Method for judging stability of polymerase
The method of reversible inactivation and reactivation of DNA polymerase simplifies the detection of DNA polymerase stability, solves the problems of radioactive contamination and high cost of existing methods, and realizes high-throughput, automated enzyme stability assessment, which is suitable for a variety of application conditions.
Patent Information
- Authority / Receiving Office
- CN · China
- Patent Type
- Applications(China)
- Current Assignee / Owner
- MGI TECH CO LTD
- Filing Date
- 2024-11-06
- Publication Date
- 2026-05-08
AI Technical Summary
Existing methods for detecting DNA polymerase stability pose risks of radioactive contamination, are costly, complex to operate, and difficult to automate with high throughput, thus failing to meet the enzyme activity determination needs of different application fields.
The principle of reversible inactivation and reactivation of DNA polymerase was adopted. The stability of the enzyme was determined by combining the first round of amplification, inactivation treatment, renaturation and second round of amplification with the detection of DNA amount under different test conditions.
It simplifies the operation process, reduces detection costs, and enables high-throughput, automated enzyme stability assessment. It is suitable for various application conditions and meets the reaction conditions for high-throughput sequencing and whole-genome amplification.
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Abstract
Description
Technical Field
[0001] This invention relates to the field of biotechnology, and more specifically to a method for determining the stability of polymerases. Background Technology
[0002] DNA polymerase, also known as DNA-dependent DNA polymerase (DNApol), is a class of enzymes that catalyze the polymerization of substrate dNTP molecules into daughter DNA using parental DNA as a template. It is a crucial tool in molecular biology, used for whole-genome amplification, isothermal amplification, rolling circle replication, DNA sequencing, biochemical analysis, virus detection, and nucleic acid detection. It plays a vital role in genetic engineering, high-throughput sequencing, molecular diagnostics, and medical testing. To ensure that modified or unmodified enzymes exhibit optimal activity and achieve the best results in these different applications, it is necessary to test the enzymes under specific biochemical reaction conditions (such as strong acidity or alkalinity, high temperature, heavy metals, high salt, and denaturing agents) to determine their stability. The catalytic activity of enzyme molecules is maintained by a delicate balance of non-covalent forces (hydrophobic, ionic, van der Waals forces, and hydrogen bonds). When enzymes are exposed to certain concentrations of denaturing agents or adverse environmental conditions, the enzyme molecule undergoes partial or complete extension, damaging its active site and resulting in decreased or lost activity. Therefore, the stability of DNA polymerase is usually determined by measuring the activity of the enzyme after treatment under different conditions.
[0003] Accurately determining the stability of DNA polymerase and ensuring optimal performance in different application fields is crucial for enzyme activity determination. Traditional polymerase determination methods are as follows: (1) Radioactive isotope labeling method: relies on the incorporation of nucleotides labeled with radioactive element 3H. That is, in the reaction system and conditions of Phi29 DNA polymerase, a nucleotide chain with radioactive labeling is synthesized, and the enzyme activity is calculated by measuring the amount of radioactive isotope in the acid-insoluble product. (2) PCR is performed using primers with cy5 fluorescent labeling at the 5' end. The reaction is terminated by adding an equal volume of stop solution at different times. The stop solution contains competitive oligonucleotides, which can prevent the fluorescent product from rebinding to the template. The results are then verified by electrophoresis using polyacrylamide containing 8M urea. (3) Fluorescent dye incorporation method: SYBR Green I is incorporated into the PCR reaction system. The fluorescence intensity gradually increases as the reaction proceeds. The initial reaction rate can be obtained from the image provided by the real-time quantitative PCR instrument. The initial rate will vary depending on the concentration of the added enzyme. Based on this characteristic, a standard curve can be plotted using enzymes with known enzyme activities. The initial reaction rate of the unknown enzyme can be substituted into the standard curve to obtain the enzyme activity of the unknown enzyme. These methods have drawbacks. The radioactive isotope labeling method is prone to radioactive contamination and requires multiple steps such as precipitation and filtration, which is time-consuming and makes it difficult to achieve high throughput and automation, thus posing challenges to screening. The method of using primers with cy5 fluorescent labeling at the 5' end for PCR requires fluorescent labeling, which is slightly more expensive. The fluorescent dye incorporation method has high requirements for primer specificity. Summary of the Invention
[0004] This invention claims protection for a method for determining the stability of polymerases.
[0005] In a first aspect, the present invention claims protection for a method for detecting the stability of DNA polymerase.
[0006] The method for detecting DNA polymerase stability claimed in this invention utilizes the principle of reversible inactivation and reactivation of DNA polymerase. Specifically, the detection of DNA polymerase stability involves determining changes in the activity of the DNA polymerase under different test conditions.
[0007] Furthermore, the method may include the following steps: (A) First round of amplification: Using parental DNA as a template, the DNA polymerase to be tested catalyzes the polymerization of substrate dNTP molecules to form daughter DNA; (B) The DNA polymerase to be tested after the first round of amplification is reversibly inactivated (in the original reaction system), and the amount of progeny DNA obtained after the first round of amplification is detected. (C) The DNA polymerase to be tested after being treated in (B) is treated under different test conditions, then renatured, and then subjected to a second round of amplification. The amount of progeny DNA obtained after the second round of amplification is detected; thereby determining the change in the activity of the DNA polymerase under the different test conditions.
[0008] Furthermore, step (C) can be performed as follows: (C1) The test group and control group were set up as follows: Test group: The DNA polymerase to be tested, which has been inactivated by (B), is treated under the different test conditions and then refolded (in the original reaction system). Control group: The DNA polymerase to be tested, which has been inactivated by (B) as described above, is directly renatured (in the original reaction system). (C2) Second round of amplification (in the original reaction system): The DNA polymerase to be tested after renaturation in the test group and the control group described in (C1) is used to continue catalyzing the polymerization of substrate dNTP molecules to form daughter DNA; (C3) The amount of progeny DNA obtained after the second round of amplification in the test group and the control group is detected and compared to determine the change in DNA polymerase activity under different test conditions.
[0009] Furthermore, in (A), the amplification intensity can be controlled by controlling the amplification time in the PCR instrument (it should not be too long, otherwise the amplification efficiency of the enzyme will deteriorate, and can be determined according to the characteristics of the enzyme or the actual situation).
[0010] Furthermore, in the method, the reversible inactivation and the refolding can be performed in any of the following ways: (a1) The reversible inactivation is performed using a chelating agent, and the refolding is performed using metal ions; (a2) The reversible inactivation is carried out under acidic conditions, and the renaturation is carried out under alkaline conditions; (a3) The reversible inactivation is performed using a denaturing agent, and the refolding is performed using an antioxidant (including a reducing agent); (a4) The reversible deactivation is performed using an ionic surfactant, and the refolding is performed using a nonionic surfactant.
[0011] Furthermore, in (a1), the chelating agent may be EDTA, and the metal ion may be Mg. 2+ .
[0012] Furthermore, in (a2), the acid may be a weak acid, and the base may be a weak base. The weak acid has a pH range of 4-6, and the weak base has a pH range of 7.5-9.5.
[0013] Furthermore, in (a3), the denaturant may be urea, guanidine hydrochloride, or guanidine isothiocyanate, and the antioxidant may be cysteine, ascorbic acid, β-mercaptoethanol, or DTT.
[0014] Furthermore, in (a4), the ionic surfactant may be SDS, and the nonionic surfactant may be Tween20 or NP-40.
[0015] Further, in step (C), the change in the activity of the DNA polymerase under different test conditions can be determined as follows: if the amount of progeny DNA obtained in the test group after the second round of amplification is not significantly different from the amount of progeny DNA obtained in the control group after the second round of amplification, it is considered that the corresponding test conditions have no significant effect on the activity of the DNA polymerase (the DNA polymerase is relatively stable under the corresponding test conditions); if the amount of progeny DNA obtained in the test group after the second round of amplification is significantly lower than the amount of progeny DNA obtained in the control group after the second round of amplification, it is considered that the corresponding test conditions will significantly reduce the activity of the DNA polymerase (the DNA polymerase is relatively weak under the corresponding test conditions).
[0016] Furthermore, in the method, the different test conditions are a series of gradients set for a certain parameter. Step (C) then yields the stability of the DNA polymerase under that parameter. For example, the different test conditions could be different temperatures, different humidity levels, different pH values, different concentrations of denaturing agents, and / or different intensities of mechanical force. Accordingly, step (C) then yields the temperature stability, humidity stability, pH stability, denaturing agent stability, and / or mechanical force stability of the DNA polymerase.
[0017] Furthermore, in the method, the detection of the amount of progeny DNA can be performed according to any of the following methods: (b1) Incorporate fluorescent molecules into the progeny DNA and detect the amount of progeny DNA by measuring the intensity of the fluorescent signal; (b2) The amount of progeny DNA was determined by measuring the absorbance at 260 nm; (b3) The amount of said progeny DNA was determined by agarose gel electrophoresis.
[0018] Furthermore, in the method, the parental DNA can be linear single-stranded DNA, circular single-stranded DNA, or double-stranded DNA.
[0019] Furthermore, in the method, the amplification is rolling circle amplification (RCA), polymerase chain reaction (PCR), or multiple displacement amplification (MDA), etc.
[0020] In one embodiment of the present invention, the DNA polymerase is phi29 DNA polymerase.
[0021] Secondly, the present invention claims protection for the application of the method described in the first aspect above in any of the following: (A1) Screening for DNA polymerase variants; (A2) Determine the conditions for using DNA polymerase; (A3) Develop DNA polymerase-related reagents; (A4) Indirectly determine the activity of DNA polymerase under different conditions.
[0022] Furthermore, in (A2), the relevant reagents may be high-throughput sequencing reagents, whole-genome amplification reagents, rolling circle amplification reagents, polymerase chain reaction reagents, or multiple strand displacement amplification reagents, etc.
[0023] This invention, based on widely used DNA polymerase-induced amplification techniques such as rolling circle amplification (RCA), polymerase chain reaction (PCR), and multiple displacement amplification (MDA), utilizes the principle of polymerase transient inactivation (reversible inactivation) followed by reactivation to determine DNA polymerase stability. The proposed method is simple to operate, requires minimal raw materials for amplification, and eliminates the need for adding radioactive isotopes or fluorescent labels during the amplification process, thus avoiding fluorescence-related contamination and interference. Furthermore, this invention better aligns with the reaction conditions required for reagent development in applications such as high-throughput sequencing, whole-genome amplification, isothermal amplification, and rolling circle replication, and is more suitable for assessing enzyme stability. It provides a reference for selecting modified enzymes or determining the usage conditions of specific enzymes, offering convenience, saving time and costs. Detailed Implementation
[0024] This invention provides a method for determining the stability of DNA polymerase based on the principle of transient inactivation (reversible inactivation) followed by reactivation. The method includes: using linear single-stranded DNA, circular single-stranded DNA, or double-stranded DNA as a template; using amplification primers, dNTPs, and buffer; and initiating replication and amplification with DNA polymerase under suitable conditions using a PCR instrument. The amplification intensity is controlled by adjusting the amplification time in the PCR instrument (it should not be too long, otherwise the amplification efficiency of the enzyme will decrease; this can be determined according to the characteristics of the enzyme or the actual situation). After amplification, the amplification system is immediately transferred from the PCR instrument, and certain methods are used to temporarily weaken or eliminate the amplification ability of the amplification enzyme. The main methods used are: 1. Adding an appropriate amount of chelating agent, such as EDTA, to form a coordination complex with metal ions, preventing the polymerase from exerting its polymerization activity. 2. Adding a certain amount of acidic buffer, such as a citric acid mixture at pH 4.6; hydrochloric acid at pH 1.6 will stop the amplification. 3. Adding a certain amount of denaturing agent such as urea, guanidine hydrochloride, or guanidine isothiocyanate. 4. Add a certain amount of ionic surfactant, such as sodium dodecyl sulfate (SDS). After treatment using the above methods, the polymerase temporarily loses its amplification activity. Then, the content of the amplification product can be quantified using the following methods: 1. Incorporate a fluorescent molecule into a trace amount of the amplification product and quantify the number of single-stranded or double-stranded DNA copies by detecting the fluorescence signal of the amplification product. 2. Use Nanodrop to measure the absorbance at 260 nm. 3. Run agarose gel electrophoresis to determine the DNA content. Then, place the temporarily inactivated polymerase reaction system under different test conditions, such as different temperatures, different pH values, different concentrations of denaturing agents, and different intensities of mechanical force. After treatment according to the stability conditions to be tested, different methods of enzyme reactivation can be adopted based on the above methods of enzyme inactivation: 1. After treatment under different conditions, add a certain amount of metal ions, such as Mg2+, to the reaction system containing the chelating agent EDTA. 2+ 2. After treatment under different conditions, add a certain amount of alkaline solution, such as potassium citrate buffer or sodium hydroxide solution, to the reaction system containing a certain acid. 3. After treatment under different conditions, add a certain amount of cysteine, ascorbic acid, β-mercaptoethanol, or DTT to the reaction system containing a denaturing agent. 4. After treatment under different conditions, add a certain amount of nonionic surfactant, such as 0.05% Tween 20 and 0.5% NP-40, to the reaction system containing SDS. Then, place the reaction system with the reactivating agent into a PCR instrument and allow it to continue amplification under suitable conditions and within a predetermined amplification time, i.e., secondary amplification. After the amplification time is complete, use the same method described above to detect the DNA content after secondary amplification. By comparing the amount of DNA after secondary amplification, the temperature stability, pH stability, and mechanical stability of the polymerase can be determined.
[0025] The present invention will now be described in further detail with reference to specific embodiments. The given embodiments are merely illustrative of the invention and not intended to limit its scope. The embodiments provided below can serve as a guide for further improvements by those skilled in the art and do not constitute a limitation on the invention in any way.
[0026] Unless otherwise specified, the experimental methods used in the following examples are conventional methods, performed according to the techniques or conditions described in the literature in this field or according to the product instructions. Unless otherwise specified, the materials and reagents used in the following examples are commercially available.
[0027] Example 1: DNA polymerase stability assay (EDTA chelation inactivates the enzyme, and the addition of metal ions reactivates the enzyme). I. Experimental Materials 1. Equipment PCR instrument, 8-tube PCR kit, Qubit® 3.0 real-time fluorescence instrument, one set of pipettes, 200μL wide-mouth pipette tips, ice box, oven.
[0028] 2. Reagents As shown in Tables 1 to 7.
[0029] Table 1. Key Reagents Used in This Example
[0030] Preparation of DNB loading buffer IV in Table 2 and Table 1
[0031] Preparation of 10% PF68 solutions in Tables 3 and 2
[0032] Preparation of 1M MgCl2 in Tables 4 and 2
[0033] Preparation of 10×Phi29 buffers in Tables 5 and 2
[0034] Preparation of 1M ammonium sulfate in Tables 6 and 5
[0035] Preparation of 1M DTT in Tables 7 and 5
[0036] II. Experimental Methods 1. Preparation of DNA nanoballs (DNB) Take 40 fmol of a single-stranded circular DNA library (specifically, an E. coli single-stranded circular library with the insert fragment main band at 300 bp, i.e., E. coli library V3.0 in Table 1), then add 20 μL of DNB preparation buffer (the product in DNB Rapid Preparation Kit V2.0 in Table 1). Incubate the PCR instrument at 95℃ for 1 min → 65℃, 1 min → 40℃, 1 min → 4℃ for 1 min. Then add 40 μL of DNB rapid polymerase mixture II (the product in DNB Rapid Preparation Kit V2.0 in Table 1) and 1.6 μL of DNB polymerase mixture II (LC), i.e., phi29 polymerase (the product in DNB Rapid Preparation Kit V2.0 in Table 1). Incubate the PCR instrument at 30℃ for 10 min for rolling circle amplification. Immediately after amplification, place the PCR instrument on an ice pack and add 10 μL of DNB rapid polymerase mixture II (LC). Mix the DNB stop buffer (0.1M EDTA) slowly by pipetting with a wide-mouth pipette tip 5-8 times. Do not shake or vigorously pipet. After the DNB is prepared, take 2μL of DNB and use the ssDNA Assay Kit and Qubit® Fluorometer to detect the concentration. The DNB concentration was found to be 11.5ng / μL.
[0037] 2. Verify that the addition of 10 μL of DNB stop buffer can inactivate phi29 DNA polymerase. Take 45 μL of the DNB obtained in step 1 and perform rolling circle amplification on a PCR instrument at 30℃ for 30 min. After amplification, take 2 μL of DNB and use the ssDNA Assay Kit and Qubit® Fluorometer to detect the concentration. The DNB concentration was found to be 11.8 ng / µL, which proves that the amount of DNB and the amount of termination buffer added in step 1 can inactivate phi29 DNA polymerase.
[0038] 3. DNB was treated under different conditions and then subjected to secondary rolling circle amplification. Untreated group, i.e. control group: Take 30 μL of DNB obtained in step 1 without any treatment, and directly add 15 μL of DNB loading buffer IV (DNB loading buffer IV contains 75.5 mM MgCl2). The concentration of the DNB mixture at this time is calculated to be 7.67 ng / μL. After mixing, place it on a PCR instrument, set it to 30℃, and continue rolling circle amplification for 30 min. After the amplification is completed, immediately place it on an ice box, take 2 μL of DNB, and use the ssDNA Assay Kit and Qubit® Fluorometer to detect the concentration. The detection result is 21.5 ng / μL.
[0039] 4℃ treatment group: Take 30μL of DNB obtained in step 1 and place them in a 4℃ refrigerator for 2h, 4h and 6h respectively. Then add 15μL of DNB loading buffer IV (DNB loading buffer IV contains 75.5mM MgCl2), mix well and place on a PCR instrument. Set the temperature to 30℃ and continue rolling circle amplification for 30min. After amplification, immediately place on an ice box. Take 2μL of DNB and use the ssDNAAssay Kit and Qubit® Fluorometer to detect the concentration. The concentrations of DNB mixture in the 4℃ 2h group, 4℃ 4h group and 4℃ 6h group were 21.1ng / μL, 21.6ng / μL and 21.8ng / μL respectively.
[0040] 30℃ treatment group: Take 30μL of DNB obtained in step 1, place them in a PCR instrument, set the temperature to 30℃, and place them for 2h, 4h, and 6h respectively. Then add 15μL of DNB loading buffer IV (DNB loading buffer IV contains 75.5mM MgCl2), mix well, and place them in the PCR instrument. Set the temperature to 30℃ and continue rolling circle amplification for 30min. After amplification, immediately place them on an ice box, take 2μL of DNB, and use an ssDNA Assay Kit and Qubit® Fluorometer to detect the concentration. The concentrations of the DNB mixture in the 30℃ 2h group, 30℃ 4h group, and 30℃ 6h group were 23.7ng / μL, 26.0ng / μL, and 26.6ng / μL, respectively.
[0041] 50℃ treatment group: Take 30μL of DNB obtained in step 1 and place them in a 50℃ oven for 2h, 4h and 6h respectively. Then add 15μL of DNB loading buffer IV (DNB loading buffer IV contains 75.5mM MgCl2), mix well and place on a PCR instrument. Set the temperature to 30℃ and continue rolling circle amplification for 30min. After rolling circle amplification is completed, immediately place on an ice box. Take 2μL of DNB and use the ssDNA Assay Kit and Qubit® Fluorometer to detect the concentration. The concentrations of DNB mixture in the 50℃ 2h group, 50℃ 4h group and 50℃ 6h group are 14.5ng / μL, 8.98ng / μL and 7.8ng / μL respectively.
[0042] III. Results and Analysis The results are shown in Table 8: By comparing the theoretical DNB concentration of DNB+DLB Ⅳ before the secondary amplification with the DNB concentration of the untreated group after the secondary amplification, it was found that adding a certain amount of Mg to the DNB with the chelating agent EDTA showed that... 2+This allows for the renaturation of phi29 polymerase, enabling secondary amplification. By comparing treatment times (4℃, 30℃, 50℃) for the same duration (2h, 4h, or 6h), a certain amount of Mg was added. 2+ The concentration of DNB after secondary amplification showed that the stability of phi29 polymerase at 50℃ was worse than that at 30℃. Further analysis of the DNB concentration after treatment at 50℃ for different times (2h, 4h, and 6h) and the addition of a certain amount of Mg... 2+ The concentration of DNB after secondary amplification showed that the stability of phi29 polymerase gradually decreased until it was inactivated at 50°C over time. All the above results indicate that the method of this invention can determine the stability of DNA polymerase. In addition to temperature stability, this invention can also determine the stability affected by factors such as humidity, pH, and denaturant treatment.
[0043] Table 8. DNB concentration after secondary rolling circle amplification
[0044] Example 2: DNA polymerase stability test (acidic solution inactivates the enzyme, and alkaline solution reactivates the enzyme). I. Experimental Materials 1. Equipment PCR instrument, 8-tube PCR kit, Qubit® 3.0 real-time fluorescence instrument, one set of pipettes, 200μL wide-mouth pipette tips, ice box, oven.
[0045] 2. Reagents As shown in Table 9.
[0046] Table 9. Key Reagents Used in This Example
[0047] II. Experimental Methods 1. Preparation of DNA nanoballs (DNB) Take 40 fmol of a single-stranded circular DNA library (specifically, an E. coli single-stranded circular library with the insert fragment main band at 300 bp, i.e., E. coli library V3.0 in Table 9), then add 20 μL of DNB preparation buffer (the product from the DNB rapid preparation kit V2.0 in Table 9), and perform the reaction on a PCR instrument at 95℃, 1 min → 65℃, 1 min → 40℃, 1 min → 4℃, 1 min. Then add 40 μL of DNB rapid polymerase mixture II (the product in DNB rapid preparation kit V2.0 in Table 9) and 1.6 μL of DNB polymerase mixture II (LC), i.e., phi29 polymerase (the product in DNB rapid preparation kit V2.0 in Table 9). Perform rolling circle amplification on a PCR instrument at 30℃ for 10 min. Immediately after rolling circle amplification, place the instrument on an ice pack and add 20 μL of freshly prepared hydrochloric acid solution (100 mM). Gently pipette the mixture 5-8 times using a wide-mouth pipette tip, avoiding shaking or vigorous pipetting. Measure 2 μL of DNB and analyze the concentration using an ssDNA Assay Kit and a Qubit® Fluorometer. The result was 9.33 ng / μL.
[0048] 2. Verify that the addition of 20 μL of freshly prepared hydrochloric acid solution (100 mM) can inactivate phi29 DNA polymerase. Take 38 μl of the DNB obtained in step 1 and perform rolling circle amplification on a PCR instrument at 30℃ for 30 min. After amplification, take 2 μL of DNB and use the ssDNA Assay Kit and Qubit® Fluorometer to detect the concentration. The DNB concentration was found to be 8.31 ng / μL, which proves that the amount of DNB added in step 1 and the amount of freshly prepared hydrochloric acid (100 mM) can inactivate phi29 DNA polymerase.
[0049] 3. DNB was treated at different temperatures and for different times before undergoing a second rolling circle amplification. Untreated group (control group): 30 μL of DNB obtained in step 1 was added directly to 8 μL of freshly prepared sodium hydroxide solution (25 mM). After mixing, 2 μL of DNB was taken and the concentration was measured using an ssDNA Assay Kit and a Qubit® Fluorometer. The concentration was 7.64 ng / μL. The remaining DNB mixture was placed on a PCR instrument and subjected to rolling circle amplification at 30℃ for 30 min. After amplification, the mixture was immediately placed on an ice box. 2 μL of DNB was taken and the concentration was measured using an ssDNA Assay Kit and a Qubit® Fluorometer. The concentration of the DNB mixture was 20.3 ng / μL.
[0050] 4℃ treatment group: Take 30μL of DNB obtained in step 1 and place them in a 4℃ refrigerator for 2h, 4h and 6h respectively. Then add 8μL of freshly prepared sodium hydroxide solution (25mM) and mix well. Take 2μL of DNB mixture and use ssDNA Assay Kit and Qubit® Fluorometer to detect the concentration. The concentrations of DNB mixture in the 4℃ 2h group, 4℃ 4h group and 4℃ 6h group were 7.75ng / μL, 7.58ng / μL and 7.58ng / μL respectively. The remaining DNB mixture was placed on a PCR instrument and subjected to rolling circle amplification at 30°C for 30 minutes. After amplification, the mixture was immediately placed on an ice box. 2 μL of DNB was taken and the concentration was measured using an ssDNA Assay Kit and a Qubit® Fluorometer. The concentrations of the DNB mixture in the 4°C 2h, 4°C 4h, and 4°C 6h groups were 19 ng / μL, 18.9 ng / μL, and 15.8 ng / μL, respectively.
[0051] 30℃ treatment group: Take 30μL of DNB obtained in step 1, place it in a PCR instrument, set it to 30℃, and place it for 2h, 4h and 6h respectively. Then add 8μL of freshly prepared sodium hydroxide solution (25mM), mix well, and take 2μL of DNB mixture. Use ssDNAAssay Kit and Qubit® Fluorometer to detect the concentration. The concentrations of DNB mixture in the 30℃ 2h group, 30℃ 4h group and 30℃ 6h group were 6.24ng / μL, 6.42ng / μL and 6.58ng / μL respectively. The remaining DNB mixture was placed on a PCR instrument and subjected to rolling circle amplification at 30°C for 30 minutes. After amplification, the mixture was immediately placed on an ice box. 2 μL of DNB was taken and the concentration was measured using an ssDNA Assay Kit and a Qubit® Fluorometer. The concentrations of the DNB mixture in the 30°C 2h, 30°C 4h, and 30°C 6h groups were 7.36 ng / μL, 7.53 ng / μL, and 6.94 ng / μL, respectively.
[0052] 50℃ treatment group: Take 30μl portions of DNB obtained in step 1 and place them in a 50℃ oven for 2h, 4h, and 6h respectively. Then, add 8μL of freshly prepared sodium hydroxide solution (25mM) and mix well. Take 2μL of the DNB mixture and use an ssDNA AssayKit and Qubit® Fluorometer to detect the concentration. The concentrations of the DNB mixture in the 50℃ 2h group, 50℃ 4h group, and 50℃ 6h group were 5.27ng / μL, 4.65ng / μL, and 4.96ng / μL, respectively. The remaining DNB mixture was placed on a PCR instrument and subjected to rolling circle amplification at 30°C for 30 minutes. After amplification, the mixture was immediately placed on an ice box. 2 μL of DNB was taken and the concentration was measured using an ssDNA Assay Kit and a Qubit® Fluorometer. The concentrations of the DNB mixture in the 50°C 2h, 50°C 4h, and 50°C 6h groups were 5.82 ng / μL, 5.16 ng / μL, and 5.62 ng / μL, respectively.
[0053] III. Results Analysis The results are shown in Table 10. By comparing the DNB concentrations before and after secondary amplification in the untreated group (control group) after adding sodium hydroxide solution, it is shown that adding a certain amount of sodium hydroxide solution to DNB treated with hydrochloric acid solution can renature the phi29 polymerase, allowing for secondary amplification. By comparing the DNB concentrations after secondary amplification with the addition of a certain amount of sodium hydroxide solution after treatment at different temperatures (4℃, 30℃, 50℃) for the same time (2h, 4h, or 6h), it is shown that the stability of phi29 polymerase at 50℃ and 30℃ is worse than at 4℃, because the DNB concentration after secondary amplification is significantly reduced after treatment at 30℃. However, the phi29 enzyme is actually stable at 30℃. It is suspected that hydrochloric acid, being a strong acid, causes irreversible damage to the protease at slightly higher temperatures. Therefore, weak acids and bases, such as citric acid and potassium citrate, should be used to determine enzyme stability. All the above results indicate that the method of this invention can determine the stability of DNA polymerase. In addition to temperature stability, this invention can also determine the stability affected by humidity, pH, denaturant treatment, and other factors.
[0054] Table 10. DNB concentration after secondary rolling circle amplification
[0055] The present invention has been described in detail above. Those skilled in the art will recognize that the invention can be practiced in a wide range of ways with equivalent parameters, concentrations, and conditions without departing from its spirit and scope, and without requiring unnecessary experiments. While specific embodiments have been provided, it should be understood that further modifications can be made to the invention. In summary, according to the principles of the invention, this application is intended to include any changes, uses, or improvements to the invention, including changes made using conventional techniques known in the art that depart from the scope disclosed herein.
Claims
1. A method for detecting the stability of DNA polymerase, characterized in that: The method described above utilizes the principle of reversible inactivation and reactivation of DNA polymerase to detect DNA polymerase stability.
2. The method according to claim 1, characterized in that: The method includes the following steps: (A) First round of amplification: Using parental DNA as a template, the DNA polymerase to be tested catalyzes the polymerization of substrate dNTP molecules to form daughter DNA; (B) The DNA polymerase to be tested after the first round of amplification is reversibly inactivated, and the amount of progeny DNA obtained after the first round of amplification is detected. (C) The DNA polymerase to be tested after being treated in (B) is treated under different test conditions, then renatured, and then subjected to a second round of amplification. The amount of progeny DNA obtained after the second round of amplification is detected; thereby determining the change in the activity of the DNA polymerase under the different test conditions.
3. The method according to claim 1 or 2, characterized in that: The procedure (C) is as follows: (C1) The test group and control group were set up as follows: Test group: The DNA polymerase to be tested, which has been inactivated by (B), is treated under the different test conditions and then renatured. Control group: The DNA polymerase to be tested, which had been inactivated by (B), was directly renatured; (C2) Second round of amplification: The DNA polymerase to be tested, after being renatured in the test group and the control group described in (C1), is used to continue catalyzing the polymerization of substrate dNTP molecules to form daughter DNA; (C3) The amount of progeny DNA obtained after the second round of amplification in the test group and the control group is detected and compared to determine the change in DNA polymerase activity under different test conditions.
4. The method according to claim 2 or 3, characterized in that: The reversible inactivation and the refolding are performed in any of the following ways: (a1) The reversible inactivation is performed using a chelating agent, and the refolding is performed using metal ions; (a2) The reversible inactivation is carried out under acidic conditions, and the renaturation is carried out under alkaline conditions; (a3) The reversible inactivation is performed using a denaturant, and the refolding is performed using an antioxidant; (a4) The reversible deactivation is performed using an ionic surfactant, and the refolding is performed using a nonionic surfactant.
5. The method according to claim 4, characterized in that: In (a1), the chelating agent is EDTA, and the metal ion is Mg. 2+ ;or In (a2), the acid is a weak acid, and the base is a weak base; or In (a3), the denaturing agent is urea, guanidine hydrochloride, or guanidine isothiocyanate, and the antioxidant is cysteine, ascorbic acid, β-mercaptoethanol, or DTT; or In (a4), the ionic surfactant is SDS, and the nonionic surfactant is Tween20 or NP-40.
6. The method according to any one of claims 1-5, characterized in that: The different test conditions are different temperatures, different humidity levels, different pH levels, different concentrations of denaturants, and / or different intensities of mechanical force. or In the method, the amount of progeny DNA is detected according to any of the following methods: (b1) Incorporate fluorescent molecules into the progeny DNA and detect the amount of progeny DNA by measuring the intensity of the fluorescent signal; (b2) The amount of progeny DNA was determined by measuring the absorbance at 260 nm; (b3) The amount of said progeny DNA was determined by agarose gel electrophoresis.
7. The method according to any one of claims 1-6, characterized in that: The parental DNA is linear single-stranded DNA, circular single-stranded DNA, or double-stranded DNA.
8. The method according to any one of claims 1-7, characterized in that: The amplification is performed by rolling circle amplification, polymerase chain reaction, or multiple strand displacement amplification.
9. The application of the method according to any one of claims 1-8 in any of the following: (A1) Screening for DNA polymerase variants; (A2) Determine the conditions for using DNA polymerase; (A3) Develop DNA polymerase-related reagents; (A4) Indirectly determine the activity of DNA polymerase under different conditions.
10. The application according to claim 9, characterized in that: The relevant reagents are high-throughput sequencing reagents, whole genome amplification reagents, rolling circle amplification reagents, polymerase chain reaction reagents, or multiple strand displacement amplification reagents.