Ammonolysis treating fluid for gas-phase ammonolysis of oligonucleotide and application of ammonolysis treating fluid

Abstract

The invention discloses ammonolysis treating fluid for gas-phase ammonolysis of oligonucleotide and application of the ammonolysis treating fluid. The ammonolysis treating fluid is anhydrous n-butylamine. The components of the ammonolysis treating fluid are optimized, water and absolute methanol contained in a conventional ammonolysis treating fluid are removed, the ammonolysis treating fluid is applied to gas-phase ammonolysis of oligonucleotide connected to a solid-phase carrier, the ammonolysis treating fluid can efficiently cut connection between an oligonucleotide synthesis chip and the oligonucleotide, the components of the ammonolysis treating fluid are simplified, and the cost of the ammonolysis treating fluid is reduced. And the problems of point location in a single chip and product crosstalk between chips can be improved.

Description

Technical Field

[0001] This invention relates to the field of oligonucleotide technology, and more specifically, to an ammonolysis treatment solution for gas-phase ammonolysis of oligonucleotides and its application. Background Technology

[0002] In the life sciences, there is an urgent need for biochips capable of high-throughput synthesis of oligonucleotide sequences. Taking oligonucleotide synthesis as an example, the chip surface needs to be modified before entering the oligonucleotide chemical synthesis stage. After synthesis, ammonolysis is used to ensure the complete cleavage of the oligonucleotide sequence, while simultaneously removing the protecting groups carried by the nucleotide monomers during the cleavage process. Therefore, cleavage ammonolysis is a crucial step in oligonucleotide synthesis using oligonucleotide synthesis chips, and it is a significant factor affecting the quality, yield, and purity of the oligonucleotide synthesis chip products.

[0003] Currently, ammonolysis mainly employs two operating modes: liquid phase and gas phase. While liquid phase ammonolysis is simple and convenient, its low chip yield prevents subsequent purification processes, thus affecting product quality and purity. Furthermore, liquid phase ammonolysis cannot meet the requirements for automated batch ammonolysis and cannot resolve crosstalk issues within a single chip. Therefore, to ensure product quality and efficient automated production, gas phase ammonolysis has become the optimal mode for oligonucleotide chip dicing and ammonolysis.

[0004] The ammonolysis solution commonly used in gas-phase ammonolysis of oligonucleotide synthesis chips cannot avoid crosstalk between different oligonucleotide sequences at different synthesis sites on the oligonucleotide chip and crosstalk between products between chips during the ammonolysis process. Ultimately, this affects the sampling of ammonolysis in a single chip partition and the simultaneous ammonolysis and cutting of multiple chips.

[0005] In view of this, the present invention is proposed. Summary of the Invention

[0006] The purpose of this invention is to provide an ammonolysis treatment solution for gas-phase ammonolysis of oligonucleotides and its application. The ammonolysis treatment solution provided by this invention can improve the problem of crosstalk between single-chip sites and products.

[0007] This invention is implemented as follows: In a first aspect, the present invention provides an ammonolysis treatment solution for gas-phase ammonolysis of oligonucleotides, comprising anhydrous n-butylamine, anhydrous triethylamine and anhydrous methanol; The volume ratio of anhydrous n-butylamine, anhydrous triethylamine, and anhydrous methanol is 1:(0.2~2):(0.2~4).

[0008] In some embodiments, the volume ratio of anhydrous n-butylamine, anhydrous triethylamine, and anhydrous methanol in the ammonolysis treatment solution is 1:(0.2~1):(0.2~1.5).

[0009] In some embodiments, the volume ratio of anhydrous n-butylamine, anhydrous triethylamine, and anhydrous methanol in the ammonolysis treatment solution is 1:(0.2~0.8):(0.2~0.8).

[0010] Secondly, the present invention provides the application of the above-mentioned ammonolysis treatment solution in the preparation of synthetic nucleic acid products.

[0011] Thirdly, the present invention provides the application of the above-mentioned ammonolysis treatment solution in the gas-phase ammonolysis of oligonucleotides linked to a solid support.

[0012] Fourthly, the present invention provides a gas-phase ammonolysis method for an oligonucleotide synthesis chip, comprising: placing the oligonucleotide synthesis chip connected to the oligonucleotide in a gas-phase ammonolysis container, and adding the above-mentioned ammonolysis treatment solution for ammonolysis treatment.

[0013] In some embodiments, the temperature of the ammonolysis treatment is 55°C to 100°C, and the reaction time is 120 min to 960 min.

[0014] In some embodiments, during ammonolysis, the ratio of oligonucleotide synthesis chip to ammonolysis solution is 1~20:10~300 (chips / mL).

[0015] In some embodiments, the ammonolysis process is carried out under sealed conditions, and the oligonucleotide synthesis chip has no substantial contact with the ammonolysis solution.

[0016] The present invention has the following beneficial effects: This invention optimizes the composition of the ammonolysis solution, removing water and methanol found in conventional ammonolysis solutions, and applies it to the gas-phase ammonolysis of oligonucleotides linked to a solid support. This ammonolysis solution not only enables high-throughput cleavage of oligonucleotide synthesis chips and simplifies the ammonolysis solution preparation process, but also improves the problems of intra-chip site crosstalk and inter-chip product crosstalk. Attached Figure Description

[0017] To more clearly illustrate the technical solutions of the embodiments of the present invention, the accompanying drawings used in the embodiments will be briefly introduced below. It should be understood that the following drawings only show some embodiments of the present invention and should not be regarded as a limitation on the scope. For those skilled in the art, other related drawings can be obtained based on these drawings without creative effort.

[0018] Figure 1 This is a schematic diagram of the solid-phase detachment of oligonucleotide sequences in this invention; Figure 2 This is a schematic diagram of oligonucleotide sequence deprotection in this invention; Figure 3This is a schematic diagram of the oligonucleotide synthesis chip structure and sequence design in Example 2; Figure 4 This is the sequence design diagram of the oligonucleotides in Example 2; Figure 5 The image shows the FISH results of the ammonia hydrolysate from Example 1 in Experiment 1 before and after treatment of the 60 / 80nt chip synthesis product. Figure 6 The image shows the FISH results of the ammonia hydrolysate from Example 2 in Experiment 1 before and after treatment of the 60 / 80nt chip synthesis product. Figure 7 The image shows the FISH results of the ammonia hydrolysate from Example 3 in Experiment 1 before and after treatment of the 60 / 80nt chip synthesis product. Figure 8 The image shows the FISH results of the ammonia hydrolysate from Example 4 in Experiment 1 before and after treatment of the 60 / 80nt chip synthesis product. Figure 9 The image shows the FISH results of the ammonia hydrolysate from Example 5 in Experiment 1 before and after treatment of the 60 / 80nt chip synthesis product. Figure 10 The image shows the FISH results of the 60 / 80nt chip synthesis product before and after treatment by the ammonolysis method in Comparative Example 5. Figure 11 The image shows the FISH results of the 60 / 80nt chip synthesis product before and after treatment using the ammonolysis method in Comparative Example 6. Figure 12 The image shows the FISH results of the 150 / 160nt chip synthesis product in Experiment Example 2. Figure 13 The images show the FISH results of the chip synthesis product in Comparative Example 1 before ammonolysis, after ammonolysis without cleaning, and after ammonolysis with cleaning; where AC represents the results of the first experiment before ammonolysis, after ammonolysis without cleaning, and after ammonolysis with cleaning, and DF represents the results of the second experiment before ammonolysis, after ammonolysis without cleaning, and after ammonolysis with cleaning (the same applies below). Figure 14 The images show the FISH results of the chip synthesis product in Comparative Example 2 before ammonolysis, after ammonolysis without cleaning, and after cleaning following ammonolysis. Figure 15 The images show the FISH results of the chip synthesis product in Comparative Example 3 before ammonolysis, after ammonolysis without cleaning, and after cleaning following ammonolysis. Figure 16 The images show the FISH results of the chip synthesis product in Comparative Example 4 before ammonolysis, after ammonolysis without cleaning, and after cleaning following ammonolysis. Figure 17 This is a capillary electrophoresis result of the 60nt chip synthesis product in Experiment Example 4; Figure 18This is a capillary electrophoresis result of the 80nt chip synthesis product in Experiment Example 4; Figure 19 The image shows the capillary electrophoresis results of the 150 and 160nt chip synthesis products in Experiment Example 5. Detailed Implementation

[0019] definition As used herein, the following terms are intended to have the meanings set forth below. It should be understood that these definitions are provided to aid in understanding the invention and are not intended to be limiting. Unless the context clearly specifies otherwise, the singular forms “an,” “a,” and “the” include plural referents. For example, the term “a reagent” includes a plurality of reagents, including mixtures thereof.

[0020] When a term is not specifically defined, it shall be given its common and customary meaning as understood by one of ordinary skill in the art at the time of this invention. In the event of any conflict between any definition generally accepted in the art and the definition provided herein, the definition provided herein shall prevail.

[0021] Various aspects of this invention are presented in scope form. It should be understood that the scope format is merely for convenience and brevity and should not be construed as a rigid limitation on the scope of this invention. Therefore, the scope description should be considered as having specifically disclosed all possible sub-scopes and common individual values ​​within those scopes.

[0022] The term "oligonucleotide" can include any polymer consisting of a limited number of covalently linked nucleotide monomers. In fact, the invention considers any type of nucleotide monomer, whether its backbone is a natural deoxyribose (DNA) or ribose (RNA) phosphodiester bond, or any form of chemical modification or artificially synthesized backbone, with non-limiting examples including phosphorothioates, peptide nucleic acids (PNAs), morpholino compounds, and locked nucleic acids (LNAs). Furthermore, oligonucleotides may contain natural purine and pyrimidine bases, or any chemical variants thereof and analogs, such as methylated, fluorescently labeled, or universal bases. The oligonucleotides of the present invention are preferably synthesized by chemical, electrochemical, biochemical, photochemical, and enzymatic methods, and can be used as primers, probes, aptamers, antisense drugs, or building blocks for assembling longer gene fragments. They can exist as single strands or combine with complementary sequences to form double strands or other higher-order structures.

[0023] The term "synthetic chip" specifically refers to any solid, semi-solid, or composite substrate used for the parallel, site-specific synthesis of oligonucleotides or longer nucleic acid molecules. In fact, this invention considers any form of substrate, whether its material is organic (e.g., various polymers, hydrogels) or inorganic (e.g., glass, quartz, silicon, ceramics, or metals), and its morphology can be rigid, flexible, porous, or three-dimensional. Furthermore, the substrate surface can be planar or contain microscopic or nanoscopic topologies formed by any means such as etching, molding, printing, or deposition, such as micropores, micropillars, microarrays, nanopores, or microfluidic channels. The substrate can be made of various materials, such as conventional glass slides, glass plates, or silicon wafers that can be processed using semiconductor techniques. Its surface has multiple discrete reaction sites, which can be defined by physical structures (such as micropores or micropillars) directly formed on the substrate (e.g., on a silicon wafer with an etched structure); or, these reaction sites can be defined and independently addressed by functional regions, for example, on a glass or silicon wafer with electrodes, where each electrode can be precisely controlled by underlying CMOS or TFT semiconductor circuitry, thereby creating a highly localized chemical environment to trigger specific steps in the synthesis reaction. Furthermore, the nucleic acid molecules synthesized on this chip can be DNA, RNA, or any chemical variant or analogue thereof, and their synthesis process can be based on any iterative chemical, electrochemical, biochemical, photochemical, and enzymatic methods, ultimately forming a high-density, sequence-programmable oligonucleotide array on the chip.

[0024] To make the objectives, technical solutions, and advantages of the embodiments of the present invention clearer, the technical solutions in the embodiments of the present invention will be clearly and completely described below. Where specific conditions are not specified in the embodiments, conventional conditions or conditions recommended by the manufacturer shall apply. Reagents or instruments whose manufacturers are not specified are all conventional products that can be purchased commercially.

[0025] The inventors discovered that in traditional techniques, the ammonolysis solution for gas-phase ammonolysis of oligonucleotide synthesis chips is often concentrated ammonia. This cannot avoid crosstalk between different oligonucleotide sequences at different synthesis sites on the oligonucleotide synthesis chip due to the presence of an aqueous phase that easily dissolves oligonucleotides in concentrated ammonia, as well as crosstalk between chip products caused by the aqueous phase. Ultimately, this affects the sampling of individual chip sections during ammonolysis and the simultaneous ammonolysis and dicing of multiple chips. To improve this problem, this invention optimizes the ammonolysis solution used in gas-phase ammonolysis. The optimized ammonolysis solution consists of anhydrous n-butylamine, anhydrous triethylamine, and anhydrous methanol, with a volume ratio of 1:(0.2~2):(0.2~4).

[0026] In the ammonolysis treatment solution of the present invention, anhydrous n-butylamine (C4H) 11N) is a nucleophilic reactant. Anhydrous n-butylamine acts as a nucleophile, directly attacking the potentiophilic sites on the target molecule (such as the carbonyl carbon of an ester group, the carbon atom of an epoxide, etc.) and forming new chemical bonds with them, thereby breaking the original chemical bonds and achieving the purpose of "dissociation" or "removal" of the target substance.

[0027] Anhydrous triethylamine (C6H) 15 N (or Et3N) is a non-nucleophilic base / acid scavenger. Many ammonolysis reactions produce acidic byproducts. For example, in the ammonolysis of acyl chlorides or certain activated esters, hydrochloric acid or the corresponding acid is produced. These acids react with butylamine (a reactant) or amines (products) to form ammonium salts, thus consuming the reactants and stopping the reaction. Triethylamine, as a base, immediately neutralizes these produced acids, protecting the main reactants and driving the reaction forward.

[0028] Anhydrous methanol (CH3OH) is used as the solvent. A mixture of n-butylamine, triethylamine, and the target material to be treated (such as a polymer film on a substrate) is dissolved in a homogeneous liquid system. Furthermore, methanol also plays the following role in this invention: Providing a suitable reaction environment: Chemical reactions can only occur when molecules come into contact with each other. Methanol, as a polar solvent, can effectively dissolve polar amines and many organic compounds, ensuring that all components can be mixed uniformly, thus greatly improving the reaction rate and efficiency.

[0029] Adjusting polarity and solubility: Methanol can help penetrate into the material to be treated (such as polymers), causing it to swell, thus allowing n-butylamine to more effectively contact the reaction site.

[0030] Dissolution of products: The products generated in the reaction also need to be dissolved and carried away. Methanol plays the same role, making the treated surface clean.

[0031] For the ammonolysis treatment solution of the present invention, the condition that the three reagents are "anhydrous" is crucial, because water molecules are also nucleophiles. If water is present, it will compete with n-butylamine and undergo a hydrolysis reaction, generating undesirable byproducts, thereby reducing the ammonolysis efficiency and potentially corroding the substrate.

[0032] To further improve the problem of product crosstalk, the present invention further optimizes the proportion of each component in the ammonolysis treatment solution based on the aforementioned proportion. More preferably, the volume ratio of anhydrous n-butylamine, anhydrous triethylamine and anhydrous methanol in the ammonolysis treatment solution is 1:(0.2~1):(0.2~1.5); more preferably, the corresponding volume ratio of the three is 1:(0.2~0.8):(0.2~0.8).

[0033] The above-mentioned ammonolysis solution can be used to gas-phase ammonoly decompose oligonucleotides linked to a solid support, wherein the solid support is preferably an oligonucleotide synthesis chip.

[0034] Based on this ammonolysis treatment solution, the present invention also provides a gas-phase ammonolysis method for oligonucleotide synthesis chips, the method comprising the following steps: S1. Preparation of ammonolysis treatment solution Anhydrous n-butylamine, anhydrous triethylamine and anhydrous methanol were mixed in a certain proportion, and the resulting mixed solution was used as the ammonolysis treatment solution.

[0035] S2. Gas-phase ammonolysis The microarray chip linked with oligonucleotides was placed in an ammonolysis container, and then the ammonolysis solution was added.

[0036] Ammonolysis refers to the process by which oligonucleotides containing various protecting groups are deprotected under the action of a high-temperature ammonolysis solution, generating free primers or probes. The principle of solid-phase deprotection of oligonucleotide sequences is as follows: Figure 1 As shown, the principle of oligonucleotide sequence deprotection is as follows: Figure 2 As shown.

[0037] The ammonolysis treatment liquid of the present invention is an effective component in the cutting and deprotection process of gas-phase ammonolysis, and the cutting and deprotection processes can be carried out simultaneously through this ammonolysis treatment liquid.

[0038] In this invention, ammonolysis is carried out in a sealed environment, and the oligonucleotide synthesis chip has no substantial contact with the ammonolysis solution. During ammonolysis, the high temperature can cause the ammonolysis solution to vaporize, causing the ammonolysis reaction to occur on the chip surface. At the same time, the sealed ammonolysis chamber effectively avoids the evaporation loss of the ammonolysis solution, which helps to complete the ammonolysis reaction efficiently.

[0039] The relative positions of the oligonucleotide synthesis chip and the ammonolysis treatment solution within the ammonolysis container can be vertical, parallel, or other relative positions, as long as there is no substantial contact between the oligonucleotide synthesis chip and the ammonolysis treatment solution. In an embodiment of the present invention, the ammonolysis treatment solution is located at the bottom of the ammonolysis device.

[0040] During the ammonolysis reaction, the temperature of the ammonolysis treatment is 55℃~100℃, and the reaction time is 120 min~960 min.

[0041] Specifically, the temperature for ammonolysis can be 55℃, 60℃, 65℃, 70℃, 75℃, 80℃, 85℃, 90℃, 95℃, or 100℃, or any value between 55℃ and 100℃. The reaction time can be 120 min, 150 min, 200 min, 300 min, 400 min, 500 min, 600 min, 700 min, 800 min, 900 min, or 960 min, or any value between 120 min and 960 min.

[0042] During ammonolysis, the ratio of oligonucleotide synthesis chips to ammonolysis solution is (1~20) chips : (10~300) mL.

[0043] In one embodiment of the present invention, the oligonucleotide synthesis chip used is a silicon-based chip with a conventional size of 25.4 * 76.2 * 0.5 mm. For chips of different sizes, those skilled in the art can adjust the amount of ammonolysis solution according to the actual chip size.

[0044] The above-mentioned gas-phase ammonolysis method can be used to cut oligonucleotides from oligonucleotide synthesis chips, and can also improve the problems of site crosstalk within a single chip and product crosstalk between chips.

[0045] The features and performance of the present invention will be further described in detail below with reference to embodiments.

[0046] Example 1 This embodiment provides an ammonolysis treatment solution for the gas-phase ammonolysis of oligonucleotides, the components of which are anhydrous n-butylamine, anhydrous triethylamine and anhydrous methanol in a ratio of 2:1:1.

[0047] The above-mentioned ammonolysis treatment solution is prepared by mixing 20 mL of anhydrous n-butylamine, 10 mL of anhydrous triethylamine, and 10 mL of anhydrous methanol.

[0048] Example 2 This embodiment provides an ammonolysis treatment solution for the gas-phase ammonolysis of oligonucleotides, the components of which are anhydrous n-butylamine, anhydrous triethylamine and anhydrous methanol, in a volume ratio of 1:0.2:0.2.

[0049] The above-mentioned ammonolysis treatment solution is prepared by mixing 10 mL of anhydrous n-butylamine, 2 mL of anhydrous triethylamine, and 2 mL of anhydrous methanol.

[0050] Example 3 This embodiment provides an ammonolysis treatment solution for the gas-phase ammonolysis of oligonucleotides, the components of which are anhydrous n-butylamine, anhydrous triethylamine and anhydrous methanol, in a volume ratio of 1:2:4.

[0051] The above-mentioned ammonolysis treatment solution is prepared by mixing 10 mL of anhydrous n-butylamine, 20 mL of anhydrous triethylamine, and 40 mL of anhydrous methanol.

[0052] Example 4 This embodiment provides an ammonolysis treatment solution for the gas-phase ammonolysis of oligonucleotides, the components of which are anhydrous n-butylamine, anhydrous triethylamine and anhydrous methanol, in a volume ratio of 2:2:3.

[0053] The above-mentioned ammonolysis treatment solution is prepared by mixing 20 mL of anhydrous n-butylamine, 20 mL of anhydrous triethylamine, and 30 mL of anhydrous methanol.

[0054] Example 5 This embodiment provides an ammonolysis treatment solution for the gas-phase ammonolysis of oligonucleotides, the components of which are anhydrous n-butylamine, anhydrous triethylamine and anhydrous methanol, in a volume ratio of 1:0.8:0.8.

[0055] The above-mentioned ammonolysis treatment solution is prepared by mixing 10 mL of anhydrous n-butylamine, 8 mL of anhydrous triethylamine, and 8 mL of anhydrous methanol.

[0056] Example 6 This embodiment describes a gas-phase ammonolysis method for oligonucleotide synthesis chips, the steps of which are as follows: (1) Take two oligonucleotide synthesis chips and synthesize two oligonucleotide sequence chips with more than 4,000 different lengths and sequence information in parallel through electrochemical principle.

[0057] The structure of the chip is as follows Figure 3 As shown, the red area represents a 60 nt oligonucleotide sequence, and the blue area represents an 80 nt oligonucleotide sequence. The oligonucleotide sequence design is as follows: Figure 4 As shown, the 3' end contains a primer region capable of polymerase chain reaction (PCR), and the 5' end contains a probe region capable of fluorescence in situ hybridization (FISH) (the primer region and probe region have the same sequence information).

[0058] (2) Place the oligonucleotide synthesis chip in an ammonolysis apparatus, and then pour 40 mL of the ammonolysis solution prepared in Examples 1-5 into the ammonolysis apparatus, ensuring that the chip does not come into contact with the ammonolysis solution. The ammonolysis temperature is 80℃ and the ammonolysis time is 5 h.

[0059] Example 7 This embodiment describes a gas-phase ammonolysis method for oligonucleotide synthesis chips, the steps of which are as follows: (1) Using the synthesis method in Example 6, oligonucleotide sequences of 150nt and 160nt were synthesized on two oligonucleotide synthesis chips respectively.

[0060] The chip structure and oligonucleotide sequence design are the same as in Example 6.

[0061] (2) Place the oligonucleotide synthesis chip in an ammonolysis apparatus, and then pour 40 mL of the ammonolysis solution prepared in Example 1 into the ammonolysis apparatus, ensuring that the chip does not come into contact with the ammonolysis solution. The ammonolysis temperature is 80℃ and the ammonolysis time is 5 h.

[0062] Comparative Example 1 The difference between this comparative example and Example 6 is that the ammonolysis treatment solution is the first ammonolysis treatment solution disclosed in CN116102599A, that is, the volume ratio of tert-butylamine, methanol and water is 1:1:2, and the steps are as follows: (1) Take two oligonucleotide synthesis chips and synthesize two oligonucleotide sequence chips with more than 4,000 different lengths and sequence information in parallel through electrochemical principle.

[0063] The structure of the chip is as follows Figure 3 As shown, the red area represents a 60 nt oligonucleotide sequence, and the blue area represents an 80 nt oligonucleotide sequence. The oligonucleotide sequence design is as follows: Figure 4 As shown, the 3' end contains a primer region capable of polymerase chain reaction (PCR), and the 5' end contains a probe region capable of fluorescence in situ hybridization (FISH) (the primer region and probe region have the same sequence information).

[0064] (2) Place the oligonucleotide synthesis chip in the ammonolysis apparatus, and then pour 40 mL of the prepared ammonolysis solution into the ammonolysis apparatus, ensuring that the chip does not come into contact with the ammonolysis solution. The ammonolysis temperature is 80℃ and the ammonolysis time is 3 h.

[0065] Comparative Example 2 The difference between this comparative example and Example 6 is that the ammonolysis treatment solution is concentrated ammonia water, and the steps are as follows: (1) Take two oligonucleotide synthesis chips and synthesize two oligonucleotide sequence chips with more than 4,000 different lengths and sequence information in parallel through electrochemical principle.

[0066] The structure of the chip is as follows Figure 3 As shown, the red area represents a 60 nt oligonucleotide sequence, and the blue area represents an 80 nt oligonucleotide sequence. The oligonucleotide sequence design is as follows: Figure 4 As shown, the 3' end contains a primer region capable of polymerase chain reaction (PCR), and the 5' end contains a probe region capable of fluorescence in situ hybridization (FISH) (the primer region and probe region have the same sequence information).

[0067] (2) Place the oligonucleotide synthesis chip in an ammonolysis apparatus, and then pour 40 mL of ammonolysis solution into the apparatus, ensuring that the chip does not come into contact with the ammonolysis solution. The ammonolysis temperature is 80℃ and the ammonolysis time is 3 h.

[0068] Comparative Example 3 The difference between this comparative example and Example 6 is that the ammonolysis treatment solution is tert-butylamine, and the steps are as follows: (1) Take two oligonucleotide synthesis chips and synthesize two oligonucleotide synthesis chips with more than 4,000 different lengths and sequence information in parallel by means of electrochemistry.

[0069] The structure of the chip is as follows Figure 3 As shown, the red area represents a 60 nt oligonucleotide sequence, and the blue area represents an 80 nt oligonucleotide sequence. The oligonucleotide sequence design is as follows: Figure 4 As shown, the 3' end contains a primer region capable of polymerase chain reaction (PCR), and the 5' end contains a probe region capable of fluorescence in situ hybridization (FISH) (the primer region and probe region have the same sequence information).

[0070] (2) Place the oligonucleotide synthesis chip in an ammonolysis apparatus, and then pour 40 mL of ammonolysis solution into the apparatus, ensuring that the chip does not come into contact with the ammonolysis solution. The ammonolysis temperature is 80℃ and the ammonolysis time is 5 h.

[0071] Comparative Example 4 The difference between this comparative example and Example 6 is that the ammonolysis treatment solution is pure triethylamine, and the steps are as follows: (1) Take two oligonucleotide synthesis chips and synthesize two oligonucleotide sequence chips with more than 4,000 different lengths and sequence information in parallel through electrochemical principle.

[0072] The structure of the chip is as follows Figure 3 As shown, the red area represents a 60 nt oligonucleotide sequence, and the blue area represents an 80 nt oligonucleotide sequence. The oligonucleotide sequence design is as follows: Figure 4 As shown, the 3' end contains a primer region capable of polymerase chain reaction (PCR), and the 5' end contains a probe region capable of fluorescence in situ hybridization (FISH) (the primer region and probe region have the same sequence information).

[0073] (2) Place the oligonucleotide synthesis chip in an ammonolysis apparatus, and then pour 40 mL of ammonolysis solution into the apparatus, ensuring that the chip does not come into contact with the ammonolysis solution. The ammonolysis temperature is 80℃ and the ammonolysis time is 5 h.

[0074] Comparative Example 5 The ammonia hydrolysate of Example 1 was used for gas-phase ammonia hydrolysis. The difference between Example 6 and Example 6 was that the ammonia hydrolysis time was different, which was 90 min.

[0075] Comparative Example 6 The ammonia hydrolysate of Example 1 was used for gas-phase ammonia hydrolysis. The difference between Example 6 and Example 6 is that the ammonia hydrolysis temperature is different, which is 50°C.

[0076] Experimental Example 1 Fluorescence in situ hybridization of the oligonucleotide synthesis chip surface before ammonolysis in Example 6 was performed using the CY3 fluorescent probe. After hybridization, the chip surface was washed sequentially with 10 ml each of anhydrous acetonitrile, anhydrous methanol, and double-distilled water, and then dried with nitrogen gas. Fluorescence scanning was then performed, and the results were saved.

[0077] After ammonolysis of the oligonucleotide synthesis chip in Example 6, the ammonolysis chamber was cooled to room temperature before the ammonolysis apparatus was opened, the oligonucleotide chip was removed, and the chip surface was then cleaned sequentially with anhydrous acetonitrile and anhydrous methanol, dried with nitrogen, and finally eluted with double-distilled water. The eluted products were stored, and the surface of the eluted oligonucleotide synthesis chip was subjected to fluorescence in situ hybridization with a CY3 fluorescent probe. After hybridization, the chip surface was cleaned sequentially with 10 ml each of anhydrous acetonitrile, anhydrous methanol, and double-distilled water, dried with nitrogen, and then subjected to fluorescence scanning. The results were stored.

[0078] according to Figure 5 The FISH results showed that after the ammonolysis of the chip was completed, the CY3 fluorescent probe did not hybridize successfully, indicating that the ammonolysis treatment solution in Example 1 was effective.

[0079] according to Figure 6 The FISH results showed that the hybridization scan before ammonolysis indicated that the synthesis was effective, the non-hybridization scan after washing after ammonolysis indicated that the hybridization probe in the previous step had been cleaned, and no fluorescence was seen in the hybridization scan after washing after ammonolysis, indicating that the ammonolysis treatment solution in Example 2 was effective. Figure 7 The test results are for Example 3. Figure 8 The test results are for Example 4. Figure 9 The results are from Example 5. All the above results show that the ammonolysis treatment solution is effective.

[0080] The same verification method was used to compare the effect of the gas-phase ammonolysis method in Comparative Examples 5-6 on the ammonolysis effect, and the results are as follows: Figures 10-11 As shown in the two figures, it can be seen that both the ammonolysis time and temperature affect the ammonolysis effect; if the range is exceeded, the ammonolysis will be incomplete.

[0081] Experiment Example 2 Fluorescence in situ hybridization of the oligonucleotide synthesis chip surface before ammonolysis in Example 7 was performed using the CY3 fluorescent probe. After hybridization, the chip surface was washed sequentially with 10 ml each of anhydrous acetonitrile, anhydrous methanol, and double-distilled water, and then dried with nitrogen gas. Fluorescence scanning was then performed, and the results were saved.

[0082] After ammonolysis of the oligonucleotide synthesis chip in Example 7, the ammonolysis chamber was cooled to room temperature before the ammonolysis apparatus was opened, the oligonucleotide chip was removed, and the surfaces of the two chips were then cleaned sequentially with anhydrous acetonitrile and anhydrous methanol, dried with nitrogen, and finally eluted with double-distilled water. The eluted products were stored, and fluorescence in situ hybridization with the CY3 fluorescent probe was performed on the surface of the eluted oligonucleotide synthesis chip. After hybridization, the chip surface was cleaned sequentially with 10 ml each of anhydrous acetonitrile, anhydrous methanol, and double-distilled water, dried with nitrogen, and then subjected to fluorescence scanning. The results were stored.

[0083] according to Figure 12 The FISH results showed that after the ammonolysis of the chip was completed, the CY3 fluorescent probe did not hybridize successfully, indicating that the ammonolysis of the ammonolysis solution in this experiment was effective.

[0084] Experimental Example 3 Comparative Examples 1-4 were subjected to fluorescence in situ hybridization using CY3 fluorescent probes on the surface of the oligonucleotide synthesis chip before ammonolysis. After hybridization, the chip surface was washed sequentially with 10 ml each of anhydrous acetonitrile, anhydrous methanol, and double-distilled water, and then dried with nitrogen gas. Fluorescence scanning was then performed, and the results were saved.

[0085] After ammonolysis of the oligonucleotide synthesis chips in Comparative Examples 1-4, the ammonolysis chamber was cooled to room temperature before the ammonolysis apparatus was opened. The oligonucleotide chips were removed and subjected to fluorescence in situ hybridization (FISH) followed by scanning. The results were saved. Then, the surfaces of the two chips were cleaned sequentially with anhydrous acetonitrile and anhydrous methanol, dried with nitrogen, and finally eluted with double-distilled water. The eluted products were saved. The surface of the eluted oligonucleotide synthesis chips was subjected to FISH using the CY3 fluorescent probe. After hybridization, the chip surface was cleaned sequentially with 10 ml each of anhydrous acetonitrile, anhydrous methanol, and double-distilled water, dried with nitrogen, and then subjected to fluorescence scanning. The results were saved.

[0086] Each comparative example was repeated twice. According to the FISH results, the results of gas-phase ammoniation of the hybridization probes on the two chips in Comparative Example 1 were consistent. However, two chips had a large amount of water stains on the chip surface after ammoniation, indicating crosstalk and poor ammoniation effect. Therefore, they were not used for subsequent PCR quality control. Figure 13In Comparative Example 2, the results of ammonia hydrolysis on the chips carrying hybridization probes were consistent in both cases. Normally, after hydrolysis, the entire surface would still fluoresce after FISH due to the presence of the probes. However, ammonia hydrolysis with water resulted in spot impurities on the chips due to the presence of water. Therefore, ammonia is not suitable for this method. The absence of fluorescence after washing and FISH following hydrolysis indicates that ammonia hydrolysis with water is still effective; the main issue is crosstalk. Due to the poor hydrolysis effect, subsequent PCR quality control was not performed. Figure 14 The results of gas-phase ammonolysis of the hybridization probes on the chips in Comparative Example 3 were consistent in both cases. The chip ammonolysis solution still showed fluorescence signals after washing following ammonolysis, indicating that the ammonolysis effect was poor and therefore it was not used for subsequent PCR quality control. Figure 15 The results of gas-phase ammonolysis of the hybridization probes on the chips in Comparative Example 4 were consistent in both cases, indicating poor ammonolysis efficiency and significant damage to the chip surface; therefore, it was not used for subsequent PCR quality control. Figure 16 ).

[0087] Experiment Example 4 In this experiment, the oligonucleotide chip products after ammonolysis in Example 7 were subjected to PCR quality control, as detailed below: The surface of the oligonucleotide chip was sampled in sections according to the synthesis region. The sampling buffer used was 10 mM Tris-HCl solution, pH 8.0.

[0088] The PCR reaction system is as follows:

[0089] The PCR reaction procedure is as follows:

[0090] After the PCR reaction was completed, the PCR products were subjected to capillary electrophoresis for quality control. The quality control results are as follows: Figure 17 and Figure 18 As shown, capillary electrophoresis results indicate that different oligonucleotide sequence length regions only amplify the single sequence length of their respective regions. This phenomenon demonstrates that the gas-phase ammonolysis method of the present invention does not cause site crosstalk within a single chip, further illustrating the feasibility of the gas-phase ammonolysis method.

[0091] Experimental Example 5 In this experiment, the oligonucleotide synthesis chip after ammonolysis in Example 8 was subjected to PCR quality control, as detailed below: The surface of the oligonucleotide synthesis chip was sampled in sections according to the synthesis region. The sampling buffer was 10 mM Tris-HCl solution, pH 8.0.

[0092] The PCR reaction system is as follows:

[0093] The PCR reaction procedure is as follows:

[0094] After the PCR reaction was completed, the PCR products were subjected to capillary electrophoresis for quality control. The quality control results are as follows: Figure 19 As shown, capillary electrophoresis results indicate that different oligonucleotide sequence length regions only amplify the single sequence length of their respective regions. This phenomenon demonstrates that the gas-phase ammonolysis method of the present invention does not cause site crosstalk within a single chip, further illustrating the feasibility of the gas-phase ammonolysis method.

[0095] In summary, the gas-phase ammonolysis treatment solution for oligonucleotide synthesis chips and the method for gas-phase ammonolysis treatment using the ammonolysis treatment solution provided by the present invention have certain advantages in the automated cutting and ammonolysis of oligonucleotide chips and in solving the problem of crosstalk between sites within a single chip.

[0096] The above description is merely a preferred embodiment of the present invention and is not intended to limit the invention. Various modifications and variations can be made to the present invention by those skilled in the art. Any modifications, equivalent substitutions, improvements, etc., made within the spirit and principles of the present invention should be included within the scope of protection of the present invention.

Claims

1. An ammonolysis treatment solution for the gas-phase ammonolysis of oligonucleotides, characterized in that, The ammonolysis treatment solution includes anhydrous n-butylamine, anhydrous triethylamine and anhydrous methanol; The volume ratio of anhydrous n-butylamine, anhydrous triethylamine and anhydrous methanol is 1:(0.2~2):(0.2~4).

2. The ammonolysis treatment solution according to claim 1, characterized in that, The volume ratio of anhydrous n-butylamine, anhydrous triethylamine, and anhydrous methanol in the ammonolysis treatment solution is 1:(0.2~1):(0.2~1.5).

3. The ammonolysis treatment solution according to claim 2, characterized in that, The volume ratio of anhydrous n-butylamine, anhydrous triethylamine, and anhydrous methanol in the ammonolysis treatment solution is 1:(0.2~0.8):(0.2~0.8).

4. The use of the ammonolysis solution according to any one of claims 1-3 in the preparation of synthetic nucleic acid products.

5. The use of the ammonolysis solution according to any one of claims 1-3 in the gas-phase ammonolysis of oligonucleotides linked to a solid support.

6. A gas-phase ammonolysis method for an oligonucleotide synthesis chip, characterized in that, include: An oligonucleotide synthesis chip connected to oligonucleotides is placed in a gas-phase ammonolysis container, and the ammonolysis treatment solution according to any one of claims 1-3 is added for ammonolysis treatment.

7. The gas-phase ammonolysis method according to claim 6, characterized in that, The temperature for ammonolysis is 55℃~100℃.

8. The gas-phase ammonolysis method according to claim 6, characterized in that, The reaction time for ammonolysis is 120 min to 960 min.

9. The gas-phase ammonolysis method according to claim 6, characterized in that, During ammonolysis, the ratio of oligonucleotide synthesis chip to ammonolysis solution is 1~20:10~300 (chips / mL).

10. The gas-phase ammonolysis method according to any one of claims 6-9, characterized in that, The ammonolysis treatment is carried out under sealed conditions, and the oligonucleotide synthesis chip has no substantial contact with the ammonolysis treatment solution.

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