阅读说明：本技术 一种引物纯化方法 (Primer purification method ) 是由 樊梓龙 刘俊 张钦 曹志旋 于 2021-07-30 设计创作，主要内容包括：本发明提供一种引物纯化方法,包括：步骤S1:将氨解后的核苷酸粗品采用有机溶剂进行第一次过柱洗涤,得到预产品；步骤S2：将所述预产品采用有机溶剂与水的混合溶剂进行过柱洗涤,得到纯化产品；其中,所述有机溶剂的体积百分比为87％～92％。与现有技术相比,本发明的有益效果在于：本发明采用先用纯乙腈作为洗脱液洗脱,在采用87％～92％的有机溶剂与水的混合溶液进行洗脱过柱,在有效的提高了合成产物的同时又确保了产物的产量。(The invention provides a primer purification method, which comprises the following steps: step S1, carrying out first column-passing washing on the aminolyzed crude nucleotide product by using an organic solvent to obtain a pre-product; step S2: carrying out column-passing washing on the pre-product by adopting a mixed solvent of an organic solvent and water to obtain a purified product; wherein the volume percentage of the organic solvent is 87-92%. Compared with the prior art, the invention has the beneficial effects that: the invention adopts pure acetonitrile as eluent for elution, and adopts a mixed solution of 87-92% of organic solvent and water for elution and column passing, thereby effectively improving the synthetic product and ensuring the yield of the product.)

1. A method of purifying a primer, comprising: step S1, carrying out first column-passing washing on the aminolyzed crude nucleotide product by using an organic solvent to obtain a pre-product; step S2: carrying out column-passing washing on the pre-product by adopting a mixed solvent of an organic solvent and water, and finally carrying out column-passing washing on the pre-product by adopting water to desorb a product so as to obtain a purified product; wherein the volume percentage of the organic solvent is 87-92%.

2. The method of claim 1, wherein the organic solvent is acetonitrile.

3. The method for purifying a primer as claimed in claim 1 or 2, wherein the volume percentage of the organic solvent is 90%.

4. A method of primer synthesis comprising:

step A1: synthesizing oligonucleotides using a solid phase phosphoramidite triester method;

step A2: carrying out ammonolysis on the synthetic column to obtain a synthetic crude product;

step A3, purifying the synthesized crude product by the primer purification method of any one of claims 1 to 3 to obtain a purified product.

5. The method for synthesizing primers according to claim 4, wherein in step A2, the synthesis column is wetted with a wetting solution, the wetting solution is composed of water and acetonitrile, and the volume percentage of acetonitrile is 70-80%.

6. The method for synthesizing a primer according to claim 4, wherein the aminolysis time is 90min and the aminolysis temperature is 90 ℃.

7. The method for synthesizing primers according to claim 4, wherein the step A1 is performed by a DNA synthesizer; the ammonolysis step in step A2 is performed by using an ammonolysis apparatus.

Technical Field

The invention belongs to the field of primer synthesis, and particularly relates to a primer purification method.

Background

The current primer synthesis basically adopts a solid-phase phosphoramidite triester method. The DNA fragment synthesized by the phosphoramidite triester method has the characteristics of high efficiency, quick coupling and relatively stable initial reactant. The phosphoramidite triester method is a method in which DNA is immobilized on a solid support to complete the synthesis of a DNA strand in the direction from the 3 'end to the 5' end of a primer to be synthesized, and adjacent nucleotides are linked by a 3'→ 5' phosphodiester bond.

After synthesis, desorption is carried out from the solid phase carrier by grafting, and finally desalination and purification are carried out. Desalting, also called as simple reverse phase column, ammonia salt can be eluted by organic solution but not by water, salt can be effectively removed, the purity of the purified oligonucleotide can meet the requirements of most molecular biology experiments, but the method can not effectively remove small primer fragments which are slightly shorter than the target fragments.

Disclosure of Invention

In order to solve the technical problems, the invention provides a primer purification method.

The specific technical scheme is as follows:

a method for purifying a primer, which is different from the method, comprises the following steps: step S1, carrying out first column-passing washing on the aminolyzed crude nucleotide product by using an organic solvent to obtain a pre-product; step S2: carrying out column-passing washing on the pre-product by adopting a mixed solvent of an organic solvent and water, and finally carrying out column-passing washing on the pre-product by using water to desorb a product so as to obtain a purified product; wherein the volume percentage of the organic solvent is 87-92%.

Further, the organic solvent is acetonitrile.

Further, the volume percentage of the organic solvent was 90%.

A method for synthesizing a primer, which is different from the method, comprises the following steps:

step A1: synthesizing oligonucleotides using a solid phase phosphoramidite triester method;

step A2: wetting a synthesis column and then carrying out ammonolysis to obtain a synthetic crude product;

and step A3, purifying the synthesized crude product according to the primer purification method to obtain a purified product.

Further, in the step a2, a wetting liquid is used to wet the synthesis column, wherein the wetting liquid is composed of water and acetonitrile, and the volume percentage of the acetonitrile is 70% to 80%.

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

Further, the step A1 is completed by a DNA synthesizer; the ammonolysis step in step A2 is performed by using an ammonolysis apparatus.

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

(1) the invention adopts pure acetonitrile as eluent for elution, and adopts a mixed solution of 87-92% of organic solvent and water for elution and column passing, thereby effectively improving the synthetic product and ensuring the yield of the product.

(2) Acetonitrile is selected as an organic solvent, so that the polarity is high, the purification effect is good, the price is low, and the preparation cost is reduced.

(3) The invention provides a primer synthesis method adopting gradient purification, which improves the purity, and simultaneously, after solid phase synthesis, humidification and ammonolysis are carried out, thus further improving the yield.

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.

In the examples of the present invention, the raw materials used were all conventional commercially available products

Example 1

This example provides a method for synthesizing oligonucleotides by solid phase phosphoramidite triester method, which comprises the following steps:

t10mers (TTTTTTTTTT (5 '-3')) was selected as the sequence for the oligonucleotide, a 50nmol synthesis column was used as the universal synthesis column and synthesized using a Mermade192E DNA synthesizer, with the following parameters:

the loading of the synthetic column is 78 nmol; CV value: 9.1 percent; the gas resistance value is 18-22 mmHg; CPG Frits volume: 50.20ul, brand: deep comma biotechnology limited.

Example 2

This example provides a method for purification of the solid phase composition of example 1 by aminolysis, which comprises the following steps:

(1) adding a wetting solution of 70% v/v acetonitrile and 30% v/v water into a synthetic column, wherein the volume of the wetting solution is larger than that of the synthetic column; and vacuumizing the synthetic column until the wetting 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) And (3) performing first column chromatography elution on the synthetic column subjected to ammonolysis by using 200ul of 100% acetonitrile, performing second column chromatography elution on the synthetic column subjected to ammonolysis by using 200ul of 90% (v/v) acetonitrile and 10% (v/v) high-purity water, throwing off residual liquid by using a centrifugal machine, performing secondary elution by using 200ul of sterilized high-purity water, and recovering the oligonucleotide to a 96-hole deep-hole plate.

Example 3

This example provides a method for purification of the solid phase composition of example 1 by aminolysis, which comprises the following steps:

(1) adding a wetting solution of 80% v/v acetonitrile and 20% v/v water into a synthetic column, wherein the volume of the wetting solution is larger than that of the synthetic column; and vacuumizing the synthetic column until the wetting 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) And (3) performing first column chromatography elution on the synthetic column subjected to ammonolysis by using 200ul of 100% acetonitrile, performing second column chromatography elution on the synthetic column subjected to ammonolysis by using 200ul of 90% (v/v) acetonitrile and 10% (v/v) high-purity water, throwing off residual liquid by using a centrifugal machine, performing secondary elution by using 200ul of sterilized high-purity water, and recovering the oligonucleotide to a 96-hole deep-hole plate.

Example 4

This example provides a method for purification of the solid phase composition of example 1 by aminolysis, which comprises the following steps:

(1) putting the synthetic column into a gas phase ammonolysis instrument, adding 400pa of ammonia gas and water vapor, sealing, reacting for 90min, and reacting at 90 ℃.

(3) And (3) performing first column chromatography elution on the synthetic column subjected to ammonolysis by using 200ul of 100% acetonitrile, performing second column chromatography elution on the synthetic column subjected to ammonolysis by using 200ul of 90% (v/v) acetonitrile and 10% (v/v) high-purity water, throwing off residual liquid by using a centrifugal machine, performing secondary elution by using 200ul of sterilized high-purity water, and recovering the oligonucleotide to a 96-hole deep-hole plate.

Comparative example 1

This comparative example provides a method for purification of the solid phase composition of example 1 by aminolysis, which was carried out as follows:

(1) adding a wetting solution of 80% v/v acetonitrile and 20% v/v water into a synthetic column, wherein the volume of the wetting solution is larger than that of the synthetic column; and vacuumizing the synthetic column until the wetting 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) And (3) performing first column chromatography elution on the synthetic column subjected to ammonolysis by using 200ul 100% acetonitrile, performing second column chromatography elution by using 200ul 100% acetonitrile, throwing off residual liquid by using a centrifugal machine, performing twice elution by using 200ul volume of sterilized high-purity water, and recovering the oligonucleotide to a 96-hole deep-hole plate.

Comparative example 2

This comparative example provides a method for purification of the solid phase composition of example 1 by aminolysis, which was carried out as follows:

(1) adding a wetting solution of 80% v/v acetonitrile and 20% v/v water into a synthetic column, wherein the volume of the wetting solution is larger than that of the synthetic column; and vacuumizing the synthetic column until the wetting 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 ammonolysis was eluted with 200ul 100% acetonitrile, the residual liquid was centrifuged off, and the column was eluted twice with 200ul volume of sterilized high purity water and the oligonucleotide was recovered to a 96-well deep-well plate.

Comparative example 3

This comparative example provides a method for purification of the solid phase composition of example 1 by aminolysis, which was carried out as follows:

(1) adding a wetting solution of 80% v/v acetonitrile and 20% v/v water into a synthetic column, wherein the volume of the wetting solution is larger than that of the synthetic column; and vacuumizing the synthetic column until the wetting 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) And (3) performing first column chromatography elution on the synthetic column subjected to ammonolysis by using 200ul of 100% acetonitrile, performing second column chromatography elution on the synthetic column subjected to ammonolysis by using 200ul of 80% (v/v) acetonitrile and 20% (v/v) high-purity water, throwing off residual liquid by using a centrifugal machine, performing secondary elution by using 200ul of sterilized high-purity water, and recovering the oligonucleotide into a 96-hole deep-hole plate.

Comparative example 4

This comparative example provides a method for purification of the solid phase composition of example 1 by aminolysis, which was carried out as follows:

(1) adding a wetting solution of 80% v/v acetonitrile and 20% v/v water into a synthetic column, wherein the volume of the wetting solution is larger than that of the synthetic column; and vacuumizing the synthetic column until the wetting 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 ammonolysis was eluted with 200ul 100% acetonitrile for the first time, 200ul 50% (v/v) acetonitrile + 50% (v/v) high purity water for the second time, the residual liquid was centrifuged off, and the liquid was eluted with 200ul volume of sterilized high purity water twice and the oligonucleotide was recovered to a 96-well deep-well plate.

The oligonucleotides of examples 2-4 and comparative examples 1-4 were detected by UV spectrophotometer at 230nm and 260nm, and the detection results are shown in Table 1, wherein the detection results are used to detect the content of small molecule and other impurities at 230nm, and the detection results are 260 nm:

TABLE 1 detection results of 230nm UV spectrophotometer for oligonucleotides of examples 2-4 and comparative examples 1-4

Oligonucleotides	A230
		Example 2	4.368
Example 3	4.524
		Example 4	4.823
Comparative example 1	6.586
		Comparative example 2	5.093
Comparative example 3	1.074
		Comparative example 4	0.810

TABLE 2 detection results of oligonucleotides 260nm UV spectrophotometer in examples 2-4 and comparative examples 1-4

The difference between the embodiment of the invention and the comparative example 1 is that the mixed solution of the organic solvent and the water adopted by the second elution solution is compared with the comparative example 1, the A230 value of the mixed solvent adopted by the second elution solution is reduced to be less than 5.

The difference between the embodiment of the invention and the comparative example 2 is that the A230 value is reduced by adopting acetonitrile and mixed solvent to carry out twice elution.

The difference between the present embodiment and comparative examples 3-4 is that the volume percent of the organic solvent is 87% -92%, the volume percent of the organic solvent in comparative example 3 is 80%, and the volume percent of the organic solvent in comparative example 4 is 50%, as can be seen from the test results in table 2, the ratio of the present invention can maintain the value of a260 above 12 while the value of a230 is reduced to 5 or less, while the values of a260 are greatly reduced in comparative examples 3-4, which affects the product yield.

Meanwhile, after the primer synthesis, the value of A230 can be reduced to below 4.8 and the value of A260 can be increased to above 13 by adopting a method of wetting first and then ammonolysis, and furthermore, when the components of the wetting solution are 80% v/v acetonitrile and 20% v/v water, the yield is further improved.

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.