A chip-based oligonucleotide chain synthesis device and synthesis method

Abstract

This invention discloses a chip-based oligonucleotide chain synthesis apparatus and method, relating to the field of oligonucleotide synthesis technology. The apparatus includes: a syringe pump module to extract chemical reagents; a rotary valve module to deliver chemical reagents via a rotary cutting operation; a chip module to synthesize oligonucleotide chains based on the chemical reagents; a bottom-in, top-out liquid-to-chemicals interface to control the flow rate and velocity of the chemical reagents; and a top-in, bottom-out gas-to-gas interface to regulate the flow rate and velocity of the gas flow to remove unreacted chemical reagents. This invention, while synthesizing oligonucleotide chains, can stably control the flow rate and velocity of reagents in the chip-based reaction chamber, as well as the flow rate and velocity of the gas flow, thereby ensuring that residual reagents within the chip-based reaction chamber are thoroughly cleaned.

Description

Technical Field

[0001] This invention relates to the field of oligonucleotide synthesis technology, and in particular to a chip-based oligonucleotide chain synthesis apparatus and method. Background Technology

[0002] Currently, the commercially available oligonucleotide synthesis technology is column synthesis. The principle involves filling a packed column with controlled-powder (CPG) beads. Because the column has upper and lower sieves, the CPG powder can be sealed within. The sealed CPG-filled column is then placed in a column synthesizer, and finally, the corresponding computer program is started to synthesize the oligonucleotides. However, this column synthesis technology uses a positive pressure liquid supply method, which does not allow for control of the liquid flow rate. ， Therefore, it cannot be guaranteed that the residual reagents will be completely cleaned. Summary of the Invention

[0003] The purpose of this invention is to provide a chip-based oligonucleotide chain synthesis device. Under the premise of synthesizing oligonucleotide chains, the reagent flow rate and reagent speed of the chip reaction chamber can be stably controlled, as can the gas flow rate and gas speed, thereby ensuring that residual reagents in the chip reaction chamber are cleaned.

[0004] To achieve the above objectives, the present invention provides the following solution:

[0005] This invention provides a chip-based oligonucleotide chain synthesis device, comprising: an injection pump module, a rotary cutting valve module, and a chip module;

[0006] The syringe pump module is used to draw chemical reagents;

[0007] The rotary cutting valve module is used to deliver chemical reagents through a rotary cutting operation;

[0008] The chip module is used for:

[0009] Synthesize oligonucleotide chains using chemical reagents;

[0010] The flow rate and speed of chemical reagents are controlled by a bottom-inlet and top-outlet method to synthesize oligonucleotide chains from chemical reagents;

[0011] The air flow rate and speed are controlled by using an air intake at the top and an air outlet at the bottom to expel unreacted chemical reagents.

[0012] The present invention also provides a synthesis method for use in a chip-based oligonucleotide chain synthesis device, comprising:

[0013] Step 10: TCA deprotection step; The TCA deprotection step involves adding TCA reagent to the reaction chamber of the chip to carry out the deprotection reaction, and then adding ACN reagent to clean it after the reaction is completed.

[0014] Step 20: Mononucleotide reagent coupling step; The mononucleotide reagent coupling step involves simultaneously adding the required mononucleotide reagent and ACT reagent to the reaction chamber of the chip to carry out the coupling reaction, and then adding ACN reagent to clean it after the reaction is completed.

[0015] Step 30: Capping reaction step; The capping reaction step involves simultaneously adding CAPA and CAPB reagents to the reaction chamber of the chip to carry out the capping reaction, and then adding ACN reagent to clean it after the reaction is completed.

[0016] Step 40: Oxidation reaction step; The oxidation reaction step involves adding OXI reagent to the reaction chamber of the chip to carry out the oxidation reaction, and after the reaction is completed, adding ACN reagent to clean it;

[0017] Step 50: Repeat steps 10 to 40 to obtain an oligonucleotide chain; wherein the number of cycles is determined by the length of the oligonucleotide chain.

[0018] According to specific embodiments provided by the present invention, the present invention discloses the following technical effects:

[0019] This invention employs a bottom-in, top-out liquid-controlling method to regulate the flow rate and speed of chemical reagents, thereby synthesizing oligonucleotide chains that meet the requirements. It also employs a top-in, bottom-out air-controlling method to regulate the airflow rate and speed, expelling any unreacted chemical reagents. This allows for stable regulation of both the reagent flow rate and speed within the chip-like reaction chamber, as well as the airflow rate and speed, ensuring that any residual reagents within the chip-like reaction chamber are thoroughly cleaned. Attached Figure Description

[0020] To more clearly illustrate the technical solutions in the embodiments of the present invention or the prior art, the drawings used in the embodiments will be briefly introduced below. Obviously, the drawings described below are only some embodiments of the present invention. For those skilled in the art, other drawings can be obtained based on these drawings without creative effort.

[0021] Figure 1 A structural block diagram of a chip-based oligonucleotide chain synthesis device provided in an embodiment of the present invention;

[0022] Figure 2 This is a schematic diagram of the chip-based oligonucleotide chain synthesis device provided in an embodiment of the present invention. Detailed Implementation

[0023] The technical solutions of the embodiments of the present invention will be clearly and completely described below with reference to the accompanying drawings. Obviously, the described embodiments are only some embodiments of the present invention, and not all embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those skilled in the art without creative effort are within the scope of protection of the present invention.

[0024] This invention replaces CPG powder with a sheet-like chip reaction chamber. Because the surface of the sheet-like chip reaction chamber is a monolayer, it is easy to clean and therefore can synthesize longer oligonucleotide chains.

[0025] Furthermore, research revealed that the liquid flow pattern on the surface of the chip-based reaction chamber differs from that in a packed column. In a packed column, liquid enters from the top and exits from the bottom, while in a chip-based reaction chamber, to ensure that the entire surface width is filled with reagents, liquid enters from the bottom and exits from the top. Moreover, due to the surface tension of the chip-based reaction chamber, the flow rate of each reagent needs to be controlled to ensure that the chip-based reaction chamber can be thoroughly cleaned. Column synthesizers use a positive pressure liquid supply method, which cannot control the liquid flow rate. Therefore, this invention utilizes equipment such as injection pumps, rotary cutting valves, and solenoid valves to construct a chip-based oligonucleotide chain synthesis device.

[0026] To make the above-mentioned objects, features and advantages of the present invention more apparent and understandable, the present invention will be further described in detail below with reference to the accompanying drawings and specific embodiments.

[0027] like Figure 1 As shown in the figure, an embodiment of the present invention provides a chip-based oligonucleotide chain synthesis device, comprising:

[0028] The syringe pump module is used to extract chemical reagents.

[0029] The rotary cutting valve module is used to deliver chemical reagents through a rotary cutting operation.

[0030] The chip module is used for:

[0031] Oligonucleotide chains are synthesized using chemical reagents.

[0032] The flow rate and speed of chemical reagents are controlled by a bottom-inlet and top-outlet method to synthesize oligonucleotide chains from chemical reagents.

[0033] The air flow rate and speed are controlled by using an air intake at the top and an air outlet at the bottom to expel unreacted chemical reagents.

[0034] like Figure 2As shown, the rotary valve module described in this embodiment of the invention is connected to the chip module via a three-way valve 7.

[0035] The present invention uses a high-precision syringe pump module for reagent extraction, such as... Figure 2 As shown, the system includes a first injection pump 1 and a second injection pump 2, which, together with a rotary cutting valve module, enable precise delivery of chemical reagents. The rotary cutting valve module includes a first rotary cutting valve 3, a second rotary cutting valve 4, a third rotary cutting valve 5, and a fourth rotary cutting valve 6. The chip module includes a first two-position three-way valve 8, a second two-position three-way valve 10, and a sheet-like chip reaction chamber 9. The inner surface of the sheet-like chip reaction chamber is a monolayer, and the liquid flow direction and airflow direction in the sheet-like chip reaction chamber 9 are controlled by two two-position three-way valves.

[0036] The fixed end of the first rotary cutting valve 3 is used to connect with the switching end of the second rotary cutting valve 4; the switching end of the first rotary cutting valve 3 is used to extract OXI (iodine solution) reagent, extract CAPA reagent, extract ACT (4,5-dicyanimidazolium) reagent, extract TCA (trichloroacetic acid) reagent or discharge waste liquid according to the rotary cutting operation, that is, it is connected to the OXI end, CAPA end, ACT end, TCA end or WASTE3 end according to the rotary cutting operation.

[0037] The fixed end of the second rotary valve 4 is connected to the first injection pump 1; the switching end of the second rotary valve 4 is used to connect to the fixed end of the first rotary valve 3, the first end of the three-way valve 7, to extract argon gas, extract ACN (acetonitrile) reagent, or discharge waste liquid according to the rotary operation, that is, to connect to the fixed end of the first rotary valve 3, the first end of the three-way valve 7, the AR1 end, the ACN1 end, or the WASTE1 end according to the rotary operation.

[0038] The fixed end of the third rotary valve 5 is connected to the second injection pump 2. The switching end of the third rotary valve 3 is used to connect to the third end of the three-way valve 7, the fixed end of the fourth rotary valve 6, to extract argon gas, extract ACN reagent, or discharge waste liquid according to the rotary operation. That is, according to the rotary operation, it connects to the third end of the three-way valve 7, the fixed end of the fourth rotary valve 6, the AR2 end, the ACN2 end, or the WASTE2 end.

[0039] The fixed end of the fourth rotary cutting valve 6 is used to connect with the switching end of the third rotary cutting valve 5. The switching end of the fourth rotary cutting valve 6 is used to extract CAPB reagent, extract G (DMF-DG-CE phosphorus amide monomer) reagent, extract C (5'-O-(4,4'-dimethoxytriphenyl)-N4-benzoyl-2'-deoxycytidine-3'-(2-cyanoethyl-N,N-diisopropyl)phosphamide) reagent, and extract T (5'- (4,4'-Dimethoxytriphenyl)-3'-deoxythymidine 2'-(2-cyanoethyl-N,N-diisopropyl)phosphamide) reagent, extract A (5'-O-(4,4'-dimethoxytriphenyl)-N6-benzoyl-2'-deoxyadenosine-3'-(2-cyanoethyl-N,N-diisopropyl)phosphamide) reagent or discharge waste liquid, i.e., according to the rotary cutting operation, CAPB end, G end, C end, T end, A end or WASTE4 end.

[0040] The second end of the three-way valve 7 is connected to the first end of the first two-position three-way valve 8. The second end of the first two-position three-way valve 8 is connected to the lower port of the chip reaction chamber 9. The third end of the first two-position three-way valve 8 is used to discharge waste liquid, i.e., this third end is the WASTE5 end. The first end of the second two-position three-way valve 10 is connected to the upper port of the chip reaction chamber 9. The second end of the second two-position three-way valve 10 is used to input external gas, such as argon, into the chip reaction chamber 9, i.e., this second end is the AR3 end. The third end of the second two-position three-way valve 10 is used to discharge waste liquid, i.e., this third end is the WASTE6 end. The chip reaction chamber 9 is a container for synthesizing oligonucleotide chains.

[0041] The lower port is used to deliver chemical reagents through the first two-position three-way valve 8 into the sheet-like chip reaction chamber 9 to synthesize oligonucleotide chains or to receive unreacted chemical reagents in the sheet-like chip reaction chamber 9, and to discharge unreacted chemical reagents through the third end of the first two-position three-way valve 8.

[0042] The upper port is used to deliver external gas through the second two-position three-way valve 10 into the sheet-like chip reaction chamber 9 to deliver unreacted chemical reagents in the sheet-like chip reaction chamber 9 to the lower port, or to deliver unreacted chemical reagents in the sheet-like chip reaction chamber 9 to the third end of the second two-position three-way valve 10 to discharge unreacted chemical reagents.

[0043] In this embodiment of the invention, the first two-position three-way valve 8 is connected to the lower port of the chip reaction chamber 9, and the second two-position three-way valve 10 is connected to the upper port of the chip reaction chamber 9. This allows control over the liquid flow direction in the chip reaction chamber 9. In order for the chemical reagent to fill the entire chip reaction chamber 9, the chemical reagent needs to flow from the lower port to the upper port. After the chemical reagent reaction is completed, in order to empty the chemical reagent, argon gas needs to be blown in from the upper port so that the chemical reagent flows out from the lower port.

[0044] Example 2

[0045] Oligonucleotide chains are formed by the sequential stacking of multiple mononucleotide reagents. The stacking principle of each mononucleotide reagent involves a four-step chemical synthesis reaction, and the entire stacking process is a cyclical repetition of these four chemical synthesis reactions. To synthesize a 60nt oligonucleotide chain, the four-step chemical synthesis reaction is repeated 60 times, with the corresponding mononucleotide reagent sequence added in each cycle.

[0046] Therefore, embodiments of the present invention provide a synthesis method applicable to the apparatus described in the embodiments, namely, the above-described four-step chemical synthesis reaction, including:

[0047] Step 10: TCA deprotection step; The TCA deprotection step involves adding TCA reagent to the reaction chamber of the chip to carry out the deprotection reaction, and then adding ACN reagent to clean it after the reaction is completed.

[0048] Step 20: Mononucleotide reagent coupling step; The mononucleotide reagent coupling step involves simultaneously adding the required mononucleotide reagent and ACT reagent to the chip reaction chamber for coupling reaction, and then adding ACN reagent for cleaning after the reaction is completed.

[0049] Step 30: Capping reaction step; The capping reaction step involves simultaneously adding CAPA and CAPB reagents to the reaction chamber of the chip to carry out the capping reaction, and then adding ACN reagent for cleaning after the reaction is completed.

[0050] Step 40: Oxidation reaction step; The oxidation reaction step involves adding OXI reagent to the reaction chamber of the chip to carry out the oxidation reaction, and then adding ACN reagent to clean it after the reaction is completed.

[0051] Step 50: Repeat steps 10 to 40 to obtain an oligonucleotide chain; wherein the number of cycles is determined by the length of the oligonucleotide chain.

[0052] The specific operation process from steps 10 to 40 is as follows:

[0053] The detailed process is as follows:

[0054] a. Adding TCA reagent: First, reset the first injection pump 1, rotate the switching end of the second rotary valve 4 to the fixed end of the first rotary valve 3, rotate the switching end of the first rotary valve 3 to the TCA end of the first rotary valve 3, and then pull down the first injection pump 1 to draw a certain amount of TCA reagent into the first injection pump 1; next, rotate the switching end of the second rotary valve 4 to the WASTE1 end of the second rotary valve 4, push the first injection pump 1 up, and push the TCA reagent to the WASTE1 end of the second rotary valve 4. This process makes the first rotary valve 3 and the second rotary valve 4... The tubing is filled with TCA reagent; then the switching end of the second rotary valve 4 is rotated to the fixed end of the first rotary valve 3, and the first syringe pump 1 is pulled down to draw TCA reagent; next, the switching end of the second rotary valve 4 is rotated to the first end of the three-way valve 7. Since the second end of the three-way valve 7 is connected to the first end (i.e., normally open end) of the first two-position three-way valve 8, the second end of the first two-position three-way valve 8 is connected to the lower port of the chip reaction chamber 9, the third end of the first two-position three-way valve 8 is the WASTE5 end, and the first end of the second two-position three-way valve 10 is connected to the upper port of the chip reaction chamber 9. Connecting the two ends of the second two-position three-way valve 10 (AR3 terminal) and the third end (normally open terminal) of the second two-position three-way valve 10 (WASTE6 terminal), closing the AR3 terminal of the second two-position three-way valve 10 and the WASTE5 terminal of the first two-position three-way valve 8, and opening the WASTE6 terminal of the second two-position three-way valve 10 and the first end of the first two-position three-way valve 8, the first injection pump 1 is pushed upwards, thus pushing the TCA reagent into the chip reaction chamber 9; finally, after the TCA reagent reacts in the chip reaction chamber 9 for 40 seconds, the AR3 terminal of the second two-position three-way valve 10 and the first two-position three-way valve 8 are opened. The first two-position three-way valve 8 is closed at its WASTE5 terminal, and the second two-position three-way valve 10 is closed at its WASTE6 terminal and the first terminal of the first two-position three-way valve 8. This creates a conductive path between the AR3 terminal of the second two-position three-way valve 10 and the WASTE5 terminal of the first two-position three-way valve 8. Argon gas enters the chip reaction chamber 9 through the AR3 terminal of the second two-position three-way valve 10 and blows the remaining TCA reagent in the chip reaction chamber 9 to the WASTE5 terminal of the first two-position three-way valve 8, whereby it is discharged. The pressure of the argon gas is 30 kJ.

[0055] b. Blowing the common pipeline: First, set both the first injection pump 1 and the second injection pump 2 to the reset position. Rotate the switching ends of the second rotary valve 4 and the third rotary valve 5 to the AR1 and AR2 ends respectively. Pull down the first injection pump 1 and the second injection pump 2 to extract a certain amount of argon gas. Next, rotate the switching ends of the second rotary valve 4 and the third rotary valve 5 to the first and third ends of the three-way valve 7 respectively. Close the AR3 end of the second two-position three-way valve 10 and the WASTE5 end of the first two-position three-way valve 8. Open the WASTE6 end of the second two-position three-way valve 10 and the first end of the first two-position three-way valve 8. Push the first injection pump 1 and the second injection pump 2 upwards to push the argon gas into the chip reaction chamber 9, while simultaneously blowing the common pipeline. TCA reagent is pushed into the chip reaction chamber 9 and discharged through the WASTE6 end of the second two-position three-way valve 10. Then, the AR3 end of the second two-position three-way valve 10 and the WASTE5 end of the first two-position three-way valve 8 are opened, and the WASTE6 end of the second two-position three-way valve 10 and the first end of the first two-position three-way valve 8 are closed. In this way, the AR3 end of the second two-position three-way valve 10 and the WASTE5 end of the first two-position three-way valve 8 form a conductive path. Argon gas enters the chip reaction chamber 9 through the AR3 end of the second two-position three-way valve 10 and blows the remaining TCA reagent in the chip reaction chamber 9 to the WASTE5 end of the first two-position three-way valve 8, and is discharged from the WASTE5 end of the first two-position three-way valve 8.

[0056] c. Cleaning the rotary valves: First, set both the first injection pump 1 and the second injection pump 2 to the reset position. Rotate the switching ends of the second rotary valve 4 and the third rotary valve 5 to the ACN1 and ACN2 ends respectively. Pull down the first injection pump 1 and the second injection pump 2 to draw ACN reagent. Next, rotate the switching ends of the second rotary valve 4 and the third rotary valve 5 to the AR1 and AR2 ends respectively. Continue to pull down the first injection pump 1 and the second injection pump 2 to draw some argon gas. Then, turn the second rotary valve 4 and the third rotary valve 5... Rotate the switching ends of the first rotary valve 3 and the fourth rotary valve 6 to their fixed ends respectively. Rotate the switching ends of the first rotary valve 3 and the fourth rotary valve 6 to the WASTE3 and WASTE4 ends respectively. Push the first injection pump 1 and the second injection pump 2 upwards until the amount of ACN reagent extracted is 1 / 2 of the total amount. Then rotate the switching ends of the second rotary valve 4 and the third rotary valve 5 to the WASTE1 and WASTE2 ends respectively. Push the first injection pump 1 and the second injection pump 2 upwards to their reset points.

[0057] d. Cleaning the reaction chamber of the chip: First, set both the first injection pump 1 and the second injection pump 2 to the reset position. Rotate the switching ends of the second rotary valve 4 and the third rotary valve 5 to the ACN1 and ACN2 ends respectively. Pull down the first injection pump 1 and the second injection pump 2 to draw ACN reagent. Next, rotate the switching ends of the second rotary valve 4 and the third rotary valve 5 to the first and third ends of the three-way valve 7 respectively. Close the AR3 end of the second two-position three-way valve 10 and the WASTE5 end of the first two-position three-way valve 8. Open the WASTE6 end of the second two-position three-way valve 10 and the first end of the first two-position three-way valve 8. Push the first injection pump 1 and the second injection pump 2 up to the reset point to clean the ACN reagent. The ACN reagent is pushed into the chip reaction chamber 9; then, the AR3 terminal of the second two-position three-way valve 10 and the WASTE5 terminal of the first two-position three-way valve 8 are opened, and the WASTE6 terminal of the second two-position three-way valve 10 and the first terminal of the first two-position three-way valve 8 are closed. This creates a conductive path between the AR3 terminal of the second two-position three-way valve 10 and the WASTE5 terminal of the first two-position three-way valve 8. Argon gas enters the chip reaction chamber 9 through the AR3 terminal of the second two-position three-way valve 10, blowing the remaining ACN reagent in the chip reaction chamber 9 to the WASTE5 terminal of the first two-position three-way valve 8 and exiting from the WASTE5 terminal of the first two-position three-way valve 8; the argon gas pressure is 30 kJ. (This cleaning step needs to be repeated 3 times).

[0058] e. Adding mononucleotide and ACT reagents: First, set both the first and second injection pumps 1 and 2 to the reset position. Rotate the second and third cleaving valves 4 and 5 to their respective positions connected to the fixed ends of the first and fourth cleaving valves 3 and 6. Rotate the first cleaving valve 3 to the ACT end and the fourth cleaving valve 6 to the mononucleotide reagent (one of A, T, C, and G) required for this cycle. Next, pull down the first and second injection pumps 1 and 2 to draw the ACT and mononucleotide reagents respectively. Rotate the second and third cleaving valves 4 and 5 to the WASTE1 and WASTE2 ends respectively. Push the first and second injection pumps 1 and 2 back to the reset position. This process is called pre-filling the reagents. Then, rotate the second and third cleaving valves 4 and 5 to their respective positions connected to the fixed ends of the first and fourth cleaving valves 3 and 6. Pull down the first and second injection pumps 1 and 2 again to draw the reagents. The switching ends of the third rotary valve 5 are rotated to the first and third ends of the three-way valve 7, respectively, closing the AR3 end of the second two-position three-way valve 10 and the WASTE5 end of the first two-position three-way valve 8, opening the WASTE6 end of the second two-position three-way valve 10 and the first end of the first two-position three-way valve 8, pushing the first injection pump 1 and the second injection pump 2 upwards, pushing the reagent into the chip reaction chamber 9, reacting for 60 seconds, and then opening the AR3 end of the second two-position three-way valve 10 and the first two-position three-way valve 8. By closing the WASTE5 terminal of the second two-position three-way valve 10 and the first terminal of the first two-position three-way valve 8, the AR3 terminal of the second two-position three-way valve 10 and the WASTE5 terminal of the first two-position three-way valve 8 form a conductive path. Argon gas can then blow the remaining ACT reagent in the chip reaction chamber 9 through the AR3 terminal of the second two-position three-way valve 10 to the WASTE5 terminal of the first two-position three-way valve 8 and discharge it from the WASTE5 terminal of the first two-position three-way valve 8.

[0059] f. Blowing the common pipeline: First, set both the first injection pump 1 and the second injection pump 2 to the reset position. Rotate the switching ends of the second rotary valve 4 and the third rotary valve 5 to the AR1 and AR2 ends respectively. Pull down the first injection pump 1 and the second injection pump 2 to extract a certain amount of argon gas. Next, rotate the switching ends of the second rotary valve 4 and the third rotary valve 5 to the first and third ends of the three-way valve 7 respectively. Close the AR3 end of the second two-position three-way valve 10 and the WASTE5 end of the first two-position three-way valve 8. Open the WASTE6 end of the second two-position three-way valve 10 and the first end of the first two-position three-way valve 8. Push the first injection pump 1 and the second injection pump 2 upwards to push the argon gas into the chip reaction chamber 9, while simultaneously blowing the common pipeline. TCA reagent is pushed into the chip reaction chamber 9 and discharged through the WASTE6 end of the second two-position three-way valve 10. Then, the AR3 end of the second two-position three-way valve 10 and the WASTE5 end of the first two-position three-way valve 8 are opened, and the WASTE6 end of the second two-position three-way valve 10 and the first end of the first two-position three-way valve 8 are closed. In this way, the AR3 end of the second two-position three-way valve 10 and the WASTE5 end of the first two-position three-way valve 8 form a conductive path. Argon gas enters the chip reaction chamber 9 through the AR3 end of the second two-position three-way valve 10 and blows the remaining TCA reagent in the chip reaction chamber 9 to the WASTE5 end of the first two-position three-way valve 8, and is discharged from the WASTE5 end of the first two-position three-way valve 8.

[0060] g. Cleaning the rotary valves: First, set both the first injection pump 1 and the second injection pump 2 to the reset position. Rotate the switching ends of the second rotary valve 4 and the third rotary valve 5 to the ACN1 and ACN2 ends respectively. Pull down the first injection pump 1 and the second injection pump 2 to draw ACN reagent. Next, rotate the switching ends of the second rotary valve 4 and the third rotary valve 5 to the AR1 and AR2 ends respectively. Continue to pull down the first injection pump 1 and the second injection pump 2 to draw some argon gas. Then, turn the second rotary valve 4 and the third rotary valve 5... Rotate the switching ends of the first rotary valve 3 and the fourth rotary valve 6 to their fixed ends respectively. Rotate the switching ends of the first rotary valve 3 and the fourth rotary valve 6 to the WASTE3 and WASTE4 ends respectively. Push the first injection pump 1 and the second injection pump 2 upwards until the amount of ACN reagent extracted is 1 / 2 of the total amount. Then rotate the switching ends of the second rotary valve 4 and the third rotary valve 5 to the WASTE1 and WASTE2 ends respectively. Push the first injection pump 1 and the second injection pump 2 upwards to their reset points.

[0061] h. Cleaning the reaction chamber of the chip: First, set both the first injection pump 1 and the second injection pump 2 to the reset position. Rotate the switching ends of the second rotary valve 4 and the third rotary valve 5 to the ACN1 and ACN2 ends respectively. Pull down the first injection pump 1 and the second injection pump 2 to draw ACN reagent. Next, rotate the switching ends of the second rotary valve 4 and the third rotary valve 5 to the first and third ends of the three-way valve 7 respectively. Close the AR3 end of the second two-position three-way valve 10 and the WASTE5 end of the first two-position three-way valve 8. Open the WASTE6 end of the second two-position three-way valve 10 and the first end of the first two-position three-way valve 8. Push the first injection pump 1 and the second injection pump 2 up to the reset point to clean the ACN reagent. The ACN reagent is pushed into the chip reaction chamber 9; then, the AR3 terminal of the second two-position three-way valve 10 and the WASTE5 terminal of the first two-position three-way valve 8 are opened, and the WASTE6 terminal of the second two-position three-way valve 10 and the first terminal of the first two-position three-way valve 8 are closed. This creates a conductive path between the AR3 terminal of the second two-position three-way valve 10 and the WASTE5 terminal of the first two-position three-way valve 8. Argon gas enters the chip reaction chamber 9 through the AR3 terminal of the second two-position three-way valve 10, blowing the remaining ACN reagent in the chip reaction chamber 9 to the WASTE5 terminal of the first two-position three-way valve 8 and exiting from the WASTE5 terminal of the first two-position three-way valve 8; the argon gas pressure is 30 kJ. (This cleaning step needs to be repeated 3 times).

[0062] i. Capping the reagent: First, set both the first injection pump 1 and the second injection pump 2 to the reset position. Rotate the second rotary valve 4 and the third rotary valve 5 to the positions connected to the fixed ends of the first rotary valve 3 and the fourth rotary valve 6, respectively. Rotate the first rotary valve 3 to the CAPA end and the fourth rotary valve 6 to the CAPB end. Next, pull down the first injection pump 1 and the second injection pump 2 to draw CAPA and CAPB reagents, respectively. Rotate the second rotary valve 4 and the third rotary valve 5 to the WASTE1 and WASTE2 ends, respectively. Push the first injection pump 1 and the second injection pump 2 to the reset position. This process is called pre-filling the reagent. Then, rotate the second rotary valve 4 and the third rotary valve 5 to the positions connected to the fixed ends of the first rotary valve 3 and the fourth rotary valve 6, respectively. Pull down the first injection pump 1 and the second injection pump 2 again to draw reagents. Rotate the switching ends of the second rotary valve 4 and the third rotary valve 5 to the three... The first and third ends of valve 7 are closed, the AR3 end of the second two-position three-way valve 10 and the WASTE5 end of the first two-position three-way valve 8 are closed, the WASTE6 end of the second two-position three-way valve 10 and the first end of the first two-position three-way valve 8 are opened, the first injection pump 1 and the second injection pump 2 are pushed up to push the reagent into the chip reaction chamber 9 and react for 40 seconds. Then, the AR3 end of the second two-position three-way valve 10 and the WASTE5 end of the first two-position three-way valve 8 are opened, and the WASTE6 end of the second two-position three-way valve 10 and the first end of the first two-position three-way valve 8 are closed. In this way, the AR3 end of the second two-position three-way valve 10 and the WASTE5 end of the first two-position three-way valve 8 form a conductive path. Argon gas can then blow the CAPA and CAPB reagents in the chip reaction chamber 9 to the WASTE5 end of the first two-position three-way valve 8 through the AR3 end of the second two-position three-way valve 10 and discharge from the WASTE5 end of the first two-position three-way valve 8.

[0063] j. Blowing the common pipeline: First, set both the first injection pump 1 and the second injection pump 2 to the reset position. Rotate the switching ends of the second rotary valve 4 and the third rotary valve 5 to the AR1 and AR2 ends respectively. Pull down the first injection pump 1 and the second injection pump 2 to extract a certain amount of argon gas. Next, rotate the switching ends of the second rotary valve 4 and the third rotary valve 5 to the first and third ends of the three-way valve 7 respectively. Close the AR3 end of the second two-position three-way valve 10 and the WASTE5 end of the first two-position three-way valve 8. Open the WASTE6 end of the second two-position three-way valve 10 and the first end of the first two-position three-way valve 8. Push the first injection pump 1 and the second injection pump 2 upwards to push the argon gas into the chip reaction chamber 9, while simultaneously blowing the common pipeline. TCA reagent is pushed into the chip reaction chamber 9 and discharged through the WASTE6 end of the second two-position three-way valve 10. Then, the AR3 end of the second two-position three-way valve 10 and the WASTE5 end of the first two-position three-way valve 8 are opened, and the WASTE6 end of the second two-position three-way valve 10 and the first end of the first two-position three-way valve 8 are closed. In this way, the AR3 end of the second two-position three-way valve 10 and the WASTE5 end of the first two-position three-way valve 8 form a conductive path. Argon gas enters the chip reaction chamber 9 through the AR3 end of the second two-position three-way valve 10 and blows the remaining TCA reagent in the chip reaction chamber 9 to the WASTE5 end of the first two-position three-way valve 8, and is discharged from the WASTE5 end of the first two-position three-way valve 8.

[0064] k. Cleaning the rotary valves: First, set both the first injection pump 1 and the second injection pump 2 to the reset position. Rotate the switching ends of the second rotary valve 4 and the third rotary valve 5 to the ACN1 and ACN2 ends respectively. Pull down the first injection pump 1 and the second injection pump 2 to draw ACN reagent. Next, rotate the switching ends of the second rotary valve 4 and the third rotary valve 5 to the AR1 and AR2 ends respectively. Continue to pull down the first injection pump 1 and the second injection pump 2 to draw some argon gas. Then, turn the second rotary valve 4 and the third rotary valve 5... Rotate the switching ends of the first rotary valve 3 and the fourth rotary valve 6 to their fixed ends respectively. Rotate the switching ends of the first rotary valve 3 and the fourth rotary valve 6 to the WASTE3 and WASTE4 ends respectively. Push the first injection pump 1 and the second injection pump 2 upwards until the amount of ACN reagent extracted is 1 / 2 of the total amount. Then rotate the switching ends of the second rotary valve 4 and the third rotary valve 5 to the WASTE1 and WASTE2 ends respectively. Push the first injection pump 1 and the second injection pump 2 upwards to their reset points.

[0065] 1. Cleaning the reaction chamber of the chip: First, set both the first injection pump 1 and the second injection pump 2 to the reset position. Rotate the switching ends of the second rotary valve 4 and the third rotary valve 5 to the ACN1 and ACN2 ends respectively. Pull down the first injection pump 1 and the second injection pump 2 to draw ACN reagent. Next, rotate the switching ends of the second rotary valve 4 and the third rotary valve 5 to the first and third ends of the three-way valve 7 respectively. Close the AR3 end of the second two-position three-way valve 10 and the WASTE5 end of the first two-position three-way valve 8. Open the WASTE6 end of the second two-position three-way valve 10 and the first end of the first two-position three-way valve 8. Push the first injection pump 1 and the second injection pump 2 up to the reset point to clean the ACN reagent. The ACN reagent is pushed into the chip reaction chamber 9; then, the AR3 terminal of the second two-position three-way valve 10 and the WASTE5 terminal of the first two-position three-way valve 8 are opened, and the WASTE6 terminal of the second two-position three-way valve 10 and the first terminal of the first two-position three-way valve 8 are closed. This creates a conductive path between the AR3 terminal of the second two-position three-way valve 10 and the WASTE5 terminal of the first two-position three-way valve 8. Argon gas enters the chip reaction chamber 9 through the AR3 terminal of the second two-position three-way valve 10, blowing the remaining ACN reagent in the chip reaction chamber 9 to the WASTE5 terminal of the first two-position three-way valve 8 and exiting from the WASTE5 terminal of the first two-position three-way valve 8; the argon gas pressure is 30 kJ. (This cleaning step needs to be repeated 3 times).

[0066] m. Adding oxidant: First, reset the first injection pump 1. Then, switch the second rotary valve 4 to the position connected to the fixed end of the first rotary valve 3. Rotate the first rotary valve 3 to the OXI end, and pull down the first injection pump 1 to draw a certain amount of OXI reagent into the first injection pump 1. Next, switch the second rotary valve 4 to the WASTE1 end, push the first injection pump 1 up, and push the OXI reagent to the WASTE1 end, so that the tubing between the first rotary valve 3 and the second rotary valve 4 is filled with OXI reagent. Continue to switch the second rotary valve 4 to the position connected to the fixed end of the first rotary valve 3. The first rotary valve 3 is fixed at its end, and the first syringe pump 1 is pulled down to draw OXI reagent. Then, the second rotary valve 4 is rotated to the position connected to the three-way valve 7, that is, connected to the first end of the three-way valve 7. The second end of the three-way valve 7 is connected to the first end (normally open) of the first two-position three-way valve 8. The second end of the first two-position three-way valve 8 is connected to the lower port of the chip reaction chamber 9. The third end of the first two-position three-way valve 8 is used to discharge waste liquid. The first end of the second two-position three-way valve 10 is connected to the upper port of the chip reaction chamber 9. The second end of the two-position three-way valve 10, AR3, is used to supply external gas to the chip reaction chamber 9. The third end of the two-position three-way valve 10, another normally open end, is used to discharge waste liquid. Therefore, by closing AR3 of the two-position three-way valve 10 and WASTE5 of the first two-position three-way valve 8, and opening WASTE6 of the two-position three-way valve 10 and the first end of the first two-position three-way valve 8, the first syringe pump 1 is pushed upwards, thus pushing the OXI reagent into the chip reaction chamber 9. Finally, after reacting in the chip reaction chamber 9 for 40 seconds... Open the AR3 terminal of the second two-position three-way valve 10 and the WASTE5 terminal of the first two-position three-way valve 8, and close the WASTE6 terminal of the second two-position three-way valve 10 and the first terminal of the first two-position three-way valve 8. In this way, the AR3 terminal of the second two-position three-way valve 10 and the WASTE5 terminal of the first two-position three-way valve 8 form a conductive path. The external argon gas can then blow the remaining OXI reagent in the chip reaction chamber 9 through the second terminal of the second two-position three-way valve 10 to the third terminal of the first two-position three-way valve 8, and then discharge it from the third terminal of the first two-position three-way valve 8.

[0067] n. The full cycle is simply a repeated cycle of a-m, and only step e is needed to select the current cycle's single nucleotide reagent pathway.

[0068] The various embodiments in this specification are described in a progressive manner, with each embodiment focusing on the differences from other embodiments. The same or similar parts between the various embodiments can be referred to each other.

[0069] This document uses specific examples to illustrate the principles and implementation methods of the present invention. The descriptions of the above embodiments are only for the purpose of helping to understand the method and core ideas of the present invention. Furthermore, those skilled in the art will recognize that, based on the ideas of the present invention, there will be changes in the specific implementation methods and application scope. Therefore, the content of this specification should not be construed as a limitation of the present invention.

Claims

1. A chip-based oligonucleotide chain synthesis device, characterized in that, include: Injection pump module, rotary valve module, and chip module; The syringe pump module is used to draw chemical reagents; The rotary cutting valve module is used to deliver chemical reagents through a rotary cutting operation; the rotary cutting valve module is connected to the chip module via a three-way valve; The chip module is used for: Synthesize oligonucleotide chains using chemical reagents; The flow rate and speed of chemical reagents are controlled by a bottom-inlet and top-outlet method to synthesize oligonucleotide chains from chemical reagents; The air flow rate and speed are controlled by using an air intake at the top and an air outlet at the bottom to expel unreacted chemical reagents. The chip module includes a first two-position three-way valve, a second two-position three-way valve, and a chip-like reaction chamber. The inner surface of the chip-like reaction chamber is a monolayer. The second end of the three-way valve is connected to the first end of the first two-position three-way valve, and the second end of the first two-position three-way valve is connected to the lower opening of the chip-like reaction chamber. The third end of the first two-position three-way valve is used to discharge waste liquid. The first end of the second two-position three-way valve is connected to the upper opening of the chip-like reaction chamber. The second end of the second two-position three-way valve is used to input external gas into the chip-like reaction chamber, and the third end of the second two-position three-way valve is used to discharge waste liquid. The chip-like reaction chamber is a synthetic oligonucleus. The container for the nucleotide chain; the lower port is used to deliver chemical reagents through the first two-position three-way valve into the chip-shaped reaction chamber to synthesize oligonucleotide chains or to receive unreacted chemical reagents in the chip-shaped reaction chamber, and to discharge unreacted chemical reagents through the third end of the first two-position three-way valve; the upper port is used to deliver external gas through the second two-position three-way valve into the chip-shaped reaction chamber to deliver unreacted chemical reagents in the chip-shaped reaction chamber to the lower port or to deliver unreacted chemical reagents in the chip-shaped reaction chamber to the third end of the second two-position three-way valve to discharge unreacted chemical reagents.

2. The chip-based oligonucleotide chain synthesis device according to claim 1, characterized in that, The rotary cutting valve module includes a first rotary cutting valve, a second rotary cutting valve, a third rotary cutting valve, and a fourth rotary cutting valve; the injection pump module includes a first injection pump and a second injection pump; The fixed end of the first rotary cutting valve is used to connect with the switching end of the second rotary cutting valve; the switching end of the first rotary cutting valve is used to extract OXI reagent, extract CAPA reagent, extract ACT reagent, extract TCA reagent, or discharge waste liquid according to the rotary cutting operation; The fixed end of the second rotary cutting valve is connected to the first injection pump; the switching end of the second rotary cutting valve is used to connect to the fixed end of the first rotary cutting valve, to the first end of the three-way valve, to extract argon gas, to extract ACN reagent, or to discharge waste liquid according to the rotary cutting operation; The fixed end of the third rotary cutting valve is connected to the second injection pump, and the switching end of the third rotary cutting valve is used to connect to the third end of the three-way valve, connect to the fixed end of the fourth rotary cutting valve, extract argon gas, extract ACN reagent, or discharge waste liquid according to the rotary cutting operation. The fixed end of the fourth rotary cutting valve is used to connect with the switching end of the third rotary cutting valve. The switching end of the fourth rotary cutting valve is used to extract CAPB reagent, extract G reagent, extract C reagent, extract T reagent, extract A reagent, or discharge waste liquid according to the rotary cutting operation.

3. A synthesis method applied to the chip-based oligonucleotide chain synthesis apparatus of claim 2, characterized in that, The synthesis method includes: Step 10: TCA deprotection step; The TCA deprotection step involves adding TCA reagent to the reaction chamber of the chip to carry out the deprotection reaction, and then adding ACN reagent to clean it after the reaction is completed. Step 20: Mononucleotide reagent coupling step; The mononucleotide reagent coupling step involves simultaneously adding the required mononucleotide reagent and ACT reagent to the reaction chamber of the chip to carry out the coupling reaction, and then adding ACN reagent to clean it after the reaction is completed. Step 30: Capping reaction step; The capping reaction step involves simultaneously adding CAPA and CAPB reagents to the reaction chamber of the chip to carry out the capping reaction, and then adding ACN reagent to clean it after the reaction is completed. Step 40: Oxidation reaction step; The oxidation reaction step involves adding OXI reagent to the reaction chamber of the chip to carry out the oxidation reaction, and after the reaction is completed, adding ACN reagent to clean it; Step 50: Repeat steps 10 to 40 to obtain an oligonucleotide chain; wherein the number of cycles is determined by the length of the oligonucleotide chain.

4. The synthesis method of the chip-based oligonucleotide chain synthesis device according to claim 3, characterized in that, In the TCA deprotection step, the process of adding the TCA reagent is as follows: First, reset the first injection pump, rotate the switching end of the second rotary valve to the fixed end of the first rotary valve, rotate the switching end of the first rotary valve to the TCA end of the first rotary valve, pull down the first injection pump, and draw a certain amount of TCA reagent into the first injection pump. Next, rotate the switching end of the second rotary valve to the WASTE1 end of the second rotary valve, push the first injection pump upward, and push the TCA reagent to the WASTE1 end of the second rotary valve. Then rotate the switching end of the second rotary valve to the fixed end of the first rotary valve, and pull down the first syringe pump to draw TCA reagent; Next, rotate the switching end of the second rotary valve to the first end of the three-way valve, close the AR3 end of the second two-position three-way valve and the WASTE5 end of the first two-position three-way valve, open the WASTE6 end of the second two-position three-way valve and the first end of the first two-position three-way valve, push the first injection pump up, and push the TCA reagent into the chip reaction chamber. Finally, after the TCA reagent reacts in the chip reaction chamber for 40 seconds, the AR3 end of the second two-position three-way valve and the WASTE5 end of the first two-position three-way valve are opened, and the WASTE6 end of the second two-position three-way valve and the first end of the first two-position three-way valve are closed. Argon gas enters the chip reaction chamber through the AR3 end of the second two-position three-way valve and blows the remaining TCA reagent in the chip reaction chamber to the WASTE5 end of the first two-position three-way valve, and then discharges from the WASTE5 end of the first two-position three-way valve. Among them, the AR3 end of the second two-position three-way valve is the second end of the second two-position three-way valve, the WASTE6 end of the second two-position three-way valve is the third end of the second two-position three-way valve, and the WASTE5 end of the first two-position three-way valve is the third end of the first two-position three-way valve.

5. The synthesis method of the chip-based oligonucleotide chain synthesis device according to claim 3, characterized in that, In the single nucleotide reagent coupling step, the process of adding the single nucleotide reagent and the ACT reagent is as follows: First, set both the first and second injection pumps to the reset position. Then, rotate the second and third cleavage valves to the positions connected to the fixed ends of the first and fourth cleavage valves, respectively. Rotate the first cleavage valve to the ACT end and rotate the fourth cleavage valve to the mononucleotide reagent required for this cycle. Next, pull down the first and second injection pumps to draw ACT reagent and mononucleotide reagent respectively, rotate the second and third cleavage valves to the WASTE1 end of the second cleavage valve and the WASTE2 end of the third cleavage valve respectively, and push the first and second injection pumps to the reset position. Then rotate the second and third rotary valves to the positions where they are connected to the fixed ends of the first and fourth rotary valves, and pull down the first and second injection pumps again to draw out the reagents; Next, rotate the switching ends of the second rotary valve and the third rotary valve to the first and third ends of the three-way valve, respectively. Close the AR3 end of the second two-position three-way valve and the WASTE5 end of the first two-position three-way valve. Open the WASTE6 end of the second two-position three-way valve and the first end of the first two-position three-way valve. Push the first injection pump and the second injection pump upward. Finally, after the reagent is pushed into the chip reaction chamber and reacted for 60 seconds, the AR3 end of the second two-position three-way valve and the WASTE5 end of the first two-position three-way valve are opened, and the WASTE6 end of the second two-position three-way valve and the first end of the first two-position three-way valve are closed. Argon gas enters the chip reaction chamber through the AR3 end of the second two-position three-way valve and blows the remaining ACT reagent in the chip reaction chamber to the WASTE5 end of the first two-position three-way valve, and then discharges from the WASTE5 end of the first two-position three-way valve. Among them, the AR3 end of the second two-position three-way valve is the second end of the second two-position three-way valve, the WASTE6 end of the second two-position three-way valve is the third end of the second two-position three-way valve, and the WASTE5 end of the first two-position three-way valve is the third end of the first two-position three-way valve.

6. The synthesis method of the chip-based oligonucleotide chain synthesis device according to claim 3, characterized in that, In the capping reaction step, the process of adding the capping reagent is as follows: First, set both the first and second injection pumps to the reset position. Then, rotate the second and third rotary valves to positions connected to the fixed ends of the first and fourth rotary valves, respectively. Rotate the first rotary valve to its CAPA end and the fourth rotary valve to its CAPB end. Next, pull down the first and second injection pumps to draw CAPA and CAPB reagents respectively, rotate the second and third rotary valves to the WASTE1 end of the second rotary valve and the WASTE2 end of the second rotary valve respectively, and push the first and second injection pumps to the reset position. Then rotate the second and third rotary valves to the positions where they are connected to the fixed ends of the first and fourth rotary valves, and pull down the first and second injection pumps again to draw out the reagents; Next, rotate the switching ends of the second rotary valve and the third rotary valve to the first and third ends of the three-way valve, respectively. Close the AR3 end of the second two-position three-way valve and the WASTE5 end of the first two-position three-way valve. Open the WASTE6 end of the second two-position three-way valve and the first end of the first two-position three-way valve. Push the first injection pump and the second injection pump upward. Finally, after the reagents are pushed into the chip reaction chamber and reacted for 40 seconds, the AR3 end of the second two-position three-way valve and the WASTE5 end of the first two-position three-way valve are opened, and the WASTE6 end of the second two-position three-way valve and the first end of the first two-position three-way valve are closed. The AR3 end of the second two-position three-way valve and the WASTE5 end of the first two-position three-way valve form a conductive path. Argon gas blows the CAPA and CAPB reagents in the chip reaction chamber to the third end of the first two-position three-way valve through the second end of the second two-position three-way valve and is discharged from the third end of the first two-position three-way valve. Among them, the AR3 end of the second two-position three-way valve is the second end of the second two-position three-way valve, the WASTE6 end of the second two-position three-way valve is the third end of the second two-position three-way valve, and the WASTE5 end of the first two-position three-way valve is the third end of the first two-position three-way valve.

7. The synthesis method of the chip-based oligonucleotide chain synthesis device according to claim 3, characterized in that, In the oxidation reaction step, the process of adding the oxidant is as follows: First, reset the first injection pump, switch the second rotary valve to the position connected to the fixed end of the first rotary valve, rotate the first rotary valve to the OXI end of the first rotary valve, pull down the first injection pump, and draw a certain amount of OXI reagent into the first injection pump. Next, switch the second rotary valve to the WASTE1 end of the second rotary valve, push the first injection pump up, and push the OXI reagent to the WASTE1 end of the second rotary valve; Then switch the second rotary valve to the position connected to the fixed end of the first rotary valve, and pull down the first syringe pump to draw OXI reagent; Next, rotate the second rotary valve to the first end of the three-way valve, close the AR3 end of the second two-position three-way valve and the WASTE5 end of the first two-position three-way valve, open the WASTE6 end of the second two-position three-way valve and the first end of the first two-position three-way valve, push the first injection pump upward, and push the OXI reagent into the chip reaction chamber. Finally, after the OXI reagent reacts in the chip reaction chamber for 40 seconds, the AR3 end of the second two-position three-way valve and the WASTE5 end of the first two-position three-way valve are opened, and the WASTE6 end of the second two-position three-way valve and the first end of the first two-position three-way valve are closed. Argon gas blows the remaining OXI reagent in the chip reaction chamber through the AR3 end of the second two-position three-way valve to the WASTE5 end of the first two-position three-way valve and is discharged from the WASTE5 end of the first two-position three-way valve. Among them, the AR3 end of the second two-position three-way valve is the second end of the second two-position three-way valve, the WASTE6 end of the second two-position three-way valve is the third end of the second two-position three-way valve, and the WASTE5 end of the first two-position three-way valve is the third end of the first two-position three-way valve.

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