CPG powder packing liquid for DNA synthesis and packing method

Abstract

The invention relates to the field of solid-phase DNA synthesis, in particular to a CPG powder packing liquid for DNA synthesis and a packing method. The CPG powder column packing liquid for DNA synthesis provided by the invention is composed of acetonitrile and other organic solvents. The column packing method comprises the following steps: (1) pouring CPG powder to be packed into a beaker, and adding CPG powder column packing liquid into the beaker for dissolving; (2) stirring at a low speed by using a magnetic stirrer or a stirring paddle to enable solid and liquid to be uniform; (3) calculating the column packing volume according to the scale of the synthetic column and the CPG powder load, and packing the column according to the volume by using a pipette; and (4) washing the CPG powder column filled in batches with acetonitrile for 5 times, and drying in a drying oven for later use. Compared with the prior art, the device has the beneficial effects that the CPG powder type composite columns can be manufactured in batches, the packing efficiency of the composite columns is improved, the packing uniformity of the composite columns can be kept, and the batch-to-batch difference of the CPG powder type composite columns is reduced.

Description

Technical Field

[0001] The invention relates to the field of solid phase DNA synthesis, and in particular to a CPG powder column packing liquid for DNA synthesis and a column packing method. Background Art

[0002] DNA synthesis basically adopts the solid phase phosphoramidite triester method, which is a synthesis technique performed on an insoluble solid phase support. It gradually constructs phosphoramidite monomers through repeated deprotection, coupling, oxidation and capping steps. This method can efficiently synthesize long-chain oligonucleotides with high yield and purity.

[0003] There are two types of existing solid phase carriers for DNA synthesis: the first is to use ultra-high molecular sintering technology to sinter CPG powder in the filter membrane, but because the process involves high temperature and other processes, it is often used to make ordinary synthesis columns without modification, and the process is mostly customized and developed by professional companies, and the cost is relatively high. The second is to load the filter membrane up and down, and load CPG powder in the middle of the filter membrane. The existing CPG powder filling method generally uses dry powder filling, but this method is cumbersome to operate and relies heavily on manpower to complete. And because CPG is in a dry powder state, it is easy to remain and difficult to quantify during packaging. There are obvious differences between individuals in the same batch or different batches, which affects the subsequent DNA synthesis yield and quality. Summary of the invention

[0004] In view of this, the present invention provides a CPG powder column packing liquid and a column packing method for DNA synthesis, which can be used to batch produce CPG powder synthetic columns, improve the efficiency of synthetic column packing, maintain the uniformity of synthetic column packing, and reduce the batch-to-batch differences of CPG powder synthetic columns.

[0005] In order to achieve the above-mentioned object of the invention, the present invention provides the following technical solutions:

[0006] The invention provides application of diiodomethane or dibromomethane in CPG powder column packing.

[0007] The present invention also provides a CPG powder column liquid, which is composed of acetonitrile and any of the following items:

[0008] (i) diiodomethane;

[0009] (ii) dibromomethane;

[0010] Wherein, the volume percentage of acetonitrile is 0%, 2%, 4%, 6%, 8%, 10%, 12%, 14%, 16%, 18% or 20%.

[0011] In some specific embodiments of the present invention, the CPG powder column loading liquid is composed of acetonitrile and dibromomethane, wherein the volume percentage of acetonitrile is 0%, 2%, 4%, 6%, 8%, 10%, 12%, 14%, 16%, 18% or 20%.

[0012] In some specific embodiments of the present invention, the volume percentage of acetonitrile in the CPG powder column liquid is 0%.

[0013] In some specific embodiments of the present invention, the volume percentage of acetonitrile in the CPG powder column liquid is 10%.

[0014] In some specific embodiments of the present invention, the volume percentage of acetonitrile in the CPG powder column liquid is 20%.

[0015] The present invention also provides a method for CPG powder column packing, comprising: column packing based on the CPG powder column packing liquid.

[0016] In some specific embodiments of the present invention, the above method comprises the following steps:

[0017] S1: Mix the CPG powder to be loaded into the column with the CPG powder loading liquid, and stir at a low speed to make the solid and liquid uniform to obtain a suspension;

[0018] S2: Calculate the column packing volume according to the scale of the synthetic column and the CPG powder loading capacity, and use a pipette to pack the suspension into the column according to the column packing volume to obtain a packed CPG powder column;

[0019] S3: The loaded CPG powder column is rinsed with acetonitrile and dried to complete the CPG powder column loading.

[0020] In some specific embodiments of the present invention, the column packing method of the above method includes: taking a synthetic column and adding a lower membrane; taking a CPG powder suspension and adding it to the synthetic column according to an appropriate volume to allow the liquid to flow out naturally; adding an upper membrane to fix the CPG powder between the two membranes; adding acetonitrile to the packed synthetic column to rinse it multiple times, and drying it for later use.

[0021] In some specific embodiments of the present invention, the above method comprises the following steps:

[0022] S1: Mix the CPG powder to be loaded into the column with the CPG powder loading liquid and stir to obtain a suspension;

[0023] S2: taking a synthetic column, setting a lower membrane, and obtaining a synthetic column with a lower membrane set;

[0024] S3: Calculate the column packing volume, add the suspension into the synthetic column with the lower membrane set according to the column packing volume, set the upper membrane, and obtain a synthetic column filled with CPG powder;

[0025] S4: The synthetic column loaded with CPG powder is rinsed with acetonitrile and dried to complete the CPG powder column loading.

[0026] In some specific embodiments of the present invention, the calculation of the above method is based on the following formula: column packing volume = column packing scale / CPG powder loading to be packed / CPG powder mass to be packed*CPG powder column packing liquid volume.

[0027] In some specific embodiments of the present invention, in the above method, every 30 mL of the CPG powder column loading liquid contains 0.1 g, 0.2 g, 0.3 g, 0.4 g, 0.5 g, 0.6 g, 0.7 g, 0.8 g, 0.9 g, 1 g, 1.1 g, 1.2 g, 1.3 g, 1.4 g, 1.5 g, 1.6 g, 1.7 g, 1.8 g, 1.9 g, 2 g, 3 g, 4 g, 5 g, 6 g, 7 g, 8 g, 9 g or 10 g of the CPG powder to be loaded.

[0028] The present invention also provides a CPG powder suspension, comprising CPG powder and the CPG powder column loading liquid.

[0029] In some specific embodiments of the present invention, each 30 mL of the CPG powder suspension contains 0.1 g, 0.2 g, 0.3 g, 0.4 g, 0.5 g, 0.6 g, 0.7 g, 0.8 g, 0.9 g, 1 g, 1.1 g, 1.2 g, 1.3 g, 1.4 g, 1.5 g, 1.6 g, 1.7 g, 1.8 g, 1.9 g, 2 g, 3 g, 4 g, 5 g, 6 g, 7 g, 8 g, 9 g or 10 g of CPG powder.

[0030] Compared with the prior art, the present invention has the following beneficial effects:

[0031] The invention provides a CPG powder column filling liquid for DNA synthesis and a column filling method. The CPG powder column filling liquid with a reasonable ratio of acetonitrile and an organic solvent is prepared and batch-dissolved and stirred, so that accurate quantification and rapid filling of the CPG powder can be achieved; the operation is simple, the dependence on personnel is greatly reduced, and the production quality and efficiency are improved. BRIEF DESCRIPTION OF THE DRAWINGS

[0032] In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings required for use in the embodiments or the description of the prior art are briefly introduced below.

[0033] Figure 1 1. An electron microscope image of a solid phase CPG powder column packed according to the method shown in Example 2 is shown;

[0034] Figure 2 The electron microscope image of the solid phase CPG powder column loaded according to the method shown in Comparative Example 1 is shown. DETAILED DESCRIPTION

[0035] The present invention discloses a CPG powder column packing liquid and column packing method for DNA synthesis. Those skilled in the art can refer to the content of this article and appropriately improve the process parameters to achieve it. It should be particularly noted that all similar replacements and modifications are obvious to those skilled in the art, and they are all considered to be included in the present invention. The method and application of the present invention have been described through preferred embodiments, and relevant personnel can obviously modify or appropriately change and combine the methods and applications described herein without departing from the content, spirit and scope of the present invention to implement and apply the technology of the present invention.

[0036] The CPG powder column-filling liquid for DNA synthesis provided by the invention is composed of acetonitrile and other organic solvents.

[0037] Furthermore, the volume percentage of the other solvent is 80% to 100%, and the other solvent is selected from one or more of diiodomethane or dibromomethane, more preferably dibromomethane.

[0038] Furthermore, the organic solvent is acetonitrile.

[0039] The CPG powder column packing method for DNA synthesis provided by the present invention comprises: (1) pouring the CPG powder to be packed into a beaker, and adding the CPG powder column packing liquid into the beaker to dissolve it; (2) using a magnetic stirrer or a stirring paddle to stir at a low speed to make the solid and liquid uniform; (3) calculating the column packing volume according to the scale of the synthesis column and the CPG powder loading, and using a pipette to pack the column according to the volume; (4) washing the CPG powder column filled in batches with acetonitrile for 5 times, drying it in an oven and setting it aside.

[0040] Further, the steps are as follows:

[0041] Step 1: Pour the CPG powder to be loaded into a beaker, and add the CPG powder loading solution into the beaker;

[0042] Step 2: Use a stirring paddle to stir at a low speed to make the solid and liquid uniform;

[0043] Step 3: Calculate the column volume according to the scale of the synthetic column and the CPG powder loading capacity, and use a pipette to load the column according to the volume;

[0044] Step 4: Rinse the batch-filled CPG powder column with acetonitrile for 5 times, and then dry it in an oven for later use.

[0045] Furthermore, in step 4, the washing liquid is acetonitrile, and the washing times are 3 to 10 times.

[0046] It should be understood that the expression "one or more of..." includes each of the items recited after the expression individually and in various different combinations of two or more of the recited items, unless otherwise understood from the context and usage. The expression "and / or" in combination with three or more recited items should be understood to have the same meaning, unless otherwise understood from the context.

[0047] The use of the terms "comprising", "having" or "containing", including their grammatical synonyms, should generally be understood as open and non-restrictive, for example not excluding other unrecited elements or steps, unless otherwise specifically stated or otherwise understood from the context.

[0048] It should be understood that the order of steps or the order in which certain actions are performed is not important as long as the application is still operable. In addition, two or more steps or actions can be performed simultaneously.

[0049] The use of any and all examples or exemplary language such as "for example" or "including" herein is intended only to better illustrate the present application and is not intended to limit the scope of the present application. No language in this specification should be construed as indicating that any unclaimed element is essential to the practice of the present application.

[0050] In addition, the numerical ranges and parameters used to define the present application are approximate values, and the relevant values ​​in the specific embodiments have been presented as accurately as possible. However, any numerical value inherently inevitably contains standard deviations due to individual test methods. Therefore, unless otherwise expressly stated, it should be understood that all ranges, quantities, values ​​and percentages used in this disclosure are modified by "about". Here, "about" generally refers to the actual value within plus or minus 10%, 5%, 1% or 0.5% of a specific value or range.

[0051] The raw materials involved in the embodiments of the present invention are described as follows:

[0052] The crude solid phase DNA was synthesized by Dr.Oligo 48 DNA synthesizer. The synthesis column was a general synthesis column, the upper membrane of the synthesis column was a large sand core, and the lower membrane of the synthesis column was a hard membrane. Brand: Hebei Dinaxingke Biotechnology Co., Ltd. The CPG powder loading was 33 μmol / g, the pore size was 1000 A, and the brand was Glen Research.

[0053] Unless otherwise specified, the raw materials, reagents, consumables and instruments involved in the present invention are all common commercial products and can be purchased from the market.

[0054] The present invention will be further described below in conjunction with the embodiments.

[0055] Comparative Example 1

[0056] Calculate the packaging mass according to the column scale and CPG powder loading, packaging mass = column scale / CPG powder loading to be packaged. Rinse the loaded CPG powder column with acetonitrile 5 times and use it after drying in an oven. Install the synthetic column loaded by the above method on the synthesizer as required for synthesis. After the synthesis is completed, use fresh ammonia water for aminolysis. After aminolysis, use a vacuum concentrator to dry it into powder for use. After the dried powder is re-dissolved with TE, the concentration is measured using a micro-UV instrument to determine the DNA sample yield.

[0057] Example 1

[0058] This embodiment provides a CPG powder column liquid for DNA synthesis, the specific components of which are as follows:

[0059] Acetonitrile 20% (v / v);

[0060] Diiodomethane 80% (v / v).

[0061] This embodiment also provides a method for column packing using the CPG powder column packing liquid for DNA synthesis, and the specific operations are as follows:

[0062] ① Take an appropriate amount of CPG powder and add it to the above CPG powder column liquid, and transfer the CPG powder to a beaker;

[0063] ② Use a magnetic stirrer to stir until the CPG powder is in a dispersed state;

[0064] ③ Calculate the filling volume according to the column scale and CPG powder loading capacity, filling volume = column filling scale / CPG powder loading capacity to be filled / CPG powder mass to be filled*CPG powder column filling liquid volume, use a pipette to quantitatively fill the CPG powder suspension. Taking the raw materials used in the present invention as an example (CPG powder loading capacity is 33 μmol / g, pore size 1000 A, brand: Glen Research), 30 mL column filling liquid is used to dissolve every 1 g of CPG powder, and the filling volume of the 0.2 μmol synthetic column is calculated as follows: filling volume = 0.2 μmol÷33 μmol / g÷1 g×30 ml≈0.18 mL=180 μL;

[0065] ④ Rinse the batch-filled CPG powder column with acetonitrile 5 times, dry it in an oven and set it aside.

[0066] The synthetic column filled by the above method is installed on the synthesizer as required for synthesis. After the synthesis is completed, fresh ammonia water is used for aminolysis. After aminolysis, a vacuum concentrator is used to dry it into powder for standby use. The dried powder is re-dissolved with TE, and the concentration is measured with a micro-UV instrument to determine the DNA sample yield.

[0067] Example 2

[0068] This embodiment provides a CPG powder column liquid for DNA synthesis, the specific components of which are as follows:

[0069] Acetonitrile 10% (v / v);

[0070] Dibromomethane 90% (v / v).

[0071] This embodiment also provides a method for column packing using the CPG powder column packing liquid for DNA synthesis, and the specific operations are as follows:

[0072] ① Take an appropriate amount of CPG powder and add it to the above CPG powder column liquid, and transfer the CPG powder to a beaker;

[0073] ② Use a magnetic stirrer to stir until the CPG powder is in a dispersed state;

[0074] ③ Calculate the filling volume according to the column scale and CPG powder loading capacity, filling volume = column filling scale / CPG powder loading capacity to be filled / CPG powder mass to be filled*CPG powder column filling liquid volume, use a pipette to quantitatively fill the CPG powder suspension. Taking the raw materials used in the present invention as an example (CPG powder loading capacity is 33 μmol / g, pore size 1000 A, brand: Glen Research), 30 mL column filling liquid is used to dissolve every 1 g of CPG powder, and the filling volume of the 0.2 μmol synthetic column is calculated as follows: filling volume = 0.2 μmol÷33 μmol / g÷1 g×30 ml≈0.18 mL=180 μL;

[0075] ④ Rinse the batch-filled CPG powder column with acetonitrile 5 times, dry it in an oven and set it aside.

[0076] The synthetic column filled by the above method is installed on the synthesizer as required for synthesis. After the synthesis is completed, fresh ammonia water is used for aminolysis. After aminolysis, a vacuum concentrator is used to dry it into powder for standby use. The dried powder is re-dissolved with TE, and the concentration is measured with a micro-UV instrument to determine the DNA sample yield.

[0077] Example 3

[0078] This embodiment provides a CPG powder column liquid for DNA synthesis, the specific components of which are as follows:

[0079] Dibromomethane 100% (v / v).

[0080] This embodiment also provides a method for column packing using the CPG powder column packing liquid for DNA synthesis, and the specific operations are as follows:

[0081] ① Take an appropriate amount of CPG powder and add it to the above CPG powder column liquid, and transfer the CPG powder to a beaker;

[0082] ② Use a magnetic stirrer to stir until the CPG powder is in a dispersed state;

[0083] ③ Calculate the filling volume according to the column scale and CPG powder loading capacity, filling volume = column filling scale / CPG powder loading capacity to be filled / CPG powder mass to be filled*CPG powder column filling liquid volume; use a pipette to quantitatively fill the CPG powder suspension. Taking the raw materials used in the present invention as an example (CPG powder loading capacity is 33 μmol / g, pore size 1000 A, brand: Glen Research), 30 ml column filling liquid is used to dissolve every 1 g of CPG powder, and the filling volume of the 0.2 μmol synthetic column is calculated as follows: filling volume = 0.2 μmol÷33 μmol / g÷1 g×30 mL≈0.18 mL=180 μL;

[0084] ④ Rinse the batch-filled CPG powder column with acetonitrile 5 times, dry it in an oven and set it aside.

[0085] The synthetic column filled by the above method is installed on the synthesizer as required for synthesis. After the synthesis is completed, fresh ammonia water is used for aminolysis. After aminolysis, a vacuum concentrator is used to dry it into powder for standby use. The dried powder is re-dissolved with TE, and the concentration is measured with a micro-UV instrument to determine the DNA sample yield.

[0086] Comparative Example 2

[0087] This embodiment provides a CPG powder column liquid for DNA synthesis, the specific components of which are as follows:

[0088] Acetonitrile 100% (v / v).

[0089] This embodiment also provides a method for column packing using the CPG powder column packing liquid for DNA synthesis, and the specific operations are as follows:

[0090] ① Take an appropriate amount of CPG powder and add it to the above CPG powder column liquid, and transfer the CPG powder to a beaker;

[0091] ② Use a magnetic stirrer to stir until the CPG powder is in a dispersed state;

[0092] ③ Calculate the filling volume according to the column scale and CPG powder loading capacity, filling volume = column filling scale / CPG powder loading capacity to be filled / CPG powder mass to be filled*CPG powder column filling liquid volume, use a pipette to quantitatively fill the CPG powder suspension. Taking the raw materials used in the present invention as an example (CPG powder loading capacity is 33 μmol / g, pore size 1000 A, brand: Glen Research), 30 mL column filling liquid is used to dissolve every 1 g of CPG powder, and the filling volume of the 0.2 μmol synthetic column is calculated as follows: filling volume = 0.2 μmol÷33 μmol / g÷1 g×30 mL≈0.18 mL=180 uL;

[0093] ④ Rinse the batch-filled CPG powder column with acetonitrile 5 times, dry it in an oven and set it aside.

[0094] The synthetic column filled with the above method is installed on the synthesizer as required for synthesis. After the synthesis is completed, fresh ammonia water is used for aminolysis. After aminolysis, it is dried into powder using a vacuum concentrator and dryer for standby use. The dried DNA sample is re-dissolved with 200 μL of TE solution, and the concentration is measured using a micro-UV instrument to determine the DNA sample yield.

[0095] Effect example

[0096] The test results of the DNA sample yields of Examples 1-3 and Comparative Examples 1-2 are shown in Table 1.

[0097] Table 1

[0098]

[0099] As can be seen from Table 1, compared with Comparative Example 1, the DNA sample yield of Examples 1 to 3 is not affected, and the intra-batch consistency is good. Compared with Comparative Example 2, the DNA sample yield of Examples 1 to 3 is significantly reduced, and the HPLC purity is also slightly reduced.

[0100] The CPG powders of Example 2 and Comparative Example 1 were taken out again and observed using a scanning electron microscope. Figure 1 As shown, through the 100,000 times electron microscopic scanning image, it can be observed that the CPG powder in Example 2 is relatively uniform ( Figure 1 ), while the CPG powder in Comparative Example 1 agglomerated ( Figure 2 ). Analysis suggests that the CPG powder may be broken due to inappropriate column loading liquid, which may cause some pores to be blocked, resulting in poor reagent flow and incomplete reaction, ultimately affecting the overall yield and purity of the DNA sample.

[0101] In summary, through experimental comparison, the newly developed CPG powder column packing method for DNA synthesis has some advantages over the commonly used column packing method: first, the CPG powder column packing liquid described in this method will not cause CPG to break and affect the final yield; second, the column packing method described in this method can achieve quantitative column packing of CPG powder, with good batch consistency, simple operation and high efficiency. Therefore, the column packing method provided by the present invention is a very effective CPG powder filling method, which can be used in laboratories and large-scale oligonucleotide production.

[0102] The above is only a preferred embodiment of the present invention. It should be pointed out that for ordinary technicians in this technical field, several improvements and modifications can be made without departing from the principle of the present invention. These improvements and modifications should also be regarded as the scope of protection of the present invention.

Claims

1. Application of diiodomethane or dibromomethane in CPG powder column packing. 2.CPG powder column liquid, characterized in that: Consists of acetonitrile and any of: (i) diiodomethane; (ii) dibromomethane; Wherein, the volume percentage of acetonitrile is 0%~20%.

3. The CPG powder column liquid as claimed in claim 2, characterized in that: It is composed of acetonitrile and dibromomethane, wherein the volume percentage of acetonitrile is 0% to 20%.

4. The CPG powder column liquid as claimed in claim 3, characterized in that: The volume percentage of acetonitrile is 0%.

5. The CPG powder column liquid as claimed in claim 3, characterized in that: The volume percentage of acetonitrile is 10%.

6. The CPG powder column liquid as claimed in claim 3, characterized in that: The volume percentage of acetonitrile is 20%.

7. A method for packing CPG powder into a column, characterized in that: include: Column packing based on the CPG powder column packing liquid described in any one of claims 2 to 6.

8. The method according to claim 7, characterized in that The steps include: S1: mixing the CPG powder to be loaded into the column with the CPG powder loading liquid according to any one of claims 2 to 6, and stirring to obtain a suspension; S2: Calculate the column packing volume, and pack the suspension into the column according to the column packing volume to obtain a packed CPG powder column; S3: The loaded CPG powder column is rinsed with acetonitrile and dried to complete the CPG powder column loading.

9. The method according to claim 8, characterized in that The calculation is based on the following formula: column packing volume = column packing scale / CPG powder loading to be packed / CPG powder mass to be packed*CPG powder column packing liquid volume. 10.CPG powder suspension, characterized in that: It comprises CPG powder and the CPG powder column-packing liquid according to any one of claims 2 to 6.

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