A high-purity dNTP monomer freeze-dried powder and its preparation process

Abstract

This application relates to the field of freeze-drying of biological products, specifically disclosing a high-purity dNTP monomer freeze-dried powder and its preparation process. The preparation process includes the following steps: under normal pressure, a dNTP aqueous solution is pre-frozen at -10 to -40°C for 3-5 hours to obtain pre-frozen crystals, then freeze-dried under vacuum at -30 to -40°C for 20-30 hours, followed by nitrogen purging to restore normal pressure, resulting in the dNTP monomer freeze-dried powder. The dNTP freeze-dried powder prepared by this application has an HPLC purity of not less than 99%, clear PCR bands, a moisture content of less than 10%, is easy to store and transport, has a long shelf life, and is easily soluble, requiring short reconstitution time with pure water, resulting in a clear liquid after reconstitution.

Description

Technical Field

[0001] This application relates to the field of freeze-drying of biological products, and more specifically, to a freeze-drying process for a dNTP monomer. Background Technology

[0002] Since the 1970s, researchers have been conducting research on DNA synthesis, which is of great significance for the development of drugs, fuels, and other chemical products. With continuous technological development and innovation, artificially synthesized DNA fragments can now be widely used in life science fields such as genetic engineering and molecular biology.

[0003] The fundamental building blocks of DNA synthesis are deoxyribonucleoside triphosphates (dNTPs), which can be classified into dATP, dTTP, dCTP, dGTP, and dUTP based on their structure. Currently, most commercially available dNTP solutions are 10-100 mmol aqueous solutions, which can be directly prepared for PCR and other experiments. However, dNTP aqueous solutions suffer from poor stability and require significant storage space.

[0004] Vacuum freeze-drying is a recognized method for enhancing the storage stability of temperature-sensitive biomolecules and has been widely used in the production of food, pharmaceuticals, and biological products. The principle involves first freezing the aqueous compound to be dried into a solid state, and then sublimating the solvent under low-temperature vacuum conditions to achieve the drying purpose. Compared with other drying methods, this method has the advantage of good thermal stability and is particularly suitable for heat-sensitive compounds, such as nucleic acids.

[0005] Current vacuum freeze-drying techniques often cause some damage to nucleic acid materials, such as strand breaks and supercoiling damage. Therefore, in practice, multiple cryoprotectants, lyophilization protectants, or excipients are often added to prevent this damage. A relevant Chinese patent, CN 107567497A, provides a method for freeze-drying RNA, in which at least one RNA and at least one cryoprotectant are mixed before freeze-drying. However, the addition of cryoprotectants in this method can interfere with the application of dNTPs in PCR, affecting the clarity of PCR bands. Summary of the Invention

[0006] To address the aforementioned technical problems, this application provides a process for preparing dNTP monomer freeze-dried powder without adding any freeze-drying protectant or excipient, and the resulting dNTP monomer freeze-dried powder with a purity higher than 99%.

[0007] Firstly, this application provides a process for preparing high-purity dNTP monomer lyophilized powder, which adopts the following technical solution:

[0008] A process for preparing high-purity dNTP monomer lyophilized powder includes the following steps:

[0009] Under normal pressure, the dNTP aqueous solution is pre-frozen at -10 to -40℃ for 3-5 hours to obtain pre-frozen crystals, and then vacuum freeze-dried at -30 to -40℃ for more than 20 hours. After that, nitrogen gas is introduced to restore normal pressure to obtain dNTP monomer freeze-dried powder.

[0010] By adopting the above technical solution, the vacuum freeze-drying process in this application maintains a constant temperature range throughout, eliminating the need for heating and heat exchange. This significantly reduces the possibility of damage to dNTPs during the vacuum freeze-drying process, thus eliminating the need for any cryoprotectants, lyophilization agents, or excipients in the freeze-drying process. This greatly reduces the likelihood of these reagents affecting the HPLC purity of dNTPs, facilitating successful PCR. Furthermore, the use of nitrogen to break the vacuum in this application reduces the possibility of degradation due to direct collisions between the compound and water or oxygen, thereby improving the HPLC purity of the dNTP monomer freeze-dried powder. In addition, the preparation process of this application is simple, easy to operate, time-efficient, and low-cost, making it suitable for large-scale industrial production.

[0011] Preferably, the temperature change during the low-temperature pre-freezing is specifically as follows:

[0012] First, pre-freeze the dNTP aqueous solution to -30 to -40°C for 1 to 1.5 hours, then raise the temperature to -10 to -20°C for 1 to 2 hours, and then lower the temperature to -30 to -40°C for 1 to 1.5 hours.

[0013] By adopting the above technical solution, this application uses a zigzag temperature change for low-temperature pre-freezing, which can obtain relatively loose pre-frozen crystals and shorten the subsequent vacuum freeze-drying time. Furthermore, it can shorten the reconstitution time of the dNTP monomer freeze-dried powder, to approximately 3-30 seconds.

[0014] Preferably, the vacuum degree of the vacuum freeze-drying is 0.15-0.20 mbar.

[0015] Preferably, the vacuum freeze drying includes sublimation drying and desorption drying, wherein the sublimation drying time is 10-15 hours and the desorption drying time is 10-15 hours.

[0016] By adopting the above technical solution, the pre-frozen crystals first undergo a sublimation drying process for a period of time. As the free water sublimates or evaporates, dNTPs gradually crystallize and precipitate into ice crystals. Then, a period of desorption drying process is carried out, and dNTPs gradually transform from ice crystals into powder. Finally, the free water content in the dNTP freeze-dried powder is 4.8-9.2%.

[0017] Preferably, the concentration of the dNTP aqueous solution is 95-105 mmol / L. In one specific embodiment of this application, the concentration of the dNTP aqueous solution is 95 mmol / L. In another specific embodiment of this application, the concentration of the dNTP aqueous solution is 100 mmol / L. In yet another specific embodiment of this application, the concentration of the dNTP aqueous solution is 105 mmol / L.

[0018] Secondly, this application provides a high-purity dNTP lyophilized powder prepared by the above-described preparation process.

[0019] Preferably, the HPLC purity of the dNTP lyophilized powder is not less than 99%.

[0020] Preferably, the moisture content of the dNTP freeze-dried powder is 4.8-9.2%.

[0021] By adopting the above technical solution, the dNTP lyophilized powder of this application has a purity of over 99%, extremely low impurity content, and a moisture content of less than 10%, making it easier to store and transport compared to dNTP aqueous solutions. While general dNTP aqueous solutions can be stored at -20℃ for 12 months, the dNTP lyophilized powder of this application can be stored at -20℃ for at least 3 years, exhibiting a longer shelf life. Furthermore, when applied to PCR, the bands are clear, it dissolves easily, and the reconstitution time in pure water is approximately 3-30 seconds, which is relatively short. After reconstitution, the liquid is clear, and the HPLC purity does not significantly decrease compared to before lyophilization, allowing for large-scale industrial production.

[0022] Thirdly, this application provides an application of high-purity dNTP lyophilized powder in gene amplification, sequencing, and labeling.

[0023] More specifically, the high-purity dNTP lyophilized powder of this application can be used in gene amplification, sequencing and labeling processes such as PCR amplification, real-time PCR (qPCR), RT-PCR, long-fragment PCR, high-fidelity PCR, cDNA synthesis, primer extension reaction, DNA sequencing, and DNA labeling.

[0024] In summary, this application has the following beneficial technical effects:

[0025] 1. The dNTP lyophilized powder prepared by the process of this application has high HPLC purity, clear PCR bands, low moisture content, is easy to store and transport, has a long shelf life, is easy to dissolve, and takes a short time to reconstitute with pure water, resulting in a clear liquid after reconstitution; 2. The preparation process of this application is simple, easy to operate, takes a short time, and has a low cost, making it suitable for large-scale industrial production. Attached Figure Description

[0026] Figure 1(a) is the HPLC chromatogram of dATP before lyophilization;

[0027] Figure 1(b) HPLC chromatogram of the lyophilized dATP monomer powder prepared in this application;

[0028] Figure 2(a) is the HPLC chromatogram of dTTP before lyophilization;

[0029] Figure 2(b) is the HPLC chromatogram of the dTTP monomer lyophilized powder obtained in this application;

[0030] Figure 3(a) is the HPLC chromatogram of dCTP before lyophilization;

[0031] Figure 3(b) is the HPLC chromatogram of the lyophilized dCTP monomer powder prepared in this application;

[0032] Figure 4(a) is the HPLC chromatogram of dGTP before lyophilization;

[0033] Figure 4(b) is the HPLC chromatogram of the lyophilized dGTP monomer powder obtained in this application;

[0034] Figure 5(a) is the HPLC chromatogram of dUTP before lyophilization;

[0035] Figure 5(b) is the HPLC chromatogram of the dUTP monomer lyophilized powder obtained in this application;

[0036] Figure 6 This is the HPLC spectrum of the lyophilized dGTP monomer powder prepared in Comparative Example 1.

[0037] Figure 7 This is the HPLC spectrum of the lyophilized dATP monomer powder prepared in Comparative Example 2.

[0038] Figure 8 This is the HPLC spectrum of the lyophilized dTTP monomer powder prepared in Comparative Example 3.

[0039] Figure 9 This is the HPLC spectrum of the lyophilized dCTP monomer powder prepared in Comparative Example 4.

[0040] Figure 10 This is the HPLC spectrum of the lyophilized dGTP monomer powder prepared in Comparative Example 5.

[0041] Figure 11 This is the HPLC spectrum of the lyophilized dUTP monomer powder prepared in Comparative Example 6.

[0042] Figure 12 This is the HPLC spectrum of the lyophilized dGTP monomer powder prepared in Comparative Example 7.

[0043] Figure 13 (Left) is a photograph of the lyophilized dGTP monomer powder obtained in Example 6 after reconstitution;

[0044] Figure 13 (Right) is a photo of the lyophilized dGTP monomer powder prepared in Comparative Example 1 after reconstitution.

[0045] Figure 14 This is a PCR band image of Application Example 1 of this application. Detailed Implementation

[0046] The present application will be further described in detail below with reference to the accompanying drawings, embodiments, comparative examples and application examples.

[0047] The dNTPs in this application can be dATP, dTTP, dCTP, dGTP, and dUTP;

[0048] The preparation process of this application uses a metal freeze-drying tray with an inner diameter of 48.1×29×2.75cm.

[0049] The vacuum freeze-drying time of this application corresponds to the liquid height of the dNTP aqueous solution in the metal freeze-drying pan. The vacuum freeze-drying time can be appropriately extended or shortened depending on the liquid height.

[0050] Example 1

[0051] A process for preparing high-purity dATP monomer lyophilized powder includes the following steps:

[0052] S1. Under normal pressure, add a 95 mmol / L dATP aqueous solution to a metal freeze-drying pan to a liquid height of 1.0 cm, then cool to -30℃ for 1.5 h, then heat to -10℃ for 2 h, and then cool to -30℃ for 1.5 h to obtain pre-freezed crystals.

[0053] S2. The pre-frozen crystals were first sublimated and dried for 15 hours under a vacuum of 0.15 mbar and a temperature of -30°C, and then desorbed and dried for 15 hours. After that, nitrogen gas was introduced to restore the atmospheric pressure, and the crystals were transferred in a sealed glove box under nitrogen protection for weighing and packaging to obtain dATP monomer freeze-dried powder.

[0054] Example 2

[0055] A process for preparing high-purity dATP monomer lyophilized powder includes the following steps:

[0056] S1. Under normal pressure, a 105 mmol / L dATP aqueous solution was added to a metal freeze-drying pan to a liquid level of 0.7 cm. The pan was then cooled to -40°C for 1 hour, then heated to -20°C for 1 hour, and then cooled to -40°C for 1 hour to obtain pre-frozen crystals. S2. The pre-frozen crystals were dried by sublimation for 10 hours under a vacuum of 0.2 mbar and a temperature of -40°C, followed by desorption drying for 10 hours. Nitrogen gas was then introduced to restore normal pressure. The crystals were then transferred in a sealed glove box under nitrogen protection for weighing and packaging to obtain dATP monomer freeze-dried powder.

[0057] Example 3

[0058] A process for preparing high-purity dATP monomer lyophilized powder includes the following steps:

[0059] S1. Under normal pressure, add a 100 mmol / L dATP aqueous solution to a metal freeze-drying pan to a liquid height of 0.8 cm, then cool to -35℃ for 1.2 h, then heat to -15℃ for 1.5 h, and then cool to -35℃ for 1.3 h to obtain pre-frozen crystals.

[0060] S2. The pre-frozen crystals were first sublimated and dried for 12 hours under a vacuum of 0.18 mbar and a temperature of -35°C, and then desorbed and dried for 14 hours. After that, nitrogen gas was introduced to restore the atmospheric pressure, and the crystals were transferred in a sealed glove box under nitrogen protection for weighing and packaging to obtain dATP monomer freeze-dried powder.

[0061] Example 4

[0062] A process for preparing high-purity dTTP monomer lyophilized powder includes the following steps:

[0063] S1. Under normal pressure, add a 100 mmol / L dTTP aqueous solution to a metal freeze-drying pan to a liquid height of 1.0 cm, then cool to -30℃ for 1.5 h, then heat to -20℃ for 1 h, and then cool to -30℃ for 1.5 h to obtain pre-freezed crystals.

[0064] S2. The pre-frozen crystals were first sublimated and dried for 15 hours under a vacuum of 0.2 mbar and a temperature of -30°C, and then desorbed and dried for 15 hours. After that, nitrogen gas was introduced to restore the atmospheric pressure, and the crystals were transferred in a sealed glove box under nitrogen protection for weighing and packaging to obtain dTTP monomer freeze-dried powder.

[0065] Example 5

[0066] A process for preparing high-purity dCTP monomer lyophilized powder includes the following steps:

[0067] S1. Under normal pressure, add a 100 mmol / L dCTP aqueous solution to a metal freeze-drying pan to a liquid height of 1.0 cm, then cool to -30℃ for 1.5 h, then heat to -15℃ for 1.5 h, then cool to -30℃ for 1.5 h to obtain pre-frozen crystals.

[0068] S2. The pre-frozen crystals were first sublimated and dried for 15 hours under a vacuum of 0.17 mbar and a temperature of -30°C, and then desorbed and dried for 15 hours. After that, nitrogen gas was introduced to restore the atmospheric pressure, and the crystals were transferred in a sealed glove box under nitrogen protection for weighing and packaging to obtain dCTP monomer freeze-dried powder.

[0069] Example 6

[0070] A process for preparing high-purity dGTP monomer lyophilized powder includes the following steps:

[0071] S1. Under normal pressure, add a 100 mmol / L dGTP aqueous solution to a metal freeze-drying pan to a liquid height of 1.0 cm, then cool to -30℃ for 1.5 h, then heat to -20℃ for 1 h, and then cool to -40℃ for 1 h to obtain pre-freezed crystals.

[0072] S2. The pre-frozen crystals were first sublimated and dried for 15 hours under a vacuum of 0.17 mbar and a temperature of -40°C, and then desorbed and dried for 10 hours. After that, nitrogen gas was introduced to restore the atmospheric pressure, and the crystals were transferred in a sealed glove box under nitrogen protection for weighing and packaging to obtain dGTP monomer freeze-dried powder.

[0073] Example 7

[0074] A process for preparing high-purity dUTP monomer lyophilized powder includes the following steps:

[0075] S1. Under normal pressure, add a 100 mmol / L dUTP aqueous solution to a metal freeze-drying pan to a liquid height of 1.0 cm, then cool to -40℃ for 1 h, then heat to -20℃ for 1 h, and then cool to -30℃ for 1.5 h to obtain pre-freezed crystals.

[0076] S2. The pre-frozen crystals were first sublimated and dried for 15 hours under a vacuum of 0.2 mbar and a temperature of -30°C, and then desorbed and dried for 15 hours. After that, nitrogen gas was introduced to restore the atmospheric pressure, and the crystals were transferred in a sealed glove box under nitrogen protection for weighing and packaging to obtain dUTP monomer freeze-dried powder.

[0077] Comparative Example 1

[0078] The difference from Example 6 is that a heat exchange process is involved in step S2. Specifically, the pre-frozen crystals are first sublimated and dried for 10 hours under a vacuum of 0.17 mbar and a temperature of -40°C. Then, the temperature is increased to 10°C at a heating rate of 20°C / h for desorption and drying. After that, nitrogen is introduced to restore the atmospheric pressure. The crystals are then transferred in a sealed glove box protected by nitrogen for weighing and packaging to obtain dGTP monomer freeze-dried powder.

[0079] Comparative Example 2

[0080] The difference from Example 3 is that the temperature of vacuum freeze drying in step S2 is different; specifically, the pre-frozen crystals are first sublimated and dried for 12 hours under a vacuum of 0.18 mbar and a temperature of -20°C, and then desorbed and dried for 14 hours. After that, nitrogen gas is introduced to restore normal pressure, and the crystals are transferred in a sealed glove box protected by nitrogen gas for weighing and packaging to obtain dATP monomer freeze-dried powder.

[0081] Comparative Example 3

[0082] The difference from Example 4 is that the vacuum freeze-drying temperature is different in step S2; specifically, the pre-frozen crystals are first sublimated and dried for 15 hours under a vacuum of 0.2 mbar and a temperature of -20°C, and then desorbed and dried for 15 hours. After that, nitrogen gas is introduced to restore normal pressure, and the crystals are transferred in a sealed glove box protected by nitrogen gas for weighing and packaging to obtain dTTP monomer freeze-dried powder.

[0083] Comparative Example 4

[0084] The difference from Example 5 is that the temperature of vacuum freeze drying in step S2 is different; specifically, the pre-frozen crystals are first sublimated and dried for 15 hours under a vacuum of 0.17 mbar and a temperature of -20°C, and then desorbed and dried for 15 hours. After that, nitrogen gas is introduced to restore normal pressure, and the crystals are transferred in a sealed glove box protected by nitrogen gas for weighing and packaging to obtain dCTP monomer freeze-dried powder.

[0085] Comparative Example 5

[0086] The difference from Example 6 is that the temperature of vacuum freeze drying in step S2 is different; specifically, the pre-frozen crystals are first sublimated and dried for 15 hours under a vacuum of 0.17 mbar and a temperature of -20°C, and then desorbed and dried for 10 hours. After that, nitrogen gas is introduced to restore normal pressure, and the crystals are transferred in a sealed glove box protected by nitrogen gas for weighing and packaging to obtain dGTP monomer freeze-dried powder.

[0087] Comparative Example 6

[0088] The difference from Example 7 is that the temperature of vacuum freeze drying in step S2 is different; specifically, the pre-frozen crystals are first sublimated and dried for 15 hours under a vacuum of 0.2 mbar and a temperature of -20°C, and then desorbed and dried for 15 hours. After that, nitrogen gas is introduced to restore normal pressure, and the crystals are transferred in a sealed glove box protected by nitrogen gas for weighing and packaging to obtain dUTP monomer freeze-dried powder.

[0089] Comparative Example 7

[0090] The difference from Example 6 is that in step S1, the low-temperature pre-freezing is not performed using a broken-line temperature change; specifically, under normal pressure, a 100 mmol / L dGTP aqueous solution is added to a metal freeze-drying pan to a liquid height of 1.5 cm, and then the temperature is lowered to -50°C for 3 hours to obtain pre-frozen crystals.

[0091] Performance testing

[0092] 1. The purity and moisture content of the dNTP monomer freeze-dried powders prepared in Examples 1-7 and Comparative Examples 1-7 were tested respectively and recorded in Table 1. Each example included multiple samples, and the recorded purity was the average value of multiple samples.

[0093] Table 1 Performance Test Results

[0094] project Purity (%) before freeze-drying Purity (%) after freeze-drying Moisture content (%) Example 1 99.59 99.08 4.875 Example 2 99.58 99.09 4.878 Example 3 99.58 99.07 4.879 Example 4 99.48 99.35 5.025 Example 5 99.82 99.85 5.693 Example 6 99.64 99.29 9.154 Example 7 99.58 99.07 4.865 Comparative Example 1 99.64 81.44 9.137 Comparative Example 2 99.58 96.27 4.632 Comparative Example 3 99.48 94.87 4.729 Comparative Example 4 99.82 93.00 5.386 Comparative Example 5 99.64 98.54 9.574 Comparative Example 6 99.58 96.27 4.287 Comparative Example 7 99.64 98.22 9.061

[0095] As can be seen from Table 1 and Figures 1-5, the dATP monomer lyophilized powders obtained in Examples 1-3 of this application have a purity of 99.07-99.09% and a moisture content of 4.875-4.879%; the dTTP monomer lyophilized powder obtained in Example 4 of this application has a purity of 99.35% and a moisture content of 5.025%; the dCTP monomer lyophilized powder obtained in Example 5 of this application has a purity of 99.85% and a moisture content of 5.693%; and the dGTP obtained in Example 6 of this application... The purity of the lyophilized monomer powder is 99.29%, and the moisture content is 9.154%. The purity of the dUTP monomer lyophilized powder prepared in Example 7 of this application is 99.07%, and the moisture content is 4.865%. The test data and figures show that the dNTP monomer lyophilized powder prepared by the process of this application has high purity, almost no decrease compared with the purity before lyophilization, and can still reach more than 99%. The biological activity is not reduced, and the moisture content is low, the shelf life is long, and it is easy to store and transport.

[0096] Since dGTP is structurally the most unstable of the dNTPs and is most prone to deterioration during lyophilization, Comparative Example 1 of this application uses dGTP as an example to demonstrate the effect of heat exchange on dNTPs in the absence of lyophilization protectants or excipients. (See Table 1 and...) Figure 6It can be seen that the purity of the dGTP freeze-dried powder prepared in Comparative Example 1 is 81.44%, and the moisture content is 9.137%. Although the moisture content is slightly lower than that in Example 6, the purity of Comparative Example 1 decreased by 18.27% compared with that before freeze-drying, which is significantly worse than that in Example 6. The experimental data show that without the presence of freeze-drying protectant or excipient, heat exchange will have a significant impact on the purity of the dNTP monomer freeze-dried powder.

[0097] The difference between Comparative Example 2 and Example 3 lies in the temperature of the vacuum freeze-drying process. This can be seen from the data in Table 1 and Figure 1. Figure 7 It can be seen that the purity of the dATP monomer freeze-dried powder in Comparative Example 2 is 96.27%, which is 2.83% lower than that in Example 3. The difference between Comparative Example 3 and Example 4 lies in the temperature of the vacuum freeze-drying process. This can be seen from the data in Table 1 and Figure 2. Figure 8 It can be seen that the purity of the dTTP monomer freeze-dried powder in Comparative Example 3 is 94.87%, which is 4.51% lower than that in Example 4. The difference between Comparative Example 4 and Example 5 lies in the temperature of the vacuum freeze-drying process. This can be seen from the data in Table 1 and Figure 3. Figure 9 It can be seen that the purity of the dCTP monomer freeze-dried powder in Comparative Example 4 is 93%, which is 6.86% lower than that in Example 5. The difference between Comparative Example 5 and Example 6 lies in the temperature of the vacuum freeze-drying process. This can be seen from the data in Table 1 and Figure 4. Figure 10 It can be seen that the purity of the dGTP monomer freeze-dried powder in Comparative Example 5 is 98.54%, which is 0.76% lower than that in Example 6. The difference between Comparative Example 6 and Example 7 lies in the temperature of the vacuum freeze-drying process. This can be seen from the data in Table 1 and Figure 5. Figure 11 It can be seen that the purity of the dNTP monomer freeze-dried powder in Comparative Example 6 is 96.27%, which is 2.83% lower than that in Example 7. The above experimental data fully demonstrate that controlling the temperature of the vacuum freeze-drying process can improve the purity of the dNTP monomer freeze-dried powder. When the temperature of the vacuum freeze-drying process changes from -30°C to -20°C, the purity of the dNTP monomer freeze-dried powder will decrease.

[0098] The difference between Comparative Example 7 and Example 6 lies in the pre-freezing process, as shown in Table 1 and Figure 4. Figure 12 It can be seen that the purity of the dGTP monomer freeze-dried powder in Comparative Example 7 is 98.22%, which is 1.08% lower than that in Example 6. The experimental data shows that not using a zigzag temperature change for pre-freezing will affect the porosity of the pre-frozen crystals, thus making it impossible for the pre-frozen crystals to achieve a good freeze-drying effect within the freeze-drying time of this application, thereby affecting the purity of the dNTP monomer freeze-dried powder.

[0099] 2. The dNTP monomer lyophilized powders obtained in Examples 1-7, Comparative Example 1, and Comparative Example 7 were reconstituted with pure water. The reconstitution time of the dNTP monomer lyophilized powders obtained in Examples 1-7 was observed to be 3-30 seconds, while the reconstitution time of the dGTP monomer lyophilized powder obtained in Comparative Example 7 was approximately 45 seconds. The photographs of the reconstituted powders in Examples 6 and 1 are shown below. Figure 13 As shown.

[0100] From the reconstitution time and Figure 13 It can be seen that the reconstitution time of the lyophilized dNTP monomer powder prepared in this application is 3-30s, which is relatively short, and the liquid is clear after reconstitution. The reconstitution time of Comparative Example 7 is slightly longer than that of Examples 1-7, which indicates that the pre-freezing method using a zigzag temperature change method in this application can obtain a more porous pre-frozen crystal, thereby shortening the reconstitution time.

[0101] Application Example 1

[0102] This application uses PCR amplification as an example to verify the application of the dNTP lyophilized powder prepared in this application.

[0103] The application of a high-purity dATP lyophilized powder in PCR amplification includes the following steps:

[0104] a. Prepare dATP Mix with a final concentration of 2.5 mmol / L using the dATP lyophilized powder obtained in Example 3;

[0105] b. Thaw primers, template, DNA polymerase, and PCR buffer on ice, gently vortex to mix, and centrifuge. Prepare the sample loading PCR premix solution with the dATP Mix to be tested and water as shown in the table below.

[0106] Table 2

[0107]

[0108] c. Turn on the gene amplification instrument, set the PCR program, and perform the PCR reaction.

[0109] d. In the running page, set the reaction program. Set the reaction system to 50 μL, and the reaction conditions as follows: first stage pre-denaturation 95℃·3min; second stage denaturation 95℃·20s, annealing 55℃·20s, extension 72℃·4min, 30 cycles; third stage 72℃·5min; and finally, incubate at 12℃.

[0110] e. Prepare agarose gel for electrophoresis experiments, and the results are as follows: Figure 14 As shown.

[0111] Application Example 2

[0112] The difference from Application Example 1 is that in step a, the dTTP lyophilized powder obtained in Example 4 is formulated into a dTTP Mix with a final concentration of 2.5 mmol / L.

[0113] Application Example 3

[0114] The difference from Application Example 1 is that in step a, the dCTP lyophilized powder obtained in Example 5 is formulated into a dCTP Mix with a final concentration of 2.5 mmol / L.

[0115] Application Example 4

[0116] The difference from Application Example 1 is that in step a, the dGTP lyophilized powder obtained in Example 6 is formulated into a dGTP Mix with a final concentration of 2.5 mmol / L.

[0117] Application Example 5

[0118] The difference from Application Example 1 is that in step a, the dUTP lyophilized powder obtained in Example 7 is formulated into a dUTP Mix with a final concentration of 2.5 mmol / L.

[0119] The PCR band images obtained in Application Examples 2-5 above are similar to those in Application Example 1. From... Figure 14 As can be seen, the high-purity dNTP lyophilized powder prepared in this application produces clear bands when applied to PCR.

[0120] The embodiments described in this specific implementation are preferred embodiments of this application and are not intended to limit the scope of protection of this application. Therefore, all equivalent changes made in accordance with the structure, shape and principle of this application should be covered within the scope of protection of this application.

Claims

1. A process for preparing high-purity dNTP monomer lyophilized powder, characterized in that, Includes the following steps: Under normal pressure, the dNTP aqueous solution is pre-frozen at -10 to -40℃ for 3-5 hours to obtain pre-frozen crystals, and then vacuum freeze-dried at -30 to -40℃ for more than 20 hours. After that, nitrogen gas is introduced to restore normal pressure, and dNTP monomer freeze-dried powder with HPLC purity of not less than 99% and moisture content of 4.8-9.2% is obtained.

2. The preparation process of a high-purity dNTP monomer lyophilized powder according to claim 1, characterized in that, The specific temperature change during the low-temperature pre-freezing is as follows: First, pre-freeze the dNTP aqueous solution to -30 to -40°C for 1 to 1.5 hours, then raise the temperature to -10 to -20°C for 1 to 2 hours, and then lower the temperature to -30 to -40°C for 1 to 1.5 hours.

3. The preparation process of a high-purity dNTP monomer lyophilized powder according to claim 1, characterized in that: The vacuum degree of the vacuum freeze-drying is 0.15-0.20 mbar.

4. The preparation process of a high-purity dNTP monomer lyophilized powder according to claim 1, characterized in that: The vacuum freeze drying includes sublimation drying and desorption drying, wherein the sublimation drying time is 10-15 hours and the desorption drying time is 10-15 hours.

5. The preparation process of a high-purity dNTP monomer lyophilized powder according to claim 1, characterized in that: The concentration of the dNTP aqueous solution is 95-105 mmol / L.

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