阅读说明：本技术 一种氨解液及氨解方法 (Ammonolysis solution and ammonolysis method ) 是由 曹春艳 赵双 靳开宝 纪磊 韩承昊 朱知浩 于 2020-08-18 设计创作，主要内容包括：本发明提供了一种氨解液及氨解方法,所述氨解液包括胺、丁醇、氨基醇和碱性水溶液,所述氨解方法包括：(1)向装有固相载体的固相合成柱中加入氨解液；(2)将所述固相合成柱置于装有氨解缓冲液的密封容器内,并保持所述固相合成柱不接触氨解缓冲液；(3)加热所述密封容器,使固相载体处于氨解缓冲液的蒸汽气氛中,随后冷却；(4)从所述密封容器中取出固相合成柱,用洗脱液处理固相合成柱中的固相载体,并收集流出液。本发明的氨解液配方温和,氨解缓冲液不仅提供氨解氛围,而且辅助氨解反应,氨解工艺不使用有毒气体、安全性高、环境友好、不破坏引物结构,同时后处理步骤简单,避免交叉污染,可以实现大规模工业化应用。(The invention provides an ammonolysis solution and an ammonolysis method, wherein the ammonolysis solution comprises amine, butanol, amino alcohol and an alkaline aqueous solution, and the ammonolysis method comprises the following steps: (1) adding the ammonolysis solution into a solid-phase synthesis column filled with a solid-phase carrier; (2) placing the solid phase synthesis column in a sealed container filled with an ammonolysis buffer solution, and keeping the solid phase synthesis column not in contact with the ammonolysis buffer solution; (3) heating the sealed container to expose the solid support to a vapor atmosphere of an aminolysis buffer, followed by cooling; (4) and taking the solid phase synthesis column out of the sealed container, treating the solid phase carrier in the solid phase synthesis column with eluent, and collecting the effluent. The ammonolysis solution disclosed by the invention is mild in formula, the ammonolysis buffer solution not only provides ammonolysis atmosphere, but also assists in ammonolysis reaction, the ammonolysis process does not use toxic gas, is high in safety and environment-friendly, does not damage a primer structure, and meanwhile, the post-treatment step is simple, so that cross contamination is avoided, and large-scale industrial application can be realized.)

1. The ammonolysis solution is characterized in that the ammonolysis solution comprises amine, butanol, amino alcohol and an alkaline aqueous solution;

the volume ratio of the amine to the butanol to the amino alcohol to the alkaline aqueous solution is (5-10): (1-5): 1: (0.01-0.1).

2. The ammonolysis solution according to claim 1, wherein said amine comprises propylamine and/or butylamine.

3. The ammonolysis liquid according to claim 2, wherein said propylamine comprises n-propylamine and/or isopropylamine.

4. The ammonolysis solution according to claim 2, wherein said butylamine comprises any one or a combination of at least two of n-butylamine, isobutylamine, sec-butylamine, or tert-butylamine.

5. The ammonolysis solution according to claim 1, wherein the butanol comprises any one of n-butanol, iso-butanol, sec-butanol or tert-butanol, or a combination of at least two thereof.

6. The ammonolysis solution according to claim 1, wherein the amino alcohol comprises amino butanol and/or amino pentanol.

7. The ammonolysis solution according to claim 6, wherein the aminopentanol comprises 5-amino-1-pentanol and/or 4-amino-1-pentanol.

8. The ammonolysis solution according to claim 1, wherein the aqueous alkaline solution comprises an aqueous LiOH solution, an aqueous NaOH solution, an aqueous KOH solution, Be (OH)₂Aqueous solution, Mg (OH)₂Aqueous solutions or Ca (OH)₂Any one of or a combination of at least two of the aqueous solutions.

9. The ammonolysis solution according to claim 1, wherein the concentration of said aqueous alkaline solution is 1 to 10M.

10. The ammonolysis solution according to claim 1, wherein the volume ratio of the amine, butanol, amino alcohol and the alkaline aqueous solution is (6-8): 2-3): 1: (0.04-0.06).

11. The ammonolysis solution according to any one of claims 4 to 8, wherein the volume ratio of n-butylamine, n-butanol, 5-amino-1-pentanol to the aqueous alkaline solution is (5-10): 1-5): 1 (0.01-0.1).

12. The ammonolysis solution according to claim 11, wherein the volume ratio of n-butylamine, n-butanol, 5-amino-1-pentanol to the aqueous alkaline solution is (6-8): 2-3): 1: (0.04-0.06).

13. An ammonolysis process, characterized in that it comprises the following steps:

(1) adding the ammonolysis solution of any one of claims 1 to 12 to a solid phase synthesis column loaded with a solid phase support;

(2) placing the solid phase synthesis column in a sealed container filled with an ammonolysis buffer solution, and keeping the solid phase synthesis column not in contact with the ammonolysis buffer solution;

(3) heating the sealed container to expose the solid support to a vapor atmosphere of an aminolysis buffer, followed by cooling;

(4) and taking the solid phase synthesis column out of the sealed container, treating the solid phase carrier in the solid phase synthesis column with eluent, and collecting the effluent.

14. The method of claim 13, wherein the solid support of step (1) comprises controlled pore glass spheres.

15. The method of claim 13, wherein the solid support of step (1) has a synthetic oligonucleotide attached thereto.

16. The method of claim 13, wherein the ammonolysis solution of step (1) is impregnated with a solid support.

17. The method of claim 13, wherein step (2) the sealed container comprises a metal sealed box.

18. The method of claim 13, wherein the ammonolysis buffer of step (2) comprises ammonia and/or an organic amine.

19. The method of claim 18, wherein the organic amine comprises any one of diethylamine, ethylenediamine, or pyridine, or a combination of at least two thereof.

20. The method according to claim 13, wherein the heating temperature in the step (3) is 45-100 ℃.

21. The method according to claim 13, wherein the heating time in the step (3) is 30-60 min.

22. The method of claim 13, wherein the cooling in step (3) is performed in an ice bath for 5-15 min.

23. The method of claim 13, wherein the eluent of step (4) comprises water and/or Tris-HCl.

24. The method of claim 23, wherein the concentration of Tris-HCl is 10-30 mM.

25. The method of claim 24, wherein the Tris-HCl has a pH of 7 to 8.

26. The method of claim 13, further comprising the step of drying and washing the solid phase synthesis column prior to treating the solid phase support in the solid phase synthesis column with the eluent in step (4).

27. The method according to claim 26, wherein the drying is carried out at 50 to 60 ℃ for 1 to 5 min.

28. The method according to claim 26, wherein the washing is performed by using a washing solution comprising 100% acetonitrile and/or 80-95% acetonitrile.

29. The method of claim 28, wherein the washing comprises: and washing the dried solid-phase synthesis column by using 100% acetonitrile and 80-95% acetonitrile in sequence.

30. The method of claim 26, further comprising a step of centrifugation after washing the solid phase synthesis column.

31. The method according to claim 13, characterized in that it comprises the steps of:

(1) adding the ammonolysis solution of any one of claims 1 to 12 to a solid phase synthesis column containing controlled pore glass spheres to which synthetic oligonucleotides are attached, and immersing the controlled pore glass spheres;

(2) placing the solid phase synthesis column in a metal sealed container filled with an ammonolysis buffer solution, and keeping the solid phase synthesis column not to contact the ammonolysis buffer solution, wherein the ammonolysis buffer solution comprises ammonia and/or organic amine;

(3) heating the metal sealed container at 45-100 ℃ for 30-60 min to enable the glass spheres connected with the synthetic oligonucleotides and with the controlled apertures to be in the steam atmosphere of an aminolysis buffer solution, and then cooling for 5-15 min in an ice bath;

(4) and taking the solid phase synthesis column out of the sealed container, drying at 50-60 ℃ for 1-5 min, washing with 100% acetonitrile and 80-95% acetonitrile in sequence, centrifuging, treating the solid phase carrier in the solid phase synthesis column with water and/or 10-25 mM Tris-HCl with pH of 7-8, and collecting the effluent.

Technical Field

The invention belongs to the technical field of nucleic acid synthesis, and relates to an ammonolysis solution and an ammonolysis method.

Background

Short-chain DNA, also known as oligonucleotides (oligos), is usually synthesized by solid-phase synthesis. In the solid phase synthesis, a controlled pore glass sphere (CPG) is used as a solid phase carrier, phosphoramidite is used as a monomer, and a target sequence is gradually connected to the solid phase carrier through four reaction cycles of deprotection, condensation, oxidation and capping according to the 3 '-5' direction. The advantages of the solid phase synthesis method are that: each step of reaction is carried out on a solid phase carrier, unreacted raw materials can be removed in a suction filtration mode after the reaction is finished, and the post-treatment step is simple. Therefore, solid phase synthesis is widely used for Oligo and polypeptide synthesis. In most of the existing Oligo synthesis processes, 5' -OH of phosphoramidite monomer is protected by Dimethoxytriphenyl (DMT) group, and can be in 2-3% CCl₃COOH/CH₂Cl₂Is cut off in the solution, and is called deprotection reaction; carrying out nucleophilic substitution reaction, namely condensation reaction, on the exposed free hydroxyl after deprotection and phosphoramidite; after the condensation reaction is finished, in order to prevent unreacted hydroxyl from entering the next condensation, the unreacted free hydroxyl needs to be acetylated, which is commonly called as a 'cap'; finally, since phosphoramidites are very unstable, oxidation of the phosphoramidite to +5 valent phosphorus is required with an oxidizing agent such as iodine. Four reactions are required for each base ligation, and after the target base is completely synthesized, the Oligo is cleaved from the CPG. Meanwhile, since A, C, G bp contains an active amino group and interferes with the condensation reaction, the amino group is generally protected with an acetyl group (Ac), a benzoyl group (Bz) and a diazomethide amidine group (dmf) in advance, and the Ac, Bz and dmf groups are cleaved after the reaction is completed. Herein, the reaction of cleaving Oligo from CPG and removing the protecting group is referred to as aminolysis. The ammonolysis reaction mainly comprises the following steps: (1) cleaving the Oligo from the solid support; (2) exposing the 3' -OH of Oligo; (3) cutting off protecting groups Ac, Bz and dmf at A, C, G base; (4) the cyanoethyl group on the phosphoric acid was cleaved off.

The ammonolysis reaction is carried out by adopting ammonia water at first, and the problems mainly comprise that the reaction time is long, 16 hours are needed for the reaction at 55 ℃, and 8 hours are needed for the reaction at 65 ℃; in the reaction process, the synthetic column needs to be soaked in ammonia water, so that cross contamination is easily caused; because the Oligo can be dissolved by ammonia water, the post-treatment steps are complicated; impurities in the ammonia water can accumulate, and Oligo pollution is caused. The ammonolysis reaction system of methylamine with ammonia =1:1 (AMA) which is developed later greatly reduces the reaction time (M.P. Reddy, N.B. Hanna, and F. Farooqui, Nucleos. Nucleot., 1997, 16, 1589-. The gas-phase ammonolysis adopts ammonia gas as an active medium, takes a high-pressure resistant ammonolysis pot as a reaction vessel, and carries out ammonolysis reaction under the reaction conditions of high temperature and high pressure. The gas phase ammonolysis has the advantages of quick and efficient reaction and simple operation, but has the defects of high equipment requirement, need of using toxic gas, potential safety hazard, environmental pollution and great structural damage to primers. Microwave ammonolysis is a process widely applied in recent years, mainly uses microwaves as a heating means, but in the actual operation process, microwave heating easily causes uneven heating, continuous heating can cause liquid bumping, loss and cross contamination.

CN109956987A discloses a method for ammonolysis after solid phase synthesis of DNA, comprising the steps of: 1) adding an ammonolysis composition to a synthesis column comprising a solid support for synthesizing DNA fragments; 2) placing the synthesis column in a sealable container containing an ammonolysis buffer solution and maintaining the synthesis column above the surface of the ammonolysis buffer solution; and 3) microwaving the sealable container such that the solid support is in a vapor atmosphere of the aminolysis buffer; 4) the synthesis column is removed, the solid phase support is treated with an eluent and the effluent is collected. However, the method still has the problems that the microwave heating is not uniformly heated, and the microwave heating is easy to cause damage or cause cross contamination.

Therefore, it is necessary to develop a new ammonolysis method, which has mild reaction conditions, does not damage the structure of the primer, and solves the problems existing in the prior art.

Disclosure of Invention

Aiming at the defects and actual requirements of the prior art, the invention provides the ammonolysis solution and the ammonolysis method, the formula of the ammonolysis solution is mild, the ammonolysis buffer solution not only provides ammonolysis atmosphere, but also assists ammonolysis reaction, the ammonolysis process does not use toxic gas, has high safety, is environment-friendly, does not damage a primer structure, and meanwhile, has simple post-treatment steps and avoids cross contamination.

In order to achieve the purpose, the invention adopts the following technical scheme:

in a first aspect, the present invention provides an ammonolysis solution comprising an amine, butanol, an aminoalcohol and an aqueous alkaline solution.

In the present invention, an amine and two alcohols are coordinated with each other to cleave the synthesized oligonucleotide from the solid support and remove the protecting groups Ac, Bz, dmf at A, C, G bases, wherein the amine serves as the active ingredient of the ammonolysis solution, butanol serves as the reaction solvent, and the amino alcohol serves both functions.

The invention adds a trace amount of alkaline aqueous solution into the ammonolysis solution to play the role of a catalyst, and the catalyst is matched with amine, butanol and alkamine, thereby being beneficial to accelerating the ammonolysis reaction rate, playing the role of ammonolysis catalysis and not influencing the yield and the quality of oligonucleotide.

Preferably, the amine comprises propylamine and/or butylamine.

Preferably, the propylamine comprises n-propylamine and/or isopropylamine.

Preferably, the butylamine comprises any one or a combination of at least two of n-butylamine, iso-butylamine, sec-butylamine, or tert-butylamine, preferably n-butylamine.

In the invention, propylamine and/or butylamine are/is used as an active ingredient of the ammonolysis solution and are/is matched with butanol, amino alcohol and an alkaline aqueous solution to exert high-efficiency ammonolysis activity, so that high-efficiency ammonolysis reaction is realized at a specific ratio.

Preferably, the butanol includes any one or a combination of at least two of n-butanol, iso-butanol, sec-butanol or tert-butanol, preferably n-butanol.

Preferably, the amino alcohol comprises amino butanol and/or amino pentanol.

Preferably, the aminopentanol comprises 5-amino-1-pentanol and/or 4-amino-1-pentanol, preferably 5-amino-1-pentanol.

Preferably, the basic aqueous solution includes an aqueous LiOH solution, an aqueous NaOH solution, an aqueous KOH solution, Be (OH)₂Aqueous solution, Mg (OH)₂Aqueous solutions or Ca (OH)₂Any one of or a combination of at least two of the aqueous solutions.

Preferably, the concentration of the alkaline aqueous solution is 1-10M, for example, 1M, 2M, 3M, 4M, 5M, 6M, 7M, 8M, 9M or 10M, preferably 5M.

According to the invention, the concentration of the alkaline aqueous solution is limited within the range of 1-10M, the ammonolysis reaction is facilitated to be efficiently carried out, the concentration of the alkaline aqueous solution cannot be too low or too high, the effect of promoting the ammonolysis reaction cannot be achieved when the concentration is too low, the yield and the purity of the oligonucleotide are influenced when the concentration is too high, the eluted oligonucleotide is easy to generate a salt peak during mass spectrometry detection, and the appearance of the dried oligonucleotide is likely to be whitish.

Preferably, the volume ratio of the amine, the butanol, the amino alcohol and the alkaline aqueous solution is (5-10): 1 (1-5): 1: (0.01-0.1), and may be, for example, 5:1:0.01, 5:2:1:0.05, 5:5:1:0.1, 6:1:1:0.01, 6:2:1:0.05, 6:5:1:0.1, 7:1: 0.01, 7:2:1:0.05, 7:5:1:0.1, 8:1:1:0.01, 8:2:1:0.05, 8:5:1:0.1, 9:1: 0.01, 9:2:1:0.05, 9:5:1:0.1, 10:1: 0.01, 10:2:1:0.05 or 10:5:1: 0.06, preferably, 6:1: 0.01-0.06.

In the invention, amine, butanol, amino alcohol and alkaline aqueous solution are matched with each other in a specific ratio, the amine, butanol and amino alcohol are used as main functional components of the ammonolysis solution, the synthesized oligonucleotide is cut from the solid phase carrier, protective groups Ac, Bz and dmf on A, C, G basic groups are removed, a trace amount of alkaline aqueous solution plays a role in ammonolysis catalysis, the yield of the oligonucleotide is improved, the phenomenon that the appearance of the oligonucleotide is whitish is avoided, and the purity of the obtained oligonucleotide is high.

Preferably, the volume ratio of the n-butylamine, the n-butanol, the 5-amino-1-pentanol to the NaOH/KOH aqueous solution is (5-10): 1-5): 1, (0.01-0.1), preferably (6-8): 2-3): 1, (0.04-0.06).

In the present invention, the ratio of the alkaline aqueous solution in the ammonolysis solution also cannot be too low or too high, and too low a ratio cannot play a role in promoting ammonolysis reaction, and too high a ratio may affect the yield and purity of the oligonucleotide.

In a second aspect, the present invention provides an ammonolysis buffer comprising ammonia and/or an organic amine.

In the invention, the ammonolysis buffer solution is gasified into steam under the heating condition, so that a proper ammonolysis environment is provided, and the ammonolysis efficiency is improved; when ammonia water is used as an ammonolysis buffer solution, gasified ammonia gas can provide a proper ammonolysis environment and can assist liquid-phase ammonolysis to carry out gas-phase ammonolysis reaction, ammonolysis efficiency and oligonucleotide yield are remarkably improved, and reaction time is shortened.

Preferably, the organic amine comprises any one of diethylamine, ethylenediamine or pyridine or a combination of at least two thereof.

In the invention, when organic amine is used as an ammonolysis buffer solution, the gasified organic amine can provide a proper ammonolysis environment and can assist liquid phase ammonolysis to carry out gas phase ammonolysis reaction, thereby obviously improving ammonolysis efficiency and oligonucleotide yield and shortening reaction time.

In a third aspect, the present invention provides an ammonolysis process comprising the steps of:

(1) adding the ammonolysis solution of the first aspect to a solid phase synthesis column loaded with a solid phase support;

(2) placing said solid phase synthesis column in a sealed container containing an ammonolysis buffer according to the second aspect and keeping said solid phase synthesis column free from contact with the ammonolysis buffer;

(3) heating the sealed container to expose the solid support to a vapor atmosphere of an aminolysis buffer, followed by cooling;

(4) and taking the solid phase synthesis column out of the sealed container, treating the solid phase carrier in the solid phase synthesis column with eluent, and collecting the effluent.

In the invention, the solid phase carrier is immersed in the ammonolysis solution, ammonolysis reaction can be carried out under mild heating conditions, ammonolysis buffer solution in a sealed container is gasified, the ammonolysis reaction is promoted to be carried out continuously, synthesized oligonucleotide is cut off from the solid phase carrier, and amino protecting groups on basic groups are removed, thus being beneficial to obtaining oligonucleotide products with high purity and high yield.

Preferably, the solid phase carrier in step (1) comprises a glass sphere with controlled pore size.

According to the present invention, there are gaps between the spheres of Controlled Pore Glass (CPG) in which the ammonolysis solution flows to cleave the oligonucleotide from the CPG.

Preferably, the solid phase carrier in step (1) is linked with a synthetic oligonucleotide, the oligonucleotide is an artificially synthesized DNA short chain, generally comprises 20-60 bases, and can be used as a primer, a sequencing linker, a probe and the like, the ammonolysis solution can cut the artificially synthesized DNA short chain from the solid phase carrier, and the amino protecting groups Ac, Bz and dmf on A, C, G bases are cut off.

Preferably, the ammonolysis solution of step (1) is impregnated with a solid support.

Preferably, the sealed container of step (2) comprises a metal sealed box.

In the invention, the metal sealing box is used as the ammonolysis box, an atmosphere slightly higher than the atmospheric pressure is provided, and metal is a good thermal conductor, so that the heating temperature provided by the heating device can be rapidly transferred into the ammonolysis box, and the gasification of the ammonolysis buffer solution and the ammonolysis reaction can be efficiently initiated; meanwhile, the sealed ammonolysis box effectively avoids the volatilization loss of the ammonolysis solution and the ammonolysis buffer solution, and is beneficial to efficiently finishing the ammonolysis reaction.

Preferably, the heating temperature in step (3) is preferably 45 to 100 ℃, for example, 45 ℃, 50 ℃, 55 ℃, 60 ℃, 65 ℃, 70 ℃, 75 ℃, 80 ℃, 81 ℃, 82 ℃, 83 ℃, 84 ℃, 85 ℃, 86 ℃, 87 ℃, 88 ℃, 89 ℃, 90 ℃, 91 ℃, 92 ℃, 93 ℃, 94 ℃, 95 ℃, 96 ℃, 97 ℃, 98 ℃, 99 ℃ or 100 ℃, and more preferably 90 to 95 ℃, by bringing the ammonolysis buffer solution to a vaporization temperature and forming a steam atmosphere of the ammonolysis buffer solution in a sealed container, thereby exposing the solid phase synthesis column to the steam atmosphere of the ammonolysis buffer solution and promoting the ammonolysis of the ammonolysis solution, and shearing the synthesized oligonucleotides from the solid phase carrier.

Preferably, the heating time in step (3) is related to synthesis specification, ammonolysis solution formula, heating temperature, sealing container volume and completion degree of desired ammonolysis reaction, and is adjusted according to specific implementation conditions, and the heating time is preferably 30-60 min, such as 30 min, 35 min, 40 min, 45 min, 50 min, 55 min or 60 min.

Preferably, the cooling in the step (3) is ice bath for 5-15 min, preferably ice bath cooling for 10 min.

Preferably, the eluent in step (4) comprises water and/or Tris-HCl.

Preferably, the concentration of Tris-HCl is 10-30 mM, such as 10 mM, 11 mM, 12 mM, 13mM, 14 mM, 15 mM, 16 mM, 17 mM, 18 mM, 19 mM, 20 mM, 21 mM, 22 mM, 23 mM, 24 mM, 25 mM, 26mM, 27 mM, 28 mM, 29 mM or 30 mM, preferably 25 mM.

Preferably, the Tris-HCl has a pH of 7 to 8, and may be, for example, 7, 7.1, 7.2, 7.3, 7.4, 7.5, 7.6, 7.7, 7.8, 7.9 or 8, preferably 7.2.

Preferably, before the step (4) of treating the solid phase carrier in the solid phase synthesis column with the eluent, the method further comprises the steps of drying and washing the solid phase synthesis column.

Preferably, the drying is carried out at 50-60 ℃ for 1-5 min to remove the ammonolysis buffer liquid drops condensed on the surface of the solid phase synthesis column, the drying temperature can be 50 ℃, 51 ℃, 52 ℃, 53 ℃, 54 ℃, 55 ℃, 56 ℃, 57 ℃, 58 ℃, 59 ℃ or 60 ℃, preferably 55 ℃, and the drying time can be 1 min, 2 min, 3 min, 4 min or 5min, preferably 3 min.

Preferably, the washing solution used for washing comprises 100% acetonitrile and/or 80-95% acetonitrile, and is used for removing unreacted reagents and reaction byproducts.

Preferably, the washing comprises: and washing the dried solid-phase synthesis column by using 100% acetonitrile and 80-95% acetonitrile in sequence.

Preferably, a centrifugation step is further included after washing the solid phase synthesis column.

As a preferred technical solution, the present invention provides an ammonolysis process, which comprises the steps of:

(1) adding the aminolysis solution of the first aspect to a solid phase synthesis column containing a controlled pore size glass sphere to which a synthetic oligonucleotide is attached, and immersing the controlled pore size glass sphere;

(2) placing the solid phase synthesis column in a metal sealed container filled with an ammonolysis buffer solution, and keeping the solid phase synthesis column not to contact the ammonolysis buffer solution, wherein the ammonolysis buffer solution comprises ammonia and/or organic amine;

(3) heating the metal sealed container at 45-100 ℃ for 30-60 min to enable the glass spheres connected with the synthetic oligonucleotides and with the controlled apertures to be in the steam atmosphere of an aminolysis buffer solution, and then cooling for 5-15 min in an ice bath;

(4) and taking the solid phase synthesis column out of the sealed container, drying at 50-60 ℃ for 1-5 min, washing with 100% acetonitrile and 80-95% acetonitrile in sequence, centrifuging, treating the solid phase carrier in the solid phase synthesis column with water and/or 10-25 mM Tris-HCl with pH of 7-8, and collecting the effluent.

Compared with the prior art, the invention has the following beneficial effects:

(1) the ammonolysis solution has a simple formula, the butylamine, the butanol and the aminopentanol are matched with one another, the synthesized oligonucleotide is cut from the solid phase carrier, protecting groups Ac, Bz and dmf on A, C, G basic groups are removed, and alkaline aqueous solution is further added into the ammonolysis solution, so that the ammonolysis reaction rate is accelerated;

(2) the ammonolysis reaction is carried out in the sealed metal ammonolysis box, the internal ammonolysis buffer solution is gasified under the heating condition, a proper ammonolysis environment is provided for the ammonolysis reaction, and ammonia water serving as the ammonolysis buffer solution not only provides the proper ammonolysis environment, but also has the effect of gas-phase ammonolysis and can assist the liquid-phase ammonolysis reaction;

(3) the ammonolysis process has the advantages of mild conditions, low cost, environmental friendliness, high safety and important application value.

Drawings

FIG. 1 is an HPLC chromatogram of an oligonucleotide of 23 bases in length;

FIG. 2 is an ESI mass spectrum of an oligonucleotide of 23 bases in length;

FIG. 3 is an HPLC chromatogram of an oligonucleotide 60 bases in length;

FIG. 4 is an ESI mass spectrum of an oligonucleotide 60 bases in length;

FIG. 5 is an HPLC chromatogram of an oligonucleotide of 86 bases in length;

FIG. 6 is an ESI mass spectrum of an oligonucleotide of 86 bases in length.

Detailed Description

To further illustrate the technical means adopted by the present invention and the effects thereof, the present invention is further described below with reference to the embodiments and the accompanying drawings. It is to be understood that the specific embodiments described herein are merely illustrative of the invention and are not limiting of the invention.