CN113373141A

CN113373141A - Extraction method of free DNA

Info

Publication number: CN113373141A
Application number: CN202110834748.1A
Authority: CN
Inventors: 郑建萍; 钱四化
Original assignee: Ningbo Institute of Material Technology and Engineering of CAS
Current assignee: Ningbo Institute of Material Technology and Engineering of CAS
Priority date: 2021-07-23
Filing date: 2021-07-23
Publication date: 2021-09-10

Abstract

The invention discloses a method for extracting free DNA. The extraction method comprises the following steps: mixing a sample to be processed, a lysate and magnetic beads for cracking treatment to obtain a cracked sample, wherein the lysate comprises guanidine isothiocyanate, polyoxyethylene ether Brij58, NaAc-HAc buffer solution, isopropanol and water; washing the cracked sample by using a washing solution and an ethanol solution to obtain a washed sample, wherein the washing solution comprises guanidinium isothiocyanate and a Tris-HCl buffer solution; and mixing the washed sample with an eluent to carry out elution treatment to obtain free DNA, wherein the eluent comprises TE buffer. The method can extract free DNA through 3 steps of cracking, washing and eluting, is more efficient and convenient, and meanwhile, the lysate in the method can well destroy proteins and form a high-salt environment, so that the free DNA is better combined with magnetic beads.

Description

Extraction method of free DNA

Technical Field

The invention belongs to the technical field of extraction and separation, and particularly relates to a method for extracting free DNA.

Background

Mandel et al first reported in 1948 the presence of free DNA (cfDNA) in plasma, which is an extracellular DNA, consisting mainly of single-or double-stranded DNA and a mixture of single-and double-stranded DNA. cfDNA exists in the peripheral blood of both healthy people and tumor patients, and the content of cfDNA in the peripheral blood of tumor patients is more than 10 times that of the cfDNA of healthy people. Clinical studies show that cfDNA can effectively reflect the overall tumor load, malignancy degree, metastatic capacity and real-time gene mutation information of patients, and has a certain correlation with the gene information of tumor tissues. The plasma cfDNA sequencing is used for detecting gene copy number change, methylation change, nucleotide mutation change, chromosome rearrangement change and the like related to tumors, and can be used for clinical diagnosis, medication guidance, detection of tumorigenesis and the like. Therefore, extracting high-quality cfDNA is a key for performing accurate analysis on the cfDNA, and the sensitivity and specificity of the extraction method are directly related to success or failure of subsequent experiments.

The nucleic acid extraction technology has undergone the processes from the 1 st generation chemical method to the 2 nd generation adsorption column method, and the 3 rd generation magnetic bead method developed in recent years, and the extraction technology and efficiency thereof are continuously improved and optimized. The current nucleic acid isolation and purification requires four important steps: disruption and lysis of tissue or cells, denaturation of nuclear protein complexes, inactivation of nucleases and removal of contaminants. How to provide a simple, rapid, and efficient method for separating cfDNA is a considerable problem to be studied.

Disclosure of Invention

The main object of the present invention is to provide a solution to the drawbacks of the prior art.

In order to achieve the purpose, the technical scheme adopted by the invention comprises the following steps:

the embodiment of the invention provides a method for extracting free DNA, which comprises the following steps:

(1) mixing a sample to be processed, a lysate and magnetic beads for cracking treatment to obtain a cracked sample, wherein the lysate comprises guanidine isothiocyanate, polyoxyethylene ether Brij58, NaAc-HAc buffer solution, isopropanol and water;

(2) washing the sample cracked in the step (1) by using a washing solution and an ethanol solution to obtain a washed sample, wherein the washing solution comprises guanidinium isothiocyanate and a Tris-HCl buffer solution;

(3) and (3) mixing the washed sample obtained in the step (2) with an eluent to carry out elution treatment, so as to obtain free DNA, wherein the eluent comprises TE buffer solution.

Further, the TE buffer has a pH of 10.

Compared with the prior art, the invention has the beneficial effects that: the method can extract free DNA through 3 steps of cracking, washing and eluting, is more efficient and convenient, and meanwhile, the lysate in the method can well destroy proteins and form a high-salt environment, so that the free DNA is better combined with magnetic beads.

Drawings

In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings used in the description of the embodiments or the prior art will be briefly described below, it is obvious that the drawings in the following description are only some embodiments described in the present invention, and for those skilled in the art, other drawings can be obtained according to the drawings without creative efforts.

FIG. 1 is a graph showing the results of molecular weight measurement of free DNA prepared in example 1 of the present invention.

Detailed Description

In view of the defects of the prior art, the inventor of the present invention has long studied and largely practiced to propose the technical solution of the present invention, which will be clearly and completely described below, and it is obvious that the described embodiments are a part of the embodiments of the present invention, but not all of the embodiments. All other embodiments, which can be derived by a person skilled in the art from the embodiments given herein without making any creative effort, shall fall within the protection scope of the present invention.

One aspect of the embodiments of the present invention provides a method for extracting free DNA, which includes:

In some specific embodiments, the volume ratio of the sample, the lysis solution and the magnetic beads is 100-200: 150-250: 0.5-3.

Furthermore, the concentration of the magnetic beads in the solution containing the magnetic beads is 10-100 mg/mL.

Further, the extraction method further comprises the following steps: and (2) centrifuging the cracked sample obtained in the step (1) for 1-2 min at the rotating speed of 1000-3000 rpm, and then separating under the action of a magnetic field.

In some more specific embodiments, the method of preparing the lysate comprises: guanidine isothiocyanate, polyoxyethylene ether Brij58 and NaAc-HAc buffer solution were mixed, and then water and isopropanol were added to obtain the lysate.

Further, the concentration of the NaAc-HAc buffer solution is 50-200 mmol/L.

Further, the pH value of the NaAc-HAc buffer solution is 4.0-6.0.

Furthermore, the volume content of isopropanol in the lysate is 10-50%.

Further, the volume ratio of the NaAc-HAc buffer solution to the isopropanol to the water is 1-10: 1-5.

In some more specific embodiments, the concentration of guanidinium isothiocyanate in the lysis solution is 5.0-6.0 mol/L.

Furthermore, the concentration of polyoxyethylene ether Brij58 in the lysis solution is 0.1-1.0 mmol/L.

In some more specific embodiments, step (2) specifically includes:

washing the cracked sample obtained in the step (1) by using a washing solution, and then separating under the action of a magnetic field;

and washing the mixed solution obtained by the separation treatment by using an ethanol solution, and then performing the separation treatment under the action of a magnetic field to obtain the washed sample.

In some more specific embodiments, the concentration of guanidinium isothiocyanate in the wash solution is 5.0-6.0 mol/L.

Furthermore, the concentration of the Tris-HCl buffer solution is 0.1-1 mol/L.

Further, the pH value of the Tris-HCl buffer solution is 4.0-8.0.

Further, the volume content of ethanol in the ethanol solution is 50-80%.

In some more specific embodiments, step (3) specifically includes: and (3) mixing the washed sample obtained in the step (2) with an eluent, oscillating, centrifuging, and separating under the action of a magnetic field to obtain the free DNA.

Further, the oscillation treatment time is 5-10 min.

In some more specific embodiments, the TE buffer has a pH of 10.

Further, the concentration of the TE buffer solution is 40-80 mmol/L.

Further, the sample includes plasma or serum, and is not limited thereto.

In some more specific embodiments, the method for extracting free DNA specifically comprises:

(1) taking a sample to be detected to a centrifuge tube, adding lysis solution and magnetic beads, wherein the volume ratio of the sample to be detected to the lysis solution to the magnetic beads is 100-200: 150-250: 0.5-3, uniformly mixing, and avoiding bubbles to generate as much as possible to obtain a mixed solution A;

(2) centrifuging the mixed solution A for 1-2 min at the rotating speed of 1000-3000 rpm, standing on a magnetic frame for 5-10 min until the solution is clear, and sucking the supernatant;

(3) taking the sample treated in the step (2) down from the magnetic frame, adding 1-10 mL of washing liquid, and mixing uniformly to avoid bubbles as much as possible to obtain a mixed liquid B;

(4) placing the mixed solution B on a magnetic frame, standing until the solution is clear, and sucking the supernatant;

(5) taking the sample treated in the step (4) down from the magnetic frame, adding 1-10 mL of 50% -80% ethanol, and mixing uniformly to obtain a mixture C;

(6) placing the mixed solution C on a magnetic frame, standing until the solution is clear, sucking the supernatant, opening a centrifugal tube cover, and air-drying the magnetic beads;

(7) taking the sample treated in the step (6) down from the magnetic frame, adding 10-50 mu L of eluent, and uniformly mixing to obtain a mixture D;

(8) and fully shaking the mixture D for 5-10 min, centrifuging, placing on a magnetic frame, standing until the solution is clear, and transferring the supernatant into a new centrifugal tube to obtain free DNA.

Preferably, the method for preparing the lysate in step (1) comprises: adding 1-5 mL of NaAc-HAc (pH value of 4.0-6.0) with concentration of 50-200 mmol/L into guanidinium isothiocyanate and polyoxyethylene ether Brij58, adding deionized water to 10mL, and adding 10-50% of isopropanol to obtain the product; wherein the concentration of the guanidinium isothiocyanate is 5.0-6.0 mol/L, and the concentration of the polyoxyethylene ether Brij58 is 0.1-1 mmol/L.

Preferably, the preparation method of the washing solution in the step (3) comprises the following steps: dissolving 5.0-6.0 mol/L guanidinium isothiocyanate in 0.1-1.0 mol/L LTris-HCl (pH value is 5.0-8.0) to obtain the guanidine isothiocyanate.

Preferably, the method for preparing the eluent in the step (7) comprises the following steps: 40-80 mmol/L TE buffer (pH 10).

The technical solution of the present invention is further described in detail with reference to several preferred embodiments, which are implemented on the premise of the technical solution of the present invention, and detailed embodiments and specific operation procedures are given, but the scope of the present invention is not limited to the following embodiments.

The experimental materials used in the examples used below were all available from conventional biochemical reagents companies, unless otherwise specified.

Example 1

(1) Putting 2mL of plasma into an 10/15mL centrifuge tube, adding 2.5mL of lysis solution (wherein the lysis solution comprises 5.0mol/L guanidine isothiocyanate, 0.5mmol/L polyoxyethylene ether Brij58, 100mmol/LpH NaAc-HAc buffer solution with the value of 5.0, 4mL of isopropanol and water) and 30 mu L of magnetic beads which are uniformly mixed and have the concentration of 40mg/mL, and uniformly mixing for 10min at room temperature to avoid the generation of bubbles as much as possible; then centrifuging at 1000rpm for 1-2 min; then, the solution is placed on a magnetic frame for 5min or until the solution is clarified, and the clarified solution is sucked off.

(2) Taking down the centrifugal tube from the magnetic frame, adding 1mL of washing solution (wherein the washing solution comprises 5.0mol/L guanidinium isothiocyanate and 0.1mol/L Tris-HCl buffer solution with the pH value of 6.0), and absorbing for 10 times by using a pipette gun to uniformly mix, so as to avoid the generation of bubbles as much as possible; then transferring to a new 1.5mL centrifuge tube, standing for 2min on a magnetic frame or until the solution is clear, and sucking clear liquid; and repeating the steps once.

(3) Taking the centrifugal tube off the magnetic frame, adding 1mL of 80% ethanol, shaking for 30s, centrifuging, placing the centrifugal tube on the magnetic frame for 2min or until the solution is clarified, removing the clarified solution by suction, and repeating the steps once; and opening the centrifugal tube cover, and air-drying the magnetic beads for 2-5 min.

(4) And (3) taking the centrifuge tube off the magnetic frame, adding 15 mu L of eluent (TE buffer solution with the value of 50mmol/LpH of 10.0), shaking for 5min, then simply centrifuging, placing the centrifuge tube on the magnetic frame for 2min or until the liquid is clear, and then sucking the clear liquid to a new centrifuge tube with the volume of 1.5mL to obtain free DNA.

The results of the measurement of the molecular weight of the free DNA in this example are shown in FIG. 1, and the results of the measurement of the concentration of the free DNA in this example are shown in Table 1.

Example 2

(1) Putting 2mL of plasma into an 10/15mL centrifuge tube, adding 2.5mL of lysis solution (wherein the lysis solution comprises 5.5mol/L guanidine isothiocyanate, 1.0mmol/L polyoxyethylene ether Brij58, 200mmol/L NaAc-HAc buffer solution with the pH value of 4.0, 4mL of isopropanol and water) and 30 mu L of magnetic beads which are uniformly mixed and have the concentration of 100mg/mL, and uniformly mixing for 10min at room temperature to avoid the generation of bubbles as much as possible; then centrifuging at 1000rpm for 1-2 min; then, the solution is placed on a magnetic frame for 5min or until the solution is clarified, and the clarified solution is sucked off.

(2) Taking down the centrifugal tube from the magnetic frame, adding 1mL of washing solution (wherein the washing solution comprises 6.0mol/L guanidinium isothiocyanate and 1.0mol/LpH value of Tris-HCl buffer solution of 8.0), and sucking back and forth for 10 times by using a pipette gun to uniformly mix, so as to avoid the generation of bubbles as much as possible; then transferring to a new 1.5mL centrifuge tube, standing for 2min on a magnetic frame or until the solution is clear, and sucking clear liquid; and repeating the steps once.

(3) Taking the centrifugal tube off the magnetic frame, adding 1mL of 60% ethanol, shaking for 30s, centrifuging, placing the centrifugal tube on the magnetic frame for 2min or until the solution is clarified, removing the clarified solution by suction, and repeating the steps once; and opening the centrifugal tube cover, and air-drying the magnetic beads for 2-5 min.

(4) Taking the centrifuge tube off the magnetic frame, adding 15 μ L of eluent (80mmol/L TE buffer solution with pH value of 10.0), shaking for 5min, centrifuging simply, placing the centrifuge tube on the magnetic frame for 2min or until the liquid is clear, and then sucking clear liquid into a new centrifuge tube of 1.5mL to obtain free DNA. The results of measuring the free DNA concentration in this example are shown in Table 1.

Example 3

(1) Putting 2mL of plasma into an 10/15mL centrifuge tube, adding 2.5mL of lysis solution (wherein the lysis solution comprises 5.5mol/L guanidine isothiocyanate, 0.1mmol/L polyoxyethylene ether Brij58, 100mmol/L NaAc-HAc buffer solution with the pH value of 5.0, 4mL of isopropanol and water) and 30 mu L of magnetic beads which are uniformly mixed and have the concentration of 40mg/mL, and uniformly mixing for 10min at room temperature to avoid the generation of bubbles as much as possible; then centrifuging at 1000rpm for 1-2 min; then, the solution is placed on a magnetic frame for 5min or until the solution is clarified, and the clarified solution is sucked off.

(2) Taking down the centrifugal tube from the magnetic frame, adding 1mL of washing solution (wherein the washing solution comprises 5.5mol/L guanidine isothiocyanate and 1.0mol/LpH value of 6.0 Tris-HCl buffer solution), and absorbing for 10 times by using a pipette gun to uniformly mix, so as to avoid the generation of bubbles as much as possible; then transferring to a new 1.5mL centrifuge tube, standing for 2min on a magnetic frame or until the solution is clear, and sucking clear liquid; and repeating the steps once.

(4) And (3) taking the centrifuge tube off the magnetic frame, adding 15 mu L of eluent (TE buffer solution with the value of 50mmol/LpH of 10.0), shaking for 5min, then simply centrifuging, placing the centrifuge tube on the magnetic frame for 2min or until the liquid is clear, and then sucking the clear liquid to a new centrifuge tube with the volume of 1.5mL to obtain free DNA. The results of measuring the free DNA concentration in this example are shown in Table 1.

Example 4

(1) Putting 2mL of plasma into an 10/15mL centrifuge tube, adding 2.5mL of lysis solution (wherein the lysis solution comprises 6.0mol/L guanidine isothiocyanate, 1.0mmol/L polyoxyethylene ether Brij58, 100mmol/L NaAc-HAc buffer solution with the pH value of 6.0, 4mL of isopropanol and water) and 30 mu L of magnetic beads which are uniformly mixed and have the concentration of 40mg/mL, and uniformly mixing for 10min at room temperature to avoid the generation of bubbles as much as possible; then centrifuging at 1000rpm for 1-2 min; then, the solution is placed on a magnetic frame for 5min or until the solution is clarified, and the clarified solution is sucked off.

(2) Taking down the centrifugal tube from the magnetic frame, adding 1mL of washing solution (wherein the washing solution comprises 6.0mol/L guanidinium isothiocyanate and 0.1mol/L Tris-HCl buffer solution with the pH value of 6.0), and absorbing for 10 times by using a pipette gun to uniformly mix, so as to avoid the generation of bubbles as much as possible; then transferring to a new 1.5mL centrifuge tube, standing for 2min on a magnetic frame or until the solution is clear, and sucking clear liquid; and repeating the steps once.

Comparative example 1

The method is the same as example 1, except that: the eluent used has a pH of 9;

(1) putting 2mL of plasma into an 10/15mL centrifuge tube, adding 2.5mL of lysis solution (wherein the lysis solution comprises 5.0mol/L guanidine isothiocyanate, 0.5mmol/L polyoxyethylene ether Brij58, 100mmol/L NaAc-HAc buffer solution with the pH value of 5.0, 4mL of isopropanol and water) and 30 mu L of magnetic beads which are uniformly mixed and have the concentration of 40mg/mL, and uniformly mixing for 10min at room temperature to avoid the generation of bubbles as much as possible; then centrifuging at 1000rpm for 1-2 min; then, the solution is placed on a magnetic frame for 5min or until the solution is clarified, and the clarified solution is sucked off.

(4) And (3) taking the centrifuge tube off the magnetic frame, adding 15 mu L of eluent (TE buffer solution with the value of 50mmol/LpH of 9.0), shaking for 5min, then simply centrifuging, placing the centrifuge tube on the magnetic frame for 2min or until the liquid is clear, and then sucking the clear liquid to a new centrifuge tube with the volume of 1.5mL to obtain free DNA. The results of measuring the concentration of free DNA in this comparative example are shown in Table 1.

Comparative example 2

The method is the same as example 1, except that: the eluent used has a pH of 11;

(4) And (3) taking the centrifuge tube off the magnetic frame, adding 15 mu L of eluent (TE buffer solution with the value of 50mmol/LpH of 11.0), shaking for 5min, then simply centrifuging, placing the centrifuge tube on the magnetic frame for 2min or until the liquid is clear, and then sucking the clear liquid to a new centrifuge tube with the volume of 1.5mL to obtain free DNA. The results of measuring the concentration of free DNA in this comparative example are shown in Table 1.

Comparative example 3

The method is the same as example 1, except that: the lysate lacks polyoxyethylene ether Brij 5;

(1) putting 2mL of plasma into an 10/15mL centrifuge tube, adding 2.5mL of lysis solution (wherein the lysis solution comprises 5.0mol/L guanidine isothiocyanate, 100mmol/L NaAc-HAc buffer solution with the pH value of 5.0, 4mL of isopropanol and water) and 30 mu L of 40mg/mL magnetic beads which are uniformly mixed, and uniformly mixing for 10min at room temperature to avoid the generation of bubbles as much as possible; then centrifuging at 1000rpm for 1-2 min; then, the solution is placed on a magnetic frame for 5min or until the solution is clarified, and the clarified solution is sucked off.

(4) And (3) taking the centrifuge tube off the magnetic frame, adding 15 mu L of eluent (TE buffer solution with the value of 50mmol/LpH of 10.0), shaking for 5min, then simply centrifuging, placing the centrifuge tube on the magnetic frame for 2min or until the liquid is clear, and then sucking the clear liquid to a new centrifuge tube with the volume of 1.5mL to obtain free DNA. The results of measuring the concentration of free DNA in this comparative example are shown in Table 1.

Comparative example 4

The method is the same as example 1, except that: the polyoxyethylene ether Brij5 in the lysate is replaced by SDS.

(1) Putting 2mL of plasma into an 10/15mL centrifuge tube, adding 2.5mL of lysis solution (wherein the lysis solution comprises 5.0mol/L guanidine isothiocyanate, 0.5mmol/L LSDS, 100mmol/LpH NaAc-HAc buffer solution with the value of 5.0, 4mL of isopropanol and water) and 30 μ L of 40mg/mL magnetic beads which are uniformly mixed, and uniformly mixing for 10min at room temperature to avoid the generation of bubbles as much as possible; then centrifuging at 1000rpm for 1-2 min; then, the solution is placed on a magnetic frame for 5min or until the solution is clarified, and the clarified solution is sucked off.

TABLE 1 results of determination of free DNA concentration in examples 1 to 4 and comparative examples 1 to 4

Name (R)	Concentration (ng/. mu.L)
		Example 1	3.35
Example 2	3.05
		Example 3	3.17
Example 4	3.29
		Comparative example 1	2.16
Comparative example 2	1.99
		Comparative example 3	1.11
Comparative example 4	1.02

In addition, the inventors of the present invention have also made experiments with other materials, process operations, and process conditions described in the present specification with reference to the above examples, and have obtained preferable results.

It should be understood that the technical solution of the present invention is not limited to the above-mentioned specific embodiments, and all technical modifications made according to the technical solution of the present invention fall within the protection scope of the present invention without departing from the spirit of the present invention and the protection scope of the claims.

Claims

1. A method for extracting free DNA, comprising:

2. The extraction method according to claim 1, characterized in that: the volume ratio of the sample to the lysis solution to the magnetic beads is 100-200: 150-250: 0.5-3:

and/or, the extraction method further comprises the following steps: and (2) centrifuging the cracked sample obtained in the step (1) for 1-2 min at the rotating speed of 1000-3000 rpm, and then separating under the action of a magnetic field.

3. The extraction method according to claim 1, wherein the preparation method of the lysate comprises: mixing guanidinium isothiocyanate, polyoxyethylene ether Brij58 and NaAc-HAc buffer solution, and then adding water and isopropanol to obtain lysate;

preferably, the concentration of the NaAc-HAc buffer solution is 50-200 mmol/L; preferably, the pH value of the NaAc-HAc buffer solution is 4.0-6.0; preferably, the volume content of isopropanol in the lysate is 10-50%; preferably, the volume ratio of the NaAc-HAc buffer solution to the isopropanol to the water is 1-10: 1-5.

4. The extraction method according to claim 1, characterized in that: the concentration of the guanidinium isothiocyanate in the lysate is 5.0-6.0 mol/L;

and/or the concentration of polyoxyethylene ether Brij58 in the lysis solution is 0.1-1.0 mmol/L.

5. The extraction method according to claim 1, wherein the step (2) specifically comprises:

6. The extraction method according to claim 1, characterized in that: the concentration of the guanidinium isothiocyanate in the washing liquid is 5.0-6.0 mol/L;

and/or the concentration of the Tris-HCl buffer solution is 0.1-1 mol/L;

and/or the pH value of the Tris-HCl buffer solution is 4.0-8.0.

7. The extraction method according to claim 1, wherein the step (3) specifically comprises:

and (3) mixing the washed sample obtained in the step (2) with an eluent, oscillating, centrifuging, and separating under the action of a magnetic field to obtain the free DNA.

8. The extraction method according to claim 7, characterized in that: the time of the oscillation treatment is 5-10 min.

9. The extraction method according to claim 1, characterized in that: the pH value of the TE buffer solution is 10;

and/or the concentration of the TE buffer solution is 40-80 mmol/L.

10. The extraction method according to claim 1, characterized in that: the sample to be treated comprises plasma and/or serum.