CN115819485A - Ammonolysis solution, preparation method thereof and application thereof in DNA solid phase synthesis - Google Patents

Abstract

The invention provides an ammonolysis solution, a preparation method thereof and application thereof in DNA solid phase synthesis. The ammonolysis solution comprises propane diamine, alcohol and an alkaline reagent. The preparation method of the ammonolysis solution comprises the step of mixing an alkaline reagent with water and alcohol to obtain the ammonolysis solution. The application of the ammonolysis solution in DNA solid phase synthesis comprises the following steps: (1) Adding the ammonolysis solution into a solid synthesis column combined with the target DNA; (2) Placing the solid synthesis column in a container containing an ammonolysis buffer, heating, and then cooling; (3) Taking out the solid synthetic column, and sequentially drying, washing and centrifuging; (4) Subsequently, the solid synthesis column is eluted, and the effluent containing the target DNA is collected. The new generation of ammonolysis solution adopted by the invention has simple formula, can avoid side reaction on the basic group C compared with the first generation of ammonolysis solution, has low cost and can better realize commercialization and industrialization.

Description

Ammonolysis solution, preparation method thereof and application thereof in DNA solid phase synthesis

Technical Field

The invention belongs to the field of nucleic acid solid-phase synthesis, and relates to an ammonolysis solution, a preparation method thereof and application thereof in DNA solid-phase synthesis.

Background

Short-chain DNA is also called oligonucleotide (Oligo), and its synthesis is usually achieved by solid phase synthesis technology. The solid phase synthesis technology is a process for gradually connecting target sequences by taking a glass sphere (CPG) with a controlled aperture as a solid phase carrier and phosphoramidite as a monomer through four reaction cycles of deprotection, condensation, oxidation and capping according to the 3'-5' direction. Because the product of each step is connected to the solid phase carrier, the solid phase synthesis has the advantages of simple operation and simple post-treatment. This technique has been widely used in Oligo and polypeptide synthesis.

Currently, in the solid phase synthesis process of Oligo, each base is connected to undergo four reactions, namely deprotection, condensation, oxidation and capping, and after all target bases are synthesized, oligo is cut from a solid phase carrier. However, the A, C, G base contains a reactive amino group, which interferes with the condensation reaction. Therefore, the active amino group on the base is generally protected with an acetyl group (Ac), a benzoyl group (Bz) and a diazomethid amidino group (dmf). After the reaction is complete, the Ac, bz and dmf groups are cleaved off. Herein, the reaction of cleaving Oligo from a solid support and cleaving off a protecting group is referred to as aminolysis. The ammonolysis reaction mainly comprises: (1) Cleaving the Oligo from the solid support, such that the 3' -OH of the Oligo is exposed; (2) Cutting off protecting groups Ac, bz and dmf on A, C, G base; (3) the cyanoethyl group of each phosphoramidite is cleaved.

Ammonia water is adopted for the earliest ammonolysis reaction, but the problems of long operation time, high cross contamination risk and complicated post-treatment exist; even with the subsequent use of the improved methylamine: ammonia =1:1 (AMA) system can greatly reduce reaction time, but still has the problems of cross-contamination and cumbersome post-treatment. Then, researchers develop gas phase ammonolysis, ammonia gas is used as an active medium, the reaction is carried out at high temperature and high pressure, although the reaction is rapid and efficient, the requirement on equipment is high, potential safety hazards exist, and the structure of a primer can be damaged. Therefore, it is important to find a more efficient and convenient ammonolysis process.

CN109956987A discloses a microwave ammonolysis method as an alternative process, and the reaction is carried out by taking microwave as a heating means, but the problems of uneven heating and bumping of liquid exist in the actual operation process, so that loss and cross contamination are caused.

CN111704644A discloses an ammonolysis solution and an ammonolysis method, which comprises amine, butanol, amino alcohol and an alkaline aqueous solution, has short ammonolysis time and simple steps, and can effectively solve the problems of high requirements of gas phase ammonolysis on equipment, damage to primers at high temperature and high pressure and the like. However, the ammonolysis solution uses organic amine as an active ingredient, which is up to 80%, so that part of amino on the basic group C is replaced by the organic amine in the ammonolysis solution, the structure of the basic group C is changed, and the subsequent biological processes such as PCR amplification and the like are possibly influenced. Meanwhile, the first generation of liquid phase ammonolysis solution uses 1-amino-pentanol with higher cost as a raw material, and the cost is equivalent to that of gas phase ammonolysis.

Therefore, under the condition of ensuring the yield and quality of Oligo and not increasing the reaction time, the development of a new ammonolysis solution can avoid the side reaction of the base C and reduce the formulation cost, which is a problem to be solved at present.

Disclosure of Invention

Aiming at the defects and actual requirements of the prior art, the invention provides the ammonolysis solution, the preparation method thereof and the application thereof in DNA solid phase synthesis, wherein the ammonolysis solution has the advantages of simple and mild formula, low price, rapidness and effectiveness, can avoid side reactions on bases in the ammonolysis process, and finally can obtain high-yield and high-quality oligonucleotides.

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: propane diamine, an alkaline agent and alcohol.

According to the invention, the propane diamine is matched with alcohol and an alkaline reagent, the synthesized oligonucleotide is cut from the solid phase carrier, the protecting groups Ac, bz and dmf on the A, C, G basic group are removed, and the propane diamine can provide two amine molecules, so that the reactivity of each organic amine molecule is improved, and the propane diamine has larger steric hindrance, so that the chance of side reaction of the basic group C is greatly reduced.

Preferably, the propylenediamine comprises 1,2-propylenediamine.

Preferably, the concentration of the propylenediamine in the ammonolysis solution is 5-20% by volume, for example 5%, 8%, 10%, 15% or 20% by volume, but is not limited to the values listed, and the same applies to other values within the present range.

In the invention, 1,2-propane diamine is preferably used as an active component of the ammonolysis solution, one molecule has two primary amines, the reaction activity of each organic amine molecule can be further improved, and by means of larger steric hindrance, the side reaction of the basic group C can be effectively avoided, and the basic group C is matched with alcohol and an alkaline reagent to play more efficient ammonolysis activity and realize efficient ammonolysis reaction.

Preferably, the alkaline agent includes KOH agent, liOH agent, naOH agent, be (OH) ₂ Reagent, mg (OH) ₂ Reagents or Ca (OH) ₂ Any one or combination of at least two of the reagents can be, for example, a combination of a KOH reagent and a LiOH reagent, a combination of a LiOH reagent and a NaOH reagent, and the like, and the combination of the other reagents can be selected arbitrarily, which is not repeated herein.

The concentration of the alkaline agent in the ammonolysis solution is preferably 0.02 to 0.08M, and may be, for example, 0.02M, 0.03M, 0.04M, 0.05M, 0.06M, 0.07M or 0.08M, but is not limited to the above-mentioned values, and other values within the range can be similarly applied.

In the invention, the concentration of the alkaline aqueous solution is limited within the range of 0.02-0.08M, which is beneficial to efficiently carrying out the ammonolysis reaction, the concentration of the alkaline aqueous solution cannot be too low or too high, the concentration cannot play a role in promoting the ammonolysis reaction, the yield and the purity of the oligonucleotide can be influenced by too high concentration, the eluted oligonucleotide is easy to generate a salt peak during mass spectrometric detection, and the oligonucleotide can be whitened after being dried.

Preferably, the alcohol includes any one or a combination of at least two of methanol, ethanol, propanol and butanol, the combination may be, for example, a combination of methanol and ethanol, a combination of ethanol and propanol, etc., and any combination of the remaining combinations may be selected, which is not described herein again.

Preferably, the volume fraction of alcohol in the ammonolysis solution is 50-90%, for example, 50%, 60%, 70%, 80%, or 90%, etc., but is not limited to the recited values, and the same applies to other values in the present range.

In a second aspect, the present invention provides a method for preparing the ammonolysis solution according to the first aspect, the method comprising: mixing an alkaline reagent, propylene diamine, alcohol and optional water to prepare the ammonolysis solution.

Preferably, the preparation method comprises:

mixing an alkaline reagent with water to obtain an alkaline aqueous solution; and mixing the alkaline aqueous solution with propylene diamine and alcohol to obtain the ammonolysis solution.

Preferably, the molar concentration of the alkaline agent in the alkaline aqueous solution is 0.1-0.5M, such as 0.1M, 0.3M or 0.5M, but is not limited to the recited values, and other values within the scope are equally applicable.

Preferably, the volume ratio of the propylenediamine, the basic aqueous solution and the alcohol is (0.5 to 2): (0.8 to 1.2): (7.8 to 8.2), and for example, 0.5.

According to the invention, the propane diamine and the alcohol are matched with each other at a specific ratio, the propane diamine and the alcohol are used as main functional components of the ammonolysis solution, the synthesized oligonucleotide is cut from the solid phase carrier, the protecting groups Ac, bz and dmf on the A, C, G basic group are removed, meanwhile, the propane diamine contains two amine molecules, the reaction activity of each organic amine molecule is improved, the propane diamine has larger steric hindrance, the probability of side reaction of the basic group C is greatly reduced, the trace alkaline aqueous solution plays a role in ammonolysis catalysis, the yield of the oligonucleotide is improved, the phenomenon of whitening of the oligonucleotide appearance is avoided, and the purity of the obtained oligonucleotide is high.

In a third aspect, the invention provides the use of an ammonolysis solution as described in the first aspect, including use in solid phase synthesis of DNA.

Preferably, the method for solid phase synthesis of DNA comprises the following steps:

(1) Adding the ammonolysis solution according to the first aspect to a solid synthesis column to which the DNA of interest is bound;

(2) Placing the solid synthesis column in the step (1) in a sealed container filled with ammonolysis buffer solution, heating, and then cooling;

(3) Taking out the solid synthetic column obtained in the step (2), and sequentially carrying out drying, washing and centrifuging operations;

(4) And (4) eluting the solid synthesis column obtained in the step (3), and collecting effluent liquid, wherein the effluent liquid contains the target DNA.

In the invention, the solid-phase synthesis column is immersed by the ammonolysis solution, ammonolysis reaction can be carried out under mild heating condition, ammonolysis buffer solution in a sealed container is gasified, ammonolysis reaction is promoted to be carried out continuously, synthesized oligonucleotide is cut off from a solid-phase carrier, amino protecting groups on basic groups are removed, side reaction on basic groups C is avoided, and oligonucleotide products with high purity and high yield are obtained.

Preferably, the amount ratio of the ammonolysis solution to the solid synthesis column used in step (1) is (0.4 to 2). Mu.L: 1nmol, and for example, it may be 0.4. Mu.L: 1nmol, 0.8. Mu.L: 1nmol, 1.2. Mu.L: 1nmol, 1.6. Mu.L: 1nmol, or 2. Mu.L: 1nmol, and the like, and the values are not limited to the values listed, and the same applies to other values in the present range.

For example, in the present invention, 10. Mu.L of the ammonolysis solution may be optionally added to 5nmol of the solid synthesis column in step (1).

Alternatively, 20. Mu.L of the ammonolysis solution is added to 25 or 50nmol of the solid synthesis column in step (1).

Preferably, the ammonolysis buffer solution of step (2) comprises an inorganic base or an organic amine.

Preferably, the inorganic base comprises aqueous ammonia.

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 includes one or a combination of at least two of diethylamine, ethylenediamine, and pyridine, and the combination may be, for example, a combination of diethylamine and ethylenediamine, a combination of ethylenediamine and pyridine, and the like, and any combination manner of the rest may be selected, which is not described herein again.

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

Preferably, the volume of the ammonolysis buffer solution is 16 to 24mL, for example, 16mL, 20mL or 24mL, preferably 20mL, but is not limited to the values listed, and the same applies to other values in the present range.

Preferably, the heating temperature in step (2) is 50 to 95 ℃, for example, 50 ℃, 60 ℃, 70 ℃, 80 ℃, 90 ℃ or 95 ℃, but not limited to the recited values, and the same applies to other values in the present range.

The heating time in step (2) is preferably 35 to 65min, and may be, for example, 35min, 45min, 55min, 60min, or 65min, and is not limited to the values listed above, and the same applies to other values within the present range.

The heating process can enable the ammonolysis buffer solution to reach the gasification temperature, and form the steam atmosphere of the ammonolysis buffer solution in the sealed container, so that the solid-phase synthesis column is in the steam atmosphere of the ammonolysis buffer solution, the ammonolysis solution is promoted to play the ammonolysis function, and the synthesized oligonucleotides are cut off from the solid-phase carrier; the heating time is related to the synthesis specification, the ammonolysis solution formula, the heating temperature, the volume of the sealed container and the completion degree of the expected ammonolysis reaction, and is adjusted according to the specific implementation situation.

Preferably, the cooling in step (2) comprises an ice-water bath, and the time of the ice-water bath is 8 to 15min, such as 8min, 10min, 12min or 15min, but is not limited to the enumerated values, and the same applies to other values in the present range.

Preferably, the drying temperature in step (3) is 50 to 60 ℃, for example, 50 ℃, 53 ℃, 55 ℃, 57 ℃ or 60 ℃, but not limited to the recited values, and the same applies to other values in the present range.

Preferably, the drying time in step (3) is 1 to 5min, such as 1min, 3min or 5min, but not limited to the recited values, and the same applies to other values in the present range.

Preferably, the washed washing solution of step (3) comprises acetonitrile.

Preferably, the washing liquid is pure acetonitrile or acetonitrile solution with volume fraction of 80-95% (for example, 80%, 85%, 90%, or 95%, etc., but not limited to the recited values, and the same applies to other values in the present range).

Preferably, the number of washing is not less than three.

Preferably, the rotation speed of the centrifugation in step (3) is not lower than 3000rpm, such as 3000rpm, 3500rpm, 4000rpm or 4500rpm, but not limited to the enumerated values, and the same applies to other values in the present range.

Preferably, the centrifugation time in step (3) is not less than 60s, such as 60s, 70s, 80s or 90s, but not limited to the recited values, and the same applies to other values in the present range.

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

Preferably, the concentration of Tris-HCl is 10 to 30mM, such as 10mM, 15mM, 20mM, 25mM or 30mM, but not limited to the values listed, and the same applies to other values within the scope.

Preferably, the Tris-HCl has a pH of 7 to 8, such as 7.1, 7.4, 7.8 or 8, but not limited to the values listed, and the same applies to other values within the scope.

As a preferred embodiment, the present invention provides a method for solid phase synthesis of DNA, comprising the steps of:

(1) Adding the ammonolysis solution according to the first aspect to a solid synthesis column to which a target DNA is bound, and immersing the solid synthesis column;

(2) Placing the solid synthesis column in the step (1) in a metal box filled with ammonia water or organic amine, keeping the synthesis column not in contact with an ammonolysis buffer solution, tightly covering the metal box, fixing by adopting screws, and keeping a closed environment;

(3) Heating the metal box obtained in the step (2) at 50-95 ℃ for 35-65 min, and after the reaction is finished, putting the metal box into an ice water bath for cooling for 8-15 min;

(4) Taking out the solid synthetic column, drying at 50-60 ℃ for 1-5 min, washing with pure acetonitrile or acetonitrile solution in sequence for at least 3 times, and taking out the synthetic column and centrifuging;

(5) And (3) taking out the solid synthesis column obtained in the step (4), eluting with water and/or Tris-HCl, and collecting effluent liquid, wherein the effluent liquid contains the target DNA.

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

(1) The ammonolysis solution provided by the invention has a simple formula, the propane diamine is matched with alcohol and an alkaline reagent, the synthesized oligonucleotide is cut off from a solid phase carrier, protecting groups Ac, bz and dmf on a A, C, G basic group are removed, the existence of the alkaline reagent can further accelerate the ammonolysis reaction rate, and compared with a first generation ammonolysis solution, the ammonolysis solution can avoid side reaction on a basic group C and further improve the purity of the oligonucleotide;

(2) The aminolysis solution obtained by adopting the propane diamine, the alcohol and the alkaline reagent is mixed according to the corresponding proportion, so that the synergistic effect can be better exerted, the aminolysis process can be better assisted, the cost of the aminolysis solution is low, and the commercialization and the industrialization can be better realized.

Drawings

FIG. 1 is an HPLC chromatogram of an oligonucleotide of 20 bases in length in example 1;

FIG. 2 is an ESI mass spectrum of the 20 base length oligonucleotide of example 1;

FIG. 3 is an HPLC chromatogram of the 30 base long oligonucleotide of example 1;

FIG. 4 is an ESI mass spectrum of the 30 base length oligonucleotide of example 1;

FIG. 5 is an HPLC chromatogram of an oligonucleotide of 20 bases in length in example 2;

FIG. 6 is an HPLC chromatogram of an oligonucleotide of 20 bases in length in example 3;

FIG. 7 is an HPLC chromatogram of an oligonucleotide 30 bases in length in example 3;

FIG. 8 is an HPLC chromatogram of the 20 base long oligonucleotide of example 4;

FIG. 9 is an HPLC chromatogram of the 30 base long oligonucleotide of example 4;

FIG. 10 is an HPLC chromatogram of the 20 base long oligonucleotide of example 5;

FIG. 11 is an HPLC chromatogram of the 30 base long oligonucleotide of example 5;

FIG. 12 is an HPLC chromatogram of the 20 base long oligonucleotide of example 6;

FIG. 13 is an HPLC chromatogram of the 30 base long oligonucleotide of example 6;

FIG. 14 is an HPLC chromatogram of the 20 base long oligonucleotide of example 7;

FIG. 15 is an ESI mass spectrum of the 20 base length oligonucleotide of example 7;

FIG. 16 is an HPLC chromatogram of the 30 base long oligonucleotide of example 7;

FIG. 17 is an ESI mass spectrum of a 30 base long oligonucleotide of example 7;

FIG. 18 is an HPLC chromatogram of the 20 base long oligonucleotide of example 8;

FIG. 19 is an ESI mass spectrum of the 20 base length oligonucleotide of example 8;

FIG. 20 is an HPLC chromatogram of an oligonucleotide 30 bases in length in example 8;

FIG. 21 is an ESI mass spectrum of a 30 base length oligonucleotide of example 8;

FIG. 22 is an HPLC chromatogram of the 20 base long oligonucleotide of example 9;

FIG. 23 is an ESI mass spectrum of the 20 base length oligonucleotide of example 9;

FIG. 24 is an HPLC chromatogram of the 30 base long oligonucleotide of example 9;

FIG. 25 is an ESI mass spectrum of a 30 base length oligonucleotide of example 9;

FIG. 26 is an HPLC chromatogram of the 20 base long oligonucleotide of example 10;

FIG. 27 is an ESI mass spectrum of the 20 base length oligonucleotide of example 10;

FIG. 28 is an HPLC chromatogram of the 30 base long oligonucleotide of example 10;

FIG. 29 is an ESI mass spectrum of a 30 base long oligonucleotide of example 10;

FIG. 30 is an HPLC chromatogram of a primary ammonolysis oligo (dC) 10 provided in comparative example 1;

FIG. 31 is an HPLC chromatogram of a new generation ammonolysis liquid aminolysis oligo (dC) 10 provided in example 1;

FIG. 32 is an HPLC chromatogram of the 20 base long oligonucleotide of comparative example 2;

FIG. 33 is an ESI mass spectrum of the 20 base long oligonucleotide of comparative example 2;

FIG. 34 is an HPLC chromatogram of the 30 base long oligonucleotide of comparative example 2;

FIG. 35 is an ESI mass spectrum of the 30 base long oligonucleotide of comparative example 2;

FIG. 36 is an HPLC chromatogram of the 20 base long oligonucleotide of comparative example 3;

FIG. 37 is an ESI mass spectrum of the 20 base long oligonucleotide of comparative example 3;

FIG. 38 is an HPLC chromatogram of the 30 base long oligonucleotide of comparative example 3;

FIG. 39 is an ESI mass spectrum of the 30 base long oligonucleotide of comparative example 3;

FIG. 40 is an HPLC chromatogram of the 20 base long oligonucleotide of comparative example 4;

FIG. 41 is an ESI mass spectrum of the 20 base long oligonucleotide of comparative example 4;

FIG. 42 is an HPLC chromatogram of the 30 base long oligonucleotide of comparative example 4;

FIG. 43 is an ESI mass spectrum of the 30 base long oligonucleotide of comparative example 4.

Detailed Description

To further illustrate the technical means and effects of the present invention, the present invention is further described with reference to the following embodiments and the accompanying drawings. The technical solution of the present invention is further explained by the following embodiments. It should be understood by those skilled in the art that the examples are only for the understanding of the present invention and should not be construed as the specific limitations of the present invention.

The examples do not specify particular techniques or conditions, and are to be construed in accordance with the description of the art in the literature or with the specification of the product. The reagents or apparatus used are conventional products commercially available from normal sources, not indicated by the manufacturer.

Example 1

In this example, the primers with the lengths of 10 bases, 20 bases and 30 bases were subjected to ammonolysis reaction, and the sequences were as follows:

1 (10 nt) with a target molecular weight of 2830:

CCCCCCCCCC；

2 (20 nt) with a target molecular weight of 6087:

CGGCAGCGTACCCTCGATAA；

3 (30 nt) with a target molecular weight of 9119:

CGGCCTTCAGCAGGTATCTCAGCTTGCCCA。

the method comprises the following specific steps:

1. primer synthesis

A 50nmol synthesis column was used; 0.25M acetonitrile solution of 5-ethylthio tetrazole (ETT) is used as an activator; a dichloromethane solution of 3% trichloroacetic acid is taken as a deprotection reagent; 10% acetic anhydride/acetonitrile and N-methylimidazole/pyridine/acetonitrile =14 as capping reagents; 0.05M iodine in tetrahydrofuran/pyridine/water = 70. The corresponding primer sequences were synthesized on a dr. Oligo 192 synthesizer.

2. Liquid phase ammonolysis

(1) Mixing an alkaline reagent, 1,2-propanediamine, alcohol and optional water to prepare an ammonolysis solution, wherein the formula of the ammonolysis solution is that the volume ratio of 1,2-propanediamine, 0.5M KOH and ethanol is 0.5;

namely 1,2-propane diamine volume fraction of 5%, KOH mole fraction of 0.05M, ethanol volume fraction of 80%;

(2) Adding 20 mu L of ammonolysis solution into 50nmol of solid synthetic column, and immersing the solid synthetic column;

(3) Suspending the solid synthetic column in the step (1) in a 9.5cm × 17cm × 13cm metal box filled with 20mL ammonia water, keeping the synthetic column not in contact with an ammonolysis buffer solution, tightly covering the metal box, fixing by using screws, and keeping a closed environment;

(4) Heating the metal box obtained in the step (2) at 90 ℃ for 60min, and after the reaction is finished, putting the metal box into an ice water bath for cooling for 10min;

(5) Taking out the metal box in the step (3), taking out the solid synthetic column, drying at 55 ℃ for 3min, taking out, cooling to room temperature, washing with 200 mu L of 100% acetonitrile and 90% acetonitrile in sequence, taking out the solid synthetic column, and centrifuging at 3000rpm for 60s; the 100% acetonitrile is washed twice, and the 90% acetonitrile is washed once;

(6) The solid synthesis column obtained in step (4) was taken out, eluted with 200. Mu.L of a 20mM, pH 7.2 Tris-HCl solution, and the effluent was collected for mass spectrometry.

The HPLC spectrogram and ESI mass spectrogram of the primer with the length of 20 bases are respectively shown in FIG. 1 and FIG. 2, and the purity is 95% as shown in FIG. 1, and the mass spectrum molecular weight is consistent with the target molecular weight as shown in FIG. 2; the HPLC spectrogram and ESI mass spectrogram of the primer with the length of 30 bases are respectively shown in FIG. 3 and FIG. 4, and as can be seen from FIG. 3, the purity is 98%, and as can be seen from FIG. 4, the mass spectrum molecular weight is consistent with the target molecular weight; the high performance liquid chromatogram of the primer of 10 bases in length is shown in FIG. 31, and the purity is 97%. It can be seen that the HPLC purity and mass spectrum of the primers with different lengths of the new generation of liquid phase ammonolysis treatment can reach the industrial standard.

Example 2

In this example, the aminolysis reaction was performed on a primer 20 bases in length, and the sequence was:

2 (20 nt) with a target molecular weight of 6087:

CGGCCTTCAGCAGGTATCTCAGCTTGCCCA。

the method comprises the following specific steps:

1. primer synthesis

Using a 25nmol synthesis column; 0.25M acetonitrile solution of 5-ethylthio tetrazole (ETT) is taken as an activator; a dichloromethane solution of 3% trichloroacetic acid is taken as a deprotection reagent; 10% acetic anhydride/acetonitrile and N-methylimidazole/pyridine/acetonitrile =14 as capping reagents; 0.05M iodine in tetrahydrofuran/pyridine/water = 70. The corresponding primer sequences were synthesized on a dr. Oligo 192 synthesizer.

2. Liquid phase ammonolysis

(1) Mixing an alkaline reagent, 1,2-propanediamine, alcohol and optional water to prepare an ammonolysis solution, wherein the formula of the ammonolysis solution is that the volume ratio of 1,2-propanediamine, 0.5M KOH and ethanol is 0.5;

namely 1,2-propane diamine volume fraction of 5%, KOH mole fraction of 0.05M, ethanol volume fraction of 80%;

(2) Adding 20 mu L of ammonolysis solution into 25nmol of solid synthetic column, and immersing the solid synthetic column;

(3) Suspending the solid synthetic column in the step (1) in a 9.5cm × 17cm × 13cm metal box filled with 24mL ammonia water, keeping the synthetic column not in contact with an ammonolysis buffer solution, tightly covering the metal box, fixing by using screws, and keeping a closed environment;

(4) Heating the metal box obtained in the step (2) at 91 ℃ for 45min, and after the reaction is finished, putting the metal box into ice-water bath for cooling for 12min;

(5) Taking out the metal box in the step (3), taking out the solid synthetic column, drying at 58 ℃ for 2min, taking out, cooling to room temperature, washing with 200 mu L of 100% acetonitrile and 92% acetonitrile in sequence, taking out the solid synthetic column, and centrifuging at 3200rpm for 65s;

the 100% acetonitrile is washed twice, and the 92% acetonitrile is washed once;

(6) The solid synthesis column obtained in step (4) was removed and eluted with 200. Mu.L of 25mM Tris-HCl solution at pH 7.1, and the effluent was collected for mass spectrometry.

The HPLC chromatogram of the primer of 20 bases in length is shown in FIG. 5, and the purity is 99%.

Example 3

In this example, the primers with the length of 20 bases and 30 bases were subjected to ammonolysis reaction, and the sequences were as follows:

2 (20 nt) with a target molecular weight of 6087:

CGGCAGCGTACCCTCGATAA；

3 (30 nt) with a target molecular weight of 9119:

CGGCCTTCAGCAGGTATCTCAGCTTGCCCA。

the method comprises the following specific steps:

1. primer synthesis

5nmol synthesis column was used; 0.25M acetonitrile solution of 5-ethylthio tetrazole (ETT) is used as an activator; a dichloromethane solution of 3% trichloroacetic acid is taken as a deprotection reagent; 10% acetic anhydride/acetonitrile and N-methylimidazole/pyridine/acetonitrile =14 as capping reagents; 0.05M iodine tetrahydrofuran/pyridine/water =70 as oxidant. The corresponding primer sequences were synthesized on a dr. Oligo 768 synthesizer.

2. Liquid phase ammonolysis

(1) Mixing an alkaline reagent, 1,2-propanediamine, alcohol and optional water to prepare an ammonolysis solution, wherein the formula of the ammonolysis solution is that the volume ratio of 1,2-propanediamine, 0.5MNaOH and methanol is 1;

namely 1,2-propane diamine with 10% volume fraction, naOH with 0.05M mole fraction and ethanol with 80% volume fraction;

(2) Adding 10 mu L of ammonolysis solution into 5nmol of solid synthetic column, and immersing the solid synthetic column;

(3) Suspending the solid synthesis column in the step (1) in a 9.5cm × 17cm × 13cm metal box filled with 22mL of ethylenediamine, keeping the synthesis column not in contact with an ammonolysis buffer solution, tightly covering the metal box, fixing by using screws, and keeping a closed environment;

(4) Heating the metal box obtained in the step (2) at 95 ℃ for 35min, and after the reaction is finished, putting the metal box into ice-water bath for cooling for 15min;

(5) Taking out the metal box in the step (3), taking out the solid synthetic column, drying at 60 ℃ for 1min, taking out, cooling to room temperature, washing with 200 mu L of 100% acetonitrile and 95% acetonitrile in sequence, taking out the solid synthetic column, and centrifuging at 3300rpm for 75s;

the 100% acetonitrile is washed twice, and the 95% acetonitrile is washed once;

(6) And (5) taking out the solid synthesis column obtained in the step (4), eluting with 200 mu L of 30mM Tris-HCl solution with the pH value of 7, and collecting effluent liquid for mass spectrum analysis.

The HPLC chromatogram of the primer with a length of 20 bases is shown in FIG. 6, and the purity is 91%; the HPLC chromatogram of the primer of 30 bases in length is shown in FIG. 7, and the purity is 81%.

Example 4

This example provides an ammonolysis solution and an ammonolysis method, wherein the formulation of the ammonolysis solution is 1,2-propanediamine, the volume ratio of 0.5M KOH to ethanol is 0.5.

In this example, the primers with the length of 20 bases and 30 bases were subjected to ammonolysis reaction, and the sequences were as follows:

2 (20 nt) with a target molecular weight of 6087:

CGGCAGCGTACCCTCGATAA；

3 (30 nt) with a target molecular weight of 9119:

CGGCCTTCAGCAGGTATCTCAGCTTGCCCA。

the process parameters of the ammonolysis process were the same as in example 1.

The HPLC chromatogram of the primer with a length of 20 bases is shown in FIG. 8, and the purity is 96%; the HPLC chromatogram of the 30-base-long primer is shown in FIG. 9, and the purity thereof is 87%.

Example 5

This example provides an ammonolysis solution and an ammonolysis method, wherein the formulation of the ammonolysis solution is 1,2-propanediamine, 0.5M NaOH and ethanol in a volume ratio of 2.

In this example, the primers with the length of 20 bases and 30 bases were subjected to ammonolysis reaction, and the sequences were as follows:

2 (20 nt) with a target molecular weight of 6087:

CGGCAGCGTACCCTCGATAA；

3 (30 nt) with a target molecular weight of 9119:

CGGCCTTCAGCAGGTATCTCAGCTTGCCCA。

the process parameters of the ammonolysis process were the same as in example 1.

The HPLC chromatogram of the primer with the length of 20 bases is shown in FIG. 10, and the purity is 93%; the HPLC chromatogram of the primer of 30 bases in length is shown in FIG. 11, and the purity is 85%.

Example 6

The embodiment provides an ammonolysis solution and an ammonolysis method, wherein the formula of the ammonolysis solution is that 1,2-propane diamine, 0.5M NaOH and ethanol have the volume ratio of 0.8.

In this example, the primers with the length of 20 bases and 30 bases were subjected to ammonolysis reaction, and the sequences were as follows:

1 (20 nt) in SEQ ID NO, and the target molecular weight is 6087:

CGGCAGCGTACCCTCGATAA；

2 (30 nt) with a target molecular weight of 9119:

CGGCCTTCAGCAGGTATCTCAGCTTGCCCA。

the process parameters of the ammonolysis process were the same as in example 1.

The HPLC chromatogram of the primer with the length of 20 bases is shown in FIG. 12, and the purity is 96%; the HPLC chromatogram of the primer of 30 bases in length is shown in FIG. 13, and the purity is 88%.

Example 7

This example provides an ammonolysis solution and an ammonolysis method, wherein the formulation of the ammonolysis solution is 1,2-propanediamine, 0.5M KOH and ethanol in a volume ratio of 1.5.

In this example, the primers with the length of 20 bases and 30 bases were subjected to ammonolysis reaction, and the sequences were as follows:

2 (20 nt) with a target molecular weight of 6087:

CGGCAGCGTACCCTCGATAA；

3 (30 nt) with a target molecular weight of 9119:

CGGCCTTCAGCAGGTATCTCAGCTTGCCCA。

the process parameters of the ammonolysis process were the same as in example 1.

HPLC spectrogram and ESI mass spectrogram of the primer with the length of 20 bases are respectively shown in FIG. 14 and FIG. 15, and as can be seen from FIG. 14, the purity is 95%, and as can be seen from FIG. 15, the mass spectrum molecular weight of the primer coincides with the target molecular weight; the HPLC chromatogram and ESI mass chromatogram of the primer having a length of 30 bases are shown in FIGS. 16 and 17, respectively, and it is understood from FIG. 16 that the purity is 87%, and it is understood from FIG. 17 that the mass spectrum molecular weight matches the target molecular weight.

Example 8

This example provides an ammonolysis solution and an ammonolysis method, wherein the formulation of the ammonolysis solution is 1,2-propanediamine, 0.2M KOH and ethanol in a volume ratio of 1.

In this example, the primers with the length of 20 bases and 30 bases were subjected to ammonolysis reaction, and the sequences were as follows:

2 (20 nt) with a target molecular weight of 6087:

CGGCAGCGTACCCTCGATAA；

3 (30 nt) with a target molecular weight of 9119:

CGGCCTTCAGCAGGTATCTCAGCTTGCCCA。

the process parameters of the ammonolysis process were the same as in example 1.

The HPLC spectrogram and ESI mass spectrogram of the primer with the length of 20 bases are respectively shown in FIG. 18 and FIG. 19, and as can be seen from FIG. 18, the purity is 97%, and as can be seen from FIG. 19, the mass spectrum molecular weight is consistent with the target molecular weight; as shown in FIGS. 20 and 21, the HPLC and ESI mass spectrograms of the primers having a length of 30 bases were found to have a purity of 83% in FIG. 20 and a mass spectrometric molecular weight corresponding to the target molecular weight in FIG. 21, respectively.

Example 9

This example provides an ammonolysis solution and an ammonolysis method, wherein the formulation of the ammonolysis solution is 1,2-propanediamine, 0.8M KOH and ethanol in a volume ratio of 1.

In this example, the primers with the length of 20 bases and 30 bases were subjected to ammonolysis reaction, and the sequences were as follows:

2 (20 nt) with a target molecular weight of 6087:

CGGCAGCGTACCCTCGATAA；

3 (30 nt) with a target molecular weight of 9119:

CGGCCTTCAGCAGGTATCTCAGCTTGCCCA。

the process parameters of the ammonolysis process were the same as in example 1.

An HPLC spectrogram and an ESI spectrogram of the primer with the length of 20 bases are respectively shown in FIGS. 22 and 23, and as can be seen from FIG. 22, the purity is 97%, and as can be seen from FIG. 23, the mass spectrum molecular weight of the primer conforms to the target molecular weight; the HPLC chromatogram and ESI mass chromatogram of the primer having a length of 30 bases are shown in FIG. 24 and FIG. 25, respectively, and it is understood from FIG. 24 that the purity is 88%, and it is understood from FIG. 25 that the mass spectrum molecular weight matches the target molecular weight.

Example 10

This example provides an ammonolysis solution and method, which are the same as example 1 except that the volume fraction of 1,2-propanediamine in the formulation of the ammonolysis solution is 20%.

In this example, the primers with the length of 20 bases and 30 bases were subjected to ammonolysis reaction, and the sequences were as follows:

2 (20 nt) with a target molecular weight of 6087:

CGGCAGCGTACCCTCGATAA；

3 (30 nt) with a target molecular weight of 9119:

CGGCCTTCAGCAGGTATCTCAGCTTGCCCA。

the HPLC spectrogram and ESI mass spectrogram of the primer with the length of 20 bases are respectively shown in FIG. 26 and FIG. 27, and the purity is 93% in FIG. 26, and the mass spectrum molecular weight is consistent with the target molecular weight in FIG. 27; the HPLC and ESI mass spectrograms of the primers having a length of 30 bases are shown in FIGS. 28 and 29, respectively, and it is understood from FIG. 28 that the purity is 93%, and from FIG. 29 that the mass spectrometric molecular weight corresponds to the target molecular weight.

Comparative example 1

The comparative example adopts a first generation liquid phase ammonolysis formula (the first generation ammonolysis solution formula is the ammonolysis solution disclosed in CN111704644A, correspondingly, the ammonolysis solution provided by the invention is called as a new generation ammonolysis solution), and carries out ammonolysis reaction on the primer with the length of 10 basic groups, and the sequence is as follows:

1 (10 nt) with a target molecular weight of 2830:

CCCCCCCCCC。

the method comprises the following specific steps:

1. primer synthesis

A 50nmol synthesis column was used; 0.25M acetonitrile solution of 5-ethylthio tetrazole (ETT) is used as an activator; a dichloromethane solution of 3% trichloroacetic acid is taken as a deprotection reagent; 10% acetic anhydride/acetonitrile and N-methylimidazole/pyridine/acetonitrile =14: 76 is a capping reagent; 0.05M iodine tetrahydrofuran/pyridine/water =70:20: and 10 is an oxidizing agent. The corresponding primer sequences were synthesized on a dr. Oligo 192 synthesizer.

2. Liquid phase ammonolysis

(1) Adding 20 mu L of ammonolysis solution into a 50nmol solid synthesis column, and immersing the solid synthesis column, wherein the formula of the ammonolysis solution is n-butylamine: n-butanol: 1-aminopentanol = 7;

(2) Suspending the solid synthetic column in the step (1) in a 9.5cm × 17cm × 13cm metal box filled with 20mL ammonia water, keeping the synthetic column not in contact with an ammonolysis buffer solution, tightly covering the metal box, fixing by using screws, and keeping a closed environment;

(3) Heating the metal box obtained in the step (2) at 90 ℃ for 60min, and after the reaction is finished, putting the metal box into an ice water bath for cooling for 10min;

(4) Taking out the metal box in the step (3), taking out the solid synthetic column, drying at 55 ℃ for 3min, taking out, cooling to room temperature, washing with 200 mu L of 100% acetonitrile and 90% acetonitrile in sequence, taking out the solid synthetic column, and centrifuging at 3000rpm for 65s;

the 100% acetonitrile is washed twice, and the 90% acetonitrile is washed once;

(5) The solid synthesis column obtained in step (4) was taken out, eluted with 200. Mu.L of a 20mM, pH 7.2 Tris-HCl solution, and the effluent was collected for mass spectrometry.

The high performance liquid chromatogram of fig. 30 shows that the purity is 69%, and the high performance liquid chromatogram of Oligo (dC) 10 obtained using the new generation of ammonolysis solution provided in example 1 has 97% purity (fig. 31), compared to the one generation liquid phase ammonolysis solution provided in comparative example 1, and the high performance liquid chromatogram thereof has higher purity and significantly reduced side reaction products (wherein, the peaks at 2.07 and 2.38 minutes represent side reaction products), indicating that the primer purity of the obtained Oligo can be further improved using the new generation liquid phase ammonolysis solution.

Comparative example 2

The comparative example provides an ammonolysis solution and an ammonolysis method, wherein the formula of the ammonolysis solution is 1,2-the volume ratio of propane diamine to ethanol is 1:9.

The comparative example performs ammonolysis reaction on the primers with the lengths of 20 bases and 30 bases, and the sequences are respectively as follows:

2 (20 nt) with a target molecular weight of 6087:

CGGCAGCGTACCCTCGATAA；

3 (30 nt) with a target molecular weight of 9119:

CGGCCTTCAGCAGGTATCTCAGCTTGCCCA。

the process parameters of the ammonolysis process were the same as in example 1.

The HPLC spectrogram and ESI mass spectrogram of the primer with the length of 20 bases are respectively shown in FIG. 32 and FIG. 33, and as can be seen from FIG. 32, the purity is 91%, and as can be seen from FIG. 33, the mass spectrum molecular weight coincides with the target molecular weight; the HPLC chromatogram and ESI mass chromatogram of the primer having a length of 30 bases are shown in FIG. 34 and FIG. 35, respectively, and it is seen from FIG. 34 that the purity is 82%, and from FIG. 35 that the mass spectrum molecular weight matches the target molecular weight.

Comparative example 3

The comparative example provides an ammonolysis solution and an ammonolysis method, wherein the formula of the ammonolysis solution is 1,2-propane diamine and the volume ratio of 0.5M KOH is 1:9.

The comparative example performs ammonolysis reaction on the primers with the lengths of 20 bases and 30 bases, and the sequences are respectively as follows:

2 (20 nt) with a target molecular weight of 6087:

CGGCAGCGTACCCTCGATAA；

3 (30 nt) with a target molecular weight of 9119:

CGGCCTTCAGCAGGTATCTCAGCTTGCCCA。

the process parameters of the ammonolysis process were the same as in example 1.

The HPLC spectrogram and ESI mass spectrogram of the primer with the length of 20 bases are respectively shown in FIG. 36 and FIG. 37, and the purity is 98% in FIG. 36, and the mass spectrum molecular weight is consistent with the target molecular weight in FIG. 37; the HPLC chromatogram and ESI mass chromatogram of the primer having a length of 30 bases are shown in FIGS. 38 and 39, respectively, and it is seen from FIG. 38 that the purity is 85%, and from FIG. 39 that the mass spectrum molecular weight matches the target molecular weight.

Comparative example 4

The comparative example provides an ammonolysis solution and an ammonolysis method, wherein the formula of the ammonolysis solution is 1,2-propane diamine and the volume ratio of 0.5M KOH ethanol solution is 1:9.

The primers with the lengths of 20 bases and 30 bases are subjected to ammonolysis reaction in the comparative example, and the sequences are respectively as follows:

2 (20 nt) with a target molecular weight of 6087:

CGGCAGCGTACCCTCGATAA；

3 (30 nt) with a target molecular weight of 9119:

CGGCCTTCAGCAGGTATCTCAGCTTGCCCA。

the process parameters of the ammonolysis process were the same as in example 1.

The HPLC spectrogram and ESI mass spectrogram of the primer with the length of 20 bases are respectively shown in FIG. 40 and FIG. 41, and the purity is 89% in FIG. 40, and the mass spectrum molecular weight is consistent with the target molecular weight in FIG. 41; the HPLC and ESI mass spectrograms of the primers having a length of 30 bases are shown in FIGS. 42 and 43, respectively, and it is understood from FIG. 42 that the purity is 79%, and from FIG. 43 that the mass spectrometric molecular weight corresponds to the target molecular weight.

The yields and purities of the primers obtained in examples 1 to 10 and comparative examples 1 to 4 are shown in Table 1.

TABLE 1

According to the above table, in the example 1, compared with the comparative example 1, the high performance liquid chromatography purity of the primer obtained by using the new generation of the ammonolysis solution is higher, and the side reaction products are obviously reduced, which indicates that the primer purity of the obtained Oligo can be further improved by using the new generation of the liquid ammonolysis solution;

compared with the comparative example 2, the purity of the primer obtained in the example 1 is obviously superior to that of the comparative example 2, and the results show that the roles of the propane diamine and the alkaline reagent in the ammonolysis process are complementary, and the absence of either of the two can reduce the ammonolysis effect;

example 1 compared to comparative example 3, the product of example 1 was a clear gum and the product of comparative example 3 was a white solid, indicating that excess KOH resulted in the product having more KOH incorporated and not being removed by conventional procedures; comparative example 3 has more incomplete products of ammonolysis than example 1, which shows that ethanol has certain auxiliary effect in the ammonolysis process, and the lack of the products can reduce the ammonolysis effect;

example 1 compared with comparative example 4, example 1 had less incomplete products of ammonolysis than example 4, indicating that a certain amount of water was effective in promoting ammonolysis in the reaction;

example 1 compares with examples 2-10, which show that the selection and concentration of alkaline reagent, the selection of alcohol, and the volume fraction of propane diamine can affect the aminolysis effect, and thus affect the primer purity and yield of the obtained Oligo.

In conclusion, the Oligo with different lengths processed by the new generation of liquid phase ammonolysis solution has the advantages that the HPLC purity and the mass spectrum of the obtained Oligo product can reach the delivery standard through the synergistic compounding effect of 1,2-propane diamine and alcohol and alkaline reagent in a specific ratio, and the result proves that the method can achieve better ammonolysis effect on the Oligo with different lengths.

The applicant declares that the above description is only a specific embodiment of the present invention, but the scope of the present invention is not limited thereto, and it should be understood by those skilled in the art that any changes or substitutions that can be easily conceived by those skilled in the art within the technical scope of the present invention disclosed herein fall within the scope and disclosure of the present invention.

SEQUENCE LISTING

<110> Suzhou Jin Weizhi Biotechnology Ltd

<120> ammonolysis solution, preparation method thereof and application thereof in DNA solid phase synthesis

<130> 20210915

<160> 3

<170> PatentIn version 3.3

<210> 1

<211> 10

<212> DNA

<213> Artificial Synthesis

<400> 1

cccccccccc 10

<210> 2

<211> 20

<212> DNA

<213> Artificial Synthesis

<400> 2

cggcagcgta ccctcgataa 20

<210> 3

<211> 30

<212> DNA

<213> Artificial Synthesis

<400> 3

cggccttcag caggtatctc agcttgccca 30

Claims (10)

1. An ammonolysis solution, comprising: propane diamine, an alkaline agent and alcohol.

2. The ammonolysis solution according to claim 1, wherein the propylenediamine comprises 1,2-propylenediamine;

preferably, the volume concentration of the propane diamine in the ammonolysis solution is 5-20%;

preferably, the alkaline agent includes KOH agent, liOH agent, naOH agent, be (OH) ₂ Reagent, mg (OH) ₂ Reagents or Ca (OH) ₂ Any one of or a combination of at least two of the reagents;

preferably, the concentration of the alkaline reagent in the ammonolysis solution is 0.02-0.08M;

preferably, the alcohol comprises any one of methanol, ethanol, propanol, butanol or a combination of at least two thereof;

preferably, the volume fraction of the alcohol in the ammonolysis solution is 50-90%.

3. A method for preparing the ammonolysis solution according to claim 1 or 2, wherein said method comprises:

mixing an alkaline reagent, propylene diamine, alcohol and optional water to prepare the ammonolysis solution.

4. The preparation method according to claim 3, characterized in that the preparation method specifically comprises:

mixing an alkaline reagent with water to obtain an alkaline aqueous solution; mixing the alkaline aqueous solution with propane diamine and alcohol to obtain the ammonolysis solution;

preferably, the molar concentration of the alkaline reagent in the alkaline aqueous solution is 0.1-0.5M;

preferably, the volume ratio of the propane diamine to the alkaline aqueous solution to the alcohol is (0.5-2): (0.8-1.2): (7.8-8.2).

5. Use of an aminolysis solution according to claim 1 or 2, wherein said use comprises use in solid phase synthesis of DNA.

6. The use according to claim 5, wherein the method of DNA solid phase synthesis comprises the steps of:

(1) Adding the ammonolysis solution according to claim 1 or 2 to a solid synthesis column to which a target DNA is bound;

(2) Placing the solid synthesis column in the step (1) in a sealed container filled with ammonolysis buffer solution, heating, and then cooling;

(3) Taking out the solid synthetic column obtained in the step (2), and sequentially carrying out drying, washing and centrifuging operations;

(4) And (4) eluting the solid synthesis column obtained in the step (3), and collecting effluent liquid, wherein the effluent liquid contains the target DNA.

7. The use of claim 6, wherein the use amount ratio of the ammonolysis solution to the solid synthesis column in the step (1) is (0.4-2) μ L:1nmol;

preferably, the ammonolysis buffer solution of step (2) comprises an inorganic base or an organic amine;

preferably, the inorganic base comprises aqueous ammonia;

preferably, the organic amine comprises any one or a combination of at least two of diethylamine, ethylenediamine or pyridine;

preferably, the heating temperature in the step (2) is 50-95 ℃;

preferably, the heating time in the step (2) is 35-65 min;

preferably, the cooling of step (2) comprises an ice-water bath;

preferably, the time of the ice-water bath is 8-15 min.

8. The use according to claim 6 or 7, wherein the temperature of the drying in step (3) is 50-60 ℃;

preferably, the drying time in the step (3) is 1-5 min;

preferably, the washed washing solution of step (3) comprises acetonitrile;

preferably, the washing solution is pure acetonitrile or acetonitrile solution with volume fraction of 80-95%;

preferably, the number of washing is not less than three;

preferably, the rotation speed of the centrifugation in the step (3) is not lower than 3000rpm;

preferably, the centrifugation time in step (3) is not less than 60s.

9. The use according to any one of claims 6 to 8, wherein the eluted eluent of step (4) comprises water and/or a Tris-HCl buffer;

preferably, the concentration of the Tris-HCl buffer solution is 10-30 mM;

preferably, the pH of the Tris-HCl buffer solution is 7-8.

10. The use according to any one of claims 6 to 9, wherein the method of solid phase synthesis of DNA comprises the steps of:

(1) Adding the ammonolysis solution according to claim 1 or 2 to a solid synthesis column to which a target DNA is bound, and immersing the solid synthesis column;

(2) Placing the solid synthesis column in the step (1) in a metal box filled with ammonia water or organic amine, keeping the synthesis column not in contact with an ammonolysis buffer solution, tightly covering the metal box, fixing by adopting screws, and keeping a closed environment;

(3) Heating the metal box obtained in the step (2) at 50-95 ℃ for 35-65 min, and after the reaction is finished, putting the metal box into an ice water bath for cooling for 8-15 min;

(4) Taking out the solid synthetic column, drying at 50-60 ℃ for 1-5 min, washing with a washing solution for at least 3 times, and then taking out the solid synthetic column and centrifuging;

(5) And (3) eluting the solid synthesis column obtained after the centrifugation in the step (4) by using water and/or Tris-HCl buffer solution, and collecting effluent liquid, wherein the effluent liquid contains the target DNA.

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