Method for determining absolute activity of Bst DNA polymerase

Abstract

The invention discloses a method for determining the absolute activity of Bst DNA polymerase, which comprises the following steps: by taking annular single-stranded DNA as a template, in a reaction system containing an amplification primer and dATP, initiating an extension replication reaction by using Bst DNA polymerase to be detected, and consuming dNTPs in the reaction system; the consumption of dATP is calculated according to a pre-constructed standard curve f1 representing the relationship between the concentration of dATP and the fluorescent value, the concentration of Bst DNA polymerase is calculated through a pre-constructed standard curve f2 representing the relationship between the concentration of Bst DNA polymerase and the consumption of dATP, and finally the absolute activity of the Bst DNA polymerase to be detected is calculated according to the absolute activity definition of Bst DNA polymerase. The method is free of radioactive contamination, and has the advantages of determination of the polymerization activity of the Bst DNA polymerase, absolute constant activity, simplicity and convenience in operation, rapidness, sensitivity, low cost and the like.

Description

Technical Field

[0001] The invention relates to the field of biotechnology, and in particular to a method for determining the absolute activity of Bst DNA polymerase. Background Art

[0002] Bst DNA polymerase is a DNA polymerase I cloned from Bacillus stearothermophilus. It has 5'-3' polymerase activity and strong strand displacement function, but lacks 3'-5' exonuclease activity. It catalyzes DNA replication at a temperature of 30°C. Since Bst DNA polymerase has strong strand displacement activity, it can catalyze DNA replication under constant temperature conditions and is an ideal choice for loop-mediated isothermal amplification (LAMP). The constant temperature, rapidity and high efficiency make Bst DNA polymerase the core enzyme of LAMP amplification technology and is widely used in point-of-care testing (POCT). In addition, BstDNA polymerase is also widely used in multiple strand displacement amplification (MDA), whole genome amplification (WGA), library construction and sequencing, etc.

[0003] Due to the above-mentioned important uses, it is of vital importance to accurately determine the activity of Bst DNA polymerase and ensure the stability of activity between batches. Traditional polymerase determination methods either rely on radioactive elements 3 The incorporation of H-labeled nucleotides, that is, in the reaction system and conditions of Bst DNA polymerase, synthesizes radioactively labeled nucleotide chains, and calculates the activity of the enzyme by measuring the amount of radioactive isotopes in the acid-insoluble product; or by incorporating fluorescent dyes, the enzyme activity is relatively calculated based on the amount of generated products. Among these methods, the former is prone to radioactive contamination and requires multiple steps such as precipitation and filtration, with a long cycle and poor popularity; the latter cannot measure the absolute activity of Bst DNA polymerase, that is, under specific temperature conditions, the consumption of a certain amount of dNTPs per unit time is 1 unit of enzyme activity. Summary of the invention

[0004] The technical problem to be solved by the present invention is to provide a method for measuring the absolute activity of BstDNA polymerase in view of the deficiencies in the above-mentioned prior art.

[0005] In order to solve the above technical problems, the technical solution adopted by the present invention is: a method for determining the absolute activity of Bst DNA polymerase, the method comprising: using circular single-stranded DNA as a template, in a reaction system containing an amplification primer and dATP, initiating an extension replication reaction with Bst DNA polymerase to consume dNTPs in the reaction system; then calculating the dATP consumption according to a pre-constructed standard curve f1 characterizing the relationship between the dATP concentration and the fluorescence value, then calculating the Bst DNA polymerase concentration according to a pre-constructed standard curve f2 characterizing the relationship between the Bst DNA polymerase concentration and the dATP consumption, and finally calculating the absolute activity of the Bst DNA polymerase according to the absolute activity definition of the Bst DNA polymerase;

[0006] The absolute activity of Bst DNA polymerase is defined as the amount of enzyme required to incorporate 25 nmol of dNTP into acid-insoluble matter at 65°C for 30 min, expressed in U / μL.

[0007] Preferably, the calculation formula of the absolute activity P of Bst DNA polymerase is:

[0008]

[0009] The unit of P is U / μL;

[0010] Wherein, C0 represents the initial concentration of dATP in the reaction system, in μM; C1 represents the final concentration of dATP in the reaction system after the reaction is completed, which is calculated by the standard curve f1, in μM; V d V is the volume of the reaction system, in μL; P is the amount of Bst DNA polymerase added, in μL; η T is the percentage of T in circular single-stranded DNA.

[0011] Preferably, the consumption of dATP is obtained by subtracting the remaining amount of dATP in the reaction system after the reaction is completed from the initial content of dATP in the reaction system, wherein the remaining amount of dATP is obtained by detecting the fluorescence value produced by luciferin catalyzed by firefly luciferase using the energy provided by dATP.

[0012] Preferably, the remaining amount of dATP is detected by the following method:

[0013] After the extension and replication reaction is completed, the reaction product is added with ATP detection reagent to detect the fluorescence value produced by luciferin catalyzed by firefly luciferase using the energy provided by dATP, and then the remaining amount of dATP is calculated based on the pre-constructed standard curve f1 that characterizes the relationship between the concentration of dATP and the fluorescence value.

[0014] Preferably, the construction method of the standard curve f1 is:

[0015] A series of standard reaction systems with different dNTPs concentrations and without Bst DNA polymerase were prepared, and extension reactions were first carried out under specific reaction temperature and the same reaction time. After the reaction was completed, equal amounts of ATP detection reagents were added to the product systems, and the fluorescence values ​​of each standard reaction system were detected under the same conditions. The standard curve f1 was obtained by fitting, with the dNTPs concentration as the horizontal axis and the fluorescence value as the vertical axis.

[0016] Preferably, the construction method of the standard curve f2 is:

[0017] A series of standard reaction systems with different concentrations of Bst DNA polymerase were prepared, and extension reactions were first carried out under specific reaction temperatures and the same time conditions. After the reaction was completed, equal amounts of ATP detection reagents were added to the product systems, and the fluorescence values ​​of each standard reaction system were detected under the same conditions. The consumption of dATP was calculated according to the standard curve f1, and the standard curve f2 was obtained by fitting with the concentrations of Bst DNA polymerase of different concentrations as the horizontal axis and the consumption of dATP as the vertical axis.

[0018] Preferably, the circular single-stranded DNA is M13 phage single-stranded DNA.

[0019] Preferably, the nucleic acid sequence of the amplification primer is 5'-TTTGCGGGAGAAGCCTTTATTTCAACGCA-3'.

[0020] Preferably, the temperature and reaction time of the extension and replication reaction of the Bst DNA polymerase to be tested correspond to the same temperature and reaction time of the extension and replication reaction in the process of constructing the standard curve f1.

[0021] Preferably, the reaction time of the Bst DNA polymerase extension replication reaction is 10 min to 60 min, and the reaction temperature is 65°C.

[0022] Preferably, within a certain concentration range, the repeatability precision and intermediate precision test results of the method of the present invention can both achieve RSD < 10%.

[0023] The beneficial effects of the present invention are:

[0024] The present invention utilizes the polymerization activity of Bst DNA polymerase, and quantifies the amount of dATP consumed in the extension and replication process by detecting the fluorescence value produced by luciferin catalyzed by firefly luciferase using the energy provided by the remaining dATP in the product after extension and replication, and then can quickly determine the absolute polymerization activity of Bst DNA polymerase. This method has no radioactive pollution, has the advantages of determining the polymerization activity of Bst DNA polymerase, absolute determination, simple operation, rapidity and sensitivity, low cost, etc., and can be used for high-throughput, automated polymerase activity screening. BRIEF DESCRIPTION OF THE DRAWINGS

[0025] Figure 1 It is a schematic diagram of the elongation and replication reaction process in the present invention;

[0026] Figure 2 The fluorescence value processing results of dATP, dGTP, dCTP, dTTP and dNTPs in Example 1 of the present invention;

[0027] Figure 3 The results of the effects of various components on the fluorescence value in Example 2 of the present invention;

[0028] Figure 4 This is a graph of the extension time in Example 3 of the present invention;

[0029] Figure 5 It is a detection time curve diagram in Example 4 of the present invention;

[0030] Figure 6 The standard curve f1 constructed in Example 5 of the present invention;

[0031] Figure 7 The standard curve f2 constructed in Example 6 of the present invention;

[0032] Figure 8 This is a graph showing the consumption of dATP in the precision test in Example 8 of the present invention. DETAILED DESCRIPTION

[0033] The present invention is further described in detail below in conjunction with embodiments so that those skilled in the art can implement the invention with reference to the description.

[0034] It should be understood that terms such as “having”, “including” and “comprising” used herein do not exclude the existence or addition of one or more other elements or combinations thereof.

[0035] The test methods used in the following examples are conventional methods unless otherwise specified. The materials and reagents used in the following examples are all commercially available unless otherwise specified. In the following examples, if no specific conditions are specified, the experiments were carried out under conventional conditions or conditions recommended by the manufacturer. The reagents or instruments used, if the manufacturer is not specified, are all conventional products that can be purchased commercially.

[0036] The invention provides a method for determining the absolute activity of Bst DNA polymerase. The method comprises the following steps: using circular single-stranded DNA as a template, in a reaction system containing an amplification primer and dATP, using a Bst DNA polymerase to be detected to initiate an extension replication reaction, and consuming dNTPs in the reaction system; then calculating the consumption of dATP in dNTPs according to a pre-constructed standard curve f1 that characterizes the relationship between the concentration of dATP and the fluorescence value, and then calculating the concentration of Bst DNA polymerase according to a pre-constructed standard curve f2 that characterizes the relationship between the concentration of Bst DNA polymerase and the consumption of dATP; and finally calculating the absolute activity of Bst DNA polymerase (hereinafter also referred to as polymerase activity) according to the absolute activity definition of Bst DNA polymerase.

[0037] Among them, the calculation formula of the absolute activity P of Bst DNA polymerase is:

[0038]

[0039] The unit of P is U / μL;

[0040] Wherein, C0 represents the initial concentration of dATP in the reaction system, in μM; C1 represents the final concentration of dATP in the reaction system after the reaction is completed, which is calculated by the standard curve f1, in μM; V d V is the volume of the reaction system, in μL; P is the amount of Bst DNA polymerase added, in μL; η T is the percentage of T in circular single-stranded DNA.

[0041] The absolute activity of Bst DNA polymerase is defined as the amount of enzyme required to incorporate 25 nmol of dNTP into acid-insoluble matter at 65°C for 30 min, expressed in U / μL.

[0042] Reference Figure 1In a preferred embodiment of the present invention, M13 bacteriophage single-stranded DNA (M13 bacteriophage single-stranded loop) is used as a template for extension and replication, and an amplification primer (M13 universal primer) is designed to trigger extension and replication under the action of Bst DNA polymerase, consuming a large amount of dNTPs in the reaction system; after adding the ATP detection reagent, the fluorescence signal of dATP in the remaining dNTP can be measured, and then the consumption of dATP can be calculated according to the pre-constructed standard curve f1 that characterizes the relationship between the concentration and the fluorescence value of dATP, and then the absolute activity of the Bst DNA polymerase to be tested can be calculated according to the absolute activity definition of Bst DNA polymerase. However, it should be noted that the present invention is not limited to using M13 single-stranded DNA as a template for extension reaction. In theory, any other circular, linear, single-stranded, and double-stranded DNA can be used in the present invention. Accordingly, the amplification primer is a complementary sequence to a specific sequence on the template DNA.

[0043] In a preferred embodiment, the method for constructing the standard curve f1 is:

[0044] A series of standard reaction systems with different dNTPs concentrations and without Bst DNA polymerase were prepared, and extension reactions were first carried out under specific reaction temperature and the same reaction time. After the reaction was completed, equal amounts of ATP detection reagents were added to the product systems, and the fluorescence values ​​of each standard reaction system were detected under the same conditions. The standard curve f1 was obtained by fitting, with the dNTPs concentration as the horizontal axis and the fluorescence value as the vertical axis.

[0045] In a preferred embodiment, the consumption of dATP is obtained by subtracting the remaining amount of dATP in the reaction system after the reaction is completed from the initial content of dATP in the reaction system, wherein the remaining amount of dATP is obtained by detecting the fluorescence value produced by luciferin catalyzed by firefly luciferase using the energy provided by dATP. The remaining amount of dATP is detected by the following method:

[0046] After the extension and replication reaction is completed, the reaction product is added with ATP detection reagent to detect the fluorescence value produced by luciferin catalyzed by firefly luciferase using the energy provided by dATP, and then the remaining amount of dATP is calculated based on the pre-constructed standard curve f1 that characterizes the relationship between the concentration of dATP and the fluorescence value.

[0047] In a preferred embodiment, the method for constructing the standard curve f2 is:

[0048] A series of standard reaction systems with different concentrations of Bst DNA polymerase were prepared, and extension reactions were first carried out under specific reaction temperatures and the same time conditions. After the reaction was completed, equal amounts of ATP detection reagents were added to the product systems, and the fluorescence values ​​of each standard reaction system were detected under the same conditions. The consumption of dATP was calculated according to the standard curve f1, and the standard curve f2 was obtained by fitting with the concentrations of Bst DNA polymerase of different concentrations as the horizontal axis and the consumption of dATP as the vertical axis.

[0049] In a preferred embodiment, the nucleic acid sequence of the amplification primer is 5'-TTTGCGGGAGAAGCCTTTATTTCAACGCA-3'.

[0050] In a preferred embodiment of the present invention, the consumption of dATP is detected using the Biyuntian ATP detection kit, but it should be noted that, in theory, any kit capable of detecting the consumption of dATP, dGTP, dCTP or dTTP can be used in the present invention. Therefore, based on the spirit of the present invention, the ATP detection reagent in the present invention can be a kit for detecting dATP or a kit for detecting the consumption of dGTP, dCTP or dTTP. Detailed examples are provided below to further illustrate the present invention.

[0051] Example 1

[0052] Luciferase response to dATP assay

[0053] 1. Preparation of dATP and other component solutions

[0054] Prepare 700 μM dATP, dGTP, dCTP, dTTP and dNTPs respectively and place them on ice for testing;

[0055] 2. Prepare ATP detection working solution. The specific system is shown in Table 1 (can be adjusted according to actual conditions). Add 100 μL of the prepared ATP detection working solution to a 96-well ELISA plate. After standing at room temperature for 3-5 minutes, add the sample to be tested to the 96-well ELISA plate, add 1 μL to each well, mix gently, and place in a chemiluminescence instrument. Select the chemiluminescence mode to detect the luminescence value.

[0056] Table 1 ATP reaction working solution reference system

[0057] Components Dosage ATP detection reagent 20μL ATP Detection Reagent Dilution 80μL

[0058] 3. Luminous value processing

[0059] Depend on Figure 2It can be seen that the fluorescence values ​​of dATP and dNTPs of the same concentration are high and basically consistent, while dGTP, dCTP, and dTTP have almost no fluorescence values, indicating that luciferase mainly responds to dATP and has no response to dGTP, dCTP, and dTTP.

[0060] Example 2

[0061] Interference test of other components in the extension reaction system on the luminescence value

[0062] 1. The materials required for the reaction system are as follows:

[0063] Template: M13 single-stranded DNA (ssDNA);

[0064] Amplification primer M13-R: 5'-TTTGCGGGAGAAGCCTTTATTTCAACGCA-3' (SEQ ID NO: 1).

[0065] 2. Reaction operation steps

[0066] ① Prepare primer template reaction mixture, the reaction system is shown in Table 2 (can be adjusted according to actual conditions). Place the prepared reaction mixture in a PCR instrument at 65°C for 30 min, 80°C for 20 min, and 4°C for 5 min for reaction. The extension reaction time can be adjusted to 10 min to 1 h according to actual conditions. Then use the ATP detection kit to determine the consumption of dATP in the reaction solution. The specific steps are the same as those shown in step 1.

[0067] Table 2 Extension reaction mixture reference system

[0068]

[0069] 3. Drawing of dATP standard curve

[0070] Depend on Figure 3 It can be seen that the luminescence values ​​of the four groups of different component systems are basically consistent when the dATP concentration is the same, and the linear correlation coefficient of the dNTPs standard curve of each group is good, R 2 All of them were greater than 0.99, which showed that 10×Bst Reaction Buffer, template M13 ssDNA, BSA and amplification primer M13-R had no effect on the luminescence value.

[0071] Example 3

[0072] Determination of extension reaction time

[0073] 1. The materials required for the reaction system are as follows:

[0074] Template: M13 single-stranded DNA (ssDNA);

[0075] Amplification primer M13-R: 5'-TTTGCGGGAGAAGCCTTTATTTCAACGCA-3' (SEQ ID NO: 1);

[0076] Polymerase: Bst DNA polymerase (NEB).

[0077] 2. Preparation of reaction system

[0078] ① Prepare 8 portions of primer template extension reaction mixture. The reaction system and reaction procedure are shown in Table 3 (can be adjusted according to actual conditions).

[0079] Table 3 Extension reaction mixture reference system

[0080] Components Dosage M13 ssDNA (500ng / μL) 4μL M13-R(10μM) 0.8μL 10×Bst Reaction Buffer 2μL BSA (10 mg / mL) 1μL dNTPs (2.5 mM) 5.6μL Bst DNA polymerase (0.02U / μL) 1μL <![CDATA[ddH2O]]> Up to 20μL

[0081] 3. Extension reaction

[0082] The reaction program of the gene thermal cycler was set to 65°C, and the extension time was tested at 10min, 20min, 30min, 40min, 50min, 60min, 80min, and 100min, respectively. After the reaction was completed, the reaction was terminated at 80°C and 20min. Then, the ATP detection kit was used to determine the consumption of dATP in the reaction solution. The specific steps are the same as those shown in step 1.

[0083] 4. Standard curve drawing

[0084] Depend on Figure 4 It can be seen that as the reaction time increases, the reaction rate gradually decreases and tends to be flat ( Figure 4 A) When the extension time is within 60 min, there is a good linear relationship between the consumption of dATP by Bst DNA polymerase, and the correlation coefficient R 2 Reach 0.99 or above ( Figure 4 B). To ensure that the consumption of Bst DNA polymerase is within the linear range and to reasonably shorten the experimental time, the subsequent extension time used in this experiment is 30 minutes.

[0085] Example 4

[0086] Determination of chemiluminescence detection time

[0087] 1. The materials required for the reaction system are as follows:

[0088] Template: M13 single-stranded DNA (ssDNA);

[0089] Amplification primer M13-R: 5'-TTTGCGGGAGAAGCCTTTATTTCAACGCA-3' (SEQ ID NO: 1).

[0090] 2. Preparation of reaction system

[0091] ① Prepare primer template extension reaction mixture, the reaction system is as shown in Table 4 (can be adjusted according to actual situation).

[0092] Table 4. Reference system of reaction mixture

[0093] Components Dosage 10×Bst Reaction Buffer 2μL M13ssDNA (500ng / μL) 4μL M13-R(10μM) 0.8μL

[0094] ② Add different volumes of dNTPs (2.5 mM) to each tube of the above reaction solution, and make up to 20 μL with water. The dNTPs concentration gradient setting refers to Table 5 (it can be adjusted according to the actual situation).

[0095] Table 5 Reference dNTPs dosage gradient

[0096] Tube No. 1 2 3 4 5 Amount of dNTPs (μL) 5.6 4 2.4 0.8 0

[0097] 3. Chemiluminescence instrument to detect luminescence value

[0098] Set the chemiluminescence instrument detection time to 60 minutes, and detect the luminescence values ​​at different time periods. Determine the optimal chemiluminescence instrument detection time by whether the luminescence values ​​at different time periods are stable.

[0099] 4. Luminous value processing

[0100] Depend on Figure 5 It can be seen that the luminescence value gradually increases with time, reaches stability after 15 minutes and remains stable within 60 minutes. The detection time of the chemiluminescence instrument in subsequent experiments can be determined as 25 minutes.

[0101] Example 5

[0102] Construct a standard curve f1 that characterizes the relationship between dATP concentration and fluorescence value

[0103] 1. The materials required for the reaction mixture are as follows:

[0104] Template: M13 single-stranded DNA (ssDNA);

[0105] Amplification primer M13-R: 5'-TTTGCGGGAGAAGCCTTTATTTCAACGCA-3' (SEQ ID NO: 1).

[0106] 2. Preparation of reaction system

[0107] ① Prepare the extension reaction mixture. The specific reaction system is shown in Table 4 (it can be adjusted according to the actual situation).

[0108] ②Add different volumes of dNTPs (2.5mM) to each tube of extension reaction mixture, and make up to 20μL with water. The dNTPs concentration gradient setting refers to Table 5 (can be adjusted according to actual conditions).

[0109] 3. Extension reaction

[0110] The reaction mixtures of the above-mentioned concentration gradients were placed in a PCR instrument and incubated at 65°C for 30 min, followed by 80°C for 20 min to terminate the reaction.

[0111] 4. Determination of dATP Residual Amount

[0112] The remaining amount of dATP in the reaction solution was determined using an ATP detection kit, and then the dATP consumption was obtained by subtracting the remaining amount from the initial amount of dATP. The specific detection system is shown in Table 6 below (it can be adjusted in the same way according to actual conditions).

[0113] Table 6 dATP consumption detection system

[0114] Components Dosage ATP detection reagent 20μL ATP Detection Reagent Dilution 80μL Extension reaction mixture 1μL

[0115] The above detection system was added into a 96-well microplate, placed into an ELISA reader, and the chemiluminescence mode was selected to detect the luminescence value.

[0116] 5. Standard curve drawing

[0117] With dATP concentration (i.e., residual amount, μM) as the horizontal axis and luminescence value (RLU) as the vertical axis, a standard curve f1 is drawn, as shown in Figure 6 , it can be seen that the obtained data points can fit a straight line (R 2 >0.995), which proves that the standard curve f1 made by this method is credible. The obtained standard curve f1 is specifically Y=6.290X+137.5.

[0118] Example 6

[0119] Construction of a standard curve f2 characterizing the relationship between Bst DNA polymerase concentration and dATP consumption

[0120] 1. The materials required for the reaction mixture are as follows:

[0121] Template: M13 single-stranded DNA (dsDNA);

[0122] Amplification primer M13-R: 5'-TTTGCGGGAGAAGCCTTTATTTCAACGCA-3' (SEQ ID NO: 1);

[0123] Polymerase: Bst DNA polymerase (NEB).

[0124] 2. Reaction operation steps:

[0125] ① Prepare the extension reaction mixture as shown in Table 7 (it can be adjusted according to the actual situation).

[0126] Table 7 Extension reaction mixture

[0127] Components Dosage M13 ssDNA (500ng / μL) 4μL M13-R(10μM) 0.8μL 10×Bst Reaction Buffer 2μL dNTPs (2.5 mM) 5.6μL BSA (10 mg / mL) 1μL Bst DNA polymerase 1μL <![CDATA[ddH2O]]> Up to 20μL

[0128] ② Dilute Bst DNA polymerase to different enzyme concentration gradients, and then add the above reaction solution. The enzyme concentration gradient setting refers to Table 8 below (it can be adjusted in the same way according to actual conditions).

[0129] Table 8 Bst DNA polymerase concentration gradient

[0130] Tube No. 1 2 3 4 5 6 Enzyme concentration (mg / mL) 0.02 0.01 0.008 0.005 0.002 0.001

[0131] 3. Extension reaction

[0132] The reaction mixtures of the above-mentioned concentration gradients were placed in a PCR instrument, and the extension reaction was carried out at 65°C for 30 minutes, and then the reaction was terminated at 80°C for 20 minutes.

[0133] 4. Determination of dATP Residual Amount

[0134] The residual amount of dATP in the reaction solution was determined using an ATP detection kit. The specific detection mixture is shown in Table 6 in Example 5 (it can be adjusted in the same way according to actual conditions).

[0135] The above detection mixture was added into a 96-well microplate, placed into an ELISA reader, and the chemiluminescence mode was selected to detect the luminescence value.

[0136] 5. Standard curve drawing

[0137] First, the residual amount of dATP was calculated according to the standard curve f1 constructed in Example 5, and then the dATP consumption of Bst DNA polymerase at different enzyme concentration gradients was obtained by subtracting the residual amount from the initial amount of dATP added. Then, the standard curve f2 was obtained by fitting with the concentration of Bst DNA polymerase as the horizontal axis and the consumption of dATP as the vertical axis, as shown in FIG. Figure 7 , it can be seen that the obtained data points can fit a straight line (R 2 >0.995), proving that the standard curve f2 obtained by this method is credible, and the obtained standard curve f2 is specifically Y=9618X+11.48, and the linear range of Bst DNA polymerase concentration is 0.001-0.02U / μL. This Example 6 is carried out simultaneously with Example 5 to reduce the error.

[0138] Example 7

[0139] Determination of the absolute activity of the Bst DNA polymerase to be tested

[0140] 1. The materials required for the reaction system are as follows:

[0141] Template: M13 single-stranded DNA (ssDNA);

[0142] Amplification primer M13-R: 5'-TTTGCGGGAGAAGCCTTTATTTCAACGCA-3' (SEQ ID NO: 1);

[0143] Polymerase: Bst DNA polymerase (GenEasy), which is different from the polymerase used to draw the standard curve f2 in Example 6.

[0144] 2. Extension reaction steps:

[0145] This experiment is actually carried out simultaneously with the standard curve experiment. The reaction system is as shown in Table 9 (can be adjusted in the same way according to actual conditions). The operation process is the same as that in drawing the standard curve f2.

[0146] Table 9 Extension reaction mixture

[0147] Components Dosage M13 ssDNA (500ng / μL) 4μL M13-R(10μM) 0.8μL 10×Bst Reaction Buffer 2μL dNTPs(25mM) 0.56μL BSA (10 mg / mL) 1μL Bst DNA polymerase (0.2 μg / mL) 1μL <![CDATA[ddH2O]]> Up to 20μL

[0148] 3. Determination of dATP Residual Amount

[0149] The remaining amount of dATP in the reaction solution was determined using an ATP detection kit. The specific reaction system was the same as that in Table 8 of Example 6.

[0150] The above detection mixture was added into a 96-well microplate, placed into an ELISA reader, and the chemiluminescence mode was selected to detect the luminescence value, and the measured luminescence value was 3825.83.

[0151] 4. Calculate the absolute activity of the Bst DNA polymerase to be tested

[0152] Substitute the measured luminescence value of the Bst DNA polymerase to be tested into the standard curve f1 (Y=6.290X+137.5) to calculate the remaining amount of dATP, then subtract the remaining amount from the initial amount of dATP to calculate the consumption of dATP: Y=113.62 (μM). The percentage of the binding chain T in the template M13 single-stranded DNA is 33.37%, and the consumption of dNTPs of the Bst DNA polymerase to be tested is calculated to be 6.81nmol. According to the definition of enzyme activity (the amount of enzyme required to make 25nmol of dNTPs incorporate into acid-insoluble matter in 30min at 65°C), the actual absolute specific activity of GenEasy Bst DNA polymerase under this method is 1361.96U / mg. At the same time, according to the results of Example 6, it is calculated that the absolute enzyme activity of NEB Bst DNA polymerase under this method is 184.68U / μL.

[0153] Under the above definition of the absolute enzyme activity of Bst DNA polymerase, according to the results of Example 6, it can be calculated that the absolute activity range of Bst DNA polymerase suitable for determination in the present invention is 0.023-0.46 U / μL.

[0154] Example 8

[0155] Precision testing

[0156] 1. The materials required for the extension reaction system are as follows:

[0157] Template: M13 single-stranded DNA (ssDNA);

[0158] Amplification primer: M13-R: 5'-TTTGCGGGAGAAGCCTTTATTTCAACGCA-3' (SEQ ID NO: 1);

[0159] Polymerase: Bst DNA polymerase (GenEasy).

[0160] 2. Extension reaction steps:

[0161] ① Except for the different amount of Bst DNA polymerase, the reaction system and operation process of this experiment are the same as those in Example 6 for drawing the standard curve f2;

[0162] ② Dilute Bst DNA polymerase to different enzyme concentration gradients, and then add the above reaction solution. The enzyme concentration gradient setting refers to Table 10 (it can be adjusted according to the actual situation).

[0163] Table 10 Reference Bst DNA polymerase concentration gradient

[0164] Tube No. 1 2 3 Enzyme concentration (μg / mL) 0.4 0.2 0.1

[0165] ③ Repeat precision: Under the same experimental conditions, the same experimenter independently conducted three experiments, and the activity of three concentrations of Bst DNA polymerase was detected. The RSD of the sample detection value was calculated, and the RSD should be less than 10%.

[0166] Intermediate precision: Under the same experimental conditions, three experiments were conducted independently by different experimenters. The activity of BstDNA polymerase was detected and the RSD of the relative deviation of the sample detection value was calculated. The RSD should be less than 10%.

[0167] 3. Calculation of RSD value of precision test

[0168] Depend on Figure 8 It can be seen that the relative deviations of sample detection values ​​in repeated precision tests were 1.19%, 0.76% and 2.65% respectively, and RSD<10%; the relative deviations of sample detection values ​​in intermediate precision tests were 1.96%, 3.74% and 4.74% respectively, and RSD<10%, indicating that this method has good repeated precision and intermediate precision.

[0169] Example 9

[0170] The detection limit of this method was tested

[0171] 1. The materials required for the reaction system are as follows:

[0172] Template: M13 single-stranded DNA (dsDNA);

[0173] Amplification primer: M13-R: 5'-TTTGCGGGAGAAGCCTTTATTTCAACGCA-3' (SEQ ID NO: 1);

[0174] Polymerase: Bst DNA polymerase (GenEasy).

[0175] 2. Extension reaction steps:

[0176] ① The reaction system and operation process of this experiment are the same as those of Example 8.

[0177] ② Dilute Bst DNA polymerase to different enzyme concentration gradients, and then add the above reaction solution. The enzyme concentration gradient setting refers to Table 11 (it can be adjusted according to the actual situation).

[0178] Table 11 Reference Bst DNA polymerase concentration gradient

[0179] Tube No. 1 2 3 4 Enzyme concentration (μg / mL) 0.08 0.06 0.04 0.02

[0180] 3. Calculation of RSD value of dATP consumption

[0181] As shown in Table 12, when Bst DNA polymerase was diluted to 0.02-0.08 μg / mL, the RSD was less than 10%, and when diluted to 0.02 μg / mL, the RSD was greater than 10%. Therefore, it is believed that the lower limit of the reference enzyme activity that can be quantified by this method is 0.02 μg / mL, that is, the absolute enzyme activity is 0.023 U / μL.

[0182] Table 12 Lower limit detection

[0183] Enzyme activity (μg / mL) Test 1 Test 2 Test 3 average value RSD(%) 0.08 64.68 62.45 67.12 64.74 3.61 0.06 50.50 48.50 51.87 50.29 3.37 0.04 38.38 38.52 41.15 39.35 3.97 0.02 4.94 6.54 3.57 5.02 29.66

[0184] Although the embodiments of the present invention have been disclosed as above, they are not limited to the applications listed in the specification and the implementation modes. They can be fully applied to various fields suitable for the present invention. For those familiar with the art, additional modifications can be easily implemented. Therefore, without departing from the general concept defined by the claims and the scope of equivalents, the present invention is not limited to specific details.

Claims

1. A method for determining the absolute activity of Bst DNA polymerase, characterized in that: The method comprises: using circular single-stranded DNA as a template, in a reaction system containing an amplification primer and dATP, using a Bst DNA polymerase to be tested to initiate an extension replication reaction, and consuming dNTPs in the reaction system; then calculating the dATP consumption according to a pre-constructed standard curve f1 characterizing the relationship between the dATP concentration and the fluorescence value, and then calculating the Bst DNA polymerase concentration according to a pre-constructed standard curve f2 characterizing the relationship between the Bst DNA polymerase concentration and the dATP consumption; and finally calculating the absolute activity of the Bst DNA polymerase according to the absolute activity definition of the Bst DNA polymerase; The absolute activity of Bst DNA polymerase is defined as the amount of enzyme required to incorporate 25 nmol of dNTP into acid-insoluble matter at 65°C for 30 min, expressed in U / μL.

2. The method for determining the absolute activity of Bst DNA polymerase according to claim 1, characterized in that: in, The absolute activity P of BstDNA polymerase is calculated as follows: The unit of P is U / μL; Wherein, C0 represents the initial concentration of dATP in the reaction system, in μM; C1 represents the final concentration of dATP in the reaction system after the reaction is completed, which is calculated by the standard curve f1, in μM; V d V is the volume of the reaction system, in μL; P is the amount of Bst DNA polymerase added, in μL; η T is the percentage of T in circular single-stranded DNA.

3. The method for measuring the absolute activity of Bst DNA polymerase according to claim 2, characterized in that, The consumption of dATP is obtained by subtracting the remaining amount of dATP in the reaction system after the reaction is completed from the initial content of dATP in the reaction system, wherein the remaining amount of dATP is obtained by detecting the fluorescence value produced by luciferin catalyzed by firefly luciferase using the energy provided by dATP.

4. The method for determining the absolute activity of Bst DNA polymerase according to claim 3, characterized in that: The remaining amount of dATP is detected by the following method: After the extension and replication reaction is completed, the reaction product is added with ATP detection reagent to detect the fluorescence value produced by luciferin catalyzed by firefly luciferase using the energy provided by dATP, and then the remaining amount of dATP is calculated based on the pre-constructed standard curve f1 that characterizes the relationship between the concentration of dATP and the fluorescence value.

5. The method for determining the absolute activity of Bst DNA polymerase according to claim 1, characterized in that: The construction method of the standard curve f1 is: A series of standard reaction systems with different dNTPs concentrations and without Bst DNA polymerase were prepared, and the reaction was first carried out under the same conditions of specific reaction temperature and time. After the reaction, equal amounts of ATP detection reagents were added to the product systems, and the fluorescence values ​​of each standard reaction system were detected under the same conditions. The standard curve f1 was obtained by fitting with the dNTPs concentration as the horizontal axis and the fluorescence value as the vertical axis.

6. The method for determining the absolute activity of Bst DNA polymerase according to claim 4, characterized in that: The construction method of the standard curve f2 is: A series of standard reaction systems with different concentrations of Bst DNA polymerase were prepared, and extension reactions were first carried out under specific reaction temperatures and the same time conditions. After the reaction was completed, equal amounts of ATP detection reagents were added to the product systems, and the fluorescence values ​​of each standard reaction system were detected under the same conditions. The consumption of dATP was calculated according to the standard curve f1, and the standard curve f2 was obtained by fitting with the concentrations of Bst DNA polymerase of different concentrations as the horizontal axis and the consumption of dATP as the vertical axis.

7. The method for determining the absolute activity of Bst DNA polymerase according to any one of claims 1 to 6, characterized in that: The circular single-stranded DNA is M13 bacteriophage single-stranded DNA.

8. The method for determining the absolute activity of Bst DNA polymerase according to any one of claims 1 to 6, characterized in that: The nucleic acid sequence of the amplification primer is 5'-TTTGCGGGAGAAGCCTTTATTTCAACGCA-3'.

9. The method for determining the absolute activity of Bst DNA polymerase according to claim 4, characterized in that: The temperature and reaction time of the extension and replication reaction of the tested BstDNA polymerase correspond to the same temperature and reaction time of the reaction in the process of constructing the standard curve f1.

10. The method for determining the absolute activity of Bst DNA polymerase according to claim 9, characterized in that: The reaction time of the Bst DNA polymerase extension replication reaction is 10 min to 60 min, and the reaction temperature is 65°C.

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