CN112391382B

CN112391382B - Method for rapidly extracting vesicle DNA

Info

Publication number: CN112391382B
Application number: CN202011430341.4A
Authority: CN
Inventors: 杨帆; 陈海伦; 俞飞
Original assignee: Hubei Soundny Bio Tech Co ltd
Current assignee: Hubei Soundny Bio Tech Co ltd
Priority date: 2020-12-07
Filing date: 2020-12-07
Publication date: 2022-10-11
Anticipated expiration: 2040-12-07
Also published as: CN112391382A

Abstract

The invention provides a method for rapidly extracting vesicle DNA, and belongs to the technical field of nucleic acid extraction. The method comprises the following steps: s1, placing the vesicle suspension in a water bath at a temperature of 80-100 ℃, and then carrying out ultrasonic disruption under the power of 90-110W to obtain a broken cell suspension; s2, adding chloroform into the cell disruption suspension, uniformly mixing, standing for a period of time, and centrifuging to obtain a supernatant to obtain vesicle DNA. The whole operation process is simple, the steps are fewer, the required time is less than 30min, the extraction flow of the free DNA in the vesicle is greatly simplified, and the extraction efficiency and the extraction purity of the free DNA in the vesicle are improved.

Description

Method for rapidly extracting vesicle DNA

Technical Field

The invention relates to the technical field of nucleic acid extraction, in particular to a method for quickly extracting vesicle DNA.

Background

Cytoskeleton is an important structure by which eukaryotic cells maintain their basic morphology, and cells, after being stimulated exogenously or endogenously, cause rearrangement of the cytoskeleton, resulting in uneven local stress on the cell membrane. The cytoplasm with abnormal stress expands outwards, and then is released to the outside of the cell in the form of vesicle after randomly wrapping part of the cell content, and the special subcellular structure with the diameter of about 0.1-1 μm is called as a 'cell vesicle'. Chinese patent CN102302784B and its series of patents disclose methods of using ultraviolet radiation to irradiate tumor cells or using chemotherapy drugs to co-incubate with tumor cells to induce tumor cell apoptosis, thereby obtaining cell vesicles. The cell vesicle prepared by the method carries bioactive molecules of the original tumor cells, such as DNA, RNA and protein, shows molecular biological imprinting of the original tumor cells, and can effectively activate immune response of organisms. The research shows that the DNA in the vesicle is from the endogenous DNA of the organism which is free in the cell and is partially degraded, the length of the fragment is far smaller than that of the genome DNA, the fragment is concentrated between 0.18 kb and 21kb, and the genetic information carried by the DNA is consistent with the DNA of the tumor cell to a certain degree, thereby having important significance for researching the occurrence and development, early diagnosis, prognosis and detection of the disease. The obtaining of vesicle DNA with stable properties and high purity is a prerequisite for the development of the above-mentioned research work.

At present, when extracting vesicle DNA, reference is generally made to the step of extraction of exosome contents. However, the existing exosome DNA extraction step is complicated in operation, a sample to be extracted with DNA needs to be firstly subjected to cleavage digestion under the action of lysis solution (Buffer ATL) and protease to release the DNA into the lysis solution, then the Buffer AL and ethanol are added, and the sample is transferred to a column and filtered, so that the DNA is adsorbed on a membrane of the column, and the protein is not adsorbed and is removed with the filtering of the solution. The column was washed with Buffer GW1 for proteins and other impurities, with Buffer GW2 for desalting, and finally the DNA was eluted with a low salt Buffer (Buffer AE). The time required for completing the whole operation is more than 1 hour, the extraction efficiency is low, and the method is not suitable for large-scale extraction of vesicle DNA.

Disclosure of Invention

Aiming at the problem of low vesicle DNA extraction efficiency in the prior art, the invention provides a method for rapidly extracting vesicle DNA.

In order to achieve the purpose, the invention is realized by the following technology:

a method for rapidly extracting vesicle DNA comprises the following steps:

s1, placing the vesicle suspension in a water bath at a temperature of 80-100 ℃, and then carrying out ultrasonic disruption under the power of 90-110W to obtain a broken cell suspension;

s2, adding chloroform into the cell disruption suspension, uniformly mixing, standing for a period of time, and centrifuging to obtain supernatant to obtain vesicle DNA.

Further, in step S1, ultrasonication was performed at a power of 95 to 105W.

Further, in step S1, the operation mode of the ultrasonic fragmentation selects an intermittent mode, the intermittent mode is 1-5S for operation and 1-5S for intermission, and the duty ratio is 50%.

Further, in the step S1, the time of the ultrasonic disruption is 2-10min.

Further, in the step S1, the water bath time is 2-10min.

Further, in step S1, the vesicle suspension is subjected to a water bath at a temperature of 90 ℃.

Further, in step S1, the concentration of vesicles in the vesicle suspension is 3 × 10 ¹⁰ -6×10 ¹⁰ One per mL.

Further, the vesicle suspension is produced following apoptosis of tumor cells.

Further, in step S2, the volume ratio of the cell disruption suspension to the chloroform is 1.5-5:1.

further, in step S2, the centrifugation conditions are: centrifuge at 12000g for 10min.

Has the advantages that:

1. the invention firstly adopts higher temperature water bath to promote cell membrane denaturation, so that cell membranes are initially ruptured, then vesicles are further broken by using lower-power ultrasonic waves, the vesicle breaking rate is improved, contents are released to the maximum extent, and then chloroform is used for extracting and removing the substances, and cell membrane fragments are removed by centrifugal separation, so that the DNA with higher purity can be obtained. The whole operation process is simple, the steps are fewer, the required time is less than about 30min, the extraction process of the free DNA in the vesicle is greatly simplified, and the extraction efficiency and the extraction purity of the free DNA in the vesicle are improved.

2. The invention does not need to use expensive cracking reagents which are possibly limited in supply, and the water bath kettle and the ultrasonic crusher are common instruments in a laboratory, so the operation is simple, the cost is low and the efficiency is high.

Drawings

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

FIG. 1 is a PCR amplification assay of vesicle DNA of example 4 of the present invention;

FIG. 2 is a PCR amplification assay of vesicle DNA of example 5 of the present invention.

Detailed Description

It should be noted that the embodiments and features of the embodiments may be combined with each other without conflict. In addition, the terms "comprising", "containing", "having" and "having" are intended to be non-limiting, i.e., that other steps and other ingredients can be added which do not affect the results. Materials, equipment and reagents are commercially available unless otherwise specified.

For a better understanding of the invention, and not as a limitation on the scope thereof, all numbers expressing quantities, volume ratios, and other numerical values used in the present invention are to be understood as being modified in all instances by the term "about". Accordingly, unless expressly indicated otherwise, the numerical parameters set forth in the specification and attached claims are approximations that may vary depending upon the desired properties sought to be obtained. At the very least, each numerical parameter should at least be construed in light of the number of reported significant digits and by applying ordinary rounding techniques.

In order to make the aforementioned objects, features and advantages of the present invention comprehensible, embodiments accompanied with figures are described in detail below.

The invention provides a method for rapidly extracting vesicle DNA, which comprises the following steps:

s2, adding chloroform into the cell disruption suspension, uniformly mixing, standing for a period of time, and centrifuging to obtain a supernatant to obtain vesicle DNA.

Wherein, in step S1, the vesicle suspension is generated after tumor cell apoptosis. The method for inducing the tumor cell apoptosis is to irradiate the tumor cell by using ultraviolet rays or to incubate the tumor cell by using chemotherapeutic drugs.

The vesicle obtained by the method for inducing apoptosis is extracellular particulate matter with small particle size, is formed by directly turning over and secreting a tumor cell membrane, DNA contained in the vesicle is basically a free nucleic acid segment in cytoplasm, and the size and the source of the segment are random.

Firstly, a higher-temperature water bath is adopted to promote the denaturation of cell membranes and partially decompose components such as lipoid, mannan, glucan and the like, so that the cell membranes are initially broken, and a plurality of enzymes for decomposing DNA contained in vesicles can be denatured at high temperature to inhibit the activity of the enzymes and even denature and inactivate the enzymes, so that the DNA is prevented from being degraded in the extraction process, more complete DNA molecules can be obtained, and the effect of protecting the DNA molecules is achieved. Secondly, the vesicle is further processed by ultrasonic waves with lower power, the mechanical effect of the ultrasonic waves is utilized to cause the damage of the cell membrane structure, the cell membrane damage of the vesicle is promoted, the vesicle breaking rate is improved, and the content is released as much as possible. Then removing impurities such as protein and the like through chloroform extraction, and then removing cell membrane fragments, inclusion bodies and the like through centrifugal separation to obtain DNA with higher purity. The whole operation process is simple, the steps are fewer, the required time is less than 30min, the extraction process of the free DNA in the vesicle is greatly simplified, and the extraction efficiency of the free DNA in the vesicle is improved.

It is known that, in the cell DNA extraction process, it is usually necessary to use proteinase K or various lysis solutions to crack and combine with drastic means such as high power (more than 300W) ultrasonic waves to break cells, and combine chloroform and phenol to extract and remove impurities, phenol is a strong protein deforming agent and is used for denaturing and precipitating proteins, chloroform is used for separating an aqueous phase and an organic phase, namely phenol, so as to remove phenol, but phenol is slightly soluble in water, and then, many precipitation and impurity removal operations are required to completely remove phenol, otherwise phenol residue affects downstream experiments. In the invention, as the prepared vesicle is used as a bubble membrane structure, the membrane bilayer of the vesicle is not very stable, and the breaking of the vesicle by combining high-temperature water bath and ultrasonic breaking with lower power can be ensured in shorter processing time, so that the content is released.

The strength of the cell damage effect is closely related to the ultrasonic intensity, and the cell damage degree is increased along with the increase of the ultrasonic power. Ultrasonic power is undersized, and the cell membrane damage in treating the extraction vesicle sample is less, can't effectively release the free DNA that contains in it, leads to DNA extraction volume less, and ultrasonic power is too big, though can make the cell breakage completely with a large amount of release DNA, can cause the excessive shear of sample simultaneously, arouses the fragmentation of cell membrane and free DNA easily, makes the unable characteristic that reflects tumor cell completely of the DNA of extracting, and causes impurity content among the DNA high. Preferably, in step S1, the ultrasonic disruption is performed at a power of 95-105W, and more preferably, the power of the ultrasonic disruption is 97.5W, under the condition of the power, the cell disruption rate is high, the extracted DNA has high concentration, high purity and stable property.

Preferably, in step S1, the operation mode of the ultrasonication is selected in a batch mode to prevent DNA denaturation caused by local overheating. More preferably, the intermittent mode is working for 1-5s and intermittent for 1-5s, and the duty ratio is 50%. The low duty cycle pulse has good mechanical effect, can shorten the ultrasonic time, and further reduces the damage of free DNA in the vesicle.

Setting accurate ultrasonic conditions aiming at the characteristics of different cells, particularly determining scientific ultrasonic power and ultrasonic treatment time can ensure very obvious cell membrane breaking effect. In the invention, because of the instability of the vesicle structure, the high cell membrane breakage rate can be ensured in a short time, the action time of ultrasonic waves is prolonged, and the released DNA is easy to be sheared into fragments or damaged by oxidation under instantaneous strong energy to influence the extraction effect, so that the method is not suitable for long-time ultrasonic treatment. Preferably, in step S1, the time for ultrasonication is 2 to 10min. More preferably, the time of ultrasonication is 6min.

In order to improve the efficiency of denaturing and inactivating enzymes such as DNase, the water bath time is preferably 2-10min in step S1.

Preferably, in step S1, the vesicle suspension is subjected to a water bath at a temperature of 90 ℃.

Preferably, in step S1, the concentration of vesicles in the vesicle suspensionDegree of 3X 10 ¹⁰ -6×10 ¹⁰ one/mL. The concentration of vesicles in the vesicle suspension has a small influence on the breakage rate, and the number of vesicles can be increased or decreased as appropriate depending on the amount of DNA to be extracted. For common experiments, such as PCR amplification, DNA sequencing, etc., 3X 10 ¹⁰ -6×10 ¹⁰ The number of vesicles per mL can achieve good experimental effect.

Preferably, in step S2, the volume ratio of the cell disruption suspension to the chloroform is 1.5-5:1. it is understood that, as the content of impurities increases due to the increase in vesicle concentration, cell membrane disruption degree, etc. in the vesicle suspension, the amount of chloroform may be increased to lower the content of impurities in the extracted DNA. However, the excessive chloroform content increases the waste of chloroform on the one hand, and has little influence on the purity of DNA on the other hand.

Preferably, in step S2, the centrifugation conditions are: centrifuge at 12000g for 10min. Under the centrifugal condition, impurities such as cell membrane fragments, inclusion bodies and the like can be ensured to be completely settled and separated from DNA in the liquid.

The invention will be further illustrated with reference to the following specific examples. It should be understood that these examples are for illustrative purposes only and are not intended to limit the scope of the present invention. The following examples are examples of experimental procedures not specified under specific conditions, generally according to the conditions recommended by the manufacturer. In the following examples, a new Toshiba JY92-IIN type ultrasonograph is used for ultrasonograph, the total power of the ultrasonograph is 650W, and the working mode is working 2s and gap 2s as an example.

Example 1 selection of vesicle DNA extraction conditions

1) Preparation of vesicle suspension: preparing A549 cell-derived vesicle (the extraction method is shown in Chinese patent CN 102302784B), and preparing vesicle suspension with sterilized water, wherein the number of vesicles in the vesicle suspension is about 5 × 10 ¹⁰ One per mL.

2) Pre-experiment: measuring vesicle suspension with the volume of 0.3mL, adding 200 mu L chloroform, mixing, shaking for 20s, standing for 3min, centrifuging 12000g for 10min, and collecting supernatant of about 50 mu L for later use. And adding anhydrous ethanol with the volume twice that of the supernatant of 200 mu L, uniformly mixing, standing in a refrigerator at the temperature of-20 ℃ for 1h, centrifuging at 12000g for 10min, removing the supernatant, cleaning the precipitate with 75% ethanol, removing the supernatant, slightly drying in the air, and dissolving with 200 mu L of sterilized water for later use. The DNA content and purity of the prepared 50. Mu.L supernatant and 200. Mu.L sterilized water-dissolved DNA solution were measured with Thermo NanoDrop 2000 ultramicro spectrophotometer. Wherein, the DNA content in the supernatant is 58.1 ng/mu L, and the ratio of A260/A280 is 1.43; the DNA content of the DNA solution dissolved in sterile water was 52.4 ng/. Mu.L, and the A260/A280 ratio was 1.61. The preliminary experiment shows that the prepared vesicle contains high-concentration free DNA, but the ratio of A260/A280 in the free DNA is lower, and the vesicle contains a certain amount of protein impurities.

3) Selection of vesicle DNA extraction conditions: 6 parts of vesicle suspension with the volume of 0.3mL are respectively measured and are respectively processed as follows:

experimental group 1: and (3) placing the vesicle suspension on ice, carrying out ultrasonic disruption for 5min (the ultrasonic disruption parameters are 97.5W, 2s of work and 2s of gap), then adding 200 mu L of chloroform, shaking for 20s, uniformly mixing, standing for 3min, centrifuging for 10min at 12000g, and taking the supernatant as a sample for detection.

Experimental group 2: and (3) placing the vesicle suspension in a water bath at 90 ℃ for 5min, then adding 200 mu L of chloroform, shaking for 20s, uniformly mixing, standing for 3min, centrifuging for 10min at 12000g, and taking the supernatant as a sample for detection.

Experimental group 3: adding 10 μ L of protease K into the vesicle suspension, mixing, placing in 60 deg.C water bath for 10min, adding 500 μ L of chloroform, shaking for 20s, mixing, and standing for 3min;

experimental group 4: and (3) putting the vesicle suspension in a water bath at 90 ℃ for 5min, then carrying out ultrasonication for 5min (ultrasonication parameters are 97.5W in power, 2s in work and 2s in clearance), then adding 500 mu L of chloroform, shaking for 20s, uniformly mixing, standing for 3min, centrifuging for 10min at 12000g, and taking the supernatant as a sample for detection.

Experimental group 5: adding 10 mu L of protease K into the vesicle suspension, uniformly mixing, placing in a water bath at 60 ℃ for 10min, then carrying out ultrasonication for 5min (ultrasonication parameters are 97.5W in power, 2s in work and 2s in clearance), then adding 500 mu L of chloroform, shaking for 20s, uniformly mixing, standing for 3min, centrifuging for 10min at 12000g, and taking the supernatant as a sample for detection.

Control group: the method comprises the following steps of (1) extracting DNA by using an Exosome DNA extraction Kit (HiPure Exosome DNA Kit) and specifically comprising the following steps:

a) Swirling to loosen the vesicle suspension, adding 200 mu L of Buffer ATL or sterile water into the vesicle suspension, and repeatedly sucking the vesicle suspension by using a liquid-transferring gun;

b) Adding 10 μ L of protease K, 3 μ L of Carrier RNA and 200 μ L of Buffer AL into the sample, vortexing for 15s, mixing, and water bathing at 56 deg.C for 30min;

c) Adding 200 μ L of anhydrous ethanol, mixing by vortex for 15s, and standing at room temperature for 3min;

d) Collecting droplets on the tube wall by short-time centrifugation;

e) Loading the DNA column into a collection tube, transferring the mixed solution to the column, and centrifuging at 6000g for 30-60s;

f) Pouring out effluent, putting the column back into the collecting tube, adding 500 μ L Buffer GW1 (diluted with ethanol) into the column, and centrifuging at 6000g for 30-60s; wherein before the Buffer GW1 is used, absolute ethyl alcohol is required to be used for dilution, and the indication is carried out according to a bottle label;

g) Discarding the filtrate, reloading the column into the collection tube, adding 600. Mu.L Buffer GW2 (diluted with ethanol) into the column, and centrifuging at 6000g for 30-60s;

h) The filtrate was decanted, the column was returned to the collection tube, 600. Mu.L of Buffer GW2 (diluted with ethanol) was added to the column, and 6000g was centrifuged for 30-60s;

i) Pouring the filtrate, putting the column back into the collecting pipe again, and centrifuging 13000g of the empty column for 3min to spin-dry the column;

j) The column was transferred to a new 1.5mL centrifuge tube, 50. Mu.L of Buffer AE preheated to 56 ℃ was added to the center of the membrane of the column, and left to stand for 3min, after which 13000g was centrifuged for 1min, and the tube bottom solution was the extracted DNA.

The supernatants of the experimental and control groups and the finally dissolved DNA solution were tested for DNA content and purity using a Thermo NanoDrop 2000 ultramicro spectrophotometer, the results are shown in Table 1.

TABLE 1 DNA content and purity results extracted under different conditions

Serial number	Vesicle suspension treatment conditions	DNA concentration	A260/A280
				Experimental group
1	Ultrasonic crushing for 5min	181.3ng/μL	1.72
				Experimental group 2	90 deg.C water bath for 5min	148.6ng/μL	1.54
Experimental group 3	Adding protease K, and water bath at 60 deg.C for 10min	188.4ng/μL	1.63
				Experimental group 4	Water bath at 90 deg.C for 5min, and ultrasonic crushing for 5min	207.4ng/μL	1.76
Experimental group 5	Adding protease K, water bathing at 60 deg.C for 10min, and ultrasonic crushing for 5min	203.6ng/μL	1.75
				Control group	Kit extraction	214.3ng/μL	1.78

As can be seen from Table 1, each of the different treatment methods can obtain a DNA solution with a certain concentration and purity, and compared with the kit, the ultrasonic disruption, the water bath disruption at 90 ℃ and the protease K enzymolysis disruption cell membrane can not effectively break the cell, so that the concentration and the purity of the DNA are lower. The DNA extraction efficiency can be improved to a certain extent by combining a plurality of different treatment methods, wherein the concentration and purity of the DNA obtained by the experimental group 4 are not obviously different from those of the kit, so that a 90 ℃ water bath for 5min and ultrasonic crushing for 5min are selected as the treatment conditions of the invention, and the water bath heating and ultrasonic crushing modes are convenient and fast without reagent tolerance, so that the method is suitable for extracting the DNA in a large scale.

In order to verify the influence of ethanol purification on the DNA purity of the supernatant, 200 muL of the supernatant of each experimental group 1-5 is added with two times of volume of absolute ethanol, the mixture is placed in a refrigerator at-20 ℃ for 30min after being mixed evenly, then 12000g is centrifuged for 10min, the supernatant is discarded, the precipitate is washed by 75% ethanol, the supernatant is discarded by a pipette, the supernatant is dissolved by 200 muL of sterilized water after being dried slightly, and the DNA content and purity detection in the dissolved solution are determined, and the results are shown in Table 2.

TABLE 2 DNA content and purity results of DNA extracted under different conditions after ethanol purification

Serial number	Conditions for vesicle suspension treatment	DNA concentration	A260/A280
				Experimental group
1	Ultrasonic crushing for 5min	178.6ng/μL	1.76
				Experimental group 2	90 deg.C water bath for 5min	138.3ng/μL	1.63
Experimental group 3	Adding protease K, and water bath at 60 deg.C for 10min	180.1ng/μL	1.68
				Experimental group 4	Water bath at 90 deg.C for 5min, and ultrasonic crushing for 5min	199.8ng/μL	1.76
Experimental group 5	Adding protease K, water bathing at 60 deg.C for 10min, and ultrasonic crushing for 5min	191.3ng/μL	1.77

The DNA concentration and purity after the ethanol purification are not greatly different, so the step of purifying and cleaning the DNA by ethanol precipitation is not needed in the subsequent experimental operation; on the other hand, the invention generates less impurities in the way of breaking vesicle membranes, and impurities such as cell membrane fragments, protein inclusion bodies and the like can be completely removed by chloroform extraction and centrifugation.

Example 2 optimization of ultrasound conditions

1) Optimization of ultrasonic time: respectively measuring 5 parts of vesicle suspension with the volume of 0.3mL, placing the vesicle suspension in a water bath at 90 ℃ for 5min, and then respectively carrying out ultrasonication for 2min, 4min, 6min, 8min and 10min, wherein the ultrasonication parameters are as follows: the power is 97.5W, the work time is 2s, the gap is 2s, then 500 mu L of chloroform is added, the mixture is shaken for 20s and mixed evenly, the mixture is kept stand for 3min,12000g is centrifuged for 10min, and the supernatant is taken as a sample to carry out detection on the concentration and the content of the DNA, and the detection result is shown in table 3.

TABLE 3 Effect of different sonication times on the content and purity of extracted DNA

As can be seen from Table 3, the extracted DNA content increased with the increase of the sonication time because the vesicle content was released more due to the increase of the cell membrane disruption rate, but the extracted DNA content did not increase significantly but the purity decreased with the further increase of the sonication time, probably because the cell was disrupted too much and a large amount of fragmented cell membranes were formed, so that the content of soluble impurities in the system increased. Therefore, the ultrasonication time is preferably 4 to 8min, more preferably 6min.

2) Optimization of ultrasonic power: respectively measuring 5 parts of vesicle suspension with the volume of 0.3mL, placing the vesicle suspension in a water bath at 90 ℃ for 5min, then respectively carrying out ultrasonic crushing for 5min under the conditions of the power of 90W, 95W, 97.5W, 105W and 110W, working for 2s and spacing for 2s during ultrasonic crushing, then adding 500 mu L of chloroform, shaking for 20s, uniformly mixing, standing for 3min, centrifuging for 10min at 12000g, and taking the supernatant as a sample to carry out detection on the concentration and the content of the DNA, wherein the detection results are shown in Table 4.

TABLE 4 Effect of different ultrasonic powers on the content and purity of extracted DNA

Ultrasonic crushing power	DNA concentration	A260/A280
			90W	195.3ng/μL	1.75
95W	203.4ng/μL	1.76
			97.5W	207.5ng/μL	1.76
105W	205.5ng/μL	1.76
			110W	205.1ng/μL	1.76

As can be seen from Table 4, as the ultrasonic power is increased, the change of the content and purity of the extracted DNA shows substantially the same change trend as the ultrasonic time is increased, and the increase of the ultrasonic power also causes the fragmentation of the DNA, causing the structural change of the DNA. Therefore, the preferred sonication power is 95-105W.

Example 3 optimization of Water bath conditions

1) Optimization of water bath time: respectively measuring 5 parts of vesicle suspension with the volume of 0.3mL, respectively placing the vesicle suspension in a water bath at 90 ℃ for 2min, 4min, 6min, 8min and 10min, respectively carrying out ultrasonic crushing for 5min, wherein the ultrasonic crushing parameters are as follows: the power is 97.5W, the work time is 2s, the gap is 2s, then 500 mu L of chloroform is added, the mixture is shaken for 20s and mixed evenly, the mixture is kept stand for 3min,12000g is centrifuged for 10min, and the supernatant is taken as a sample to carry out detection on the concentration and the content of the DNA, and the detection result is shown in Table 5.

TABLE 5 Effect of different bath times on extracted DNA content and purity

Time of water bath	DNA concentration	A260/A280
			2min	185.2ng/μL	1.71
4min	204.4ng/μL	1.75
			6min	207.6ng/μL	1.76
8min	204.1ng/μL	1.76
			10min	204.5ng/μL	1.75

As can be seen from Table 5, the concentration and purity of DNA extracted from the samples in the water bath for more than 4min do not change much, and the vesicle lysis degree may be already optimized at the time of 4min in the water bath, and in order to ensure the extraction efficiency and also ensure the sufficient vesicle lysis, the water bath time is preferably 4-8min, and more preferably 6min.

2) Optimizing the temperature of the water bath: respectively measuring 5 parts of vesicle suspension with the volume of 0.3mL, respectively placing the vesicle suspension in water baths at 85, 90 and 95 ℃ for 5min, and then respectively carrying out ultrasonic crushing for 5min, wherein the ultrasonic crushing parameters are as follows: the power is 97.5W, the work time is 2s, the gap is 2s, then 500 mu L of chloroform is added, the mixture is shaken for 20s and mixed evenly, the mixture is kept stand for 3min,12000g is centrifuged for 10min, and the supernatant is taken as a sample to carry out detection on the concentration and the content of the DNA, and the detection result is shown in Table 6.

TABLE 6 Effect of different bath temperatures on the content and purity of extracted DNA

Temperature of water bath	DNA concentration	A260/A280
			80℃	194.4ng/μL	1.73
85℃	203.8ng/μL	1.76
			90℃	206.2ng/μL	1.76
95℃	204.1ng/μL	1.76
			100℃	203.6ng/μL	1.75

As is clear from Table 6, the vesicle samples were well lysed in five water bath temperatures of 80, 85, 90, 95 and 100 ℃ and boiling water was likely to be splashed into the sample solution at 100 ℃ and the water bath temperature was preferably 85-95 ℃ and more preferably 90 ℃ to ensure sufficient lysis of the vesicles.

Example 4 extraction of vesicle DNA derived from A549 cells

Extracting the vesicle DNA from the A549 cell by using the optimized parameters in the embodiment, which comprises the following steps:

s1, taking 0.3mL vesicle suspension, placing the vesicle suspension in a water bath at 90 ℃ for 6min, and then ultrasonically crushing for 6min under the power of 97.5W, wherein the work mode of ultrasonic crushing is 2S of work and 2S of clearance, so as to obtain broken cell suspension;

s2, adding 200 mu L of chloroform into the cell disruption suspension, shaking for 10S, uniformly mixing, standing for 3min, then centrifuging for 10min at 12000g, taking supernatant to obtain vesicle DNA, and detecting that the content of the DNA in the supernatant is 209.2 ng/mu L and A260/A280 is 1.77. In this example, the A260/A280 ratios of the extracted DNA were all around 1.8, which indicates that the purity of the extracted DNA was high, and that the extracted DNA was free from contamination by impurities such as protein and RNA, and could be used as a template for subsequent PCR experiments. Compared with the reagent kit in the example 1 (the DNA content is 214.3 ng/. Mu. L, A260/A280 is 1.78), the extraction efficiency is more than 97 percent; moreover, the time required for the completion of the whole operation is only about 21min, and the DNA extraction efficiency is high.

PCR amplification detection was performed on two DNA solutions extracted from the kit of this example and example 1, respectively, using a PCR apparatus model of Bori TC-96/G/H (B), using 2 XTaq MIX from Takara as Taq enzyme, and using highly expressed conserved cytochrome B as a template to design primers having the following sequences:

upstream primer sequence (F): 5'-AACTTCGGCTCACTCCTTGG-3' of the formula,

downstream primer sequence (R): 5'GGAGGTGATTCCTAGGGGGT-3'.

The amplification procedure was: 94 ℃ 4min → (94 ℃ 30s → 60 ℃ 30s → 72 ℃ 60 s) × 32 → 72 ℃ 2min. The PCR amplification products were detected by electrophoresis on 1.5% agarose. The result is shown in fig. 1, where M in fig. 1 is a DNA marker, lane 1 is the amplification result of the extraction method of this embodiment, lane 2 is the amplification result of the extraction method of the kit, lane 3 is a negative control, lane 4 is the amplification result of the cell genome DNA, it can be seen that both samples can amplify a positive band, the difference is not obvious, the DNA bands are concentrated and clear, which indicates that the DNA quality is good, and no degradation or fragmentation occurs.

Example 5 extraction of D562 cell-derived vesicle DNA

Preparation of vesicle suspensions: preparing D562 cell-derived vesicles (see Chinese patent CN 102302784B) from sterile water to obtain vesicle suspension, wherein the number of vesicles in the vesicle suspension is about 3 ¹⁰ one/mL. Extracting D562 cell-derived vesicle DNA by using the optimized parameters in the above embodiment, specifically comprising the steps of:

s1, taking 0.3mL of vesicle suspension, placing the vesicle suspension in water bath at the temperature of 90 ℃ for 6min, and then ultrasonically crushing the vesicle suspension for 6min under the power of 97.5W, wherein the work mode of ultrasonic crushing is 2S of work and 2S of gap, so as to obtain broken cell suspension;

s2, adding 200 mu L of chloroform into the cell disruption suspension, shaking for 10S, mixing uniformly, standing for 3min, centrifuging for 10min at 12000g, taking supernate to obtain vesicle DNA, and detecting the content of the DNA in the supernate to be 116.5 ng/mu L, wherein A260/A280 is 1.78 _。 This exampleThe A260/A280 ratio of the extracted DNA is about 1.8, which shows that the purity of the extracted DNA is high, the extracted DNA is free from the pollution of impurities such as protein, RNA and the like, and the extracted DNA can be used as a template to carry out subsequent experiments such as PCR and the like. Compared with a kit (the DNA content is 121.7 ng/. Mu. L, A260/A280 is 1.78), the extraction efficiency is over 95 percent.

The two DNA solutions extracted by the embodiment and the kit are respectively subjected to PCR amplification detection under the PCR amplification conditions of the embodiment 4, the result is shown in FIG. 2, M in FIG. 2 is a DNA marker, a lane 1 is the amplification result of the extraction method of the embodiment, and a lane 2 is the amplification result of the extraction method of the kit, so that the positive bands can be amplified by the two samples, the difference is not obvious, the DNA bands are concentrated and clear, and the conditions of complete DNA quality, no degradation or breakage and the like are shown.

Although the present disclosure has been described above, the scope of the present disclosure is not limited thereto. Various changes and modifications may be effected therein by one of ordinary skill in the pertinent art without departing from the spirit and scope of the present disclosure, and these changes and modifications are intended to be within the scope of the present disclosure.

Claims

1. A method for rapidly extracting vesicle DNA is characterized by comprising the following steps:

s1, placing the vesicle suspension in a water bath at the temperature of 80-100 ℃, wherein the water bath time is 2-10min, and then carrying out ultrasonic crushing at the power of 90-110W for 2-10min to obtain a cell-broken suspension; wherein the vesicle suspension is produced following apoptosis of the tumor cells;

2. The method for rapidly extracting vesicle DNA according to claim 1, wherein in step S1, the ultrasonication is performed at a power of 95-105W.

3. The method for rapidly extracting vesicle DNA according to claim 1, wherein in step S1, the operation mode of the ultrasonication is selected from an intermittent mode, wherein the intermittent mode is operation for 1-5S and intermittent for 1-5S, and the duty ratio is 50%.

4. The method for rapidly extracting vesicle DNA according to claim 1, wherein in step S1, the vesicle suspension is placed in a water bath at a temperature of 90 ℃.

5. The method for rapidly extracting vesicle DNA according to claim 1, wherein the concentration of vesicles in the vesicle suspension is 3 x 10 in step S1 ¹⁰ -6×10 ¹⁰ one/mL.

6. The method for rapidly extracting vesicle DNA according to claim 1, wherein in step S2, the volume ratio of the cell disruption suspension to the chloroform is 1.5-5:1.

7. the method for rapidly extracting vesicle DNA according to claim 6, wherein in step S2, the centrifugation conditions are: centrifuge at 12000g for 10min.