CN112501161B

CN112501161B - Double-magnetic-particle-intervention DNA extraction and purification method

Info

Publication number: CN112501161B
Application number: CN202011532811.8A
Authority: CN
Inventors: 贾丽; 付云海
Original assignee: South China Normal University
Current assignee: South China Normal University
Priority date: 2020-12-23
Filing date: 2020-12-23
Publication date: 2023-03-28
Anticipated expiration: 2040-12-23
Also published as: CN112501161A

Abstract

The invention discloses a double-magnetic-particle intervention DNA extraction and purification method, which comprises the following steps: sampling lysate, adding an adsorption solution and a NaCl solution, mixing uniformly, adding GO-MPs suspension, performing magnetic separation after incubation, and taking supernatant; adding polyDOPA-MPs suspension, incubating, magnetically separating, discarding supernatant, washing the obtained precipitate with ethanol solution, adding eluate, incubating, magnetically separating, and collecting supernatant containing purified DNA. In the method, the GO-MPs can selectively remove RNA from the mixed solution of DNA and RNA, and can replace RNase used in the traditional method, thereby reducing the cost of RNA removal; meanwhile, the adsorption process is simple and quick, and the separation can be realized only by a magnetic field.

Description

Double-magnetic-particle-intervention DNA extraction and purification method

Technical Field

The invention relates to a double-magnetic-particle (graphene oxide modified magnetic particle GO-MPs and poly-DOPA modified magnetic particle polyDOPA-MPs) interventional DNA extraction and purification method.

Background

High quality DNA purification is fundamental to numerous downstream operations including PCR, qPCR, nucleic acid detection, and the like. Therefore, the development of a low-cost, high-efficiency and high-quality DNA purification method has important research value.

The phenol-chloroform method is a traditional DNA extraction method, but the method has complex operation steps and long time consumption, needs highly toxic reagents such as phenol, chloroform and the like, and is not friendly to the environment and experimenters.

Solid Phase Extraction (SPE) based methods avoid the use of toxic and hazardous reagents, but still require multiple centrifugations. The shear force caused by centrifugation is very damaging to the quality of DNA.

The existing method for purifying nucleic acid based on magnetic material utilizes an external magnetic field to carry out solid-liquid separation, and solves the problem of DNA damage caused by shearing force. However, it is often necessary to extract under strong acid conditions (pH 1.0-4.0), and the strong acid environment will destroy DNA and affect the purification quality. In addition, RNA and DNA have similar structures, and the important index of DNA purification quality is the RNA removal rate. Traditionally, ribonucleases (RNases) have been added to degrade RNA. As enzyme substances, RNase has high purification cost, poor stability and difficulty in storage. More importantly, RNases are often derived from animal pancreas extracts and cannot be used in the extraction of medical grade plasmid DNA. The existing commercial DNA extraction kit needs RNase to degrade RNA so as to remove RNA.

Disclosure of Invention

The invention aims to provide a double-magnetic-particle-mediated DNA extraction and purification method, wherein graphene oxide modified magnetic particles (GO-MPs) can effectively remove RNA, and polyDOPA modified magnetic particles (polyDOPA-MPs) can adsorb DNA to achieve the purpose of purification.

The purpose of the invention is realized by the following technical scheme:

a DNA extraction and purification method comprises the following steps:

(1) Sampling a lysate, adding an adsorption solution and a NaCl solution, mixing uniformly, adding GO-MPs (graphene oxide modified magnetic particles) suspension, incubating for at least 10min, performing magnetic separation, and taking a supernatant;

(2) Adding polyDOPA-MPs (polyDOPA modified magnetic particles) suspension into the supernatant, incubating for at least 10min, performing magnetic separation, removing the supernatant, washing the obtained precipitate with ethanol solution, and drying at room temperature; adding eluent, incubating for at least 15min to elute adsorbed DNA from polyDOPA-MPs, performing magnetic separation, and collecting supernatant containing purified DNA;

preferably, the adsorption solution in the step (1) is Tris-HCl-EDTA buffer solution, wherein the concentration of Tris is 10mmol/L, the concentration of EDTA is 1mmol/L, and the pH value is 2.8-6.8;

preferably, the eluent in the step (2) is Tris-HCl-EDTA buffer solution, wherein the concentration of Tris is 10mmol/L, the concentration of EDTA is 1mmol/L, and the pH value is 8.8-10.8;

the concentration of NaCl in the step (1) is 0.2-1 mol/L;

the volume concentration of the ethanol solution in the step (2) is preferably 70%.

The sample lysate in the step (1) is prepared by the following steps:

A. if the sample is bacteria, taking a bacteria culture solution, centrifuging to take a precipitate, and adding a lysis solution; if the sample is other solid samples, grinding the sample to powder under the protection of liquid nitrogen, and adding a lysis solution;

B. adding lysis solution, incubating in water bath at 37 deg.C for at least 1h, adding NaCl solution, mixing, centrifuging, and collecting supernatant as tissue lysis solution;

the lysate contains 25mmol/L Tris-HCl,20mmol/L NaAc,1mmol/L EDTA and 1% SDS, pH 7.8;

the NaCl solution is preferably 4mol/L;

the centrifugation is preferably carried out at 10000rpm for 1-20min.

The GO-MPs suspension and the polyDOPA-MPs suspension are respectively dispersed in Tris-HCl-EDTA buffer solution (10 mmol/L Tris,1mmol/L EDTA, pH value 5.8), and the concentration of the GO-MPs suspension and the polyDOPA-MPs suspension is 50-300 mg/mL;

the preparation method of the GO-MPs comprises the following steps:

dissolving ferric trichloride in ethylene glycol, adding anhydrous sodium acetate and polyethylene glycol (PEG) 4000, uniformly mixing, heating the obtained mixture to 160-220 ℃, and reacting for 8-22 h to obtain PEG-MPs; adding the synthesized PEG-MPs into a Graphene Oxide (GO) solution, heating and stirring, and separating to obtain GO-MPs;

in the mixture, the concentration of ferric trichloride is 0.10-0.15 mol/L.

Preferably, the prepared PEG-MPs can be separated by an external magnetic field, washed by ethanol and deionized water, and finally dried to obtain uniformly dispersed PEG-MPs;

the mass ratio of PEG-MPs to GO is (1-4) to 1; the mass ratio of PEG-MPs to GO can adjust the magnetic responsiveness of the prepared GO-MPs, and is more ideal in the range;

the heating and stirring are preferably carried out for stirring reaction at 25-70 ℃ for 2-10 h;

preferably, the prepared GO-MPs can be separated by an external magnetic field, washed by deionized water and finally dried.

The preparation method of the polyDOPA-MPs comprises the following steps:

dissolving ferric trichloride in a diethylene glycol-ethylene glycol mixed solvent, adding anhydrous sodium acetate and sodium acrylate, uniformly mixing, heating the obtained mixture to 160-220 ℃, and reacting for 10-20 h to obtain COOH-MPs; adding the synthesized COOH-MPs into an alkaline solution of dopa, heating, stirring, and separating to obtain polyDOPA-MPs;

in the mixture, the concentration of ferric trichloride is 0.10-0.15 mol/L;

in the mixture, the mass ratio of anhydrous sodium acetate to sodium acrylate is (1-3) to 1; the mass ratio of the two components can influence the particle size and the magnetic responsiveness of the prepared COOH-MPs, and is more ideal in the range;

in the diethylene glycol-ethylene glycol mixed solvent, the volume ratio of diethylene glycol to ethylene glycol is (1-4) to 1; the volume ratio of the diethylene glycol to the ethylene glycol can regulate the particle size of the prepared COOH-MPs.

Preferably, the prepared COOH-MPs can be separated by an external magnetic field, washed by ethanol and deionized water, and finally dried to obtain uniformly dispersed COOH-MPs;

the mass ratio of the dopa to the COOH-MPs is (2-4): 1, the mass ratio of the dopa to the COOH-MPs can regulate and control the thickness of the poly-dopa coating, and the thickness of the coating can influence the magnetic responsiveness of the prepared poly-DOPA-MPs and the separation capacity of the poly-DOPA-MPs on a target object;

in the alkaline solution of dopa, the concentration of dopa is 0.5-2.0 mg;

the alkaline solution is preferably Tris-HCl buffer solution, and the pH value of the alkaline solution is 7.5-9.5; the concentration of Tris (hydroxymethyl) aminomethane (Tris) water solution is 5-30 mmol/L;

the heating and stirring are preferably carried out at the temperature of 25-50 ℃ for stirring reaction for 8-24 h.

Preferably, the polyDOPA-MPs prepared can be separated by an external magnetic field, washed with deionized water and finally dried.

The invention utilizes a one-step method to prepare PEG-MPs rich in hydroxyl, the PEG-MPs have good balling property and dispersibility, and the grain diameter of magnetic grains is about 288 +/-18 nm. PEG-MPs produce GO-MPs by hydrogen bonding with GO. The preparation method has the advantages of simple process, mild conditions and good reproducibility. GO-MPs have the magnetic responsiveness of PEG-MPs and the properties of GO.

The invention adopts a one-step method to prepare COOH-MPs rich in carboxyl, and then utilizes the oxidative auto-polymerization of dopa under weak base and the adhesion capability of polyDOPA to form a coating on the surface of the COOH-MPs to obtain the polyDOPA-MPs. The preparation method has the advantages of simple process, mild conditions and good reproducibility. The prepared polyDOPA-MPs have good balling property and dispersibility, the grain diameter of magnetic grains is about 135 +/-15nm, and the thickness of the polyDOPA coating is about 8.5 +/-1.5 nm.

The GO-MPs prepared by the method can be used for replacing RNase to remove RNA, and the polyDOPA-MPs can extract DNA under the condition of ensuring the stability of the DNA.

Compared with the prior art, the invention has the following advantages and effects:

1. in the method, GO-MPs can selectively remove RNA from the mixed solution of DNA and RNA, and can replace RNase used in the traditional method, thereby reducing the cost of RNA removal; meanwhile, the adsorption process is simple and quick, and the separation can be realized only by a magnetic field.

2. In the method, polyDOPA-MPs are used as carriers to adsorb DNA, so that the existing DNA purification method is greatly improved. Compared with the traditional phenol-chloroform method and the silica gel column kit extraction method, no toxic and harmful reagent is needed in the process, and the damage of shearing force caused by repeated centrifugation to DNA is avoided. At the same time, magnetic separation-based purification also provides convenience for high-throughput nucleic acid extraction and automated nucleic acid extraction. In addition, the adsorption and elution buffers are simple in composition, and the whole adsorption process can be completed at room temperature (25 ℃). And can be widely applied to DNA extraction of bacteria and plants.

3. The method optimally considers the effective connection of RNA removal and DNA purification, can ensure that the RNA removal and the DNA purification are carried out in the same environment, and avoids the complicated operation and the waste of reagents.

4. The GO-MPs and the polyDOPA-MPs have the advantages of mild preparation conditions, simple and convenient operation, good reproducibility and stability, and long storage time under the protection of nitrogen.

Drawings

FIG. 1 is a TME map of each magnetic particle; wherein A: PEG-MPs, B: GO-MPs, C: COOH-MPs diagram, D: polyDOPA-MPs.

FIG. 2 is a FT-IR diagram and a hysteresis curve diagram of each magnetic particle; wherein A: FT-IR diagram of GO, PEG-MPs, GO-MPs, B: FT-IR chart of polyDOPA, COOH-MPs, polyDOPA-MPs, C: hysteresis curves for PEG-MPs and GO-MPs, D: hysteresis curves for COOH-MPs and polyDOPA-MPs.

FIG. 3 is a Zeta potential diagram for each magnetic particle; wherein A: zeta potential diagrams for PEG-MPs and GO-MPs, B: zeta potential plots of COOH-MPs and polyDOPA-MPs.

FIG. 4 is an agarose gel electrophoresis of the PCR amplification product; wherein, A is E.coli DH5 alpha genome DNA amplification product; b is a corn genome DNA amplification product; lane M is a standard DNA fragment; the results of the amplification in lanes 1 and 2.

Detailed Description

The present invention will be described in further detail with reference to examples and drawings, but the present invention is not limited thereto.

Example 1

The preparation of GO-MPs comprises the following steps:

(1) 2.4g of ferric chloride is accurately weighed and dissolved in 120mL of glycol for ultrasonic dissolution, and then 10.8g of sodium acetate and 4.5g of polyethylene glycol 4000 are added for full dissolution to form a uniform dark yellow solution. Uniformly subpackaging into 4 Teflon high-pressure reaction kettles, and reacting for 10 hours at 200 ℃. Cooling to room temperature, separating the obtained PEG-MPs by using an external magnetic field, washing the separated PEG-MPs by using ethanol and deionized water for a plurality of times, and drying the separated PEG-MPs in an oven at 50 ℃ under the protection of nitrogen.

(2) Taking 50mg of graphene oxide, and ultrasonically dispersing in 30mL of deionized water; meanwhile, 500mg of PEG-MPs are ultrasonically dispersed in 20mL of deionized water. Then the two are mixed and reacted in a water bath at 70 ℃ for 2h under the protection of nitrogen. And separating by an external magnetic field to obtain GO-MPs, and washing and drying by using ethanol and deionized water to obtain the GO-MPs.

TEM observation is carried out on the PEG-MPs and GO-MPs prepared in the steps (1) and (2), and the result is shown in figure 1. As can be seen from FIGS. 1A and 1B, the prepared PEG-MPs and GO-MPs have relatively average particle size distribution, the average particle size is 288 + -18 nm, and meanwhile, the prepared PEG-MPs and GO-MPs have good dispersibility.

FT-IR spectrum measurement is carried out on the PEG-MPs and GO-MPs prepared in the steps (1) and (2), and the result is shown in figure 2A. In FIG. 2A, the FT-IR spectrum of GO-MPs shows the Fe-O vibration peak (580 cm) in PEG-MPs ^-1 ) And C-O vibration peak (1100 cm) ^-1 ) At the same time at 1650cm ^-1 、1384cm ^-1 The characteristic infrared absorption peak of the graphene oxide appears, thereby indicating that GO-MPs have been successfully prepared。

And (3) determining a hysteresis loop of the PEG-MPs and the GO-MPs prepared in the steps (1) and (2), and obtaining a result shown in figure 2C. FIG. 2C shows that by comparing the magnetic responsivity of PEG-MPs and GO-MPs under different magnetic field values, the magnetic saturation of the two is 78.3emu/g and 62.2emu/g, respectively. Both the magnetic particles have good magnetic responsiveness, and the reduction of the magnetic responsiveness of the magnetic particles after GO is modified indicates that GO-MPs are successfully prepared.

Zeta potential measurement is carried out on the PEG-MPs and GO-MPs prepared in the steps (1) and (2), and the result is shown in figure 3A. FIG. 3A shows that the difference between the two different pH values of the PEG-MPs and the GO-MPs can be seen by comparing the Zeta potentials of the PEG-MPs and the GO-MPs at different pH values, thereby illustrating the difference between the surface functional groups of the PEG-MPs and the GO-MPs, and therefore, the successful preparation of GO-MPs can be judged.

Example 2

The preparation of polyDOPA-MPs comprises the following steps:

(1) 2.4g of ferric chloride was accurately weighed out and dissolved in 30mL of ethylene glycol and 90mL of diethylene glycol bis-solvent. 9g of sodium acrylate and 9g of sodium acetate are then added and stirred to form a dark yellow solution. Uniformly subpackaging into 4 Teflon high-pressure reaction kettles, and reacting for 10h at 200 ℃. The product obtained (COOH-MPs) was subsequently washed several times with water and ethanol and dried at 50 ℃ under nitrogen.

(2) 160mg of dopa was weighed and dissolved in 160mL of Tris-HCl (10 mM, pH 8.5) buffer; 40mg of COOH-MPs were then weighed out and dispersed in the dopa solution and stirred magnetically at room temperature (25 ℃) for 24h. After the reaction is finished, the product polyDOPA-MPs is obtained by magnetic separation, washed for 3 times by ethanol and deionized water, and finally dried for later use at 50 ℃ under the protection of nitrogen.

TEM observation of COOH-MPs and polyDOPA-MPs prepared in steps (1) and (2) was carried out, and the results are shown in FIGS. 1C and 1D. As can be seen from FIGS. 1C and 1D, the prepared COOH-MPs and polyDOPA-MPs have relatively average particle size distribution, the average particle sizes are respectively 115 +/-10 nm and 132 +/-8 nm, and the prepared COOH-MPs and the prepared polyDOPA-MPs have good dispersibility. The increase of the particle size after modification of the polydopa shows that the polydopa coating is successfully modified on the surface of COOH-MPs, and the average thickness of the coating is 8.5 +/-1.5 nm. The polydopa coating is also clearly visible in fig. 1D.

FT-IR spectrum measurement was performed on COOH-MPs and polyDOPA-MPs prepared in steps (1) and (2), and the results are shown in FIG. 2B. In FIG. 2B, the FT-IR spectrum of polyDOPA-MPs showed the presence of Fe-O vibration peak (580 cm) in COOH-MPs ^-1 ) And, simultaneously, at 1650cm ^-1 、1455cm ^-1 、1384cm ^-1 The characteristic infrared absorption peak of polyDOPA appears, thereby indicating that the polyDOPA-MPs are successfully prepared.

Hysteresis loops were measured for COOH-MPs and polyDOPA-MPs prepared in steps (1) and (2), and the results are shown in FIG. 2D. FIG. 2D shows that by comparing the magnetic responsivity of COOH-MPs and polyDOPA-MPs under different magnetic field values, the magnetic saturation of the two is 68.8emu/g and 65.0emu/g, respectively. Both of them are proved to have good magnetic responsiveness, and the reduction of the magnetic responsiveness after poly-dopa modification is proved to successfully prepare poly-DOPA-MPs.

Zeta potential measurements were carried out on the COOH-MPs and polyDOPA-MPs prepared in steps (1) and (2), and the results are shown in FIG. 3B. FIG. 3 shows that the difference between the charges of COOH-MPs and polyDOPA-MPs at different pH values is observed by comparing the Zeta potentials of the two at different pH values, thereby illustrating the difference between the surface functional groups of the two, and thus, it can be judged that the polyDOPA-MPs have been successfully prepared.

Example 3

A method for extracting and purifying coli DH5 alpha genome DNA, which comprises the following steps:

(1) Respectively dispersing GO-MPs prepared in example 1 and polyDOPA-MPs prepared in example 2 in Tris-HCl-EDTA buffer (10 mmol/L Tris,1mmol/L EDTA, pH value 5.8) to obtain 100mg/mL solution; respectively obtaining GO-MPs suspension and polyDOPA-MPs suspension;

(2) 1.5mL of E.coli DH5 alpha culture solution was centrifuged at 10000rpm for 1min to separate the bacteria from the medium. Subsequently, 300. Mu.L of a lysis solution (25 mmol/L Tris-HCl,20mmol/L NaAc,1mmol/L EDTA,1% SDS, pH 7.8) was added to the pellet, and the pellet was incubated in a water bath at 37 ℃ for 1 hour with occasional shaking during the incubation period to ensure the lysis effect. After the incubation was completed, 100. Mu.L of NaCl (4.0 mol/L) was added thereto and mixed well, and the mixture was centrifuged (10000 rpm) at 4 ℃ for 20min;

(3) mu.L of the supernatant (bacterial lysate) was taken, 500. Mu.L of the adsorption solution (10 mmol/L Tris-HCl and 1mmol/L EDTA, pH 5.8) and 200. Mu.L NaCl (4 mol/L) were added, and after thorough mixing, 100. Mu.L (40 mg/mL) of GO-MPs suspension was added. After incubation of the mixture at room temperature for 10min with gentle shaking, the supernatant was separated magnetically and carefully transferred to a new centrifuge tube.

(4) mu.L (40 mg/mL) of polyDOPA-MPs suspension was added to a fresh centrifuge tube. After incubation of the mixture at room temperature for 10min with gentle shaking, the mixture was magnetically separated and the supernatant was carefully removed. polyDOPA-MPs were washed twice with 70% ethanol (V/V) and left to dry at room temperature. Then, 400. Mu.L of an eluent (10 mmol/L Tris-HCl and 1mmol/L EDTA, pH 8.8) was added thereto, and incubation was performed at room temperature with gentle shaking for 15min to elute the adsorbed DNA from polyDOPA-MPs. The supernatant was carefully removed, transferred to a new centrifuge tube and sealed at 4 ℃ until use.

And (3) DNA quality detection: extraction of A of DNA ₂₆₀ /A ₂₈₀ The ratio of (A) to (B) of about 1.8 indicates that the purity and quality of the extracted DNA are better. When the extraction is carried out with double magnetic particles, A of the extracted DNA ₂₆₀ /A ₂₈₀ The ratio of (A) to (B) is 1.85, which proves that the GO-MPs well remove RNA and obtain high-quality DNA. At the same time, the DNA purified only by using polyDOPA-MPs was compared with the A of the genomic DNA of Escherichia coli extracted only by using polyDOPA-MPs ₂₆₀ /A ₂₈₀ The ratio of (A) to (B) is 2.01, which indicates that GO-MPs play a good role in purifying DNA.

PCR amplification experiments:

reagents used in the PCR amplification process include: mu.L of DNA template, 10. Mu.L of 2 XPCR mix, 1. Mu.L each of the upstream and downstream primers (10 mmol/L), and a total reaction volume of 20. Mu.L. The selected upstream and downstream primers are designed aiming at the proB A gene of E.coli DH5 alpha, and the size of a target amplification fragment is 2500bp. The sequences of the upstream and downstream primers are as follows:

an upstream primer: 5'-ATAGGCGCCGCAACCGACGACAGTCCTGC-3'

A downstream primer: 5'-TTTGGCGCCTGTTCACGAACGTGAATCAC-3'

The PCR reaction was performed on a PCR amplification apparatus. The specific amplification process is that the reaction solution is firstly pre-denatured at 94 ℃ for 3min, and then the cyclic reaction is carried out according to the following conditions: denaturation (94 ℃ C., 30 s), annealing (56 ℃ C., 30 s), extension (72 ℃ C., 30 s), and extension for 10min after 35 cycles. After the reaction is finished, the PCR product is stored in an environment of 4 ℃ for standby.

Preparing 1% agarose gel, taking 6 mu L of PCR product, uniformly mixing with 1 mu L of 6 × loading buffer, dripping into a sample application hole, and performing 90V constant voltage electrophoresis for 45min by taking a DNA marker as a reference. After staining the gel with Gold view, it was imaged with a gel imaging system, and the results are shown in FIG. 4A.

By comparing the standard DNA fragment in lane M and the PCR product of E.coli DH 5. Alpha. DNA template extracted by the double magnetic particle method in lanes 1 and 2, a bright amplified band with a size of 2500bp can be seen. It is demonstrated that the GO-MPs and polyDOPA-MPs of the present invention can be successfully used for DNA extraction.

Example 4

The corn genome DNA extracting and purifying process includes the following steps:

(1) The GO-MPs and polyDOPA-MPs suspensions were prepared as in example 3;

(2) Taking the transgenic corn kernels, and fully grinding the transgenic corn kernels to powder under the protection of liquid nitrogen. Subsequently, 300. Mu.L of lysis solution (25 mmol/L Tris-HCl,20mmol/L NaAc,1mmol/L EDTA,1% SDS, pH 7.8) was added to 5mg of the powder; the rest of the procedure was the same as in example 3;

and (3) DNA quality detection: extraction of A of DNA ₂₆₀ /A ₂₈₀ The ratio of (A) to (B) of about 1.8 indicates that the purity and quality of the extracted DNA are better. When the extraction is carried out with double magnetic particles, A of the extracted DNA ₂₆₀ /A ₂₈₀ The ratio of (A) to (B) is 1.70, which proves that the GO-MPs well remove RNA and obtain high-quality DNA. Meanwhile, the DNA purified by only using polyDOPA-MPs is compared with the A of the corn genome DNA extracted by only using the polyDOPA-MPs ₂₆₀ /A ₂₈₀ The ratio of (A) to (B) is 1.43, which indicates that GO-MPs play a good role in purifying DNA.

PCR amplification experiments:

the experimental method is the same as that in example 3, the selected upstream and downstream primers are designed aiming at the 35S gene of the transgenic corn, and the size of the target amplified fragment is 190bp. The sequences of the upstream and downstream primers are as follows:

an upstream primer: 5'-GCTCCTACAAATGCCATCA-3'

A downstream primer: 5'-GATAGTGGGATTGTGCGTCA-3'

The results are shown in FIG. 4B, and by comparing the standard DNA fragment in lane M, lanes 1 and 2 are PCR products of the DNA template of transgenic maize extracted by the double-magnetic particle method, a bright amplified band with a size of 190bp can be seen. The GO-MPs and polyDOPA-MPs can be successfully used for extracting and separating the genomic DNA of the transgenic corn.

The above embodiments are preferred embodiments of the present invention, but the present invention is not limited to the above embodiments, and any other changes, modifications, substitutions, combinations, and simplifications which do not depart from the spirit and principle of the present invention should be construed as equivalents thereof, and all such changes, modifications, substitutions, combinations, and simplifications are intended to be included in the scope of the present invention.

Claims

1. A DNA extraction and purification method is characterized by comprising the following steps:

(1) Sampling lysate, adding an adsorption solution and a NaCl solution, uniformly mixing, adding GO-MPs suspension, incubating for at least 10min, performing magnetic separation, and taking supernatant;

(2) Adding polyDOPA-MPs suspension into the supernatant, incubating for at least 10min, performing magnetic separation, discarding the supernatant, washing the obtained precipitate with ethanol solution, and drying at room temperature; adding eluent, incubating for at least 15min to elute adsorbed DNA from polyDOPA-MPs, performing magnetic separation, and collecting supernatant containing purified DNA;

the preparation method of the GO-MPs comprises the following steps:

dissolving ferric trichloride in ethylene glycol, adding anhydrous sodium acetate and polyethylene glycol 4000, uniformly mixing, heating the obtained mixture to 160-220 ℃, and reacting for 8-22 h to obtain PEG-MPs; adding the synthesized PEG-MPs into the GO solution, heating and stirring, and separating to obtain GO-MPs;

the preparation method of the polyDOPA-MPs comprises the following steps:

dissolving ferric trichloride in a diethylene glycol-ethylene glycol mixed solvent, adding anhydrous sodium acetate and sodium acrylate, uniformly mixing, heating the obtained mixture to 160-220 ℃, and reacting for 10-20 h to obtain COOH-MPs; adding the synthesized COOH-MPs into an alkaline solution of dopa, heating, stirring and separating to obtain polyDOPA-MPs;

the mass ratio of PEG-MPs to GO is (1~4) 1;

in the mixture in the preparation process of the polyDOPA-MPs, the mass ratio of anhydrous sodium acetate to sodium acrylate is (1-3) to 1;

in the diethylene glycol-ethylene glycol mixed solvent, the volume ratio of diethylene glycol to ethylene glycol is (1-4) to 1;

the mass ratio of the dopa to the COOH-MPs is (2-4) to 1.

2. The method of claim 1, wherein: the adsorption solution in the step (1) is Tris-HCl-EDTA buffer solution, wherein the concentration of Tris is 10mmol/L, the concentration of EDTA is 1mmol/L, and the pH value is 2.8-6.8.

3. The method of claim 1, wherein: the eluent in the step (2) is Tris-HCl-EDTA buffer solution, wherein the concentration of Tris is 10mmol/L, the concentration of EDTA is 1mmol/L, and the pH value is 8.8-10.8.

4. The method of claim 1, wherein: the sample lysate in the step (1) is prepared by the following steps:

B. adding the lysate, incubating in a water bath at 37 ℃ for at least 1h, adding NaCl solution, mixing, and centrifuging the mixture to obtain supernatant as tissue lysate.

5. The method of claim 4, wherein: the lysis solution contains 25mmol/L Tris-HCl,20mmol/L NaAc,1mmol/L EDTA and 1% SDS, and has a pH value of 7.8.

6. The method of claim 1, wherein: the GO-MPs suspension and the polyDOPA-MPs suspension are respectively dispersed in Tris-HCl-EDTA buffer solution, and the concentration of the GO-MPs suspension and the polyDOPA-MPs suspension is 50-300 mg/mL.