CN115069099A - Preparation method and application of polymer affinity membrane with efficient nucleic acid separation and purification function - Google Patents
Preparation method and application of polymer affinity membrane with efficient nucleic acid separation and purification function Download PDFInfo
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- C12N15/1006—Extracting or separating nucleic acids from biological samples, e.g. pure separation or isolation methods; Conditions, buffers or apparatuses therefor by means of a solid support carrier, e.g. particles, polymers
- C12N15/101—Extracting or separating nucleic acids from biological samples, e.g. pure separation or isolation methods; Conditions, buffers or apparatuses therefor by means of a solid support carrier, e.g. particles, polymers by chromatography, e.g. electrophoresis, ion-exchange, reverse phase
Abstract
The invention relates to a preparation method and application of a polymer affinity membrane with high-efficiency nucleic acid separation and purification functions, wherein the preparation method comprises the following steps: after simultaneously applying a pyrocatechol compound and a polyamine compound to the surface of a polymer porous membrane, inducing the pyrocatechol compound and the polyamine compound to generate Michael addition reaction and Schiff base reaction to obtain a polymer affinity membrane with efficient nucleic acid separation and purification functions; the application comprises the following steps: when the polymer affinity membrane with high-efficiency nucleic acid separation and purification functions is used for separating and purifying nucleic acid by a solid phase adsorption method, the recovery efficiency of the nucleic acid is more than 85 percent; the preparation method has simple process; when the product is applied to nucleic acid separation and purification, the recovery efficiency is higher.
Description
Technical Field
The invention belongs to the technical field of high molecular materials, and particularly relates to a preparation method and application of a polymer affinity membrane with efficient nucleic acid separation and purification functions.
Background
Nucleic acid is a biological macromolecule carrying important genetic information, and the nucleic acid detection technology plays a key role in the fields of genetics, pharmacology, diagnosis, forensic medicine and the like. The rapid development of biological and medical technologies cannot be separated from simple and efficient nucleic acid separation and purification technologies, but the existing nucleic acid separation and purification methods still have many problems in the actual application process, and the research and development of new technologies and new materials for separating and purifying nucleic acid are significant. Affinity membrane chromatography is used as a new separation and purification technology, has the advantages related to membrane separation technology and chromatography, is widely applied to separation and purification of various biomacromolecules, and has great potential in DNA separation and purification.
The existing biological nucleic acid separation and purification method and the problems thereof are mainly as follows:
(1) organic solvent extraction method
A method for extracting purified nucleic acid from biological sample by liquid phase extraction with organic solvent such as phenol and chloroform. During extraction and purification of nucleic acid, a large amount of toxic solvents are used, so that life and health are threatened, and the ecological environment is destroyed; the complex purification processes of extraction, centrifugation and the like are time-consuming and labor-consuming, and the automatic operation cannot be realized.
(2) Solid phase adsorption process
Different solid phases (such as silica gel membranes, cellulose, magnetic beads and the like) absorb nucleic acid molecules to the surface of the solid phase by utilizing the specific binding action between the nucleic acid molecules, and then the nucleic acid is separated and purified through the steps of washing and desorption. The solid phase adsorption method is convenient to improve in experimental operation, use of organic solvents and the like, so that the solid phase adsorption method is the most common method at present, but has the problems of poor size adaptability and low recovery efficiency in the purification process.
Therefore, it is very important to research the preparation method and application of a polymer affinity membrane with high efficiency nucleic acid separation and purification functions to solve the above problems.
Disclosure of Invention
The invention aims to solve the problems in the prior art and provides a preparation method and application of a polymer affinity membrane with high-efficiency nucleic acid separation and purification functions.
In order to achieve the purpose, the technical scheme of the invention is as follows:
a preparation method of a polymer affinity membrane with efficient nucleic acid separation and purification functions comprises the steps of simultaneously applying a catechol compound and a polyamine compound to the surface of a polymer porous membrane, and inducing Michael addition reaction and Schiff base reaction between the catechol compound and the polyamine compound to obtain the polymer affinity membrane with efficient nucleic acid separation and purification functions;
when nucleic acids are separated and purified by a solid phase adsorption method using a polymer affinity membrane having a high nucleic acid separation and purification function, the recovery efficiency of nucleic acids is 85% or more (recovery efficiency: the amount of nucleic acids produced/the amount of nucleic acids put in × 100%); when the prior art separates and purifies nucleic acid by a solid phase adsorption method, the recovery efficiency of the nucleic acid is hardly over 70 percent, for example, CN202011480478.0 mentions that the concentration of DNA before purification is 83.12nm/L, the concentration of DNA after purification by using a glass cellulose membrane or a modified silica gel membrane is 42.17nm/L, and the recovery efficiency is only 50.7 percent; CN201080020635.8 adopts a magnetic bead method to purify DNA, the average input amount of DNA is 5.43 mu g, the average output amount of DNA is 1.24 mu g, and the recovery efficiency is only 22.8%; CN201810136469.6 adopts a magnetic bead solid phase separation technology, and the recovery efficiency of small-size nucleic acid is only about 65-70%;
the principle of the invention is as follows:
one of the raw materials of the invention is a polymer porous membrane, the aperture and the type of which can be adjusted, the preparation method is not limited, and the invention can be suitable for separating and purifying nucleic acid molecules with different sizes and different forms.
The catechol compound in the invention has strong adhesive force to various base materials based on the adhesive property of marine organism mussels, and can be used for coating and modifying different membrane materials.
According to the invention, the o-phenylenediphenol compound and the polyamine compound can generate Michael addition reaction and Schiff base reaction to generate a polymer, and the polymer can be uniformly coated on a polymer porous membrane to obtain a polymer affinity membrane.
The invention can endow the polymer porous membrane with high density and high content of nucleic acid affinity ligand, namely: an amino group. The affinity ligand can realize protonation and deprotonation transformation under different buffer solution conditions (pH values), namely, the combination and release between the affinity ligand and nucleic acid molecules based on amino electrostatic interaction are realized, and thus the polymer affinity membrane with high-efficiency nucleic acid separation and purification capacity is prepared.
As a preferred technical scheme:
the preparation method of the polymer affinity membrane with the high-efficiency nucleic acid separation and purification function comprises the following specific processes: firstly dissolving an o-phenylenediphenol compound in a buffer solution with the pH value of 6.0-10.0 to prepare an o-phenylenediphenol compound solution, then adding a polyamine compound into the o-phenylenediphenol compound solution, and immediately (aiming at avoiding the Michael addition reaction and the Schiff base reaction between the o-phenylenediphenol compound and the polyamine compound before being applied to a polymer porous membrane) soaking the polymer porous membrane in the mixed solution for co-coating modification to obtain the polymer affinity membrane with the efficient nucleic acid separation and purification function after completely dissolving the o-phenylenediphenol compound solution to obtain the mixed solution.
In the method for preparing a polymer affinity membrane having a high-efficiency nucleic acid isolation and purification function, the catechol compound is catechol, gallic acid, tannic acid, protocatechuic acid, catechin or quercitrin; the polyamine compound is triethylene tetramine, tetraethylene pentamine, pentaethylene hexamine, hexaethylene heptamine, polyethylene polyamine or polyethylene imine.
According to the preparation method of the polymer affinity membrane with the efficient nucleic acid separation and purification function, the concentration of the catechol compound solution is 0.1-5 mg/ml.
According to the preparation method of the polymer affinity membrane with the efficient nucleic acid separation and purification function, the molar ratio of the catechol compound to the polyamine compound in the mixed solution is 1: 4-4: 1.
The preparation method of the polymer affinity membrane with the high-efficiency nucleic acid separation and purification function comprises the following steps of (1) preparing a polymer affinity membrane, wherein the average pore diameter of the polymer porous membrane is 0.01-5 mu m, the thickness of the polymer porous membrane is 20-2000 mu m, and the porosity of the polymer porous membrane is 40-85%; the mass volume ratio of the polymer porous membrane to the mixed solution is 0.1-10 mg: 20-100 ml.
According to the preparation method of the polymer affinity membrane with the efficient nucleic acid separation and purification function, the co-coating modification time is 0.1-12 h, and the temperature is 20-60 ℃.
The invention also provides an application of the polymer affinity membrane with the high-efficiency nucleic acid separation and purification function prepared by the preparation method, which comprises the following steps:
(1) stacking 1-15 pieces of polymer affinity membranes in a purification column;
(2) adding a mixed solution of a sample solution containing nucleic acid and an adsorption buffer solution into a purification column, wherein the volume of the mixed solution is 30-100% of the capacity of the purification column, carrying out centrifugal filtration for 20-240 s at 3000-9000 g, and removing the solution in a collection tube; the pH value of the adsorption buffer solution is 4.0-6.0;
(3) adding a washing buffer solution into the purification column, wherein the volume of the washing buffer solution is 30-100% of the capacity of the purification column, centrifuging and filtering 3000-9000 g for 20-240 s, and removing the solution in the collection pipe; the washing buffer is a solution containing a water-soluble organic solvent;
(4) adding a recovery buffer solution into the purification column, wherein the volume of the recovery buffer solution is 2-10% of the capacity of the purification column, and performing centrifugal filtration for 3000-9000 g for 20-240 s to obtain a purified nucleic acid solution; the pH value of the recovered buffer solution is 7.5-12.0 and is higher than that of the washing buffer solution.
The present invention allows a nucleic acid to be adsorbed to the affinity membrane by bringing a mixed solution of a sample solution containing the nucleic acid and an adsorption buffer into contact with the affinity membrane; washing the affinity membrane by contacting a wash buffer with the membrane, during which the nucleic acid remains adsorbed to the affinity; releasing the nucleic acid from the affinity membrane by contacting a recovery buffer with the affinity membrane; the pH value of each buffer solution is reasonably controlled to ensure that the buffer solution can play a corresponding function.
As a preferred technical scheme:
the application comprises the steps that in the step (2), the size of the nucleic acid molecule fragment in the sample solution containing the nucleic acid is 100 bp-30 kp, and the concentration is 1-200 ng/mu l; the volume ratio of the sample solution containing nucleic acid to the adsorption buffer solution is 1: 1-1: 10.
In the above application, in the step (3), the water-soluble organic solvent is a C1-C3 alcohol.
Advantageous effects
(1) The preparation method disclosed by the invention is simple and convenient to operate, low in raw material cost and suitable for industrial large-scale production.
(2) The polymer affinity membrane prepared by the invention has the recovery efficiency of nucleic acid of more than 85 percent.
(3) The polymer affinity membrane prepared by the invention has higher nucleic acid size applicability and higher nucleic acid recovery efficiency for nucleic acids with the size of 100 bp-30 kb.
Drawings
FIG. 1 is an SEM image of the surface of a polyethersulfone affinity membrane in example 1;
fig. 2 is an SEM image of the cross section of the polyethersulfone affinity membrane in example 1.
Detailed Description
The invention will be further illustrated with reference to specific embodiments. It should be understood that these examples are for illustrative purposes only and are not intended to limit the scope of the present invention. Further, it should be understood that various changes or modifications of the present invention may be made by those skilled in the art after reading the teaching of the present invention, and such equivalents may fall within the scope of the present invention as defined in the appended claims.
In the following examples and comparative examples, absorbance of a260 in a purified nucleic acid solution was measured using a NanoDrop microspectrophotometer, and the concentration of the purified nucleic acid solution and the recovery efficiency of nucleic acid were characterized, and NanoDrop is a special instrument for characterizing the concentration of nucleic acid and the test principle is that the concentration of nucleic acid was given by measuring the absorbance of a260 in a nucleic acid solution.
Example 1
The preparation method of the polymer affinity membrane with the high-efficiency nucleic acid separation and purification function comprises the following specific steps:
(1) preparation of raw materials:
catechol compound: catechol;
polyamine compounds: polyethylene polyamines;
polymer porous membrane: a polyethersulfone membrane; the average pore diameter of the polymer porous membrane is 0.1 μm, the thickness is 500 μm, and the porosity is 70%;
(2) firstly, dissolving a catechol compound in a buffer solution (a buffer solution is specifically a Tris-HCl buffer solution) with the pH value of 8.5, preparing a catechol compound solution with the concentration of 5mg/ml, then adding a polyamine compound into the catechol compound solution, completely dissolving to obtain a mixed solution, immediately soaking a polymer porous membrane in the mixed solution according to the mass-volume ratio of 0.1mg to 20ml, and coating and modifying at 50 ℃ for 12 hours to obtain a polymer affinity membrane with the efficient nucleic acid separation and purification function, specifically shown in figures 1 and 2; wherein the molar ratio of the catechol compound to the polyamine compound in the mixed solution is 2: 1.
The application of the polymer affinity membrane with the efficient nucleic acid separation and purification function prepared by the preparation method comprises the following specific steps:
(I) stacking 5 sheets of the polymer affinity membrane in a purification column;
(II) adding a mixed solution of a sample solution (the solvent is ultrapure water) of nucleic acid and an adsorption buffer solution (specifically a phosphate buffer solution) into the purification column, wherein the volume of the mixed solution is 80% of the capacity of the purification column, centrifuging and filtering by 9000g for 30s, and removing the solution in the collection tube; the pH value of the adsorption buffer solution is 5; the size of the nucleic acid molecule fragment in the sample solution of the nucleic acid is 200bp, and the concentration is 50 ng/mul; the volume ratio of the sample solution of nucleic acid to the adsorption buffer solution is 1: 5;
(III) adding a washing buffer solution into the purification column, wherein the volume of the washing buffer solution is 90% of the capacity of the purification column, and removing the solution in the collection tube by 9000g of centrifugal filtration for 60 s; the washing buffer solution is ultrapure water containing 80 wt% of ethanol, and the pH value of the washing buffer solution is 7;
(IV) adding a recovery buffer solution (specifically a glycine-sodium hydroxide buffer solution) into the purification column, wherein the volume of the recovery buffer solution is 8% of the capacity of the purification column, and centrifuging and filtering for 60s at 9000g to obtain a purified nucleic acid solution; the pH of the recovery buffer was 8.5.
The recovery efficiency of nucleic acid was 87%.
Comparative example 1
Domestic SiO 2 The procedure of applying the membrane purification cartridge was substantially the same as that of the application of the polymer affinity membrane of example 1 except that the domestic SiO was prepared in step (I) 2 Membrane Purification Column (SanPrep Column PCR Product Purification Kit,produced by bio-engineering (Shanghai) corporation).
The recovery efficiency of nucleic acid was 55%.
Compared with example 1, the recovery efficiency of the nucleic acid of comparative example 1 is much lower than that of example 1, because the high density and high number of amino groups of the polymer affinity membrane in example 1 provides stronger binding capacity for the adsorption of nucleic acid molecules on the membrane; meanwhile, the polymer affinity membrane in example 1 can improve the release efficiency of nucleic acid molecules by changing the positive and negative potentials under different buffer solution conditions (pH values) provided by amino groups.
Example 2
The preparation method of the polymer affinity membrane with the high-efficiency nucleic acid separation and purification function comprises the following specific steps:
(1) preparation of raw materials:
catechol compound: tannic acid;
polyamine compounds: a polyethyleneimine;
polymer porous membrane: a polyvinylidene fluoride membrane; the average pore diameter of the polymer porous membrane is 0.6 μm, the thickness is 180 μm, and the porosity is 80%;
(2) firstly, dissolving a catechol compound in a buffer solution (a buffer solution is specifically a Tris-HCl buffer solution) with the pH value of 8, preparing a catechol compound solution with the concentration of 1mg/ml, then adding a polyamine compound into the catechol compound solution, immediately soaking a polymer porous membrane in the mixed solution according to the mass-volume ratio of 1mg:30ml after completely dissolving to obtain a mixed solution, and coating and modifying at the temperature of 35 ℃ for 9 hours to obtain a polymer affinity membrane with the efficient nucleic acid separation and purification function; wherein the molar ratio of the catechol compound to the polyamine compound in the mixed solution is 1: 1.
The application of the polymer affinity membrane with the efficient nucleic acid separation and purification function prepared by the preparation method comprises the following specific steps:
(I) stacking 12 sheets of the polymer affinity membrane in a purification column;
(II) adding a mixed solution of a sample solution (the solvent is ultrapure water) of nucleic acid and an adsorption buffer solution (specifically a phosphate buffer solution) into the purification column, wherein the volume of the mixed solution is 80% of the capacity of the purification column, and removing the solution in the collection tube by centrifugal filtration for 90s at 5000 g; the pH value of the adsorption buffer solution is 4; the size of the nucleic acid molecule fragment in the sample solution of nucleic acid is 4.8kb, and the concentration is 75 ng/mul; the volume ratio of the sample solution of nucleic acid to the adsorption buffer solution is 1: 6;
(III) adding a washing buffer solution into the purification column, wherein the volume of the washing buffer solution is 90% of the capacity of the purification column, and removing the solution in the collection pipe by centrifugal filtration for 120s at 5000 g; the washing buffer solution is ultrapure water containing 80 wt% of ethanol, and the pH value of the washing buffer solution is 7;
(IV) adding a recovery buffer solution (specifically a glycine-sodium hydroxide buffer solution) into the purification column, wherein the volume of the recovery buffer solution is 10% of the capacity of the purification column, and performing centrifugal filtration for 5000g for 120s to obtain a purified nucleic acid solution; the pH of the recovery buffer was 7.5.
The recovery efficiency of nucleic acid was 92%.
Example 3
The preparation method of the polymer affinity membrane with the high-efficiency nucleic acid separation and purification function comprises the following specific steps:
(1) preparation of raw materials:
catechol compound: a catechin;
polyamine compounds: triethylene tetramine;
polymer porous membrane: a polysulfone membrane; the average pore diameter of the polymer porous membrane is 0.4 μm, the thickness is 250 μm, and the porosity is 66%;
(2) firstly, dissolving a catechol compound in a buffer solution (a buffer solution is specifically a Tris-HCl buffer solution) with the pH value of 6.5, preparing a catechol compound solution with the concentration of 3mg/ml, then adding a polyamine compound into the catechol compound solution, immediately soaking a polymer porous membrane in the mixed solution according to the mass-volume ratio of 2mg to 60ml after completely dissolving to obtain a mixed solution, and coating and modifying at 40 ℃ for 12 hours to obtain a polymer affinity membrane with the efficient nucleic acid separation and purification function; wherein the molar ratio of the catechol compound to the polyamine compound in the mixed solution is 1: 4.
The application of the polymer affinity membrane with the efficient nucleic acid separation and purification function prepared by the preparation method comprises the following specific steps:
(I) stacking 7 sheets of the polymer affinity membrane in a purification column;
(II) adding a mixed solution of a sample solution (the solvent is ultrapure water) of nucleic acid and an adsorption buffer solution (specifically a phosphate buffer solution) into the purification column, wherein the volume of the mixed solution is 90% of the capacity of the purification column, and removing the solution in the collection tube by 6000g centrifugal filtration for 60 s; the pH value of the adsorption buffer solution is 4.5; the size of the nucleic acid molecule fragment in the sample solution of nucleic acid is 3kb, and the concentration is 30 ng/. mu.l; the volume ratio of the sample solution of nucleic acid to the adsorption buffer solution is 1: 5;
(III) adding a washing buffer solution into the purification column, wherein the volume of the washing buffer solution is 90% of the capacity of the purification column, and removing the solution in the collection tube by 6000g centrifugal filtration for 90 s; the washing buffer solution is ultrapure water containing 80 wt% of ethanol, and the pH value of the washing buffer solution is 7;
(IV) adding a recovery buffer solution (specifically a glycine-sodium hydroxide buffer solution) into the purification column, wherein the volume of the recovery buffer solution is 10% of the capacity of the purification column, and carrying out 6000g centrifugal filtration for 90s to obtain a purified nucleic acid solution; the pH of the recovery buffer was 8.5.
The recovery efficiency of nucleic acid was 85%.
Example 4
The preparation method of the polymer affinity membrane with the high-efficiency nucleic acid separation and purification function comprises the following specific steps:
(1) preparation of raw materials:
catechol compound: gallic acid;
polyamine compounds: polyethylene polyamines;
polymer porous membrane: a polyacrylonitrile film; the average pore diameter of the polymer porous membrane is 2 mu m, the thickness is 140 mu m, and the porosity is 72%;
(2) firstly, dissolving a catechol compound in a buffer solution (a buffer solution is specifically a Tris-HCl buffer solution) with the pH value of 9 to prepare a catechol compound solution with the concentration of 0.5mg/ml, then adding a polyamine compound into the catechol compound solution, immediately soaking a polymer porous membrane in the mixed solution according to the mass-volume ratio of 0.1mg to 25ml after complete dissolution to obtain a mixed solution, and coating and modifying at 50 ℃ for 9 hours to obtain a polymer affinity membrane with efficient nucleic acid separation and purification functions; wherein the molar ratio of the catechol compound to the polyamine compound in the mixed solution is 4: 1.
The application of the polymer affinity membrane with the efficient nucleic acid separation and purification function prepared by the preparation method comprises the following specific steps:
(I) stacking 13 sheets of the polymer affinity membrane in a purification column;
(II) adding a mixed solution of a sample solution (the solvent is ultrapure water) of nucleic acid and an adsorption buffer solution (specifically a phosphate buffer solution) into the purification column, wherein the volume of the mixed solution is 90% of the capacity of the purification column, centrifuging and filtering for 150s at 4000g, and removing the solution in the collection tube; the pH value of the adsorption buffer solution is 4; the size of the nucleic acid molecule fragment in the sample solution of nucleic acid is 8.5kb, and the concentration is 100 ng/mul; the volume ratio of the sample solution of nucleic acid to the adsorption buffer solution is 1: 3;
(III) adding a washing buffer solution into the purification column, wherein the volume of the washing buffer solution is 90% of the capacity of the purification column, and removing the solution in the collection pipe by 4000g centrifugal filtration for 150 s; the washing buffer solution is ultrapure water containing 80 wt% of ethanol, and the pH value of the washing buffer solution is 7;
(IV) adding a recovery buffer solution (specifically a glycine-sodium hydroxide buffer solution) into the purification column, wherein the volume of the recovery buffer solution is 10% of the capacity of the purification column, and centrifuging 4000g for 150s to obtain a purified nucleic acid solution; the recovery buffer had a pH of 12.
The recovery efficiency of nucleic acid was 89%.
Example 5
The preparation method of the polymer affinity membrane with the high-efficiency nucleic acid separation and purification function comprises the following specific steps:
(1) preparation of raw materials:
catechol compound: quercetin;
polyamine compounds: hexaethyleneheptamine;
polymer porous membrane: a polyvinyl chloride film; the average pore diameter of the polymer porous membrane is 0.05 μm, the thickness is 1500 μm, and the porosity is 56%;
(2) firstly, dissolving a catechol compound in a buffer solution (a buffer solution is specifically a Tris-HCl buffer solution) with the pH value of 10, preparing a catechol compound solution with the concentration of 1mg/ml, then adding a polyamine compound into the catechol compound solution, immediately soaking a polymer porous membrane in the mixed solution according to the mass-volume ratio of 5mg:80ml after completely dissolving to obtain a mixed solution, and coating and modifying at 20 ℃ for 0.5h to obtain the polymer affinity membrane with the efficient nucleic acid separation and purification function; wherein the molar ratio of the catechol compound to the polyamine compound in the mixed solution is 1: 3.
The application of the polymer affinity membrane with the efficient nucleic acid separation and purification function prepared by the preparation method comprises the following specific steps:
(I) stacking 1 sheet of the polymer affinity membrane in a purification column;
(II) adding a mixed solution of a sample solution (the solvent is ultrapure water) of nucleic acid and an adsorption buffer solution (specifically a phosphate buffer solution) into the purification column, wherein the volume of the mixed solution is 90% of the volume of the purification column, performing 8000g centrifugal filtration for 60s, and removing the solution in the collection pipe; the pH value of the adsorption buffer solution is 5.5; the size of the nucleic acid molecule fragment in the sample solution of the nucleic acid is 150bp, and the concentration is 150 ng/microliter; the volume ratio of the sample solution of nucleic acid to the adsorption buffer solution is 1: 1;
(III) adding a washing buffer solution into the purification column, wherein the volume of the washing buffer solution is 90% of the capacity of the purification column, performing centrifugal filtration at 8000g for 90s, and removing a solution in the collection pipe; the washing buffer solution is ultrapure water containing 80 wt% of ethanol, and the pH value of the washing buffer solution is 7;
(IV) adding a recovery buffer solution (specifically a glycine-sodium hydroxide buffer solution) into the purification column, wherein the volume of the recovery buffer solution is 8% of the capacity of the purification column, and 8000g of centrifugal filtration is carried out for 90s to obtain a purified nucleic acid solution; the recovery buffer had a pH of 8.
The recovery efficiency of nucleic acid was 90%.
Example 6
The preparation method of the polymer affinity membrane with the high-efficiency nucleic acid separation and purification function comprises the following specific steps:
(1) preparation of raw materials:
catechol compound: catechol;
polyamine compounds: polyethylene polyamines;
polymer porous membrane: a polyphenylsulfone membrane; the average pore diameter of the polymer porous membrane is 0.2 μm, the thickness is 350 μm, and the porosity is 65%;
(2) firstly, dissolving a catechol compound in a buffer solution (a buffer solution is specifically a Tris-HCl buffer solution) with the pH value of 8.5, preparing a catechol compound solution with the concentration of 5mg/ml, then adding a polyamine compound into the catechol compound solution, immediately soaking a polymer porous membrane in the mixed solution according to the mass-volume ratio of 0.1mg to 20ml after complete dissolution to obtain a mixed solution, and coating and modifying at 30 ℃ for 2h to obtain the polymer affinity membrane with the efficient nucleic acid separation and purification function; wherein the molar ratio of the catechol compound to the polyamine compound in the mixed solution is 2: 1.
The application of the polymer affinity membrane with the efficient nucleic acid separation and purification function prepared by the preparation method comprises the following specific steps:
(I) stacking 8 sheets of the polymer affinity membrane in a purification column;
(II) adding a mixed solution of a sample solution (the solvent is ultrapure water) of nucleic acid and an adsorption buffer solution (specifically a phosphate buffer solution) into the purification column, wherein the volume of the mixed solution is 80% of the volume of the purification column, performing 8000g centrifugal filtration for 40s, and removing the solution in the collection pipe; the pH value of the adsorption buffer solution is 5; the size of the nucleic acid molecule fragment in the sample solution of the nucleic acid is 200bp, and the concentration is 50 ng/mul; the volume ratio of the sample solution of nucleic acid to the adsorption buffer solution is 1: 8;
(III) adding a washing buffer solution into the purification column, wherein the volume of the washing buffer solution is 90% of the capacity of the purification column, and performing centrifugal filtration at 8000g for 60s to remove a solution in the collection pipe; the washing buffer solution is ultrapure water containing 80 wt% of ethanol, and the pH value of the washing buffer solution is 7;
(IV) adding a recovery buffer solution (specifically a glycine-sodium hydroxide buffer solution) into the purification column, wherein the volume of the recovery buffer solution is 8% of the capacity of the purification column, and performing centrifugal filtration at 8000g for 60s to obtain a purified nucleic acid solution; the recovery buffer had a pH of 9.
The recovery efficiency of nucleic acid was 93%.
Example 7
The preparation method of the polymer affinity membrane with the high-efficiency nucleic acid separation and purification function comprises the following specific steps:
(1) preparation of raw materials:
catechol compound: a catechin;
polyamine compounds: polyethylene polyamines;
polymer porous membrane: an ethylene-vinyl alcohol copolymer film; the average pore diameter of the polymer porous membrane is 1.5 mu m, the thickness is 150 mu m, and the porosity is 75 percent;
(2) firstly, dissolving a catechol compound in a buffer solution (a buffer solution is specifically a Tris-HCl buffer solution) with the pH value of 8, preparing a catechol compound solution with the concentration of 0.1mg/ml, then adding a polyamine compound into the catechol compound solution, immediately soaking a polymer porous membrane in the mixed solution according to the mass-volume ratio of 0.5mg to 40ml after complete dissolution to obtain a mixed solution, and coating and modifying at 30 ℃ for 12 hours to obtain a polymer affinity membrane with efficient nucleic acid separation and purification functions; wherein the molar ratio of the catechol compound to the polyamine compound in the mixed solution is 3: 1.
The application of the polymer affinity membrane with the efficient nucleic acid separation and purification function prepared by the preparation method comprises the following specific steps:
(I) stacking 10 sheets of the polymer affinity membrane in a purification column;
(II) adding a mixed solution of a sample solution (the solvent is ultrapure water) of nucleic acid and an adsorption buffer solution (specifically a phosphate buffer solution) into the purification column, wherein the volume of the mixed solution is 80% of the capacity of the purification column, centrifuging and filtering for 120s at 4000g, and removing the solution in the collection tube; the pH value of the adsorption buffer solution is 4; the size of the nucleic acid molecule fragment in the sample solution of nucleic acid is 6.5kb, and the concentration is 50 ng/mul; the volume ratio of the sample solution of nucleic acid to the adsorption buffer solution is 1: 8;
(III) adding a washing buffer solution into the purification column, wherein the volume of the washing buffer solution is 90% of the capacity of the purification column, centrifuging and filtering for 120s at 4000g, and removing the solution in the collection pipe; the washing buffer solution is ultrapure water containing 80 wt% of ethanol, and the pH value of the washing buffer solution is 7;
(IV) adding a recovery buffer solution (specifically a glycine-sodium hydroxide buffer solution) into the purification column, wherein the volume of the recovery buffer solution is 8% of the capacity of the purification column, and centrifuging 4000g for 120s to obtain a purified nucleic acid solution; the recovery buffer had a pH of 10.
The recovery efficiency of nucleic acid was 85%.
Example 8
The preparation method of the polymer affinity membrane with the high-efficiency nucleic acid separation and purification function comprises the following specific steps:
(1) preparation of raw materials:
catechol compound: tannic acid;
polyamine compound: polyethylene polyamines;
polymer porous membrane: a polypropylene film; the average pore diameter of the polymer porous membrane is 4 mu m, the thickness is 1500 mu m, and the porosity is 60%;
(2) firstly, dissolving a catechol compound in a buffer solution (a buffer solution is specifically a Tris-HCl buffer solution) with the pH value of 7, preparing a catechol compound solution with the concentration of 0.2mg/ml, then adding a polyamine compound into the catechol compound solution, immediately soaking a polymer porous membrane in the mixed solution according to the mass-volume ratio of 1mg to 40ml after completely dissolving to obtain a mixed solution, and coating and modifying at 55 ℃ for 0.5h to obtain the polymer affinity membrane with the efficient nucleic acid separation and purification function; wherein the molar ratio of the catechol compound to the polyamine compound in the mixed solution is 1: 4.
The application of the polymer affinity membrane with the efficient nucleic acid separation and purification function prepared by the preparation method comprises the following specific steps:
(I) stacking 1 sheet of the polymer affinity membrane in a purification column;
(II) adding a mixed solution of a sample solution (the solvent is ultrapure water) of nucleic acid and an adsorption buffer solution (specifically a phosphate buffer solution) into the purification column, wherein the volume of the mixed solution is 90% of the capacity of the purification column, centrifuging and filtering for 160s at 4000g, and removing the solution in the collection tube; the pH value of the adsorption buffer solution is 4; the size of the nucleic acid molecule fragment in the sample solution of nucleic acid is 12kb, and the concentration is 5 ng/mul; the volume ratio of the sample solution of nucleic acid to the adsorption buffer solution is 1: 4;
(III) adding a washing buffer solution into the purification column, wherein the volume of the washing buffer solution is 90% of the capacity of the purification column, centrifuging and filtering for 160s at 4000g, and removing the solution in the collection pipe; the washing buffer solution is ultrapure water containing 80 wt% of ethanol, and the pH value of the washing buffer solution is 7;
(IV) adding a recovery buffer solution (specifically a glycine-sodium hydroxide buffer solution) into the purification column, wherein the volume of the recovery buffer solution is 10% of the capacity of the purification column, and performing centrifugal filtration for 5000g for 160s to obtain a purified nucleic acid solution; the recovery buffer had a pH of 12.
The recovery efficiency of nucleic acid was 88%.
Example 9
The preparation method of the polymer affinity membrane with the high-efficiency nucleic acid separation and purification function comprises the following specific steps:
(1) preparation of raw materials:
catechol compound: a catechin;
polyamine compounds: polyethylene polyamines;
polymer porous membrane: a polyethylene film; the average pore diameter of the polymer porous membrane is 5 mu m, the thickness is 600 mu m, and the porosity is 48 percent;
(2) firstly, dissolving a catechol compound in a buffer solution (a buffer solution is specifically a Tris-HCl buffer solution) with the pH value of 10, preparing a catechol compound solution with the concentration of 3mg/ml, then adding a polyamine compound into the catechol compound solution, immediately soaking a polymer porous membrane in the mixed solution according to the mass-volume ratio of 10mg:200ml after completely dissolving to obtain a mixed solution, and coating and modifying at 60 ℃ for 1h to obtain the polymer affinity membrane with the efficient nucleic acid separation and purification function; wherein the molar ratio of the catechol compound to the polyamine compound in the mixed solution is 4: 1.
The application of the polymer affinity membrane with the efficient nucleic acid separation and purification function prepared by the preparation method comprises the following specific steps:
(I) stacking 3 sheets of the polymer affinity membrane in a purification column;
(II) adding a mixed solution of a sample solution (the solvent is ultrapure water) of nucleic acid and an adsorption buffer solution (specifically a phosphate buffer solution) into the purification column, wherein the volume of the mixed solution is 90% of the capacity of the purification column, centrifuging and filtering for 160s at 4000g, and removing the solution in the collection tube; the pH value of the adsorption buffer solution is 4; the size of the nucleic acid molecule fragment in the sample solution of nucleic acid is 30kb, and the concentration is 150 ng/. mu.l; the volume ratio of the sample solution of nucleic acid to the adsorption buffer solution is 1: 4;
(III) adding washing buffer solution into the purification column, wherein the volume of the washing buffer solution is 90% of the capacity of the purification column, and carrying out 6000g centrifugal filtration for 120s to remove the solution in the collection tube; the washing buffer solution is ultrapure water containing 80 wt% of ethanol, and the pH value of the washing buffer solution is 7;
(IV) adding a recovery buffer solution (specifically a glycine-sodium hydroxide buffer solution) into the purification column, wherein the volume of the recovery buffer solution is 10% of the capacity of the purification column, and 6000g of the buffer solution is centrifugally filtered for 120s to obtain a purified nucleic acid solution; the recovery buffer had a pH of 10.
The recovery efficiency of nucleic acid was 86%.
Example 10
The preparation method of the polymer affinity membrane with the high-efficiency nucleic acid separation and purification function comprises the following specific steps:
(1) preparation of raw materials:
catechol compound: protocatechuic acid;
polyamine compounds: a polyethyleneimine;
polymer porous membrane: polymethyl methacrylate; the average pore diameter of the polymer porous membrane is 0.01 mu m, the thickness is 300 mu m, and the porosity is 45 percent;
(2) firstly dissolving a catechol compound in a buffer solution (a buffer solution is specifically a Tris-HCl buffer solution) with the pH value of 9 to prepare a catechol compound solution with the concentration of 5mg/ml, then adding a polyamine compound into the catechol compound solution, immediately soaking a polymer porous membrane in the mixed solution according to the mass-volume ratio of 5mg:120ml after completely dissolving to obtain a mixed solution, and coating and modifying at 25 ℃ for 8h to obtain the polymer affinity membrane with the efficient nucleic acid separation and purification function; wherein the molar ratio of the catechol compound to the polyamine compound in the mixed solution is 2: 1.
The application of the polymer affinity membrane with the efficient nucleic acid separation and purification function prepared by the preparation method comprises the following specific steps:
(I) stacking 6 sheets of the polymer affinity membrane in a purification column;
(II) adding a mixed solution of a sample solution (the solvent is ultrapure water) of nucleic acid and an adsorption buffer solution (specifically a phosphate buffer solution) into the purification column, wherein the volume of the mixed solution is 90% of the capacity of the purification column, centrifuging and filtering by 9000g for 30s, and removing the solution in the collection tube; the pH value of the adsorption buffer solution is 5; the size of the nucleic acid molecule fragment in the sample solution of the nucleic acid is 100bp, and the concentration is 150 ng/mul; the volume ratio of the sample solution of nucleic acid to the adsorption buffer solution is 1: 10;
(III) adding a washing buffer solution into the purification column, wherein the volume of the washing buffer solution is 90% of the capacity of the purification column, and removing the solution in the collection tube by 9000g of centrifugal filtration for 40 s; the washing buffer solution is ultrapure water containing 80 wt% of ethanol, and the pH value of the washing buffer solution is 7;
(IV) adding a recovery buffer solution (specifically a glycine-sodium hydroxide buffer solution) into the purification column, wherein the volume of the recovery buffer solution is 10% of the capacity of the purification column, and centrifuging and filtering 9000g for 30s to obtain a purified nucleic acid solution; the recovery buffer had a pH of 11.
The recovery efficiency of nucleic acid was 92%.
Example 11
The preparation method of the polymer affinity membrane with the high-efficiency nucleic acid separation and purification function comprises the following specific steps:
(1) preparation of raw materials:
catechol compound: gallic acid;
polyamine compounds: a polyethyleneimine;
polymer porous membrane: a polytetrafluoroethylene membrane; the average pore diameter of the polymer porous membrane is 3 mu m, the thickness is 700 mu m, and the porosity is 65%;
(2) firstly, dissolving a catechol compound in a buffer solution (a buffer solution is specifically a Tris-HCl buffer solution) with the pH value of 8, preparing a catechol compound solution with the concentration of 3mg/ml, then adding a polyamine compound into the catechol compound solution, immediately soaking a polymer porous membrane in the mixed solution according to the mass-volume ratio of 2mg to 50ml after completely dissolving to obtain a mixed solution, and coating and modifying at 30 ℃ for 6h to obtain a polymer affinity membrane with the efficient nucleic acid separation and purification function; wherein the molar ratio of the catechol compound to the polyamine compound in the mixed solution is 1: 1.
The application of the polymer affinity membrane with the efficient nucleic acid separation and purification function prepared by the preparation method comprises the following specific steps:
(I) stacking 3 sheets of the polymer affinity membrane in a purification column;
(II) adding a mixed solution of a sample solution (the solvent is ultrapure water) of nucleic acid and an adsorption buffer solution (specifically a phosphate buffer solution) into the purification column, wherein the volume of the mixed solution is 90% of the capacity of the purification column, centrifuging and filtering for 120s at 4000g, and removing the solution in the collection tube; the pH value of the adsorption buffer solution is 4; the size of the nucleic acid molecule fragment in the sample solution of nucleic acid is 13kb, and the concentration is 200 ng/. mu.l; the volume ratio of the sample solution of nucleic acid to the adsorption buffer solution is 1: 5;
(III) adding a washing buffer solution into the purification column, wherein the volume of the washing buffer solution is 90% of the capacity of the purification column, and removing the solution in the collection tube by centrifugal filtration for 120s at 5000 g; the washing buffer solution is ultrapure water containing 80 wt% of ethanol, and the pH value of the washing buffer solution is 7;
(IV) adding a recovery buffer solution (specifically a glycine-sodium hydroxide buffer solution) into the purification column, wherein the volume of the recovery buffer solution is 10% of the capacity of the purification column, and centrifuging 4000g and filtering 240s to obtain a purified nucleic acid solution; the recovery buffer had a pH of 12.
The recovery efficiency of nucleic acid was 87%.
Example 12
The preparation method of the polymer affinity membrane with the high-efficiency nucleic acid separation and purification function comprises the following specific steps:
(1) preparation of raw materials:
catechol compound: protocatechuic acid;
polyamine compounds: tetraethylenepentamine;
polymer porous membrane: a polyethersulfone membrane; the average pore diameter of the polymer porous membrane is 1 μm, the thickness is 75 μm, and the porosity is 74%;
(2) firstly dissolving a catechol compound in a buffer solution (a buffer solution is specifically a Tris-HCl buffer solution) with the pH value of 7, preparing a catechol compound solution with the concentration of 2mg/ml, then adding a polyamine compound into the catechol compound solution, immediately soaking a polymer porous membrane in the mixed solution according to the mass-volume ratio of 10mg:100ml after completely dissolving to obtain a mixed solution, and coating and modifying at 40 ℃ for 1h to obtain the polymer affinity membrane with the efficient nucleic acid separation and purification function; wherein the molar ratio of the catechol compound to the polyamine compound in the mixed solution is 1: 2.
The application of the polymer affinity membrane with the efficient nucleic acid separation and purification function prepared by the preparation method comprises the following specific steps:
(I) stacking 15 sheets of the polymer affinity membrane in a purification column;
(II) adding a mixed solution of a sample solution (the solvent is ultrapure water) of nucleic acid and an adsorption buffer solution (specifically a phosphate buffer solution) into the purification column, wherein the volume of the mixed solution is 80% of the capacity of the purification column, performing 3000g centrifugal filtration for 240s, and removing the solution in the collection tube; the pH value of the adsorption buffer solution is 5.5; the size of the nucleic acid molecule fragment in the sample solution of nucleic acid is 5.5kb, and the concentration is 15 ng/mul; the volume ratio of the sample solution of nucleic acid to the adsorption buffer solution is 1: 5;
(III) adding a washing buffer solution into the purification column, wherein the volume of the washing buffer solution is 90% of the capacity of the purification column, centrifuging and filtering 3000g for 240s, and removing the solution in the collection tube; the washing buffer solution is ultrapure water containing 80 wt% of ethanol, and the pH value of the washing buffer solution is 7;
(IV) adding a recovery buffer solution (specifically a glycine-sodium hydroxide buffer solution) into the purification column, wherein the volume of the recovery buffer solution is 2% of the capacity of the purification column, and centrifuging 3000g and filtering for 240s to obtain a purified nucleic acid solution; the recovery buffer had a pH of 8.
The recovery efficiency of nucleic acid was 90%.
Example 13
The preparation method of the polymer affinity membrane with the high-efficiency nucleic acid separation and purification function comprises the following specific steps:
(1) preparation of raw materials:
catechol compound: catechol;
polyamine compounds: pentaethylenehexamine;
polymer porous membrane: a polyvinylidene fluoride membrane; the average pore diameter of the polymer porous membrane is 1.2 mu m, the thickness is 120 mu m, and the porosity is 82%;
(2) firstly, dissolving a catechol compound in a buffer solution (a buffer solution is specifically a Tris-HCl buffer solution) with the pH value of 6, preparing a catechol compound solution with the concentration of 1mg/ml, then adding a polyamine compound into the catechol compound solution, immediately soaking a polymer porous membrane in the mixed solution according to the mass-volume ratio of 5mg to 100ml after completely dissolving to obtain a mixed solution, and coating and modifying at 30 ℃ for 3 hours to obtain a polymer affinity membrane with the efficient nucleic acid separation and purification function; wherein the molar ratio of the catechol compound to the polyamine compound in the mixed solution is 1: 3.
The application of the polymer affinity membrane with the efficient nucleic acid separation and purification function prepared by the preparation method comprises the following specific steps:
(I) stacking 12 sheets of the polymer affinity membrane in a purification column;
(II) adding a mixed solution of a sample solution (the solvent is ultrapure water) of nucleic acid and an adsorption buffer solution (specifically a phosphate buffer solution) into the purification column, wherein the volume of the mixed solution is 90% of the volume of the purification column, and removing the solution in the collection tube by 5000g centrifugal filtration for 160 s; the pH value of the adsorption buffer solution is 4.5; the size of the nucleic acid molecule fragment in the sample solution of nucleic acid is 6.0kb, and the concentration is 30 ng/mul; the volume ratio of the sample solution of nucleic acid to the adsorption buffer solution is 1: 6;
(III) adding a washing buffer solution into the purification column, wherein the volume of the washing buffer solution is 90% of the capacity of the purification column, performing centrifugal filtration for 160s at 5000g, and removing the solution in the collection tube; the washing buffer solution is ultrapure water containing 80 wt% of ethanol, and the pH value of the washing buffer solution is 7;
(IV) adding a recovery buffer solution (specifically a glycine-sodium hydroxide buffer solution) into the purification column, wherein the volume of the recovery buffer solution is 10% of the capacity of the purification column, and performing centrifugal filtration for 5000g for 160s to obtain a purified nucleic acid solution; the recovery buffer had a pH of 10.
The recovery efficiency of nucleic acid was 93%.
The preparation method of the polymer porous membrane in the invention is not limited, and the invention exemplarily provides a preparation method of the polymer porous membrane, which comprises the following specific processes: firstly, dissolving a polymer in a solvent to obtain a polymer solution, then adding a pore-forming agent into the polymer solution, stirring until the pore-forming agent is completely dissolved to obtain a membrane casting solution, and finally preparing the membrane casting solution into a polymer porous membrane according to a non-solvent induced phase separation method;
the solvent is an aprotic polar solvent, the polymer is a polymer soluble in the aprotic polar solvent, and the mass fraction of the polymer solution is 14-30%;
the aprotic polar solvent is DMAc, DMSO, NMP or DMF; the polymer soluble in the aprotic polar solvent is polyether sulfone, polysulfone, polyvinylidene fluoride, polyphenylsulfone, polyvinyl chloride or ethylene-vinyl alcohol copolymer;
the pore-foaming agent is polyvinylpyrrolidone, polyethylene glycol, polyoxyethylene and polyoxyethylene-polyoxypropylene copolymer, and the mass fraction of the pore-foaming agent in the membrane casting solution is 1-5%;
the coagulating bath in the non-solvent induced phase separation method is water or an aqueous solution containing an organic solvent;
reference will now be made to specific embodiments:
the procedure for preparing the polyethersulfone membrane of example 12 was: firstly, dissolving polyether sulfone in DMAc to prepare a polyether sulfone solution with the mass fraction of 14%, then adding pore-forming agent polyvinylpyrrolidone into the polyether sulfone solution, stirring until the pore-forming agent polyvinylpyrrolidone is completely dissolved to obtain a membrane casting solution A, wherein the pore-forming agent accounts for 2% of the mass fraction of the membrane casting solution, and finally preparing the membrane casting solution into the polymer porous membrane according to a non-solvent induced phase separation method.
The polyvinylidene fluoride membrane of example 13 was prepared by the following procedure: dissolving polyvinylidene fluoride in DMAc to prepare a polyvinylidene fluoride solution with the mass fraction of 13%, adding pore-forming agent polyvinylpyrrolidone into the polyvinylidene fluoride solution, stirring until the pore-forming agent polyvinylpyrrolidone is completely dissolved to obtain a membrane casting solution A, wherein the pore-forming agent accounts for 1% of the mass fraction of the membrane casting solution, and finally preparing the membrane casting solution into the polymer porous membrane according to a non-solvent induced phase separation method.
Claims (10)
1. The preparation method of the polymer affinity membrane with the efficient nucleic acid separation and purification function is characterized in that after a pyrocatechol compound and a polyamine compound are simultaneously applied to the surface of a polymer porous membrane, a Michael addition reaction and a Schiff base reaction are induced to occur between the pyrocatechol compound and the polyamine compound, so that the polymer affinity membrane with the efficient nucleic acid separation and purification function is obtained;
when nucleic acid is separated and purified by a solid phase adsorption method using a polymer affinity membrane having a high nucleic acid separation and purification function, the recovery efficiency of nucleic acid is 85% or more.
2. The method for preparing a polymer affinity membrane with high efficiency nucleic acid separation and purification function according to claim 1, which comprises the following steps: firstly, dissolving a catechol compound in a buffer solution with the pH value of 6.0-10.0 to prepare a catechol compound solution, then adding a polyamine compound into the catechol compound solution, completely dissolving to obtain a mixed solution, immediately soaking a polymer porous membrane in the mixed solution for co-coating modification to obtain the polymer affinity membrane with the efficient nucleic acid separation and purification function.
3. The method for preparing a polymer affinity membrane having a high efficiency of nucleic acid isolation and purification according to claim 2, wherein the catechol compound is catechol, gallic acid, tannic acid, protocatechuic acid, catechin, or quercetin; the polyamine compound is triethylene tetramine, tetraethylene pentamine, pentaethylene hexamine, hexaethylene heptamine, polyethylene polyamine or polyethylene imine.
4. The method for preparing a polymer affinity membrane with high efficiency nucleic acid separation and purification function according to claim 2, wherein the concentration of the catechol compound solution is 0.1-5 mg/ml.
5. The method for preparing the polymer affinity membrane with the efficient nucleic acid separation and purification function according to claim 2, wherein the molar ratio of the catechol compound to the polyamine compound in the mixed solution is 1: 4-4: 1.
6. The method for preparing a polymer affinity membrane with high-efficiency nucleic acid separation and purification functions according to claim 2, wherein the average pore diameter of the polymer porous membrane is 0.01-5 μm, the thickness is 20-2000 μm, and the porosity is 40-85%; the mass volume ratio of the polymer porous membrane to the mixed solution is 0.1-10 mg: 20-100 ml.
7. The method for preparing a polymer affinity membrane with high-efficiency nucleic acid separation and purification functions according to claim 2, wherein the co-coating modification time is 0.1-12 h, and the temperature is 20-60 ℃.
8. The application of the polymer affinity membrane with the high-efficiency nucleic acid separation and purification function prepared by the preparation method of any one of claims 1 to 7 is characterized by comprising the following steps:
(1) stacking 1-15 pieces of polymer affinity membranes in a purification column;
(2) adding a mixed solution of a sample solution containing nucleic acid and an adsorption buffer solution into the purification column, wherein the volume of the mixed solution is 30-100% of the capacity of the purification column, and removing the solution in the collection pipe by 3000-9000 g of centrifugal filtration for 20-240 s; the pH value of the adsorption buffer solution is 4.0-6.0;
(3) adding a washing buffer solution into the purification column, wherein the volume of the washing buffer solution is 30-100% of the capacity of the purification column, centrifuging and filtering 3000-9000 g for 20-240 s, and removing the solution in the collection pipe; the washing buffer is a solution containing a water-soluble organic solvent;
(4) adding a recovery buffer solution into the purification column, wherein the volume of the recovery buffer solution is 2-10% of the capacity of the purification column, and performing centrifugal filtration for 3000-9000 g for 20-240 s to obtain a purified nucleic acid solution; the pH value of the recovered buffer solution is 7.5-12.0 and is higher than that of the washing buffer solution.
9. The use according to claim 8, wherein in the step (2), the nucleic acid molecule fragment in the sample solution containing nucleic acid has a size of 100 bp-30 kp and a concentration of 1-200 ng/μ l; the volume ratio of the sample solution containing nucleic acid to the adsorption buffer solution is 1: 1-1: 10.
10. The use according to claim 8, wherein in step (3), the water-soluble organic solvent is a C1-C3 alcohol.
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