阅读说明：本技术 一种固相合成dna的氨解预处理液及氨解方法 (Ammonolysis pretreatment solution and ammonolysis method for solid-phase synthesis of DNA ) 是由 张钦 樊梓龙 刘俊 赵梦婷 于 2021-07-26 设计创作，主要内容包括：本发明提供了一种固相合成DNA的氨解预处理液及氨解方法,所述DNA氨解预处理液由水与有机溶剂构成；其中,有机体积百分比为70％～80％,所述有机溶剂选自乙腈、乙醇或甲醇中的一种或多种。与现有技术相比,本发明的有益效果在于：通过配制水与乙腈合理比例配制的DNA氨解预处理液,在保证可以提高氨解效率的同时又能进一步提高产率。(The invention provides an ammonolysis pretreatment liquid and an ammonolysis method for synthesizing DNA by a solid phase, wherein the DNA ammonolysis pretreatment liquid consists of water and an organic solvent; wherein the organic volume percentage is 70-80%, and the organic solvent is selected from one or more of acetonitrile, ethanol or methanol. Compared with the prior art, the invention has the beneficial effects that: the DNA ammonolysis pretreatment liquid prepared by reasonable proportion of water and acetonitrile can further improve the yield while ensuring that the ammonolysis efficiency can be improved.)

1. A DNA ammonolysis pretreatment liquid is characterized by comprising water and an organic solvent; wherein the volume percentage of the organic solvent is 70-80%, and the organic solvent is selected from one or more of acetonitrile, ethanol or methanol.

2. The DNA ammonolysis pretreatment solution according to claim 1, wherein said organic solvent is acetonitrile.

3. An ammonolysis method for solid phase synthesis of DNA, comprising the steps of:

step S1 of adding the DNA aminolysis pretreatment liquid according to claim 1 or 2 to a synthesis column for solid phase carriers containing synthetic DNA fragments for wetting;

step S2: mixed gas of ammonia gas and water vapor is adopted for ammonolysis, and then eluent is adopted for desorption, so that a DNA product is obtained.

4. The method for aminolysis of solid-phase synthesized DNA according to claim 3, wherein in step S2, the aminolysis time is 90min and the aminolysis temperature is 90 ℃.

5. The method of claim 3, wherein in step S2, after the ammonolysis treatment, the inorganic salt is washed away with a washing solution, and then the inorganic salt is desorbed with an eluent, which is water.

6. The method for ammonolysis of solid phase synthesized DNA according to claim 5, wherein said washing solution is acetonitrile.

7. The method for aminolysis of solid-phase synthesized DNA according to claim 5, wherein in the step S1, the volume of the pretreatment solution for aminolysis added is larger than the volume of the synthesis column.

Technical Field

The invention belongs to the field of solid-phase DNA synthesis, and particularly relates to an ammonolysis pretreatment solution and an ammonolysis method for solid-phase synthetic DNA.

Background

The solid phase phosphoramidite triester method is a common method for synthesizing DNA at present, and the basic principle of the reaction is that a phosphoramidite monomer in a solution forms a 3 ' -5 ' phosphodiester bond through a condensation reaction, is connected to a solid phase carrier and sequentially extends until the synthesis is finished when the last 5 ' base of the sequence is connected. The whole synthesis process is automatically completed by an instrument, and each cycle is sequentially completed according to five steps of deprotection, activation, coupling, capping and oxidation.

After the DNA synthesizer has completed the DNA synthesis step, the product is attached to a solid support. The product is then separated from the solid support by ammonolysis and purified to yield the product. In the existing ammonolysis method, ammonia water ammonolysis is directly carried out, ammonia gas and water vapor are mixed for ammonolysis, but the ammonolysis efficiency is not high, so that the subsequent DNA synthesis yield is influenced.

Disclosure of Invention

In order to solve the technical problems, the invention provides an ammonolysis pretreatment solution and an ammonolysis method for solid-phase synthesis of DNA.

The specific technical scheme is as follows:

a DNA ammonolysis pretreatment solution is characterized by comprising water and an organic solvent; wherein the volume percentage of the organic solvent is 70-80%, and the organic solvent is selected from one or more of acetonitrile, ethanol or methanol.

Further, the organic solvent is acetonitrile.

An ammonolysis method for solid phase synthesis of DNA, which is characterized by comprising the following steps:

step S1, adding the DNA ammonolysis pretreatment solution into a synthesis column filled with a solid phase carrier for synthesizing DNA fragments for wetting;

step S2: mixed gas of ammonia gas and water vapor is adopted for ammonolysis, and then eluent is adopted for desorption, so that a DNA product is obtained.

Further, in the step S2, the ammonolysis time is 90min, and the ammonolysis temperature is 90 ℃.

Further, in step S2, after the ammonolysis treatment, inorganic salts are washed away with a washing solution, and then desorption is performed with an eluent, which is water.

Further, the washing solution is acetonitrile.

Further, in the step S1, the volume of the ammonolysis pretreatment liquid added is larger than the volume of the synthesis column.

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

(1) the invention provides a pretreatment solution for ammonolysis, which is prepared by preparing a DNA ammonolysis pretreatment solution prepared by a reasonable proportion of water and an organic solvent, so that the ammonolysis efficiency can be improved, and the yield can be further improved.

(2) Acetonitrile is selected as an organic solvent, is non-toxic, large in polarity and stable in property, does not react with an unstable reagent in a crude product, is compared with other organic solvents, is selected as a component of an ammonolysis pretreatment liquid, is favorable for further improving the ammonolysis efficiency and the product yield, is low in price and is favorable for further reducing the cost.

(3) Compared with the prior art, the ammonolysis method adopts the DNA ammonolysis pretreatment liquid to firstly wet and then carry out gas phase ammonolysis, thereby improving the ammonolysis efficiency and the yield of DNA products.

(4) When the ammonolysis method is added into the ammonolysis column, the adding content of the ammonolysis pretreatment liquid does not need to be strictly controlled, and the operation is simple and convenient.

Detailed Description

The present invention is further described in detail below with reference to specific examples so that those skilled in the art can more clearly understand the present invention.

The following examples are provided only for illustrating the present invention and are not intended to limit the scope of the present invention. All other embodiments obtained by a person skilled in the art based on the specific embodiments of the present invention without any inventive step are within the scope of the present invention.

In the examples of the present invention, all the raw material components are commercially available products well known to those skilled in the art, unless otherwise specified; in the examples of the present invention, unless otherwise specified, all technical means used are conventional means well known to those skilled in the art.

The embodiment of the invention relates to the following raw material descriptions:

solid phase synthesis crude DNA was synthesized by a mercade 192E DNA synthesizer: t10mers (TTTT TTTT TT (5 '-3')) was selected as the sequence of the oligonucleotide, and the column was a 50nmol universal column (parameters: 78nmol column loading; CV value: 9.1%; vapor lock value: 18-22 mmHg; CPG Frits volume: 50.20ul, brand: deep comma biotechnology, Inc.).

Example 1

This example provides an ammonolysis pretreatment solution for solid-phase synthesis of DNA, which comprises the following components:

water: 30% (v/v); acetonitrile: 70% (v/v).

The embodiment also provides a method for carrying out ammonolysis by using the DNA ammonolysis pretreatment solution, which comprises the following specific operations:

(1) adding the ammonolysis pretreatment liquid into a synthetic column, wherein the volume of the ammonolysis pretreatment liquid is larger than that of the synthetic column; and carrying out vacuum treatment on the synthetic column until the ammonolysis pretreatment liquid is fully diffused and wetted in the synthetic column.

(2) Putting into a gas phase ammonolysis apparatus, adding 400pa ammonia gas and water vapor, sealing, reacting for 90min at 90 ℃.

(3) The synthetic column after the completion of the ammonolysis was washed with anhydrous acetonitrile to remove impurities of inorganic salt components, and eluted twice with 200ul volume of sterilized high-purity water and the oligonucleotide was recovered.

Example 2

This example provides an ammonolysis pretreatment solution for solid-phase synthesis of DNA, which comprises the following components:

water: 20% (v/v); acetonitrile: 80% (v/v).

The embodiment also provides a method for carrying out ammonolysis by using the DNA ammonolysis pretreatment solution, which comprises the following specific operations:

(1) adding the ammonolysis pretreatment liquid into a synthetic column, wherein the volume of the ammonolysis pretreatment liquid is larger than that of the synthetic column; and carrying out vacuum treatment on the synthetic column until the ammonolysis pretreatment liquid is fully diffused and wetted in the synthetic column.

(2) Putting into a gas phase ammonolysis apparatus, adding 400pa ammonia gas and water vapor, sealing, reacting for 90min at 90 ℃.

(3) The synthetic column after the completion of the ammonolysis was washed with anhydrous acetonitrile to remove impurities of inorganic salt components, and eluted twice with 200ul volume of sterilized high-purity water and the oligonucleotide was recovered.

Comparative example 1

The comparative example provides an ammonolysis pretreatment solution for solid-phase synthesis of DNA, which comprises the following specific components:

water: 90% (v/v); acetonitrile: 10% (v/v).

The comparative example also provides a method for carrying out ammonolysis by using the DNA ammonolysis pretreatment solution, which comprises the following specific operations:

(1) adding the ammonolysis pretreatment liquid into a synthesis column, wherein the volume of the ammonolysis pretreatment liquid is the same as that of the ammonolysis pretreatment liquid added in example 3; and carrying out vacuum treatment on the synthetic column until the ammonolysis pretreatment liquid is fully diffused and wetted in the synthetic column.

(2) Putting into a gas phase ammonolysis apparatus, adding 400pa ammonia gas and water vapor, sealing, reacting for 90min at 90 ℃.

(3) The synthetic column after the completion of the ammonolysis was washed with anhydrous acetonitrile to remove impurities of inorganic salt components, and eluted twice with 200ul volume of sterilized high-purity water and the oligonucleotide was recovered.

Comparative example 2

The comparative example provides an ammonolysis pretreatment solution for solid-phase synthesis of DNA, which comprises the following specific components:

water: 50% (v/v); acetonitrile: 50% (v/v).

The embodiment also provides a method for carrying out ammonolysis by using the DNA ammonolysis pretreatment solution, which comprises the following specific operations:

(1) adding the ammonolysis pretreatment liquid into a synthetic column, wherein the volume of the ammonolysis pretreatment liquid is larger than that of the synthetic column; and carrying out vacuum treatment on the synthetic column until the ammonolysis pretreatment liquid is fully diffused and wetted in the synthetic column.

(2) Putting into a gas phase ammonolysis apparatus, adding 400pa ammonia gas and water vapor, sealing, reacting for 90min at 90 ℃.

(3) The synthetic column after the completion of the ammonolysis was washed with anhydrous acetonitrile to remove impurities of inorganic salt components, and eluted twice with 200ul volume of sterilized high-purity water and the oligonucleotide was recovered.

Comparative example 3

The comparative example provides an ammonolysis pretreatment solution for solid-phase synthesis of DNA, which comprises the following specific components:

water: 100% (v/v).

The comparative example also provides a method for carrying out ammonolysis by using the DNA ammonolysis pretreatment solution, which comprises the following specific operations:

(1) adding the ammonolysis pretreatment liquid into a synthesis column, wherein the volume of the ammonolysis pretreatment liquid is the same as that of the ammonolysis pretreatment liquid added in example 3; and carrying out vacuum treatment on the synthetic column until the ammonolysis pretreatment liquid is fully diffused and wetted in the synthetic column.

(2) Putting into a gas phase ammonolysis apparatus, adding 400pa ammonia gas and water vapor, sealing, reacting for 90min at 90 ℃.

(3) The synthetic column after the completion of the ammonolysis was washed with anhydrous acetonitrile to remove impurities of inorganic salt components, and eluted twice with 200ul volume of sterilized high-purity water and the oligonucleotide was recovered.

Comparative example 4

This comparative example provides a DNA ammonolysis method which was substantially the same as that of example 2 except that the wet treatment with the ammonolysis pretreatment liquid in the step (1) was not carried out.

The contents of the oligonucleotides of examples 1 to 2 and comparative examples 1 to 4 were determined by a spectrophotometer, and the results are shown in Table 1.

TABLE 1 results of the oligonucleotide content test in examples 1 to 2 and comparative examples 1 to 4

Ammonolysis process	Oligonucleotide content (A260)
		Example 1	16.795
Example 2	17.899
		Comparative example 1	4.337
Comparative example 2	10.906
		Comparative example 3	4.538
Comparative example 4	15.429

In Table 1, A260 represents the absorbance of the oligonucleotide at 260nm ultraviolet light.

As shown in Table 1, in examples 1-2, compared with comparative examples 1-2, the volume percentage of the organic solvent is 70-80%, and the oligonucleotide content is more than 16%;

compared with the comparative example 3, the organic solvent is added in the examples 1-2, so that the yield is greatly improved compared with that of pure water for wetting.

In examples 1 to 2, compared with comparative example 4, the yield was improved by performing the wetting treatment with the pretreatment solution for ammonolysis before the ammonolysis.

It should be noted that the above examples are only for further illustration and description of the technical solution of the present invention, and are not intended to further limit the technical solution of the present invention, and the method of the present invention is only a preferred embodiment, and is not intended to limit the protection scope of the present invention. Any modification, equivalent replacement, or improvement made within the spirit and principle of the present invention should be included in the protection scope of the present invention.