enzyme is added to the diagnostic reagent through a separate method.

[0015] Enzymes used in diagnostic reagents undergo a rigorous process from cultivation to purification, followed by additional, lengthy reaction steps to impart hot-start performance. Therefore, producing hot-start enzymes of consistent quality is extremely challenging. However, if the quality of the final product, the hot-start enzyme, could be precisely analyzed and quantified, these issues could be addressed and enzyme quality improved until the final product, a consistent diagnostic reagent, could be manufactured.

[0016] Therefore, the present inventors recognized the need for developing a method capable of monitoring the activity and quality of enzymes by providing a series of methods for examining the final performance and quality of diagnostic hot start enzymes.

[0017] Numerous references and patents are cited and cited throughout this specification. The disclosures of these references and patents are incorporated herein by reference in their entirety to further clarify the state of the art and the scope of the present invention.

[0018] The present inventors have endeavored to develop a method for analyzing the performance of a hot-start nucleic acid polymerase, which can monitor the activity and quality of the hot-start nucleic acid polymerase. As a result, the present inventors have established an activity profile of a hot-start nucleic acid polymerase with respect to thermal activation time using a nucleic acid polymerase activity (unit) measurement method that does not use radioisotopes, and confirmed that the performance of a hot-start nucleic acid polymerase can be evaluated and a QC-capable hot-start nucleic acid polymerase can be provided. In addition, the present invention has been completed by providing a blended nucleic acid polymerase that mimics the performance of a target hot-start nucleic acid polymerase.

[0019] Accordingly, an object of the present invention is to provide a method for evaluating the performance of a hot start nucleic acid polymerase.

[0020] Another object of the present invention is to provide a method for obtaining a QC-able hot start nucleic acid polymerase.

[0021] Another object of the present invention is to provide a method for providing a blending hot start nucleic acid polymerase that mimics the performance of a target hot start nucleic acid polymerase.

[0022] Other objects and advantages of the present invention will become more apparent from the following examples, claims and drawings.

[0023] I. Method for evaluating the performance of a hot-start nucleic acid polymerase (first aspect)

[0024] According to one aspect of the present invention, the present invention provides a method for evaluating the performance of a hot start nucleic acid polymerase, comprising the following steps:

[0025] (a) obtaining a product comprising a hot start nucleic acid polymerase;

[0026] (b) a step of fractionating a product containing the hot start nucleic acid polymerase and performing heat activation for each of the fractionated products for different times;

[0027] (c) a step of measuring the activity of each hot start nucleic acid polymerase included in each of the heat-activated products for different times; the activity is measured from the intensity of a signal measured by adding each of the heat-activated products for different times to a composition for activity assay of nucleic acid polymerase and performing a nucleic acid polymerization reaction, wherein the nucleic acid polymerase activity assay composition includes a primer, and the primer is extended by the hot start nucleic acid polymerase included in each of the heat-activated products under polymerization conditions to form an extension duplex, and a detectable signal is provided in a manner dependent on the extension duplex, and

[0028] (d) obtaining the activity of the hot start nucleic acid polymerase for the different thermal activation times as an activity profile of the hot start nucleic acid polymerase; the activity profile of the hot start nucleic acid polymerase indicates the performance of the hot start nucleic acid polymerase.

[0029] The method of the present invention can evaluate the performance of a hot start nucleic acid polymerase by constructing an activity profile of the hot start nucleic acid polymerase with respect to the thermal activation time using a method for measuring the activity (unit) of the nucleic acid polymerase without using a radioactive isotope.

[0030] Figure 1 is a flowchart of processes for implementing the method of the present invention according to one embodiment of the present invention. The method of the present invention is described with reference to Figure 1 as follows:

[0031] Step (a): Obtaining a product comprising a hot start nucleic acid polymerase (110)

[0032] First, the method of the present invention comprises the step of (a) obtaining a product comprising a hot start nucleic acid polymerase.

[0033] As used herein, “hot start nucleic acid polymerase” means a nucleic acid polymerase whose activity is inhibited at room temperature or low temperature through chemical modification or additives prior to treatment at high temperature to prevent unintended amplification reactions.

[0034] According to one embodiment of the present invention, the hot start nucleic acid polymerase is a chemically modified hot start nucleic acid polymerase, an antibody-based hot start nucleic acid polymerase, or an aptamer-based hot start nucleic acid polymerase. Most specifically, the hot start nucleic acid polymerase is a chemically modified hot start nucleic acid polymerase.

[0035] The chemicals used to produce the above chemically modified hot start nucleic acid polymerase may be various chemicals known in the art, and examples thereof include formaldehyde, citraconic anhydride, etc.

[0036] In this specification, a nucleic acid polymerase is an enzyme that polymerizes DNA using DNA or RNA as a template, specifically a DNA polymerase, and more specifically, Taq DNA polymerase, KlenTaq DNA polymerase, Klenow fragment of E. coli DNA polymerase I, DNA polymerase I, T4 DNA polymerase, Bst DNA polymerase, Bsu DNA polymerase, MMLV reverse transcriptase, AMV reverse transcriptase, or HIV reverse transcriptase.

[0037] According to one embodiment of the present invention, the nucleic acid polymerase is prepared by a method comprising the following steps: (a-1) culturing a host cell containing a nucleic acid polymerase expression construct; and (a-2) purifying the nucleic acid polymerase by subjecting the resultant of step (a-1) to column chromatography.

[0038] The preparation process of nucleic acid polymerase largely includes (a-1) a culture process and (a-2) a purification process.

[0039] (a-1) Cultivation process

[0040] As used herein, the term “nucleic acid polymerase expression construct” means a nucleic acid molecule containing only the minimum elements for expressing nucleic acid polymerase in a cell.

[0041] According to one embodiment of the present invention, the nucleic acid polymerase expression construct is a vector comprising (i) a nucleic acid polymerase-coding nucleotide sequence and (ii) a promoter.

[0042] As used herein, the term "nucleotide" refers to a deoxyribonucleotide or ribonucleotide existing in single-stranded or double-stranded form, and includes analogs of natural nucleotides unless specifically stated otherwise (Scheit, Nucleotide Analogs, John Wiley, New York (1980); Uhlman and Peyman, Chemical Reviews, 90:543-584 (1990)).

[0043] As used herein, the term "promoter" refers to an untranslated nucleic acid sequence upstream of a coding region that contains a binding site for a polymerase and has transcription initiation activity into mRNA of a gene downstream of the promoter.

[0044] The above expression construct can be constructed by various methods known in the art, and specific methods thereof are disclosed in Sambrook et al., Molecular Cloning, A Laboratory Manual, Cold Spring Harbor Laboratory Press (2001), which is incorporated herein by reference.

[0045] When the above expression construct is typically constructed as a vector for expression, the vector can be constructed using a prokaryotic cell or a eukaryotic cell as a host cell, and specifically, can be constructed using a prokaryotic cell as a host cell. When the vector is an expression vector and uses a prokaryotic cell as a host cell, it generally includes a promoter having strong transcriptional activity (e.g., tac promoter, lac promoter, lacUV5 promoter, lpp promoter, pLλ promoter, pRλ promoter, rac5 promoter, amp promoter, recA promoter, SP6 promoter, trp promoter, T7 promoter, and phoB promoter, etc.), a ribosome binding site for translation initiation, and a transcription / translation termination sequence.

[0046] When E. coli (e.g., HB101, BL21, DH5α, etc.) is used as a host cell, the promoter and operator region of the E. coli tryptophan biosynthetic pathway (Yanofsky, C., J. Bacteriol., 158:1018-1024(1984)) and the left-hand promoter of phage λ (pL λ promoter, Herskowitz, I. and Hagen, D., Ann. Rev. Genet., 14:399-445(1980)) can be used as promoters.

[0047] When Bacillus bacteria are used as a host cell, the promoter of the toxin protein gene of Bacillus thuringiensis (Appl. Environ. Microbiol. 64:3932-3938(1998); Mol. Gen. Genet. 250:734-741(1996)) or any promoter that can be expressed in Bacillus bacteria can be used as a promoter.

[0048] When the above vector is prepared for eukaryotic cell expression, available promoters include, but are not limited to, for example, the CMV promoter, the adenovirus late promoter, the vaccinia virus 7.5K promoter, the SV40 promoter, the tk promoter of HSV, the RSV promoter, the EF1 alpha promoter, the metallothionine promoter, the beta-actin promoter, the promoter of the human IL-2 gene, the promoter of the human IFN gene, the promoter of the human IL-4 gene, the promoter of the human lymphotoxin gene, and the promoter of the human GM-CSF gene.

[0049] Eukaryotic expression vectors preferably have a polyadenylation sequence linked downstream of the nucleic acid polymerase-coding nucleotide. The polyadenylation sequence may be derived from SV40 polyadenylation sequence (Schek, N, et al., Mol. Cell Biol. 12:5386-5393(1992)), HIV-1 polyA (Klasens, BIF, et al., Nucleic Acids Res. 26:1870-1876(1998)), β-globin polyA (Gil, A., et al., Cell 49:399-406(1987)), HSV TK polyA (Cole, CN and TP Stacy, Mol. Cell. Biol. 5:2104-2113(1985)) or polyomavirus polyA (Batt, D. B and GG Carmichael, Mol. Cell. Biol. 15:4783-4790(1995)). Including, but not limited to:

[0050] The expression “nucleic acid polymerase-coding nucleotide” in this specification means the CDS (coding sequence) nucleotide of the nucleic acid polymerase, which is the target protein.

[0051] The above expression construct may have a selectable marker for selection. Specifically, the selectable marker is a polynucleotide encoding a gene that exhibits antibiotic resistance. Examples of such antibiotic resistance genes include resistance genes for ampicillin, gentamicin, carbenicillin, chloramphenicol, streptomycin, kanamycin, geneticin, neomycin, and tetracycline.

[0052] More specifically, the vector can be constructed by manipulating a plasmid (e.g., pDG1661, pSC101, pGV1106, pACYC177, ColE1, pKT230, pME290, pBR322, pUC8 / 9, pUC6, pBD9, pHC79, pIJ61, pLAFR1, pHV14, pGEX series, pET series, and pUC19, etc.), a phage (e.g., λgt4·λB, λ-Charon, λΔz1, and M13, etc.), or a virus (e.g., SV40, etc.), and most specifically, it is constructed by manipulating a plasmid.

[0053] Meanwhile, the method of introducing the above-described vector into cells can be carried out through various methods known in the art, for example, microinjection (Capecchi, MR, Cell, 22:479(1980); and Harland and Weintraub, J. Cell Biol. 101:1094-1099(1985)), calcium phosphate precipitation (Graham, FL et al., Virology, 52:456(1973); and Chen and Okayama, Mol. Cell. Biol. 7:2745-2752(1987)), electroporation (Neumann, E. et al., EMBO J., 1:841(1982); and TurKaspa et al., Mol. Cell Biol., 6:716-718(1986)), liposome-mediated transfection (Wong, TK Vectors can be introduced into cells by the DEAE-dextran treatment method (Gopal, Mol. Cell Biol., 5:1188-1190 (1985)), and gene bombardment (Yang et al., Proc. Natl. Acad. Sci., 87:9568-9572 (1990)).

[0054] Any known host cell capable of stably and continuously expressing the expression construct may be used as the host cell. The host cell may be a prokaryotic or eukaryotic cell.

[0055] When using a prokaryotic host cell, examples thereof include Bacillus strains such as E. coliJM109, E. coliBL21, E. coliRR1, E. coliLE392, E. coliB, E. coliX 1776, E. coli W3110, Bacillus subtilis, Bacillus thuringiensis, and enterobacteria and strains such as Salmonella typhimurium, Serratia marcescens, and various Pseudomonas species. When using a eukaryotic cell as a host cell, yeast (Saccharomyce cerevisiae), insect cells, and human cells (e.g., CHO cell line (Chinese hamster ovary), W138, BHK, COS-7, 293, HepG2, 3T3, RIN, and MDCK cell lines) can be used.

[0056] According to one embodiment of the present invention, the host cell is a prokaryotic cell, most specifically E. coli.

[0057] Transformed host cells can be cultured using media commonly used in the art. For example, if the transformed host cells are prokaryotic cells (e.g., E. coli), they can be cultured using LB (Luria-Bertani) media. Various methods for culturing transformed host cells are well known to those skilled in the art and are disclosed in Sambrook et al., Molecular Cloning, A Laboratory Manual, Cold Spring Harbor Laboratory Press (2001), which is incorporated herein by reference.

[0058] According to a more specific embodiment of the present invention, the step (a-1) includes the step (a-1-1) of disrupting the cultured host cell.

[0059] Cultured host cells can be disrupted by various methods known in the art, specifically, sonication or a disruption method using glass beads can be used, and more specifically, disruption of the cultured host cells is performed by sonication.

[0060] According to a more specific embodiment of the present invention, the step (a-1) includes the step (a-1-2) of heat-treating the cultured host cells or the lysate obtained by crushing the cultured host cells. More specifically, the heat treatment is performed at 60-120°C for 30-120 minutes.

[0061] (a-2) Purification process

[0062] The term "chromatography" as used herein means a process for separating a solute of interest in a mixture from other solutes in the mixture by the difference in the rate of movement of each solute as it passes through a stationary phase under the influence of a moving phase.

[0063] The above purification process can utilize a protein purification method known in the art, and for example, purification can be performed using filtration using a silica gel or celite gel column, size-exclusion chromatography using liquid column chromatography, ion-exchange chromatography, partition chromatography, affinity chromatography, or a combination of these chromatographies.

[0064] According to one embodiment of the present invention, the step (a-2) includes a step of purifying a nucleic acid polymerase by applying the result of the step (a-2-1) to ion-exchange chromatography and a step of purifying a nucleic acid polymerase by applying the result of the step (a-2-2) to affinity chromatography.

[0065] Specifically, the step (a-2-1) is performed by a purification method using an anion exchange column chromatography (more specifically, a quaternary amino group-functionalized anion exchange column chromatography) from the resultant product of the step.

[0066] Specifically, for purification by the affinity chromatography, the nucleic acid polymerase used in the present invention is in the form of a fusion protein fused with any one selected from the group consisting of glutathione-S-transferase (GST), maltose binding protein (MBP), FLAG, 6x His (hexahistidine), NusA (N utilization substance A), and Trx (thioredoxin). For example, when glutathione-S-transferase is fused to the nucleic acid polymerase, glutathione, which is a substrate of the enzyme, can be utilized, and when 6x His (His-tag) is utilized, purification can be performed quickly and easily using a column packed with Ni-NTA resin.

[0067] According to one embodiment of the present invention, the hot start nucleic acid polymerase of step (a) is a nucleic acid polymerase whose polymerization activity is suppressed, and more specifically, the hot start nucleic acid polymerase is a nucleic acid polymerase whose polymerization activity is suppressed to 1 U / ug or less.

[0068] According to one embodiment of the present invention, the method further comprises, after step (a), the step (a-3) of diluting the product(s) to a concentration of 0.02 mg / ml-2 mg / ml. More specifically, the product is diluted to a concentration of 0.03 mg / ml-2 mg / ml. Even more specifically, the product is diluted to a concentration of 0.05 mg / ml-0.8 mg / ml. When the concentration of the product exceeds 2 mg / ml, there is a problem that the real-time fluorescence value reaches the maximum point too quickly, making it difficult to derive an analysis value suitable for differentiation, and when it is less than 0.02 mg / ml, there is a disadvantage that it is difficult to measure enzyme activity because fluorescence measurement due to DNA double-strand binding is not properly performed.

[0069] The dilution in the above step (a-3) can be performed using a reaction buffer.

[0070] The concentration of hot start nucleic acid polymerase contained in the above product can be measured using various methods known in the art, for example, the concentration of hot start nucleic acid polymerase can be measured using the Bradford method, the BCA method, or A280 absorbance.

[0071] Step (b): Heat activation (120) for different times for each of the fractionated products.

[0072] Next, the method of the present invention comprises the step of (b) fractionating a product including the hot start nucleic acid polymerase and performing heat activation for each of the fractionated products for different periods of time.

[0073] The term "heat activation" as used herein means reversibly restoring the activity of a hot start nucleic acid polymerase whose activity has been inhibited by physical, chemical or other means by treating it with heat.

[0074] According to one embodiment of the present invention, the heat activation of step (b) is performed for at least two time intervals from 0 to 60 minutes for each of the fractionated products. For example, 1) heat activation for 0 minutes and heat activation for 60 minutes, 2) heat activation for 15 minutes and heat activation for 30 minutes, 3) heat activation for 0 minutes, heat activation for 15 minutes and heat activation for 60 minutes, 4) heat activation for 0 minutes, heat activation for 15 minutes and heat activation for 30 minutes, 5) heat activation for 0 minutes, heat activation for 15 minutes, heat activation for 30 minutes and heat activation for 60 minutes, or 6) heat activation for 0 minutes, heat activation for 15 minutes, heat activation for 30 minutes, heat activation for 45 minutes and heat activation for 60 minutes.

[0075] More specifically, the thermal activation of step (b) is performed for at least two time intervals selected from the group consisting of 0 minutes, 15 minutes, 30 minutes, 45 minutes, and 60 minutes for each of the fractionated products. More specifically, the at least two time intervals may be 1) 0 minutes and 15 minutes, 2) 0 minutes, 15 minutes, and 30 minutes, 3) 0 minutes, 15 minutes, and 60 minutes, 4) 0 minutes, 15 minutes, 30 minutes, and 45 minutes, 5) 0 minutes, 15 minutes, 30 minutes, and 60 minutes, or 6) 0 minutes, 15 minutes, 30 minutes, 45 minutes, and 60 minutes.

[0076] According to one embodiment of the present invention, the thermal activation of step (b) is performed at 90 to 100°C. More specifically, the thermal activation is performed at 92 to 98°C, and even more specifically, at 94 to 96°C.

[0077] According to one embodiment of the present invention, the thermal activation of step (b) is performed at 90 to 100°C for 0 minutes, 15 minutes, 30 minutes, and 60 minutes for each of the fractionated products.

[0078] According to one embodiment of the present invention, between steps (b) and (c), the method further comprises the step (b-1) of diluting each of the heat-activated products to a concentration of 6.25 ng / ul to 50 ng / ul. More specifically, each of the heat-activated products is diluted to a concentration of 6.25 ng / ul to 12.5 ng / ul. When the concentration range is as described above, the activity change value of the hot start nucleic acid polymerase due to heat activation can be discriminated and measured.

[0079] In cases where the product containing the hot start nucleic acid polymerase contains a surfactant or a buffer component having light absorption, making it impossible to measure the concentration of the hot start nucleic acid polymerase, between steps (b) and (c), a step of (b-1) diluting each of the heat-activated products by at least 1 / 4 is additionally included.

[0080] The dilution in the above step (b-1) can be performed using a reaction buffer.

[0081] Step (c): Measuring the activity of each hot start nucleic acid polymerase contained in each of the heat-activated products for different times (130)

[0082] Next, the method of the present invention comprises the step of (c) measuring the activity of each hot start nucleic acid polymerase included in each of the products that have been heat-activated for different times. The activity is measured from the intensity of a signal measured by adding each of the products that have been heat-activated for different times to a composition for activity assay of nucleic acid polymerase and performing a nucleic acid polymerization reaction, wherein the composition for activity assay of nucleic acid polymerase includes a primer, and the primer is extended by the hot start nucleic acid polymerase included in each of the heat-activated products under polymerization conditions to form an extension duplex, and a detectable signal is provided in a manner dependent on the extension duplex.

[0083] The term “activity” as used herein in reference to a hot start nucleic acid polymerase refers to the polymerization activity of the hot start nucleic acid polymerase.

[0084] The activity of each hot start nucleic acid polymerase contained in each of the products activated for different times above is measured from the intensity of the signal obtained by adding each of the products activated for different times above to an activity assay composition of the nucleic acid polymerase and performing a nucleic acid polymerization reaction.

[0085] According to one embodiment of the present invention, in the step (c), the addition of each of the products heat-activated for different periods of time to the nucleic acid polymerase activity assay composition is carried out by adding each of the products heat-activated for different periods of time to the nucleic acid polymerase activity assay composition according to the dilution factor. For example, each of the products may be diluted 100-fold, 200-fold, 300-fold, 400-fold, and 500-fold and added to the nucleic acid polymerase activity assay composition.

[0086] The above nucleic acid polymerase activity assay composition comprises a primer, wherein the primer is extended by a hot start nucleic acid polymerase contained in each of the heat-activated products under polymerization conditions to form an extension duplex, and a detectable signal is provided in a manner dependent on the extension duplex.

[0087] The term "primer" as used herein refers to an oligonucleotide that can act as an initiator of synthesis under conditions that induce the synthesis of a primer extension product complementary to a nucleic acid strand (template), i.e., the presence of nucleotides and a polymerizing agent such as DNA polymerase, and conditions of suitable temperature and pH. In particular, the primer is a single-stranded deoxyribonucleotide molecule. The primer used in the present invention may include naturally occurring dNMPs (i.e., dAMP, dGMP, dCMP, and dTMP), modified nucleotides, or non-natural nucleotides. In addition, a conventional primer may include ribonucleotides.

[0088] The primer must be sufficiently long to prime the synthesis of an extension product in the presence of a polymerization agent. The appropriate primer length depends on several factors, including, for example, temperature, the application, and the source of the primer. As used herein, the terms "annealing" or "priming" refer to the juxtaposition of an oligodeoxynucleotide or nucleic acid to a template nucleic acid, which causes a polymerase to polymerize the nucleotides to form a nucleic acid molecule complementary to the template nucleic acid or a portion thereof.

[0089] The length of the primer can be 7-150 nucleotides, 10-120 nucleotides, 12-100 nucleotides, 14-80 nucleotides, 16-60 nucleotides, 18-40 nucleotides, 20-35 nucleotides or 20-30 nucleotides.

[0090] According to one embodiment of the present invention, the activity assay composition of the nucleic acid polymerase additionally comprises (i) a template, (ii) a label, (iii) dNTP, and (iv) a buffer.

[0091] (i) template

[0092] In the polymerization reaction of the present invention, a nucleic acid polymerase can form an extended duplex by hybridizing to a template nucleic acid and polymerizing nucleotides to form a nucleic acid molecule complementary to the template nucleic acid.

[0093] Specifically, the template can be used by using M13mp18 DNA, which is generally single-stranded DNA, or by synthesizing template DNA to which a primer is linked, but is not limited thereto.

[0094] According to one embodiment of the present invention, the primer included in the activity assay composition of the nucleic acid polymerase comprises a first primer and a second primer as a dimer-forming primer pair having the functions of both a primer and a template, wherein each of the first primer and the second primer comprises a 3'-dimer forming site, and the nucleotide sequence of the 3'-dimer forming site in the first primer is complementary to the nucleotide sequence of the 3'-dimer forming site in the second primer.

[0095] According to the present embodiment, the polymerization reaction can form an extended duplex by partially hybridizing a pair of primers that form a dimer and allowing each primer to act as a template for the other primer to polymerize nucleotides by a nucleic acid polymerase to form a nucleic acid molecule complementary to each primer.

[0096] The term "extension duplex" as used herein means a duplex formed by an extension reaction in which a primer hybridized to a template (specifically, a primer acting as a template) is extended by a template-dependent nucleic acid polymerase.

[0097] The term "dimer-forming primer" as used herein refers to a primer that can partially hybridize with another dimer-forming primer that is not a template (or acts as a template) under certain conditions and be extended in the presence of a nucleic acid polymerase.

[0098] The term "dimer-forming primer pair" as used herein refers to a pair of two primers, such as a first primer and a second primer, that partially hybridize to each other and can each be extended by the polymerization activity of a nucleic acid polymerase.

[0099] The term "annealing" or "priming" as used in dimer-forming primers refers to the apposition of the 3'-dimer forming site of a second primer to the 3'-dimer forming site of a first primer, which causes a nucleic acid polymerase to polymerize nucleotides to form a nucleic acid molecule complementary to the 5'-templating site or a portion thereof, thereby forming an extended duplex.

[0100] The term "3'-dimer forming site" as used herein refers to a site located at the 3' end of a dimer-forming primer, which can form a dimer by hybridizing with the 3'-dimer forming site of another dimer-forming primer under specific hybridization conditions. That is, the 3'-dimer forming site refers to a site at the 3'-end of one dimer-forming primer that has a nucleotide sequence that can hybridize with the 3'-dimer forming site of another dimer-forming primer.

[0101] The term "hybridization," as used herein, refers to the formation of a double-stranded nucleic acid from complementary single-stranded nucleic acids. Hybridization can occur when the complementarity between the two nucleic acid strands is perfect (a perfect match), or even when some base mismatches exist. The degree of complementarity required for hybridization can vary depending on the hybridization conditions, and can be controlled, in particular, by temperature.

[0102] Hybridization between a primer and a template or between dimer-forming primers can be performed under suitable hybridization conditions, which are generally determined by optimization procedures. Conditions such as temperature, concentration of components, number of hybridization and washing cycles, buffer components, and their pH and ionic strength can vary depending on various factors, including the length and GC content of the primer or dimer-forming primer. For example, when using a relatively short primer or a dimer-forming primer pair with a dimer-forming region, it is preferable to select low stringency conditions. Detailed conditions for hybridization can be found in Joseph Sambrook et al., Molecular Cloning, A Laboratory Manual, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY (2001); and M. L. M. Anderson, Nucleic Acid Hybridization, Springer-Verlag New York Inc., NY (1999).

[0103] The terms “annealing” and “hybridization” are not different and are used interchangeably in this specification.

[0104] A primer has a hybridizing nucleotide sequence complementary to the template.

[0105] The first primer has a 3'-dimer formation site at the 3' end, the second primer has a 3'-dimer formation site at the 3' end, and the nucleotide sequence of the 3'-dimer formation site of the first primer is complementary to the nucleotide sequence of the 3'-dimer formation site of the second primer.

[0106] The term "complementary" as used herein means that the primers are sufficiently complementary to selectively hybridize to the template or the 3'-dimer forming site of the first primer to the 3'-dimer forming site of the second primer under certain annealing conditions or stringent conditions, and has a meaning that includes both "substantially complementary" and "perfectly complementary", and specifically means perfectly complementary.

[0107] The length of each of the 3'-dimer forming portions of the first primer and the second primer may be 3-50 nucleotides, 5-45 nucleotides, 8-40 nucleotides, 10-35 nucleotides, 12-35 nucleotides, 14-30 nucleotides, or 15-25 nucleotides.

[0108] Each of the first primer and the second primer includes a 3'-dimer forming portion as well as a 5'-templating portion.

[0109] As used herein, the term "5'-templating portion" of a dimer-forming primer refers to the remaining portion (region) at the 5' end of the primer, excluding the 3'-dimer-forming portion. Furthermore, the term "5'-templating portion" of a dimer-forming primer refers to the portion of a dimer-forming primer that functions as a template for the extension of another dimer-forming primer hybridized to the dimer-forming primer. The 5'-templating portion of a dimer-forming primer has a nucleotide sequence that is non-complementary to the 3'-dimer-forming portion of another dimer-forming primer.

[0110] The term "non-complementary" means that the primer is sufficiently non-complementary that it will not selectively hybridize to the template under the given annealing conditions or stringent conditions, and includes both "substantially non-complementary" and "perfectly non-complementary", and in particular means perfectly non-complementary. For example, with respect to the 5'-templating portion of a dimer-forming primer, the term "non-complementary" means that the 5'-templating portion of the dimer-forming primer is sufficiently non-complementary that it will not selectively hybridize to the entire sequence of another dimer-forming primer under the given annealing conditions or stringent conditions, and includes both "substantially non-complementary" and "perfectly non-complementary", and in particular means perfectly non-complementary.

[0111] The length of each of the 5'-templating portions of the first primer and the second primer may be 4-100 nucleotides, 6-90 nucleotides, 8-80 nucleotides, 10-70 nucleotides, 12-60 nucleotides, 14-50 nucleotides, 15-40 nucleotides, 15-30 nucleotides, or 20-30 nucleotides.

[0112] The above dimer-forming primer or dimer-forming primer pair is as disclosed in WO 2019 / 045532, and details regarding the dimer-forming primer or dimer-forming primer pair are referred to in the above document.

[0113] (ii) Cover

[0114] The nucleic acid polymerase activity assay composition may include a label to provide a detectable signal in an extension duplex dependent manner.

[0115] The expression "a detectable signal is provided in a manner dependent on the extended duplex" as used herein means that the detectable signal is provided directly or indirectly from the presence of the extended duplex, and specifically, the detectable signal may be provided from the extended duplex itself.

[0116] More specifically, the present invention may use an intercalating label that provides a signal indicating the degree of formation of an extended duplex by a nucleic acid polymerase, and the intercalating label is SYBR™ Green I, PO-PRO™-1, BO-PRO™-1, SYTO™ 43, SYTO™ 44, SYTO™ 45, SYTOX™ Blue, POPO™-1, POPO™-3, BOBO™-1, BOBO™-3, LO-PRO™-1, JO-PRO™-1, YO-PRO™ 1, TO-PRO™1, SYTO™11, SYTO™13, SYTO™15, SYTO™16, SYTO™20, SYTO™23, TOTO™-3, YOYO™3, GelStar™, thiazole orange, PicoGreen ® , EvaGreen ® and LAM Fluorescent Dye.

[0117] Intercalating labels specifically integrate into double-stranded nucleic acid molecules to generate a signal. The formation of extended duplexes increases the fluorescence intensity from a single fluorescent label. Therefore, the signal increase resulting from the formation of extended duplexes can be detected in real time.

[0118] (iii) dNTP

[0119] The nucleic acid polymerase activity assay composition may contain dNTPs, which are building blocks of DNA or RNA to be synthesized. The dNTPs comprise a mixture of four deoxyribonucleotides, namely dATP, dCTP, dGTP, and dTTP (or dUTP).

[0120] (iv) Buffer

[0121] The nucleic acid polymerase activity assay composition may include a buffer to provide a pH environment for optimal activity of the nucleic acid polymerase and to prevent rapid changes in pH due to temperature changes and chemical reactions during the polymerization reaction. An example of the buffer includes Tris-HCl.

[0122] According to another embodiment of the present invention, the activity assay composition of the nucleic acid polymerase additionally comprises (v) a salt and (vi) an additive.

[0123] (v) salt

[0124] The nucleic acid polymerase activity assay composition may include a salt to help stabilize the activity of the nucleic acid polymerase. The salt may be a magnesium or potassium salt, such as MgCl2, MgSO4, or KCl. The salt may be added at various concentrations known in the art.

[0125] (vi) Additives

[0126] The nucleic acid polymerase activity assay composition may contain various additives to stabilize the nucleic acid polymerase. Examples of such additives include BSA, DMSO, betaine, KCl, and non-ionic surfactants (e.g., Tween 20, Tritox X-100).

[0127] The nucleic acid polymerization reaction may include an isothermal polymerization reaction, PCR, quantitative real-time PCR (qPCR), melting curve analysis, high resolution melting analysis, sequencing, quantitative fluorescent PCR, multiplex PCR, digital PCR (dPCR), whole genome amplification (WGA), or reverse transcription PCR.

[0128] According to one embodiment of the present invention, the nucleic acid polymerization reaction is carried out at 35°C to 75°C for 10 to 60 minutes.

[0129] The term "polymerization conditions" as used herein refers to the presence of components necessary for polymerization, and conditions of suitable temperature and pH. The term "polymerization conditions" as used herein refers to conditions for separate steps involved in a polymerization reaction, such as primer annealing / hybridization and primer extension. These conditions can vary depending on various factors, including the length and GC content of the primer, and the length and GC content of the hybridization region. Detailed conditions can be found in the literature [Joseph Sambrook et al., Molecular Cloning, A Laboratory Manual, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY (2001); and M. L. M. Anderson, Nucleic Acid Hybridization, Springer-Verlag New York Inc. NY (1999)].

[0130] According to one embodiment of the present invention, the activity of the hot start nucleic acid polymerase measured in the step (c) is provided from the slope measured in the range in which the intensity of the signal increases linearly depending on the concentration of the hot start nucleic acid polymerase contained in the product.

[0131] Specifically, the activity of the hot start nucleic acid polymerase included in the product can be provided by measuring the slope of the first-order function portion (differential value of time-signal intensity) for the intensity of the signal over time according to the concentration of the hot start nucleic acid polymerase included in the product, and calculating the slope of the portion where the differential value of the time-signal intensity according to the concentration of the hot start nucleic acid polymerase included in the product becomes a first-order function.

[0132] According to one embodiment of the present invention, the activity of the hot start nucleic acid polymerase measured in the step (c) is the activity per unit mass of the hot start nucleic acid polymerase or the activity per unit volume of the hot start nucleic acid polymerase.

[0133] According to one embodiment of the present invention, the hot start nucleic acid polymerase is a hot start Taq DNA polymerase or a hot start KlenTaq DNA polymerase, and the activity of the hot start nucleic acid polymerase in step (c) is provided as the amount of nucleic acid polymerase determined by substituting the intensity of the signal into the following general formula 1:

[0134] General formula 1

[0135] 1 unit (enzyme activity unit) = the amount of nucleic acid polymerase required to produce a signal intensity indicating polymerization by consuming 4.4±0.56 nmole of dNTP at 72°C for 30 minutes.

[0136] According to one embodiment of the present invention, the hot start nucleic acid polymerase is a hot start Taq DNA polymerase or a hot start KlenTaq DNA polymerase, and the activity of the hot start nucleic acid polymerase in step (c) is provided as a unit number of the nucleic acid polymerase determined by substituting the intensity of the signal into the following general formula 1:

[0137] General formula 1

[0138] 1 unit (enzyme activity unit) = the amount of nucleic acid polymerase required to produce a signal intensity indicating polymerization by consuming 4.4±0.56 nmole of dNTP at 72°C for 30 minutes.

[0139] Step (d): Obtaining the activity of the hot start nucleic acid polymerase for the different thermal activation times as an activity profile of the hot start nucleic acid polymerase (140)

[0140] Finally, the method of the present invention comprises the step of (d) obtaining an activity profile of the hot start nucleic acid polymerase based on the activity of the hot start nucleic acid polymerase for the different thermal activation times. The activity profile of the hot start nucleic acid polymerase represents the performance of the hot start nucleic acid polymerase.

[0141] In this specification, the term "activity profile of a hot start nucleic acid polymerase" means a graph showing the extent to which the activity of an inhibited hot start nucleic acid polymerase is restored by thermal activation over time, with respect to the thermal activation time.

[0142] The greatest feature of the present invention is that the performance of the hot start nucleic acid polymerase can be evaluated from the activity value or aspect (morphology) of the activity profile of the hot start nucleic acid polymerase, and through this, it can be determined whether the quality status is the same between hot start nucleic acid polymerases.

[0143] For example, Fig. 4 shows the activity profile of hot start Taq DNA polymerase, and Fig. 5 shows the activity profile of hot start klentaq polymerase, a mutant of Taq DNA polymerase that excludes the exonuclease active site. As can be seen in Figs. 4 and 5, changes in the properties (mutations) of the nucleic acid polymerase can be seen in the activity profiles even after the hot start reaction, resulting in different activity values ​​and morphologies (patterns), which can have different effects on the PCR results.

[0144] According to one embodiment of the present invention, the method further comprises, after step (d), the following steps:

[0145] (d-1) A step of obtaining a product including a hot start nucleic acid polymerase obtained by a different obtaining process from the process of obtaining a product including a hot start nucleic acid polymerase of step (a);

[0146] (d-2) a step of performing steps (b) to (d) using the product obtained in step (d-1) to obtain the activity of the hot start nucleic acid polymerase included in the product of step (d-1) for the different thermal activation times as an activity profile of the hot start nucleic acid polymerase included in the product of step (d-1); the activity profile of the hot start nucleic acid polymerase included in the product of step (d-1) indicates the performance of the hot start nucleic acid polymerase included in the product of step (d-1); and

[0147] (d-3) A step of comparing the activity profile of the hot start nucleic acid polymerase included in the product of the above step (a) with the activity profile of the hot start nucleic acid polymerase included in the product in the obtaining process of the above step (d-1).

[0148] According to the present embodiment, the activity profiles of the hot start nucleic acid polymerase obtained in the first production process and the hot start nucleic acid polymerase obtained in the second production process can be compared to confirm the identity of quality between the hot start nucleic acid polymerases for each production batch.

[0149] II. Method for Obtaining a QC-Capable Hot Start Nucleic Acid Polymerase (Second Embodiment)

[0150] According to another aspect of the present invention, the present invention provides a method for obtaining a QC-capable hot start nucleic acid polymerase comprising the following steps:

[0151] (a) obtaining a product comprising a hot start nucleic acid polymerase;

[0152] (b) a step of fractionating a product containing the hot start nucleic acid polymerase and performing heat activation for each of the fractionated products for different times;

[0153] (c) a step of measuring the activity of each hot start nucleic acid polymerase included in each of the heat-activated products for different times; the activity is measured from the intensity of a signal measured by adding each of the heat-activated products for different times to a composition for activity assay of nucleic acid polymerase and performing a nucleic acid polymerization reaction, wherein the nucleic acid polymerase activity assay composition includes a primer, and the primer is extended by the hot start nucleic acid polymerase included in each of the heat-activated products under polymerization conditions to form an extension duplex, and a detectable signal is provided in a manner dependent on the extension duplex;

[0154] (d) obtaining the activity of the hot start nucleic acid polymerase for the different thermal activation times as an activity profile of the hot start nucleic acid polymerase; the activity profile of the hot start nucleic acid polymerase represents the performance of the hot start nucleic acid polymerase, and

[0155] (e) a step of providing a hot start nucleic acid polymerase having the above activity profile.

[0156] The present invention relates to a method for obtaining a QC-capable hot start nucleic acid polymerase using the method for evaluating the performance of the hot start nucleic acid polymerase of the present invention described above. Therefore, descriptions of common contents between the two methods are omitted to avoid excessive complexity of the present specification due to repetitive descriptions.

[0157] The expression "method for obtaining a QC-able nucleic acid polymerase" in this specification means a method for producing a nucleic acid polymerase capable of quality control (QC) of a hot start nucleic acid polymerase by performing thermal activation on an activity-inhibited hot start nucleic acid polymerase over time to obtain a profile of activity recovered over time, and evaluating and monitoring the performance of the hot start nucleic acid polymerase from the profile.

[0158] Figure 9 is a flowchart of processes for implementing the method of the present invention according to one embodiment of the present invention. The method of the present invention is described with reference to Figure 9 as follows:

[0159] Step (a): Obtaining a product comprising a hot start nucleic acid polymerase (210)

[0160] First, the method of the present invention comprises the step of (a) obtaining a product comprising a hot start nucleic acid polymerase.

[0161] According to one embodiment of the present invention, the hot start nucleic acid polymerase is a chemically modified hot start nucleic acid polymerase, an antibody-based hot start nucleic acid polymerase, or an aptamer-based hot start nucleic acid polymerase. Most specifically, the hot start nucleic acid polymerase is a chemically modified hot start nucleic acid polymerase.

[0162] According to one embodiment of the present invention, the nucleic acid polymerase is a DNA polymerase.

[0163] According to a more specific embodiment of the present invention, the DNA polymerase is Taq DNA polymerase, KlenTaq DNA polymerase, Klenow fragment of E. coli DNA polymerase I, DNA polymerase I, T4 DNA polymerase, Bst DNA polymerase, Bsu DNA polymerase, MMLV reverse transcriptase, AMV reverse transcriptase, or HIV reverse transcriptase.

[0164] According to one embodiment of the present invention, the nucleic acid polymerase is prepared by a method comprising the following steps: (a-1) culturing a host cell containing a nucleic acid polymerase expression construct; and (a-2) purifying the nucleic acid polymerase by subjecting the resultant of step (a-1) to column chromatography.

[0165] According to one embodiment of the present invention, the hot start nucleic acid polymerase of step (a) is a nucleic acid polymerase with suppressed polymerization activity. More specifically, the hot start nucleic acid polymerase is a nucleic acid polymerase with suppressed polymerization activity of 1 U / ug or less.

[0166] According to one embodiment of the present invention, the method further comprises, after step (a), a step (a-3) of diluting the product(s) to a concentration of 0.02 mg / ml to 2 mg / ml.

[0167] Step (b): Heat activation is performed for different times for each of the fractionated products (220).

[0168] Next, the method of the present invention comprises the step of (b) fractionating a product including the hot start nucleic acid polymerase and performing heat activation for each of the fractionated products for different periods of time.

[0169] According to one embodiment of the present invention, the thermal activation of step (b) is performed for at least two time intervals from 0 to 60 minutes for each of the fractionated products.

[0170] According to one embodiment of the present invention, the thermal activation of step (b) is performed at 90 to 100°C.

[0171] According to one embodiment of the present invention, the thermal activation of step (b) is performed at 90 to 100°C for 0 minutes, 15 minutes, 30 minutes, and 60 minutes for each of the fractionated products.

[0172] According to one embodiment of the present invention, the method further comprises, between steps (b) and (c), a step (b-1) of diluting each of the heat-activated products to a concentration of 6.25 ng / ul to 50 ng / ul.

[0173] Step (c): Measuring the activity of each hot start nucleic acid polymerase contained in each of the heat-activated products for different times (230)

[0174] Next, the method of the present invention comprises the step of (c) measuring the activity of each hot start nucleic acid polymerase included in each of the products that have been heat-activated for different times. The activity is measured from the intensity of a signal measured by adding each of the products that have been heat-activated for different times to a composition for activity assay of nucleic acid polymerase and performing a nucleic acid polymerization reaction, wherein the composition for activity assay of nucleic acid polymerase includes a primer, and the primer is extended by the hot start nucleic acid polymerase included in each of the products that have been heat-activated for different times under polymerization conditions to form an extension duplex, and a detectable signal is provided in a manner dependent on the extension duplex.

[0175] According to one embodiment of the present invention, the activity of the hot start nucleic acid polymerase measured in the step (c) is provided from the slope measured in the range in which the intensity of the signal increases linearly depending on the concentration of the hot start nucleic acid polymerase contained in the product.

[0176] According to one embodiment of the present invention, the activity of the hot start nucleic acid polymerase measured in the step (c) is the activity per unit mass of the hot start nucleic acid polymerase or the activity per unit volume of the hot start nucleic acid polymerase.

[0177] According to one embodiment of the present invention, the activity assay composition of the nucleic acid polymerase additionally comprises (i) a template, (ii) a label, (iii) dNTP, and (iv) a buffer.

[0178] According to one embodiment of the present invention, the primer included in the activity assay composition of the nucleic acid polymerase comprises a first primer and a second primer as a dimer-forming primer pair having the functions of both a primer and a template, wherein each of the first primer and the second primer comprises a 3'-dimer forming site, and the nucleotide sequence of the 3'-dimer forming site in the first primer is complementary to the nucleotide sequence of the 3'-dimer forming site in the second primer.

[0179] According to one embodiment of the present invention, in the step (c), the addition of each of the products heat-activated for different times to the nucleic acid polymerase activity assay composition is carried out by adding each of the products heat-activated for different times to the nucleic acid polymerase activity assay composition according to the dilution factor.

[0180] According to one embodiment of the present invention, the nucleic acid polymerization reaction is carried out at 35°C to 75°C for 10 to 60 minutes.

[0181] According to a more specific embodiment of the present invention, the label is an intercalating label. More specifically, the intercalating label is SYBR™ Green I, PO-PRO™-1, BO-PRO™-1, SYTO™ 43, SYTO™ 44, SYTO™ 45, SYTOX™ Blue, POPO™-1, POPO™-3, BOBO™-1, BOBO™-3, LO-PRO™-1, JO-PRO™-1, YO-PRO™ 1, TO-PRO™1, SYTO™11, SYTO™13, SYTO™15, SYTO™16, SYTO™20, SYTO™23, TOTO™-3, YOYO™3, GelStar™, thiazole orange, PicoGreen ® , EvaGreen ® or selected from the group consisting of LAM Fluorescent Dye.

[0182] According to one embodiment of the present invention, the hot start nucleic acid polymerase is a hot start Taq DNA polymerase or a hot start KlenTaq DNA polymerase, and the activity of the hot start nucleic acid polymerase in step (c) is provided as the amount of nucleic acid polymerase determined by substituting the intensity of the signal into the following general formula 1:

[0183] General formula 1

[0184] 1 unit (enzyme activity unit) = the amount of nucleic acid polymerase required to produce a signal intensity indicating polymerization by consuming 4.4±0.56 nmole of dNTP at 72°C for 30 minutes.

[0185] According to one embodiment of the present invention, the hot start nucleic acid polymerase is a hot start Taq DNA polymerase or a hot start KlenTaq DNA polymerase, and the activity of the hot start nucleic acid polymerase in step (c) is provided as a unit number of the nucleic acid polymerase determined by substituting the intensity of the signal into the following general formula 1:

[0186] General formula 1

[0187] 1 unit (enzyme activity unit) = the amount of nucleic acid polymerase required to produce a signal intensity indicating polymerization by consuming 4.4±0.56 nmole of dNTP at 72°C for 30 minutes.

[0188] Step (d): Obtaining the activity of the hot start nucleic acid polymerase for the different thermal activation times as an activity profile of the hot start nucleic acid polymerase (240)

[0189] Next, the method of the present invention comprises the step of (d) obtaining an activity profile of the hot start nucleic acid polymerase based on the activity of the hot start nucleic acid polymerase for the different thermal activation times. The activity profile of the hot start nucleic acid polymerase indicates the performance of the hot start nucleic acid polymerase.

[0190] According to one embodiment of the present invention, the method further comprises, after step (d), the following steps:

[0191] (d-1) A step of obtaining a product including a hot start nucleic acid polymerase obtained by a different obtaining process from the process of obtaining a product including a hot start nucleic acid polymerase of step (a);

[0192] (d-2) a step of performing steps (b) to (d) using the product obtained in step (d-1) to obtain the activity of the hot start nucleic acid polymerase included in the product of step (d-1) for the different thermal activation times as an activity profile of the hot start nucleic acid polymerase included in the product of step (d-1); the activity profile of the hot start nucleic acid polymerase included in the product of step (d-1) indicates the performance of the hot start nucleic acid polymerase included in the product of step (d-1); and

[0193] (d-3) A step of comparing the activity profile of the hot start nucleic acid polymerase included in the product of the above step (a) with the activity profile of the hot start nucleic acid polymerase included in the product in the obtaining process of the above step (d-1).

[0194] Step (e): Providing a hot start nucleic acid polymerase having the above activity profile (250)

[0195] Finally, the method of the present invention comprises the step of (c) providing a hot start nucleic acid polymerase having the above activity profile.

[0196] In this step, a hot start nucleic acid polymerase having an activity profile is provided.

[0197] III. Method for providing a blended hot-start nucleic acid polymerase that mimics the performance of a target hot-start nucleic acid polymerase (third aspect)

[0198] According to another aspect of the present invention, the present invention provides a method for providing a blended hot start nucleic acid polymerase that mimics the performance of a target hot start nucleic acid polymerase, comprising the following steps:

[0199] (a) obtaining i) a product comprising a target hot start nucleic acid polymerase and ii) products each comprising at least two hot start nucleic acid polymerases to be blended;

[0200] (b) a step of i) collecting a product containing the target hot start nucleic acid polymerase and performing heat activation for different times for each of the collected products, and ii) collecting each of the products containing each of the at least two hot start nucleic acid polymerases to be blended and performing heat activation for different times for each of the collected products; wherein the heat activation time of i) and the heat activation time of ii) are the same,

[0201] (c) a step of measuring the activity of each target hot start nucleic acid polymerase included in each of the products heat-activated for different times in i) of the step (b), and a step of measuring the activity of each of at least two hot start nucleic acid polymerases to be blended included in each of the products heat-activated for different times in ii) of the step (b); wherein the activity is measured from the intensity of a signal measured by adding each of the products heat-activated for different times to a composition for activity assay of nucleic acid polymerase and performing a nucleic acid polymerization reaction, wherein the composition for activity assay of nucleic acid polymerase includes a primer, and the primer is extended by the hot start nucleic acid polymerase included in each of the heat-activated products under polymerization conditions to form an extension duplex, and a detectable signal is provided in a manner dependent on the extension duplex,

[0202] (d) obtaining the activity of the target hot start nucleic acid polymerase for different thermal activation times in i) of the step (b) as an activity profile of the target hot start nucleic acid polymerase, and obtaining the activity of the at least two hot start nucleic acid polymerases to be blended for different thermal activation times in ii) of the step (b) as an activity profile of the at least two hot start nucleic acid polymerases to be blended; the activity profile of the target hot start nucleic acid polymerase represents the performance of the target hot start nucleic acid polymerase, and each of the activity profiles of the at least two hot start nucleic acid polymerases to be blended represents the performance of each of the at least two hot start nucleic acid polymerases to be blended,

[0203] (e) providing an amount of each of at least two hot start nucleic acid polymerases to be blended using the performance of the target hot start nucleic acid polymerase and the performance of at least two hot start nucleic acid polymerases to be blended to mimic the performance of the target hot start nucleic acid polymerase; and

[0204] (f) providing at least two hot start nucleic acid polymerases blended in the amounts provided in step (e) as a blended hot start nucleic acid polymerase that mimics the performance of the target hot start nucleic acid polymerase.

[0205] The present invention relates to a method for providing a blended hot-start nucleic acid polymerase that mimics the performance of a target hot-start nucleic acid polymerase, using the method for evaluating the performance of the hot-start nucleic acid polymerase of the present invention described above. Therefore, descriptions of common contents between the two are omitted to avoid excessive complexity of the present specification due to repetitive descriptions.

[0206] In this specification, the term "target hot start nucleic acid polymerase" means a target hot start nucleic acid polymerase that is intended to mimic or imitate the performance of a hot start nucleic acid polymerase by blending (mixing or combining) at least two hot start nucleic acid polymerases.

[0207] Figure 10 is a flowchart of processes for implementing the method of the present invention according to one embodiment of the present invention. The method of the present invention is described with reference to Figure 10 as follows:

[0208] Step (a): Obtaining a product comprising a hot start nucleic acid polymerase (310)

[0209] First, the method of the present invention comprises the steps of (a) obtaining i) a product comprising a target hot start nucleic acid polymerase and ii) products each comprising at least two hot start nucleic acid polymerases to be blended.

[0210] According to one embodiment of the present invention, the number of hot start nucleic acid polymerases to be blended is at most 10. More specifically, the number of hot start nucleic acid polymerases to be blended is selected from 2 to 8, even more specifically from 2 to 5, and even more specifically from 2 or 3.

[0211] According to one embodiment of the present invention, the hot start nucleic acid polymerase is a chemically modified hot start nucleic acid polymerase, an antibody-based hot start nucleic acid polymerase, or an aptamer-based hot start nucleic acid polymerase. Most specifically, the hot start nucleic acid polymerase is a chemically modified hot start nucleic acid polymerase.

[0212] According to one embodiment of the present invention, the nucleic acid polymerase is a DNA polymerase.

[0213] According to a more specific embodiment of the present invention, the DNA polymerase is Taq DNA polymerase, KlenTaq DNA polymerase, Klenow fragment of E. coli DNA polymerase I, DNA polymerase I, T4 DNA polymerase, Bst DNA polymerase, Bsu DNA polymerase, MMLV reverse transcriptase, AMV reverse transcriptase, or HIV reverse transcriptase.

[0214] According to one embodiment of the present invention, the nucleic acid polymerase is prepared by a method comprising the following steps: (a-1) culturing a host cell containing a nucleic acid polymerase expression construct; and (a-2) purifying the nucleic acid polymerase by subjecting the resultant of step (a-1) to column chromatography.

[0215] According to one embodiment of the present invention, the hot start nucleic acid polymerase of step (a) is a nucleic acid polymerase with suppressed polymerization activity. More specifically, the hot start nucleic acid polymerase is a nucleic acid polymerase with suppressed polymerization activity of 1 U / ug or less.

[0216] According to one embodiment of the present invention, the method further comprises, after step (a), a step (a-3) of diluting the product(s) to a concentration of 0.02 mg / ml to 2 mg / ml.

[0217] Step (b): Heat activation is performed for different times for each of the collected products (320).

[0218] Next, the method of the present invention comprises the steps of (b) i) collecting a product comprising the target hot start nucleic acid polymerase and performing heat activation for different times on each of the collected products, and ii) collecting each of the products comprising each of the at least two hot start nucleic acid polymerases to be blended and performing heat activation for different times on each of the collected products. The heat activation time of i) and the heat activation time of ii) are the same.

[0219] According to one embodiment of the present invention, the thermal activation of step (b) is performed for at least two time intervals from 0 to 60 minutes for each of the fractionated products.

[0220] According to one embodiment of the present invention, the thermal activation of step (b) is performed at 90 to 100°C.

[0221] According to one embodiment of the present invention, the thermal activation of step (b) is performed at 90 to 100°C for 0 minutes, 15 minutes, 30 minutes, and 60 minutes for each of the fractionated products.

[0222] According to one embodiment of the present invention, the method further comprises, between steps (b) and (c), a step (b-1) of diluting each of the heat-activated products to a concentration of 6.25 ng / ul to 50 ng / ul.

[0223] Step (c): Measuring the activity of each hot start nucleic acid polymerase contained in each of the heat-activated products for different times (330)

[0224] Next, the method of the present invention comprises the step of (c) measuring the activity of each target hot start nucleic acid polymerase included in each of the products heat-activated for different times in i) of the step (b), and the step of measuring the activity of each of at least two hot start nucleic acid polymerases to be blended included in each of the products heat-activated for different times in ii) of the step (b). The activity is measured from the intensity of a signal measured by adding each of the products heat-activated for different times to a composition for activity assay of nucleic acid polymerase and performing a nucleic acid polymerization reaction, wherein the composition for activity assay of nucleic acid polymerase comprises a primer, and the primer is extended by the hot start nucleic acid polymerase included in each of the heat-activated products under polymerization conditions to form an extension duplex, and a detectable signal is provided in a manner dependent on the extension duplex.

[0225] According to one embodiment of the present invention, the activity of the hot start nucleic acid polymerase measured in the step (c) is provided from the slope measured in the range in which the intensity of the signal increases linearly depending on the concentration of the hot start nucleic acid polymerase contained in the product.

[0226] According to one embodiment of the present invention, the activity of the hot start nucleic acid polymerase measured in the step (c) is the activity per unit mass of the hot start nucleic acid polymerase or the activity per unit volume of the hot start nucleic acid polymerase.

[0227] According to one embodiment of the present invention, the activity assay composition of the nucleic acid polymerase additionally comprises (i) a template, (ii) a label, (iii) dNTP, and (iv) a buffer.

[0228] According to one embodiment of the present invention, the primer included in the activity assay composition of the nucleic acid polymerase comprises a first primer and a second primer as a dimer-forming primer pair having the functions of both a primer and a template, wherein each of the first primer and the second primer comprises a 3'-dimer forming site, and the nucleotide sequence of the 3'-dimer forming site in the first primer is complementary to the nucleotide sequence of the 3'-dimer forming site in the second primer.

[0229] According to one embodiment of the present invention, in the step (c), the addition of each of the products heat-activated for different times to the nucleic acid polymerase activity assay composition is carried out by adding each of the products heat-activated for different times to the nucleic acid polymerase activity assay composition according to the dilution factor.

[0230] According to one embodiment of the present invention, the nucleic acid polymerization reaction is carried out at 35°C to 75°C for 10 to 60 minutes.

[0231] According to a more specific embodiment of the present invention, the label is an intercalating label. More specifically, the intercalating label is SYBR™ Green I, PO-PRO™-1, BO-PRO™-1, SYTO™ 43, SYTO™ 44, SYTO™ 45, SYTOX™ Blue, POPO™-1, POPO™-3, BOBO™-1, BOBO™-3, LO-PRO™-1, JO-PRO™-1, YO-PRO™ 1, TO-PRO™1, SYTO™11, SYTO™13, SYTO™15, SYTO™16, SYTO™20, SYTO™23, TOTO™-3, YOYO™3, GelStar™, thiazole orange, PicoGreen ® , EvaGreen ® or selected from the group consisting of LAM Fluorescent Dye.

[0232] According to one embodiment of the present invention, the hot start nucleic acid polymerase is a hot start Taq DNA polymerase or a hot start KlenTaq DNA polymerase, and the activity of the hot start nucleic acid polymerase in step (c) is provided as the amount of nucleic acid polymerase determined by substituting the intensity of the signal into the following general formula 1:

[0233] General formula 1

[0234] 1 unit (enzyme activity unit) = the amount of nucleic acid polymerase required to produce a signal intensity indicating polymerization by consuming 4.4±0.56 nmole of dNTP at 72°C for 30 minutes.

[0235] According to one embodiment of the present invention, the hot start nucleic acid polymerase is a hot start Taq DNA polymerase or a hot start KlenTaq DNA polymerase, and the activity of the hot start nucleic acid polymerase in step (c) is provided as a unit number of the nucleic acid polymerase determined by substituting the intensity of the signal into the following general formula 1:

[0236] General formula 1

[0237] 1 unit (enzyme activity unit) = the amount of nucleic acid polymerase required to produce a signal intensity indicating polymerization by consuming 4.4±0.56 nmole of dNTP at 72°C for 30 minutes.

[0238] Step (d): Obtaining the activity of the hot start nucleic acid polymerase for the different thermal activation times as an activity profile of the hot start nucleic acid polymerase (340)

[0239] Next, the method of the present invention comprises the step (d) of obtaining the activity of the target hot start nucleic acid polymerase for different thermal activation times as an activity profile of the target hot start nucleic acid polymerase in i) of the step (b), and obtaining the activity of the at least two hot start nucleic acid polymerases to be blended for different thermal activation times as an activity profile of the at least two hot start nucleic acid polymerases to be blended in ii) of the step (b). The activity profile of the target hot start nucleic acid polymerase represents the performance of the target hot start nucleic acid polymerase, and each of the activity profiles of the at least two hot start nucleic acid polymerases to be blended represents the performance of each of the at least two hot start nucleic acid polymerases to be blended.

[0240] Step (e): Providing the amount of each of at least two hot start nucleic acid polymerases to be blended using the performance of the target hot start nucleic acid polymerase and the performance of at least two hot start nucleic acid polymerases to be blended to mimic the performance of the target hot start nucleic acid polymerase (350)

[0241] Next, the method of the present invention comprises the step of (e) providing an amount of each of the at least two hot start nucleic acid polymerases to be blended using the performance of the target hot start nucleic acid polymerase and the performance of the at least two hot start nucleic acid polymerases to be blended to mimic the performance of the target hot start nucleic acid polymerase.

[0242] The most significant feature of the present invention is that the activity profiles representing the performance of a target hot start nucleic acid polymerase and at least two hot start nucleic acid polymerases to be blended can be used to provide an amount of each of at least two hot start nucleic acid polymerases to be blended that mimics the performance of the target hot start nucleic acid polymerase.

[0243] According to a more specific embodiment of the present invention, the step (e) is a step of providing the amount of each of the at least two hot start nucleic acid polymerases to be blended by substituting the performance of the target hot start nucleic acid polymerase and the performance of at least two hot start nucleic acid polymerases to be blended to mimic the performance of the target hot start nucleic acid polymerase into a linear algebraic equation.

[0244] In this embodiment, the activity values ​​of the activity profile representing the performance of the hot start nucleic acid polymerase can be substituted into a linear algebraic equation to provide the amount of each of at least two hot start nucleic acid polymerases to be blended to simulate the performance of the target hot start nucleic acid polymerase.

[0245] In this specification, a linear algebraic equation refers to a simultaneous equation in which all variables are linear terms.

[0246] According to a more specific embodiment of the present invention, the linear algebraic equation is expressed by the following mathematical expression 1:

[0247] Mathematical formula 1

[0248] [A] nxn [X] nx1 = [B]nx1

[0249] In the above mathematical formula, the matrix [A] represents the performance of at least two hot start nucleic acid polymerases to be blended, and n of the row elements representing the size of the matrix in the matrix [A] represents the order of the time of thermal activation, and n of the column elements in the matrix [A] represents the hot start nucleic acid polymerases to be blended, the matrix [X] represents the amount of at least two hot start nucleic acid polymerases to be blended, and n of the row elements representing the size of the matrix in the matrix [X] represents the hot start nucleic acid polymerases to be blended, and the matrix [B] represents the performance of the target hot start nucleic acid polymerase, and n of the row elements representing the size of the matrix in the matrix [B] represents the order of the time of thermal activation.

[0250] According to the most specific embodiment of the present invention, the above mathematical formula 1 is expressed as the following mathematical formula 2:

[0251] Mathematical formula 2

[0252]

[0253] In the above mathematical formula, the matrix element a represents the activity of at least two hot start nucleic acid polymerases to be blended, the row element n in the matrix element a represents the order of the time of thermal activation, and the column element n in the matrix element a represents the hot start nucleic acid polymerase to be blended, the matrix element x represents the amount of at least two hot start nucleic acid polymerases to be blended, the row element n in the matrix element x represents the hot start nucleic acid polymerase to be blended, and the matrix element b represents the activity of the target hot start nucleic acid polymerase, and the row element n in the matrix element b represents the order of the time of thermal activation.

[0254] In the matrix [X] and matrix component x of the above mathematical formulas 1 and 2, n of the row components representing the hot start nucleic acid polymerase to be blended is at most 10, more specifically selected from 2 to 8, even more specifically selected from 2 to 5, and even more specifically 2 or 3.

[0255] In the above mathematical expressions 1 and 2, the matrix [A] and the matrix [B], and the matrix component a and the matrix component b, the row component n indicating the order of the time of thermal activation is a natural number. For example, 1) if thermal activation was performed for 0 minutes and thermal activation was performed for 60 minutes, the order of the time of thermal activation for 0 minutes is 1, and the order of the time of thermal activation for 60 minutes is 2. 2) if thermal activation was performed for 0 minutes, 15 minutes, and 60 minutes, the order of the time of thermal activation for 0 minutes is 1, the order of the time of thermal activation for 15 minutes is 2, and the order of the time of thermal activation for 60 minutes is 3. 3) If heat activation was performed for 0 minutes, 15 minutes, 45 minutes, and 60 minutes, the order of time for heat activation for 0 minutes is 1, the order of time for heat activation for 15 minutes is 2, the order of time for heat activation for 30 minutes is 3, and the order of time for heat activation for 60 minutes is 4.

[0256] According to one embodiment of the present invention, n of the row components representing the order of the thermally activated time is selected from 1 to 10. More specifically, it is selected from 2 to 8, even more specifically, it is selected from 2 to 5, and even more specifically, it is 2 or 3.

[0257] According to one embodiment of the present invention, the performance of the at least two hot start nucleic acid polymerases to be blended and the target hot start nucleic acid polymerase is the activity of the at least two hot start nucleic acid polymerases to be blended and the target hot start nucleic acid polymerase.

[0258] In the above mathematical formulas 1 and 2, when the activity of the at least two hot start nucleic acid polymerases to be blended is activity per unit mass (Unit) and the activity of the target hot start nucleic acid polymerase is activity (Unit), the amount of the at least two hot start nucleic acid polymerases to be blended is mass, and when the activity of the at least two hot start nucleic acid polymerases to be blended is activity per unit mass (Unit) and the activity of the target hot start nucleic acid polymerase is activity per reaction (Unit), the amount of the at least two hot start nucleic acid polymerases to be blended is mass per reaction.

[0259] Step (f): providing at least two hot start nucleic acid polymerases blended in the amounts provided above as a blended hot start nucleic acid polymerase that mimics the performance of the target hot start nucleic acid polymerase (360)

[0260] Finally, the method of the present invention comprises the step of (f) providing at least two hot start nucleic acid polymerases blended in the amounts provided in step (e) as a blended hot start nucleic acid polymerase that mimics the performance of the target hot start nucleic acid polymerase.

[0261] In this way, a blended hot start nucleic acid polymerase can be provided that mimics the performance of the above target hot start nucleic acid polymerase.

[0262] The features and advantages of the present invention are summarized as follows:

[0263] (a) In the past, hot start nucleic acid polymerase was in a state of suppressed activity, so there was a problem in that the activity was measured very low or not measured at all using a general method of measuring the activity of nucleic acid polymerase.

[0264] (b) The present invention can evaluate the performance of a hot start nucleic acid polymerase and provide a QC-capable hot start nucleic acid polymerase by constructing an activity profile of a hot start nucleic acid polymerase with respect to thermal activation time using a method for measuring the activity (unit) of a nucleic acid polymerase without using a radioactive isotope.

[0265] (c) In addition, the present invention can provide a blended nucleic acid polymerase that mimics the performance of a target hot start nucleic acid polymerase by utilizing the activity profile of the hot start nucleic acid polymerase with respect to the thermal activation time.

[0266] Figure 1 is a flowchart of processes for implementing the method of the present invention according to one embodiment of the present invention.

[0267] Figure 2 shows an analysis flow chart of a hot start nucleic acid polymerase according to one embodiment of the present invention.

[0268] Figure 3 shows a real-time fluorescence measurement graph for measuring polymerization activity according to one embodiment of the present invention.

[0269] Figure 4 shows an activity profile analyzing the activity of a hot start TaqDNA polymerase produced according to one embodiment of the present invention.

[0270] Figure 5 shows an activity profile analyzing the activity that appears when hot start is applied to klentaq polymerase according to one embodiment of the present invention.

[0271] Figure 6 is a graph showing the activity of a hot start nucleic acid polymerase, the concentration of which is difficult to determine, according to thermal activation time according to one embodiment of the present invention.

[0272] FIG. 7 shows the activity profile of the hot start nucleic acid polymerase of FIG. 6 according to one embodiment of the present invention.

[0273] Figure 8 shows a graph of regression analysis of the results of a Ct-based unit measurement method and a unit measurement method according to an embodiment of the present invention.

[0274] Figure 9 is a flowchart of processes for implementing the method of the present invention according to another embodiment of the present invention.

[0275] Figure 10 is a flowchart of processes for implementing the method of the present invention according to another embodiment of the present invention.

[0276] FIG. 11 illustrates a formula for calculating the amount (e.g., mass, concentration, etc.) of a hot start polymerase to be blended to simulate a target hot start polymerase by a linear algebraic equation (matrix) according to another embodiment of the present invention.

[0277] Figure 12a) shows a formula in which the activity value per reaction among the activity profiles of the target hot start polymerase and the activity value per unit mass among the activity profiles of the hot start polymerases 1 and 2 to be blended are substituted into a linear algebraic equation (matrix) to determine the amount of any hot start polymerase 1 and hot start polymerase 2 to be blended to simulate the performance of the target hot start polymerase. Figure 12b) shows the mass per reaction of the hot start polymerases 1 and 2 to be blended calculated through the formula of Figure 12a). Figure 12c) shows the activity profiles of hot start polymerases 1 and 2 to be blended and the activity profile of the target hot start polymerase, and the activity profile of the blending hot start polymerase calculated using the above formula is superimposed on the activity profile of the target hot start polymerase at thermal activation times of 15 minutes and 30 minutes.

[0278] Figure 13 is a graph showing that the activity profile of the blended hot start polymerase mixed in the amount calculated in Figure 12 is similar to the activity profile of the target hot start polymerase to be imitated.

[0279] Hereinafter, the present invention will be described in more detail through examples. These examples are intended solely to illustrate the present invention more specifically. It will be apparent to those skilled in the art that the scope of the present invention is not limited by these examples, in accordance with the gist of the present invention.

[0280] Example

[0281] Example 1: Measurement of enzyme activity units using a fluorescence amplification method based on DNA double-strand binding.

[0282] First, two products, NEB's TaqDNA polymerase and Roche's TaqDNA polymerase, were selected as standard products. The concentrations of each enzyme were confirmed according to the literature (Jesse L. Montgomery et al. Analytical Biochemistry 2013, 441(2), 133-139). In addition, EvaEZ TM Product (EvaGreen ® To calculate the initial polymerization rate according to the protein dilution factor using a dye, a primed template, dNTPs, MgCl2, and Tris buffer, the change in fluorescence was tracked at 30-second intervals for 60 minutes using a Bio-Rad CFX96-DR instrument (Fig. 3a)), and the information on the error range was accurately confirmed through 3 repetitions within the same set for the same experiment, for a total of 7 sets.

[0283] The measured graph of Fig. 3 (x-axis - time, y-axis - fluorescence value) draws a curve that gradually becomes gentler as the dNTP, which is the substrate, gradually fails to participate 100% in the reaction for the enzyme due to consumption of the dNTP. At this time, if the concentration of the enzyme is set to a certain dilution level as in Example 1-1 or Example 1-2 below, the amount of dNTP becomes abundant compared to the enzyme within the first 10 minutes, so the graph of the fluorescence value over time becomes a linear function. The slope of the linear function at this time (differential value of the time-fluorescence value graph) is measured. Since this value is the value of fluorescence that increases over the time of DNA synthesis, it is directly related to the amount of dNTP consumed.

[0284] If this measurement value is measured at various concentrations where the initial reaction function becomes first-order, as in Example 1-1 or Example 1-2 below, this can be expressed again in the form of 'enzyme concentration' on the x-axis and 'differential value of time-fluorescence value' on the y-axis. In this case, a linear function of 'differential value of time-fluorescence value' with respect to 'enzyme concentration' is created again. Here, the slope of this graph (enzyme concentration - derivative of 'differential value of time-fluorescence value') ultimately becomes an indicator of the amount of dNTP consumption, which is directly proportional to the enzyme concentration. Therefore, through this, the unit for the polymerization activity of DNA polymerase can be defined as the amount of dNTP consumed per unit time at unit temperature.

[0285] In conclusion, the definition of 1 Unit (consumption of 15 nmole of dNTP in 30 minutes at 75°C) measured by NEB's radioisotope standard was compared with EvaEZ TMWe confirmed that the product consumed 4.4 ± 0.56 nmoles (95% confidence interval) of dNTPs over 30 minutes at 72°C, confirming the reliability of the measurement method for the standard product against changes. We also confirmed that the same level of confidence was achieved with the Roche product under the same conditions (consuming 10 nmoles of dNTP over 30 minutes at 75°C).

[0286] Example 1-1: Preparation of reagents for selection of standard products

[0287] For the selection of standard products, the concentration of TaqDNA polymerase sold at 5 U / ul was diluted with 10X ThermoPol reaction buffer and ultrapure water from New England Biolabs to concentrations of 1000 mU / ul, 500 mU / ul, 250 mU / ul, 125 mU / ul, 62.5 mU / ul, 31.3 mU / ul, 15.6 mU / ul, 7.8 mU / ul, and 3.9 mU / ul, respectively, and mixed in a volume ratio of 10:1:9 (EvaEZ reagent, enzyme, ultrapure water). All processes were performed at a low temperature using ice (Fig. 3a)). Similarly, the concentration of TaqDNA polymerase from Roche was diluted in the same manner to prepare a reagent (Fig. 3b)).

[0288] Example 1-2: Reaction of reagents

[0289] To observe the kinetic reaction, we prepared a Bio-Rad CFX96, a temperature-controlled instrument capable of real-time fluorescence measurement. The instrument was set up to measure fluorescence at 30-second intervals, with 20 μl of reagents divided into 8-strip PCR tubes, and the reaction was performed at 72°C. The extracted data were used to calculate the slope of the portion with the initial linear function.

[0290] Example 1-3: Measurement of the specific activity of 100% pure TaqDNA polymerase

[0291] Through the experiments of Example 1-2, if a consistent value was obtained each time even after changing the equipment, experimenter, and reagent, that reagent could be designated as a standard. These standards were buffer exchanged and concentrated through Amicon Ultra centrifugal filter units (30 kDa cut-off) and then subjected to SDS-PAGE analysis. The standard substance for quantification of BSA (Bovine Serum Albumin) was loaded together in the same SDS-PAGE in equal volumes at concentrations ranging from 0.1 mg / ml to 1 mg / ml, and based on this data, the actual enzyme amount in the standard was quantified and the specific activity value could be calculated.

[0292] Example 2: Preparation of polymerase

[0293] TaqDNA polymerase was expressed by culturing E. coli through transformation. Transformed E. coli cells (transformed with a plasmid containing TaqDNA polymerase and His Tag) cultured at 37°C were separated from the medium solution by centrifugation, thawed again in buffer (20 mM Tris, pH 8, 500 mM NaCl, 0.1% Triton X-100), and then sonicated while incubated on ice to break down the cell membrane and elute the protein. The sample was centrifuged at 13,000 rpm to separate the sediment and the remaining solution, and only the remaining solution was obtained. To further remove impurities from the portion, the portion was treated at 80°C for 60 minutes and then centrifuged again to remove the sediment.

[0294] To purify the heat-treated product using an anion exchange column, it was first filtered through a 0.2 micrometer syringe filter, and then purified using an anion exchange column (Cytiva HiTrap Q) using FPLC equipment (Cytiva AKTA pure). The anion exchange column separated and purified the protein by increasing the concentration of KCl in 20 mM phosphate buffer. The fraction that passed through the FPLC of each purified product was analyzed by SDS-PAGE to select the region with a high concentration of the target protein.

[0295] For additional purification, a nickel-based His-taq purification column (HisTrap, Cytiva) was used on an FPLC (AKTA pure, Cytiva) instrument. Since the enzyme was designed to have a His-tag plasmid, it was bound to the column, and after impurities were washed away through a 20 mM phosphate buffer, the concentration of imidazole was increased to 500 mM to isolate only the desired enzyme into a specific fraction. As above, each fraction was analyzed by SDS-PAGE, and the fraction from which the desired protein was eluted was selected. The solution that went through the purification process was filtered through a 0.2 micrometer syringe filter, and then using an Amicon tube (Amicon Ultra centrifugal filter units, 30 kDa cut-off), proteins larger than the filter unit (including the target enzyme) were concentrated, while small proteins smaller than the target protein size were additionally removed.

[0296] Example 3: Preparation of hot start polymerase

[0297] To hot-start the purified TaqDNA polymerase, a 100:168 dilution of formaldehyde stock solution (37%, Sigma-Aldrich) and sterile distilled water was prepared, and the mixture was slowly reacted with the enzyme at 37℃. The final concentration and reaction time of formaldehyde added to the reaction mixture varied depending on the intended hot-start enzyme product, and the hot-start polymerase was manufactured by varying the treatment from 0.0125% to 0.5% or adjusting the reaction time from 30 minutes to 16 hours. The hot-start polymerase after the reaction was completed was incubated at low temperature, and the reaction was terminated with a solution containing a mixture of 20 mM Tris solution and DTT. The final product was concentrated using an Amicon tube (Amicon Ultra centrifugal filter units, 30 kDa cut-off) to obtain the intended protein concentration.

[0298] Example 4: Analysis of the activity profile of hot start polymerase

[0299] The final concentrated hot start polymerase is ideally concentrated to a concentration of at least 0.5 mg / ml. The hot start polymerase prepared for controlled heat treatment under all conditions was diluted to a concentration of 500 ng / ul and dispensed into PCR tubes in 20 μl portions to allow for rapid temperature ramps. The tubes were placed in a heat block or PCR machine maintained at 95°C and heat-treated for 0 min (heat activation), 15 min, 30 min, and 60 min. After the heat treatment was complete, 80 μl of 1x ThermoPol reaction buffer prepared on ice was immediately added to rapidly cool the sample to prevent further heat-induced reaction and further diluted to an appropriate concentration level for analysis (50 ng / ul to 6.25 ng / ul). In the case of samples where it is difficult to measure the concentration, the same process as above is followed, but the reaction is started by initially replacing the buffer with more than 1 / 4 of the thermal stabilizer.

[0300] The solution prepared by dilution above was analyzed for the enzyme unit by fluorescence value for each temperature sample using the method of Example 1. The analyzed samples were plotted in Fig. 4, with the heat treatment time on the x-axis and the enzyme's specific activity (unit per unit enzyme mass) or unit value (unit per unit volume) on the y-axis. This becomes a unique profile for each hot start polymerase, and can be closely used to determine the homology between enzymes for each batch.

[0301] Example 5: Activity profile analysis from hot-start Klentaq enzyme

[0302] Klentaq, a mutant of TaqDNA polymerase excluding the exohydrolytic activity site, was expressed and purified using the same method as in Examples 1 to 4, and the hot start reaction was applied in the same manner to produce a hot start klentaq enzyme. It was confirmed that the activity profile analysis was also possible for the hot start klentaq enzyme (Fig. 5).

[0303] Through this, it was found that changes in the properties of the enzyme can lead to different shapes and appearances even when undergoing a hot start reaction under the same conditions, and ultimately can have different effects on the PCR results.

[0304] Example 6: Activity profile analysis for a hot-start polymerase whose concentration is difficult to determine.

[0305] In general, in order to store enzymes stably for a long time, heat stability is secured using glycerol or sugars, but this makes it impossible to measure the concentration of the enzyme itself and causes problems in the heat treatment for hot start profile analysis (activity profile of hot start polymerase). Therefore, when analyzing an external standard enzyme, AmpliTaq Gold from Applied Biosystems, whose concentration is unknown, it was diluted 1 / 4 with ThermoPol reaction buffer from NEB, which does not affect thermal stability, and after heat treatment (heat activation), the concentration was serially diluted to perform the analysis of Example 4, and the results are shown in Fig. 6. Fig. 6 a) shows the result of heat treatment at 95°C for 60 minutes, Fig. 6 b) shows the result of heat treatment at 95°C for 30 minutes, Fig. 6 c) shows the result of heat treatment at 95°C for 15 minutes, and Fig. 6 d) shows the result of heat treatment at 95°C for 0 minute.

[0306] For convenience of analysis, the initial enzyme concentration value is assumed to be 500 ng / ul and diluted, and the value indicated on the x-axis of Fig. 6 represents the concentration of the enzyme diluted from the assumed enzyme concentration value. Since the analytical value derived in this way does not know the concentration of the enzyme, it is impossible to infer the specific activity value based on the concentration. However, if the assumed enzyme concentration value of 500 ng / ul is multiplied by the final calculated assumed specific activity value, it is possible to calculate the unit per unit volume, and thus the shape of the hot start profile could be confirmed through the result (Fig. 7).

[0307] Since the hot start inhibition effect generally disappears at the 30-minute 95℃ heat treatment level and the polymerase activity tends to be strong, it was possible to diagram the trend when this was replaced with the recovery of 100% activity level (Fig. 7b)).

[0308] Example 7: Regression analysis of the activity profile of a hot start polymerase with Ct-based units.

[0309] When calculating enzyme units in actual diagnostic reagent products, it is important to produce results with identical Ct values ​​for the same target to maintain consistent quality between previously used and newly produced enzymes. Typically, Ct values ​​are assigned differently depending on the characteristics of the probe. Since probes such as TaqMan are significantly affected by external hydrolysis performance in addition to the polymerization activity of the enzyme, an additional correction factor is required to equate the Ct-based unit value with the unit value of the enzyme analyzed according to Examples 1 and 4.

[0310] For 22 samples of TaqDNA polymerase produced differently, the unit values ​​of the enzymes analyzed by the methods of Examples 1 and 4 were inserted into the x-axis and y-axis, respectively, and regression analysis was performed to determine which factors were related to the actual Ct-based unit values ​​(Fig. 8).

[0311] Unit evaluation based on Ct value is Allplex of Seegene Co., Ltd. TM It was tested with CT / NG / MG / TV assay products. The PCR protocol of the PCR reaction was a heat treatment at 95℃ for 15 minutes, followed by 45 cycles of 95℃ for 3 seconds, 60℃ for 10 seconds, and 72℃ for 10 seconds, through which the Ct value was calculated. Based on the previously used enzyme (Pre-lot) with a predetermined unit, the concentration of the template DNA was serially diluted to calculate the Ct value, and the experiment was repeated while changing the random dilution concentration of the polymerase until the same Ct value as the Ct value calculated from the Pre-lot was obtained with the newly produced DNA polymerase. Through this, the enzyme dilution ratio that produces the same Ct value was determined, and through this, the unit value of the enzyme stock solution was set to be the same as the unit value of the Pre-lot.

[0312] That is, the Ct-based unit value measurement was performed by assuming relatively the unit value of the polymerase that can produce the same Ct value within the product as the Ct value of the reference polymerase in a method of measuring the Ct of a diagnostic reagent product through exonuclease of the polymerase, such as a TaqMan probe or similar probe method.

[0313] The regression analysis results showed that the 15-minute value in the hot-start profile (activity profile of hot-start nucleic acid polymerase) was significantly correlated with the Ct value for hot-start polymerases that performed the hot-start reaction under the same conditions, and the coefficient of determination was 0.9907, and the P-value was in the range of 10^-23, confirming that there was a statistically significant correlation. Therefore, it was found that the activity unit of the enzyme analyzed by the hot-start profile can be significantly used when evaluating the product performance based on Ct.

[0314] Meanwhile, the regression analysis performed on the results of the 0-minute, 30-minute, and 60-minute values ​​in the hot start profile and the unit values ​​obtained through Ct confirmed that the correlation was low.

[0315] Example 8: Method for blending two or more polymerases to mimic the activity profile of a hot start polymerase to be produced (target)

[0316] The values ​​of 0, 15, 30, and 60 minutes of the hot start profile (activity profile of hot start polymerase) can be interpreted by substituting them for stability below the target temperature of hot start, correlation with Ct-based unit measurement, stability over the PCR reaction cycle, and long-term storage stability, respectively, and considering these, the hot start profile of the intended hot start polymerase could be designed.

[0317] However, as shown in the case of TaqDNA polymerase klentaq, each enzyme experiences changes in its hot start profile depending on its type, changes in the production process, or changes in the hot start reaction method, so there may be cases where it is impossible to produce a polymerase with the intended hot start profile. For example, when trying to produce a polymerase with the hot start profile analyzed by AmpliTaq Gold, if you want to produce a hot start profile that maintains the activity level at about half of the activity level at 15 minutes compared to the activity level at 30 minutes, you can achieve this by changing various experimental conditions, but an easier method is to blend two or more polymerases with different hot start profiles.

[0318] If there are n polymerases to be blended, a square matrix can be constructed by calculating n hot start profiles obtained through heat treatment at 95℃ for 0 to 60 minutes, and through this, the desired hot start profile can be created regardless of the number of polymerases (Fig. 11).

[0319] For example, if there are two enzymes in which the hot start profile of the analyzed enzyme 1 was 7.4 U / ug, 10.4 U / ug, and 4.1 U / ug at 15 minutes, 30 minutes, and 60 minutes, respectively, and the hot start profile of the analyzed enzyme 2 was 22.1 U / ug, 69.4 U / ug, and 88.6 U / ug at 15 minutes, 30 minutes, and 60 minutes, respectively, then the concentration of each enzyme can be obtained by solving the equation that linearly combines them (Fig. 12).

[0320] Assuming that two enzymes are blended, the target hot start polymerase was calculated as AmpliTaq Gold. When measuring the unit per reaction based on 2 ul of AmpliTaq Gold used in the PCR reaction, the activity at 0 min is analyzed as 0.4 U / rxn, at 15 min as 10.8 U / rxn, at 30 min as 20.0 U / rxn, and at 60 min as 19.43 U / rxn. In order to simulate this performance, the linear algebraic equation of a) in Fig. 12 was used for calculation, and the usage amounts (ug) of enzyme 1 and enzyme 2 with different hot start profiles could be derived.

[0321] Therefore, by mixing 1.08 ug of enzyme 1 and 0.13 ug of enzyme 2 per reaction, the desired hot start profile (10.8 U / rxn for 15 minutes of activity, 20.0 U / rxn for 30 minutes of activity) could be reproduced (c in Fig. 12).

[0322] When the enzyme was manufactured by blending in the calculated amount and the hot start profile was analyzed again, it was confirmed that the hot start profile was analyzed with a value almost similar to the expected level, as shown in Fig. 13.

[0323] While specific aspects of the present invention have been described in detail above, it should be apparent to those skilled in the art that these specific descriptions are merely preferred implementation examples and are not intended to limit the scope of the present invention. Therefore, the substantial scope of the present invention is defined by the appended claims and their equivalents.

Claims

1. A method for evaluating the performance of a hot start nucleic acid polymerase comprising the following steps: (a) obtaining a product comprising a hot start nucleic acid polymerase; (b) a step of fractionating a product containing the hot start nucleic acid polymerase and performing heat activation for each of the fractionated products for different times; (c) a step of measuring the activity of each hot start nucleic acid polymerase included in each of the heat-activated products for different times; the activity is measured from the intensity of a signal measured by adding each of the heat-activated products for different times to a composition for activity assay of nucleic acid polymerase and performing a nucleic acid polymerization reaction, wherein the nucleic acid polymerase activity assay composition includes a primer, and the primer is extended by the hot start nucleic acid polymerase included in each of the heat-activated products under polymerization conditions to form an extension duplex, and a detectable signal is provided in a manner dependent on the extension duplex, and (d) obtaining the activity of the hot start nucleic acid polymerase for the different thermal activation times as an activity profile of the hot start nucleic acid polymerase; the activity profile of the hot start nucleic acid polymerase indicates the performance of the hot start nucleic acid polymerase.

2. A method for obtaining a QC-capable hot start nucleic acid polymerase comprising the following steps: (a) obtaining a product comprising a hot start nucleic acid polymerase; (b) a step of fractionating a product containing the hot start nucleic acid polymerase and performing heat activation for each of the fractionated products for different times; (c) a step of measuring the activity of each hot start nucleic acid polymerase included in each of the heat-activated products for different times; the activity is measured from the intensity of a signal measured by adding each of the heat-activated products for different times to a composition for activity assay of nucleic acid polymerase and performing a nucleic acid polymerization reaction, wherein the nucleic acid polymerase activity assay composition includes a primer, and the primer is extended by the hot start nucleic acid polymerase included in each of the heat-activated products under polymerization conditions to form an extension duplex, and a detectable signal is provided in a manner dependent on the extension duplex; (d) obtaining the activity of the hot start nucleic acid polymerase for the different thermal activation times as an activity profile of the hot start nucleic acid polymerase; the activity profile of the hot start nucleic acid polymerase represents the performance of the hot start nucleic acid polymerase, and (e) a step of providing a hot start nucleic acid polymerase having the above activity profile.

3. A method for providing a blended hot start nucleic acid polymerase that mimics the performance of a target hot start nucleic acid polymerase, comprising the following steps: (a) obtaining i) a product comprising a target hot start nucleic acid polymerase and ii) products each comprising at least two hot start nucleic acid polymerases to be blended; (b) i) collecting a product containing the target hot start nucleic acid polymerase, and performing heat activation for different times for each of the collected products, and ii) collecting each of the products containing each of the at least two hot start nucleic acid polymerases to be blended, and performing heat activation for different times for each of the collected products; wherein the heat activation time of i) and the heat activation time of ii) are the same, (c) a step of measuring the activity of each target hot start nucleic acid polymerase included in each of the products heat-activated for different times in i) of the step (b), and a step of measuring the activity of each of at least two hot start nucleic acid polymerases to be blended included in each of the products heat-activated for different times in ii) of the step (b); wherein the activity is measured from the intensity of a signal measured by adding each of the products heat-activated for different times to a composition for activity assay of nucleic acid polymerase and performing a nucleic acid polymerization reaction, wherein the composition for activity assay of nucleic acid polymerase includes a primer, and the primer is extended by the hot start nucleic acid polymerase included in each of the heat-activated products under polymerization conditions to form an extension duplex, and a detectable signal is provided in a manner dependent on the extension duplex, (d) obtaining the activity of the target hot start nucleic acid polymerase for different thermal activation times in i) of the step (b) as an activity profile of the target hot start nucleic acid polymerase, and obtaining the activity of the at least two hot start nucleic acid polymerases to be blended for different thermal activation times in ii) of the step (b) as an activity profile of the at least two hot start nucleic acid polymerases to be blended; the activity profile of the target hot start nucleic acid polymerase represents the performance of the target hot start nucleic acid polymerase, and each of the activity profiles of the at least two hot start nucleic acid polymerases to be blended represents the performance of each of the at least two hot start nucleic acid polymerases to be blended, (e) providing an amount of each of the at least two hot start nucleic acid polymerases to be blended using the performance of the target hot start nucleic acid polymerase and the performance of at least two hot start nucleic acid polymerases to be blended to mimic the performance of the target hot start nucleic acid polymerase; and (f) providing at least two hot start nucleic acid polymerases blended in the amounts provided in step (e) as a blended hot start nucleic acid polymerase that mimics the performance of the target hot start nucleic acid polymerase.

4. A method according to any one of claims 1 to 3, wherein the hot start nucleic acid polymerase is a chemically modified hot start nucleic acid polymerase, an antibody-based hot start nucleic acid polymerase, or an aptamer-based hot start nucleic acid polymerase.

5. A method according to any one of claims 1 to 3, characterized in that the nucleic acid polymerase is a DNA polymerase.

6. A method according to claim 5, characterized in that the DNA polymerase is Taq DNA polymerase, KlenTaq DNA polymerase, Klenow fragment of E. coli DNA polymerase I, DNA polymerase I, T4 DNA polymerase, Bst DNA polymerase, Bsu DNA polymerase, MMLV reverse transcriptase, AMV reverse transcriptase, or HIV reverse transcriptase.

7. In any one of paragraphs 1 to 3, the nucleic acid polymerase is prepared by a method comprising the following steps: (a-1) a step of culturing a host cell containing a nucleic acid polymerase expression construct; and (a-2) A step of purifying a nucleic acid polymerase by applying the result of the above step (a-1) to column chromatography.

8. A method according to any one of claims 1 to 3, wherein the hot start nucleic acid polymerase of step (a) is a nucleic acid polymerase with inhibited polymerization activity.

9. A method according to claim 8, characterized in that the hot start nucleic acid polymerase is a nucleic acid polymerase whose polymerization activity is suppressed to 1 U / ug or less.

10. A method according to any one of claims 1 to 3, characterized in that the method further comprises, after step (a), a step (a-3) of diluting the product(s) to a concentration of 0.02 mg / ml to 2 mg / ml.

11. A method according to any one of claims 1 to 3, characterized in that the thermal activation of step (b) is performed for at least two time intervals from 0 to 60 minutes for each of the fractionated products.

12. A method according to any one of claims 1 to 3, characterized in that the thermal activation of step (b) is performed at 90 to 100°C.

13. A method according to any one of claims 1 to 3, characterized in that the thermal activation of step (b) is performed at 90 to 100°C for 0 minutes, 15 minutes, 30 minutes, and 60 minutes for each of the fractionated products.

14. A method according to any one of claims 1 to 3, characterized in that the method further comprises, between steps (b) and (c), a step (b-1) of diluting each of the heat-activated products to a concentration of 6.25 ng / ul to 50 ng / ul.

15. A method according to any one of claims 1 to 3, wherein the activity of the hot start nucleic acid polymerase measured in step (c) is provided from a slope measured in a range in which the intensity of the signal increases linearly depending on the concentration of the hot start nucleic acid polymerase contained in the product.

16. A method according to any one of claims 1 to 3, wherein the activity of the hot start nucleic acid polymerase measured in step (c) is the activity per unit mass of the hot start nucleic acid polymerase or the activity per unit volume of the hot start nucleic acid polymerase.

17. A method according to any one of claims 1 to 3, characterized in that the nucleic acid polymerase activity assay composition additionally comprises (i) a template, (ii) a label, (iii) dNTP, and (iv) a buffer.

18. A method according to any one of claims 1 to 3, wherein the primer included in the activity assay composition of the nucleic acid polymerase comprises a first primer and a second primer as a dimer-forming primer pair having both a primer and a template role, wherein each of the first primer and the second primer comprises a 3'-dimer forming site, and the nucleotide sequence of the 3'-dimer forming site in the first primer is complementary to the nucleotide sequence of the 3'-dimer forming site in the second primer.

19. A method according to any one of claims 1 to 3, characterized in that in step (c), each of the products heat-activated for different times is added to the nucleic acid polymerase activity assay composition by adding each of the products heat-activated for different times to the nucleic acid polymerase activity assay composition according to a dilution factor.

20. A method according to any one of claims 1 to 3, characterized in that the nucleic acid polymerization reaction is carried out at 35°C to 75°C for 10 to 60 minutes.

21. A method according to claim 17, characterized in that the label is an intercalating label.

22. In the 21st paragraph, the intercalating label is SYBR™ Green I, PO-PRO™-1, BO-PRO™-1, SYTO™ 43, SYTO™ 44, SYTO™ 45, SYTOX™ Blue, POPO™-1, POPO™-3, BOBO™-1, BOBO™-3, LO-PRO™-1, JO-PRO™-1, YO-PRO™ 1, TO-PRO™1, SYTO™11, SYTO™13, SYTO™15, SYTO™16, SYTO™20, SYTO™23, TOTO™-3, YOYO™3, GelStar™, thiazole orange, PicoGreen ® , EvaGreen ® Or a method characterized in that it is selected from the group consisting of LAM Fluorescent Dye.

23. A method according to any one of claims 1 to 3, wherein the hot start nucleic acid polymerase is a hot start Taq DNA polymerase or a hot start KlenTaq DNA polymerase, and the activity of the hot start nucleic acid polymerase in step (c) is provided as an amount of the nucleic acid polymerase determined by substituting the intensity of the signal into the following general formula 1: General formula 1 1 unit (enzyme activity unit) = the amount of nucleic acid polymerase required to produce a signal intensity indicating polymerization by consuming 4.4±0.56 nmole of dNTP at 72°C for 30 minutes.

24. A method according to any one of claims 1 to 3, wherein the hot start nucleic acid polymerase is a hot start Taq DNA polymerase or a hot start KlenTaq DNA polymerase, and the activity of the hot start nucleic acid polymerase in step (c) is provided as a unit number of the nucleic acid polymerase determined by substituting the intensity of the signal into the following general formula 1: General formula 1 1 unit (enzyme activity unit) = the amount of nucleic acid polymerase required to produce a signal intensity indicating polymerization by consuming 4.4±0.56 nmole of dNTP at 72°C for 30 minutes.

25. A method according to claim 1 or 2, characterized in that the method further comprises the following steps after step (d): (d-1) A step of obtaining a product including a hot start nucleic acid polymerase obtained by a different obtaining process from the process of obtaining a product including a hot start nucleic acid polymerase of step (a); (d-2) a step of performing steps (b) to (d) using the product obtained in step (d-1) to obtain the activity of the hot start nucleic acid polymerase included in the product of step (d-1) for the different thermal activation times as an activity profile of the hot start nucleic acid polymerase included in the product of step (d-1); the activity profile of the hot start nucleic acid polymerase included in the product of step (d-1) indicates the performance of the hot start nucleic acid polymerase included in the product of step (d-1); and (d-3) A step of comparing the activity profile of the hot start nucleic acid polymerase included in the product of the above step (a) with the activity profile of the hot start nucleic acid polymerase included in the product in the obtaining process of the above step (d-1).

26. In the third paragraph, the step (e) is characterized in that the step is a step of providing the amount of each of the at least two hot start nucleic acid polymerases to be blended by substituting the performance of the target hot start nucleic acid polymerase and the performance of at least two hot start nucleic acid polymerases to be blended to simulate the performance of the target hot start nucleic acid polymerase into a linear algebraic equation.

27. In the 26th paragraph, the linear algebraic equation is characterized in that it is expressed by the following mathematical expression 1: Mathematical formula 1 [A] nxn [X] nx1 = [B] nx1 In the above mathematical formula, the matrix [A] represents the performance of at least two hot start nucleic acid polymerases to be blended, and n of the row elements representing the size of the matrix in the matrix [A] represents the order of the time of thermal activation, and n of the column elements in the matrix [A] represents the hot start nucleic acid polymerases to be blended, the matrix [X] represents the amount of at least two hot start nucleic acid polymerases to be blended, and n of the row elements representing the size of the matrix in the matrix [X] represents the hot start nucleic acid polymerases to be blended, and the matrix [B] represents the performance of the target hot start nucleic acid polymerase, and n of the row elements representing the size of the matrix in the matrix [B] represents the order of the time of thermal activation.

28. In the 27th paragraph, the method is characterized in that the mathematical formula 1 is expressed by the following mathematical formula 2: Mathematical formula 2 In the above mathematical formula, the matrix element a represents the activity of at least two hot start nucleic acid polymerases to be blended, the row element n in the matrix element a represents the order of the time of thermal activation, and the column element n in the matrix element a represents the hot start nucleic acid polymerase to be blended, the matrix element x represents the amount of at least two hot start nucleic acid polymerases to be blended, the row element n in the matrix element x represents the hot start nucleic acid polymerase to be blended, and the matrix element b represents the activity of the target hot start nucleic acid polymerase, and the row element n in the matrix element b represents the order of the time of thermal activation.

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