A variable volume synthesis column and a synthesis system using the same

Abstract

The application discloses a variable-volume synthetic column and a synthetic system using the same. The synthetic column comprises a hollow column body, a fixed seat, an upper end cover and a hollow connecting column are arranged on the upper part of the column body, the bottom end of the hollow connecting column extends into the inside of the column body and is connected with a piston, and a lower plug is arranged on the lower end of the column body; wherein the piston is penetrated by a vertical first through hole, the lower plug is penetrated by a vertical second through hole, a hydraulic cavity is formed between the upper end cover and the piston, and a bottom outlet is arranged on the lower end of the lower plug. The variable-volume synthetic column can make the total amount of monomers and activators in a single step cycle not less than the sum of the pipeline and the residual volume of the column, can make the single step cycle be realized through one-step coupling, thereby reducing the synthesis time, improving the synthesis efficiency, and ensuring the stability of the quality of the synthetic product.

Description

Technical Field

[0001] The present invention relates to the field of biotechnology, in particular to a variable volume synthesis column and a synthesis system using the same. Background Art

[0002] In DNA synthesis technology, the synthesized fragments are often expressed in different ways, such as oligonucleotides, PCR primers, adapters, probes, short-chain primers, and long-chain primers. In the laboratory, DNA synthesis is generally completed using equipment and tools such as DNA synthesizers and DNA synthesis columns. The DNA synthesis process is briefly described as follows:

[0003] In the first step, the nucleotide with protected active group pre-connected to the solid support is reacted with trichloroacetic acid to remove the DMT protecting group of the 5′-hydroxyl group and obtain a free 5′-hydroxyl group.

[0004] In the second step, the raw materials for synthesizing DNA are protected by phosphoramidite-protected nucleotide monomers, which are mixed with the activator tetrazolium to obtain a nucleoside phosphite-activated intermediate, the 3' end of which is activated and the 5'-hydroxyl group is still protected by DMT, thereby undergoing a condensation reaction with the free 5'-hydroxyl group in the solution.

[0005] The third step is the capping reaction. There may be very few 5′-hydroxyl groups that do not participate in the condensation reaction. Acetic anhydride and 1-methylimidazole are used to terminate the reaction and then continue the reaction. This short fragment can be separated by purification in the subsequent steps according to experimental needs.

[0006] In the fourth step, under the action of the oxidant iodine, the phosphite form is transformed into a more stable phosphate triester.

[0007] The existing synthesis column consists of a column body and a solid-phase carrier. The column tube is a tubular piece with a gradually narrowing inner diameter, and the solid-phase carrier is fixed in the inner cavity of the column tube. Its disadvantages are: 1. The fixed solid-phase carrier divides the inner cavity into two, and the volume of the synthesis cavity is fixed. When the DNA raw material is added in the second step of DNA synthesis, if the total amount of DNA raw material and activator in the pipeline exceeds the sum of the residual volume of the column, it will be discharged into the waste liquid pipeline. Therefore, multi-step coupling is required to achieve, which greatly increases the time of DNA synthesis; 2. When the DNA raw material in the single-step cycle is added to the synthesis column, if the reaction is insufficient, a capping reaction is required. At the same time, the raw material will directly enter the waste liquid collection pipeline through the outlet, wasting expensive raw materials and reagents and failing to obtain good product quality; 3. The inner wall of the column tube is designed with an inclined design, with the inner diameter of the assembly position being larger at the top and smaller at the bottom. It will also shrink during sintering, resulting in large batch-to-batch variability. As a result, the degree of column tube backlog during assembly is also different, resulting in different product tightness and uniformity, and unstable product quality. Summary of the Invention

[0008] To solve the above problems, the present invention provides a variable volume synthesis column and a synthesis system using the same.

[0009] According to one aspect of the present invention, a variable volume synthetic column is provided, comprising a hollow column, wherein a fixing seat, an upper end cover and a hollow connecting column are installed above the column, the bottom end of the hollow connecting column extends into the interior of the column and is connected to a piston, and a lower plug is installed at the lower end of the column; wherein the piston is penetrated by a vertical first through hole, and the lower plug is penetrated by a vertical second through hole, a hydraulic chamber is formed between the upper end cover and the piston, and a bottom outlet is provided at the lower end of the lower plug.

[0010] In some embodiments, the fixing base is fixedly mounted on the column, the upper end cover is detachably mounted on the fixing base, and the hollow connecting column is mounted on the upper end cover. Thus, the specific installation method of the fixing base, the upper end cover, and the hollow connecting column is described.

[0011] In some embodiments, the fixing seat and the upper end cover are both hollow convex structures. Thus, the specific structures of the fixing seat and the upper end cover are described.

[0012] In some embodiments, an upper sieve plate is mounted on the lower end of the piston, and a lower sieve plate is mounted on the upper end of the lower plug, with a synthesis chamber formed between the upper and lower sieve plates. Thus, the specific structure of the synthesis column is further described.

[0013] In some embodiments, the bottom of the column is mounted on a base, and the base is provided with an outlet channel, and the outlet channel is connected to the bottom outlet, thereby enabling the column to be stably placed through the base.

[0014] In some embodiments, the fixing seat and the base are connected via a support column, thereby improving the stability of the composite column structure.

[0015] In some embodiments, a first inlet and a second inlet are provided on the outside of the fixing seat, and both the first inlet and the second inlet are connected to the hydraulic chamber. Thus, hydraulic oil can be introduced into the hydraulic chamber through the first inlet and the second inlet to adjust the hydraulic pressure.

[0016] In some embodiments, sealing rings are provided between the column and the fixing base, between the fixing base and the upper end cap, between the upper end cap and the hollow connecting column, between the hollow connecting column and the piston, between the piston and the column, and between the lower plug and the column. Thus, the synthetic column can be properly sealed by providing each gasket.

[0017] According to one aspect of the present invention, a synthesis system of the above-mentioned variable volume synthesis column is provided, which includes an inlet main pipeline, a first pipeline, a second pipeline, an outlet main pipeline, a fourth pipeline and a fifth pipeline; wherein, one end of the inlet main pipeline is connected to the hollow connecting column, and the other end is connected to the first pipeline and the second pipeline respectively; the outlet main pipeline is connected to the bottom outlet; one end of the fourth pipeline and the fifth pipeline are both connected to the hydraulic chamber, and the other end is connected to an oil tank.

[0018] In some embodiments, a first pump is provided on the first pipeline, and a second pump is provided on the second pipeline, so that the first pump and the second pump can provide power to the first pipeline and the second pipeline respectively.

[0019] In some embodiments, the first pump and the second pump are both peristaltic pumps. Thus, the types of the first pump and the second pump are set.

[0020] In some embodiments, a first valve is provided on the outlet main pipeline, thereby enabling the outlet main pipeline to be opened and closed by the first valve.

[0021] In some embodiments, a third pump and a pressure sensor are provided on the fourth pipeline, thereby enabling the flow of the hydraulic oil in the fourth pipeline to be controlled and detected by the third pump and the pressure sensor.

[0022] In some embodiments, a second valve is provided on the fifth pipeline, thereby enabling the outlet main pipeline to be opened and closed by providing the second valve.

[0023] In some embodiments, the second pipe is connected to the c-terminal of a two-position, four-way electric valve, and the outlet main pipe is connected to the a-terminal of the two-position, four-way electric valve. The d-terminal of the two-position, four-way electric valve is connected to a sixth pipe, and the b-terminal of the two-position, four-way electric valve is connected to a third pipe. Thus, the two-position, four-way electric valve can be positioned to facilitate the synthesis process.

[0024] In some embodiments, the third pipe is provided with an ultraviolet sensor, a pH sensor, and a conductivity temperature sensor, so that various parameters of the fluid passing through the third pipe can be detected by providing the ultraviolet sensor, the pH sensor, the conductivity temperature sensor, etc.

[0025] The variable-volume synthesis column and the synthesis system using the same in the present invention can ensure that the total amount of monomers and activators in a single-step cycle is not less than the sum of the residual volumes of the pipeline and the column, and can realize a single-step cycle through one-step coupling, thereby reducing the synthesis time, improving the synthesis efficiency, and ensuring the stability of the quality of the synthesized product. BRIEF DESCRIPTION OF THE DRAWINGS

[0026] Figure 1 This is a schematic structural diagram of a variable volume synthesis column according to one embodiment of the present invention;

[0027] Figure 2 for Figure 1 A cross-sectional view of the variable volume synthesis column shown;

[0028] Figure 3 For an application Figure 1 A schematic diagram of an embodiment of a synthesis system for a variable volume synthesis column is shown;

[0029] Figure 4 For another application Figure 1 Schematic diagram of an embodiment of a synthesis system showing a variable volume synthesis column.

[0030] In the figure: column 1, fixed base 2, upper end cover 3, hollow connecting column 4, piston 5, upper sieve plate 6, first through hole 7, lower plug 8, second through hole 9, lower sieve plate 10, bottom outlet 11, base 12, outlet channel 13, first inlet and outlet 14, second inlet and outlet 15, support column 16, sealing ring 17, synthesis chamber 18, hydraulic chamber 19, inlet main pipe 21, 31, first pipe 22, 32, second pipe 23, 33, first pump 24, 34 , second pump 25, 35, outlet main pipeline 26, 36, first valve 27, 37, fourth pipeline 28, 38, fifth pipeline 29, 39, third pump 210, 310, oil tank 211, 311, pressure sensor 212, 312, second valve 213, 313, sixth pipeline 314, third pipeline 315, UV sensor 316, pH sensor 317, conductivity temperature sensor 318, two-position four-way electric valve 319. DETAILED DESCRIPTION

[0031] In an embodiment of the present invention, the monomer is a modified deoxyribonucleoside triphosphate with a 3'-O-reversible protecting group, namely, a phosphoramidite-protected nucleotide monomer, or a phosphoramidite-protected deoxynucleotide monomer.

[0032] The solid phase carrier is generally controlled pore glass beads (CPG), to which nucleotides with protected active groups are attached.

[0033] The different reagents include:

[0034] Trichloroacetic acid, which is used to remove the protecting group DMT of its 5′-hydroxyl group to obtain the free 5′-hydroxyl group;

[0035] An example of an activating agent is a tetrazole mixture, which can activate the 3′ end of the monomer while the 5′-hydroxyl group is still protected by DMT;

[0036] iodine oxidation, by which the phosphite form can be converted into the more stable phosphotriester;

[0037] Ammonia is used to cut off the primer connected to CPG.

[0038] The present invention will be further described in detail below with reference to the accompanying drawings.

[0039] Figure 1 The structure of a variable volume synthesis column according to one embodiment of the present invention is schematically shown. Figure 2 Shows Figure 1 The cross-sectional structure of the variable volume synthetic column in . Figure 1-2 As shown, the synthesis column comprises a column 1, which is hollow inside and filled with a solid phase carrier (not shown in the figure). A fixing base 2, an upper end cap 3, and a hollow connecting column 4 are mounted above the column 1. The fixing base 2, upper end cap 3, and hollow connecting column 4 can jointly seal the upper end of the column 1.

[0040] Among them, the fixing seat 2 is a hollow convex structure, which is fixedly connected to the column 1 through threads; the upper end cover 3 is also a hollow convex structure, which is detachably mounted on the fixing seat 2 and is connected to the fixing seat 2 through threads; the hollow connecting column 4 is installed on the upper end cover 3, and its lower end passes through the upper end cover 3 and the fixing seat 2 and extends into the interior of the column 1.

[0041] The lower end of the hollow connecting column 4 is connected to a piston 5. The piston 5 has a convex structure and can slide up and down along the inner wall of the column 1. The lower end of the piston 5 is fixedly mounted with an upper screen plate 6. The piston 5 is provided with a vertical first through hole 7. The first through hole 7 passes through the upper and lower ends of the piston 5 and is connected to the hollow connecting column 4.

[0042] A lower plug 8 is fixedly mounted at the lower end of the column 1 and is threadedly connected to the inner wall of the column 1. A lower sieve plate 10 is also fixedly mounted at the upper end of the lower plug 8. The lower sieve plate 10 is located within the column 1, and a bottom outlet 11 is provided at the lower end of the lower plug 8. A second vertical through hole 9 is also provided in the lower plug 8, extending through the upper and lower ends of the plug 8 and communicating with the bottom outlet 11.

[0043] The bottom of the column 1 is mounted on a base 12 , and an outlet channel 13 is provided on the base 12 , and the outlet channel 13 is communicated with a bottom outlet 11 .

[0044] The inner cavity of the column 1 , specifically the hollow portion between the upper sieve plate 6 and the lower sieve plate 10 , is called a synthesis cavity 18 .

[0045] A first inlet and outlet 14 and a second inlet and outlet 15 are provided on the outside of the fixing seat 2 , which are respectively connected to a portion of the cavity of the column 1 between the upper end cover 3 and the piston 5 , which is called a hydraulic cavity 19 .

[0046] The fixing seat 2 and the base 12 are further connected via a support column 16 to prevent the fixing seat 2 from moving.

[0047] In addition, sealing rings 17 are provided between the column 1 and the fixing seat 2, between the fixing seat 2 and the upper end cover 3, between the upper end cover 3 and the hollow connecting column 4, between the hollow connecting column 4 and the piston 5, between the piston 5 and the inner wall of the column 1, and between the lower plug 8 and the inner wall of the column 1 to ensure the airtightness between the various components.

[0048] Figure 3 One of the applications is shown Figure 1 The structure of an embodiment of a synthesis system of a variable volume synthesis column. Figure 3 As shown, in this synthesis system, the upper end of the hollow connecting column 4 of the synthesis column is connected to one end of an inlet main pipeline 21, while the other end of the inlet main pipeline 21 is connected to the outlet ends of a first pipeline 22 and a second pipeline 23, respectively. The first pipeline 22 is used to transport different monomers and is equipped with a first pump 24. The second pipeline 23 transports different reagents and is equipped with a second pump 25. Both the first pump 24 and the second pump 25 are peristaltic pumps.

[0049] The bottom outlet 11 of the synthesis column is connected to the inlet end of an outlet main pipeline 26 . The outlet main pipeline 26 is used to transport waste liquid, recovered liquid and synthesis product, and a first valve 27 is provided on the outlet main pipeline 26 .

[0050] The first inlet and outlet 14 of the synthesis column is connected to an oil tank 211 via a fourth pipe 28. A third pump 210 and a pressure sensor 212 are provided on the fourth pipe 28. The third pump 210 is a plunger pump, and the signal output terminal of the pressure sensor 212 is connected to the signal acquisition terminal of the third pump 210.

[0051] The second inlet and outlet 15 of the synthesis column is also connected to the oil tank 211 through a fifth pipe 29, and a second valve 213 is provided on the fifth pipe 29. The first valve 27 and the second valve 213 are both two-position two-way solenoid valves for controlling the flow or cutoff of the flow path.

[0052] The process of synthesizing DNA using this synthesis system is as follows.

[0053] In the first step, trichloroacetic acid is added.

[0054] Close first valve 27, open second valve 213, and start second pump 25, allowing trichloroacetic acid to flow through second conduit 23 and inlet main conduit 21 into the synthesis chamber 18 of the synthesis column. As trichloroacetic acid is added, piston 5 moves upward, and hydraulic oil enters oil tank 211 through fourth conduit 28. After the active group-protected nucleotides pre-attached to the solid support fully react with trichloroacetic acid, the DMT protecting group on the 5'-hydroxyl group is removed, releasing the free 5'-hydroxyl group.

[0055] Then, the second pump 25 and the second valve 213 are closed, and the first valve 27 and the third pump 210 are opened. The hydraulic oil then enters the hydraulic chamber 19 of the synthesis column through the fourth pipe 28. At this time, the piston 5 moves downward, and the reacted reagent is discharged and collected through the outlet main pipe 26.

[0056] The second step is to synthesize the raw materials of DNA.

[0057] Close first valve 27, open second valve 213, start second pump 25 and first pump 24, and introduce the phosphoramidite-protected nucleotide monomer and the activating agent tetrazolium through first conduit 22 and second conduit 23, respectively, and then through inlet main conduit 21 into the synthesis chamber 18 of the synthesis column. The phosphoramidite-protected nucleotide monomer is mixed with the activating agent tetrazolium to produce a nucleoside phosphorous acid-activated intermediate, whose 3'-end is activated and whose 5'-hydroxyl group remains protected by DMT. This intermediate then undergoes a condensation reaction with the free 5'-hydroxyl group on the solid support. A predetermined ratio of activator to monomer can be achieved by setting different flow rates for second pump 25 and first pump 24.

[0058] As the monomers and the activator tetrazole are added, piston 5 moves upward, and hydraulic oil enters tank 211 through fourth conduit 28. When the reaction is complete, first pump 24, second pump 25, and second valve 213 are closed, and first valve 27 and third pump 210 are opened. Hydraulic oil then flows through fourth conduit 28 into hydraulic chamber 19 of the synthesis column. Piston 5 then moves downward to its initial position, and the reacted reagents can be discharged and collected through outlet main conduit 26.

[0059] The third step is to add iodine oxide.

[0060] Close first valve 27, open second valve 213, and start second pump 25, allowing iodine oxide to flow through second conduit 23 and inlet main conduit 21 into synthesis chamber 18 of the synthesis column. As iodine oxide is added, piston 5 moves upward, and hydraulic oil enters oil tank 211 through fourth conduit 28. The iodine oxide then converts the phosphite produced in step 2 into a more stable phosphate triester.

[0061] Then, the second pump 25 and the second valve 213 are closed, and the first valve 27 and the third pump 210 are opened. The hydraulic oil then flows through the fourth pipe 28 into the hydraulic chamber 19 of the synthesis column. At this time, the piston 5 moves downward, and the reacted reagents are discharged and collected through the outlet main pipe 26.

[0062] After these three steps, a deoxynucleotide is attached to the primer on the solid support, and the DMT protecting group on its 5′-hydroxyl group is removed using trichloroacetic acid. The above steps are repeated until all the bases to be synthesized have been attached.

[0063] The fourth step is to introduce high-temperature ammonia water.

[0064] Close the first valve 27, open the second valve 213, start the second pump 25, and let the high-temperature ammonia water pass through the second pipe 23 and the inlet main pipe 21 into the synthesis chamber 18 of the synthesis column. With the addition of high-temperature ammonia water, the piston 5 moves upward, and the hydraulic oil enters the oil tank 211 through the fourth pipe 28. The high-temperature ammonia water then fully reacts with the primer on the solid phase carrier, and the primer connected to the solid phase carrier can be cut off.

[0065] Then close the second pump 25 and the second valve 213, open the first valve 27 and the third pump 210, and the hydraulic oil will enter the hydraulic chamber 19 of the synthesis column through the fourth pipe 28. At this time, the piston 5 moves downward, and the reacted reagents are discharged and collected through the outlet main pipe 26, and the synthesis products are collected at the same time.

[0066] Figure 4 Another application is shown Figure 1 The structure of another embodiment of the synthesis system of the variable volume synthesis column. Figure 4As shown, in this synthesis system, the upper end of the hollow connecting column 4 of the synthesis column is connected to one end of an inlet main pipeline 31, while the other end of the inlet main pipeline 31 is connected to the outlet ends of a first pipeline 32 and a second pipeline 33, respectively. First pipeline 32 is used to transport different monomers and is equipped with a first pump 34. Second pipeline 33 transports different reagents and is equipped with a second pump 35. Both first pump 34 and second pump 35 are peristaltic pumps.

[0067] The inlet end of the second pipe 33 is connected to the c end of a two-position four-way electric valve 319, and the d end of the two-position four-way electric valve is connected to the outlet end of a sixth pipe 314, and the inlet ends of the sixth pipe 314 are respectively connected to reagent bottles containing different reagents;

[0068] The bottom outlet 11 of the synthesis column is connected to the inlet end of an outlet main pipeline 36 . The outlet main pipeline 36 is used to transport waste liquid, recovered liquid and synthetic products, and a first valve 37 is provided on the outlet main pipeline 36 .

[0069] The outlet end of the outlet main pipe 36 is also connected to the a end of the two-position four-way electric valve 319, and the b end of the two-position four-way electric valve 319 is connected to the inlet end of a third pipe 315, and the outlet end of the third pipe 315 is connected to multiple different collection bottles.

[0070] The first position of the two-position four-way electric valve 319 is that the ends a and b are connected, and the ends c and d are connected; the second position is that the ends a and c are connected, and the ends b and d are connected.

[0071] In addition, the third pipe 315 is also provided with an ultraviolet sensor 316 , a pH sensor 317 and a conductivity temperature sensor 318 , which can be used to detect various parameters of the fluid passing through the third pipe 315 .

[0072] The first inlet and outlet 14 of the synthesis column is connected to an oil tank 311 via a fourth pipe 38. A third pump 310 and a pressure sensor 312 are provided on the fourth pipe 38. The third pump 310 is a plunger pump, and the signal output terminal of the pressure sensor 312 is connected to the signal acquisition terminal of the third pump 310.

[0073] The second inlet and outlet 15 of the synthesis column is also connected to the oil tank 311 through a fifth pipe 39, and a second valve 313 is provided on the fifth pipe 39. The first valve 37 and the second valve 313 are both two-position two-way solenoid valves for controlling the flow or cutoff of the flow path.

[0074] The process of synthesizing DNA using this synthesis system is as follows.

[0075] In the first step, trichloroacetic acid is added.

[0076] The two-position, four-way electric valve 319 is set to the first position, connecting terminals C and D, and terminals A and B. The first valve 37 is closed, the second valve 313 is opened, and the second pump 35 is started. Trichloroacetic acid is introduced into the synthesis column's synthesis chamber 18 via the sixth pipe 314 and the second pipe 33, and then via the inlet main pipe 31. As the trichloroacetic acid is added, the piston 5 moves upward, and hydraulic oil enters the oil tank 311 via the fourth pipe 38.

[0077] When a certain amount of trichloroacetic acid has been added, the two-position, four-way electric valve 319 is adjusted to the second position, connecting terminals C and A, and terminals D and B. Simultaneously, the second valve 313 is closed and the first valve 37 is opened. Trichloroacetic acid then circulates through the second pump 35 along the second conduit 33, the inlet main conduit 31, the synthesis chamber 18 of the synthesis column, and the outlet main conduit 36. After the active group-protected nucleotides pre-attached to the solid support fully react with trichloroacetic acid, the DMT protecting group on the 5'-hydroxyl group is removed, resulting in a free 5'-hydroxyl group.

[0078] Then, the second pump 35 is turned off, the two-position four-way electric valve 319 is adjusted to the first position, and the third pump 310 is turned on. The hydraulic oil will enter the hydraulic chamber 19 of the synthesis column through the fourth pipe 38. At this time, the piston 5 moves downward, and the reacted reagents are discharged and collected through the outlet main pipe 36 and the third pipe 315.

[0079] The second step is to synthesize the raw materials of DNA.

[0080] Close first valve 37, open second valve 313, start second pump 35 and first pump 34, adjust two-position four-way electric valve 319 to the first position, and then pass the phosphoramidite-protected nucleotide monomer through first conduit 32, the activator tetrazole through sixth conduit 314 and second conduit 33, and finally into the synthesis chamber 18 of the synthesis column through inlet main conduit 31. The phosphoramidite-protected nucleotide monomer is mixed with the activator tetrazole to produce a nucleoside phosphorous acid activated intermediate. Its 3' end is activated, and the 5'-hydroxyl group remains protected by DMT, which then undergoes a condensation reaction with the free 5'-hydroxyl group on the solid support. A predetermined ratio of activator to monomer can be achieved by setting different flow rates of second pump 35 and first pump 34.

[0081] As the monomers and the activating agent tetrazole are added, piston 5 moves upward, and hydraulic oil enters tank 311 through fourth conduit 38. When a certain amount of hydraulic oil is reached, first pump 34 and second valve 313 are closed, two-position four-way electric valve 319 is adjusted to the second position, and first valve 37 is opened. The activated nucleoside phosphite intermediate then circulates along second conduit 33, inlet main conduit 31, synthesis chamber 18 of the synthesis column, and outlet main conduit 36.

[0082] When the reaction is complete, the second pump 35 is turned off and the third pump 310 is turned on. The hydraulic oil then flows through the fourth pipe 38 into the hydraulic chamber 19 of the synthesis column. The piston 5 then moves down to its original position, and the reacted reagents can be discharged and collected through the outlet main pipe 36 and the third pipe 315.

[0083] The third step is to add iodine oxide.

[0084] Open the second valve 313, start the second pump 35, and adjust the two-position four-way electric valve 319 to the first position. Iodine oxide is then introduced through the sixth pipe 314 and the second pipe 33, and then through the inlet main pipe 31 into the synthesis chamber 18 of the synthesis column. As iodine oxide is added, the piston 5 moves upward, and hydraulic oil enters the oil tank 311 through the fourth pipe 38. When a certain amount of iodine oxide is introduced, the two-position four-way electric valve 319 is adjusted to the second position, while the second valve 313 is closed and the first valve 37 is opened. Iodine oxide is then circulated by the second pump 35 along the second pipe 33, the inlet main pipe 31, the synthesis chamber 18 of the synthesis column, and the outlet main pipe 36. Iodine oxide converts the phosphite in the second step into a more stable phosphate triester.

[0085] When the reaction is complete, the second pump 35 is closed, the two-position four-way electric valve 319 is adjusted to the first position, and the third pump 310 is opened. The hydraulic oil then flows through the fourth pipe 38 into the hydraulic chamber 19 of the synthesis column. At this time, the piston 5 moves downward, and the reacted reagents are discharged and collected through the outlet main pipe 36 and the third pipe 315.

[0086] After these three steps, a deoxynucleotide is attached to the primer on the solid support, and the DMT protecting group on its 5′-hydroxyl group is removed using trichloroacetic acid. The above steps are repeated until all the bases to be synthesized have been attached.

[0087] The fourth step is to introduce high-temperature ammonia water.

[0088] The two-position, four-way electric valve 319 is adjusted to the first position, the first valve 37 is closed, the second valve 313 is opened, and the second pump 35 is started. The high-temperature ammonia solution is introduced through the sixth pipe 314 and the second pipe 33, and then through the inlet main pipe 31 into the synthesis chamber 18 of the synthesis column. As the high-temperature ammonia solution is added, the piston 5 moves upward, and the hydraulic oil enters the oil tank 311 through the fourth pipe 38. When a certain amount of hydraulic oil is reached, the two-position, four-way electric valve 319 is adjusted to the second position, the second valve 313 is closed, and the first valve 37 is opened. The high-temperature ammonia solution is then circulated by the second pump 35 along the second pipe 33, the inlet main pipe 31, the synthesis chamber 18 of the synthesis column, and the outlet main pipe 36. The high-temperature ammonia solution then fully reacts with the primers on the solid support, allowing the primers attached to the solid support to be cleaved.

[0089] Then, the second pump 35 is closed, the two-position four-way electric valve 319 is adjusted to the first position, and the third pump 310 is opened. The hydraulic oil will enter the hydraulic chamber 19 of the synthesis column through the fourth pipe 38. At this time, the piston 5 moves downward, and the reacted reagents are discharged and collected through the outlet main pipe 36 and the third pipe 315. At the same time, the synthesis product is collected.

[0090] The above are only some embodiments of the present invention. For those skilled in the art, several modifications and improvements can be made without departing from the inventive concept of the present invention, which all fall within the scope of protection of the present invention.

Claims

1. A variable volume synthesis column, characterized in that: The invention comprises a hollow column (1), a fixing seat (2), an upper end cover (3) and a hollow connecting column (4) are installed above the column (1), the fixing seat (2) is fixedly installed on the column (1), the upper end cover (3) is detachably installed on the fixing seat (2), the hollow connecting column (4) is installed on the upper end cover (3), the bottom end of the hollow connecting column (4) extends into the interior of the column (1) and is connected to a piston (5), and a lower plug (8) is installed at the lower end of the column (1); wherein the piston (5) is penetrated by a vertical first through hole (7), and the lower plug (8) A vertical second through hole (9) runs through the piston (5), an upper sieve plate (6) is installed at the lower end of the piston (5), a lower sieve plate (10) is installed at the upper end of the lower plug (8), and a synthetic chamber (18) is formed between the upper sieve plate (6) and the lower sieve plate (10); a hydraulic chamber (19) is formed between the upper end cover (3) and the piston (5), a bottom outlet (11) is provided at the lower end of the lower plug (8), and a first inlet and outlet (14) and a second inlet and outlet (15) are provided on the outer side of the fixing seat (2), and the first inlet and outlet (14) and the second inlet and outlet (15) are both connected to the hydraulic chamber (19).

2. The variable volume synthesis column according to claim 1, characterized in that: The fixing seat (2) and the upper end cover (3) are both hollow convex structures.

3. The variable volume synthesis column according to claim 1, characterized in that: The bottom of the column (1) is mounted on a base (12), an outlet channel (13) is provided on the base (12), and the outlet channel (13) is communicated with the bottom outlet (11).

4. The variable volume synthesis column according to claim 3, characterized in that: The fixing seat (2) and the base (12) are connected via a support column (16).

5. The variable volume synthesis column according to claim 1, characterized in that: Sealing rings (17) are provided between the column (1) and the fixing seat (2), between the fixing seat (2) and the upper end cover (3), between the upper end cover (3) and the hollow connecting column (4), between the hollow connecting column (4) and the piston (5), between the piston (5) and the column (1), and between the lower plug (8) and the column (1).

6. A synthesis system using the variable volume synthesis column according to any one of claims 1 to 5, characterized in that: It includes an inlet main pipeline (21, 31), a first pipeline (22, 32), a second pipeline (23, 33), an outlet main pipeline (26, 36), a fourth pipeline (28, 38) and a fifth pipeline (29, 39); One end of the inlet main pipe (21, 31) is connected to the hollow connecting column (4), and the other end is connected to the first pipe (22, 32) and the second pipe (23, 33), respectively; a first pump (24, 34) is provided on the first pipe (22, 32), and a second pump (25, 35) is provided on the second pipe (23, 33); The outlet main pipe (26, 36) is connected to the bottom outlet (11), and a first valve (27, 37) is provided on the outlet main pipe (26, 36); One end of each of the fourth pipeline (28, 38) and the fifth pipeline (29, 39) is connected to the hydraulic chamber (19), and the other end is connected to an oil tank (211, 311). A second valve (213, 313) is provided on the fifth pipeline (29, 39), and a third pump (210, 310) and a pressure sensor (212, 312) are provided on the fourth pipeline (28, 38).

7. A synthesis system according to claim 6, characterized in that: The first pump (24, 34) and the second pump (25, 35) are both peristaltic pumps.

8. A synthesis system according to claim 6, characterized in that: The second pipe (33) is connected to the c-end of a two-position four-way electric valve (319), and the outlet main pipe (36) is connected to the a-end of the two-position four-way electric valve (319); wherein the d-end of the two-position four-way electric valve (319) is connected to a sixth pipe (314), and the b-end is connected to a third pipe (315).

9. A synthesis system according to claim 8, characterized in that: The third pipe (315) is provided with an ultraviolet sensor (316), a pH value sensor (317), and a conductivity temperature sensor (318).

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