CN112662665A - Method for extracting plasmid DNA by paramagnetic particle method and kit for extracting plasmid DNA - Google Patents

Abstract

The invention relates to a kit for extracting plasmid DNA, which comprises a magnetic separation medium and at least one plasmid extraction reagent, wherein the magnetic separation medium is a magnetic nano microsphere with the particle size of 50-1000 nm. The invention also provides a method for extracting plasmid DNA by a paramagnetic particle method. The method of the invention realizes magnetic separation by utilizing superparamagnetism of a magnetic separation medium, omits a centrifugation step, is quick, simple and convenient, has low cost and is easy to realize automation.

Description

Method for extracting plasmid DNA by paramagnetic particle method and kit for extracting plasmid DNA

Technical Field

The invention relates to the technical field of molecular biology, in particular to purification of nucleic acid, especially plasmid. More particularly, the invention relates to a magnetic separation technology, which is a new method for quickly, simply and conveniently purifying plasmid DNA. The invention also includes the use of the method can be purified plasmid DNA kit.

Background

The plasmid is a circular small molecule double-stranded DNA independently existing in cells such as bacteria or molds, exists in host cells in a supercoiled state, and has a size of 1-200 kb. Each plasmid is a stable genetic element that is independent of the cell extrachromosomal entity, is usually constantly episomal, but under certain conditions is reversibly integrated into the host chromosome, replicated as the chromosome replicates, and is transmitted to progeny through cell division. The plasmid can maintain a constant copy number in the daughter cells and express the carried genetic information. However, they rely on host-encoded enzymes and proteins for replication and transcription, and cannot survive if leaving the host cell (see: Sunjahan, principles and methods of genetic engineering. Beijing: civil military Press, 2001). Plasmid vectors are constructed artificially on the basis of natural plasmids for adaptation to laboratory procedures, and are important tools for carrying foreign genes into bacteria for amplification or expression. At present, the vector used in genetic engineering is mainly plasmid, and the extraction and purification of plasmid DNA are important preconditions for genetic engineering, so that the efficient and rapid purification of plasmid has important significance.

Plasmid DNA extraction methods are numerous and all comprise the following 3 main steps: 1) culturing the bacteria to amplify the plasmid; 2) collecting and lysing bacteria; 3) isolating and purifying the plasmid DNA. The first problem encountered in plasmid purification is the release of DNA by lysing the bacteria. Cell lysis methods are many, and currently, alkali lysis methods (Birnborn and Dohly, Nucl. acids Res.,7: 1513-. The first two methods are more vigorous and are applicable to smaller plasmids (<15 kb). Detergent lysis rules are mild and are generally used for isolation of large plasmids (>15 kb). The organic solvent method, which is most commonly phenol/chloroform extraction, can effectively remove various protein contaminants, but because it does not use salts, endotoxin contamination is easily generated, and RNA and ssDNA are also difficult to remove. In addition, treatment of nucleic acids with phenols tends to produce nicked nucleic acids, thereby affecting the stability of the nucleic acids.

A second consideration is how to effectively isolate plasmid DNA from other bacterial components such as proteins, chromosomal DNA and metabolites, etc., using various methods. All purification methods commonly used take advantage of the relatively small size and covalently closed circular nature of plasmid DNA. Generally, the simplest method is to isolate the nucleic acid by adding salts, such as LiCl, to precipitate the proteins in the cells, but this method is inefficient. In addition, ethanol precipitation is also commonly used for nucleic acid isolation, but this method is prone to contamination by RNA and cannot completely remove ssDNA and proteins. Cesium chloride-ethidium bromide gradient equilibrium centrifugation is the first choice for preparing large amounts of plasmid DNA. However, this method is only suitable for large scale extraction, requires expensive ultracentrifugation equipment and is relatively time-consuming, usually requires at least 48 hours for purification at a time, and moreover uses toxic materials such as toxic reagents and mutagens, and many alternatives such as chromatography have been developed subsequently. The principle of chromatography is to introduce the target substance into the mobile phase contacting the stationary phase by using the difference of physical or chemical properties of the components of the sample to be separated, and then to achieve the separation and purification through a series of interactions between the stationary phase and the mobile phase. Currently, anion exchange chromatography (WO99/63076(The Immune Response Corp.), hydrophobic chromatography (US6441160(Tosoh Corp.) and gel chromatography (US6011148(Megabios Corp.) are mainly used, but these methods are relatively expensive, time-consuming and difficult to automate, and Ronganine et al (CN1313478C) disclose a new method for rapid and mass purification of plasmids, which uses specially treated diatomaceous earth to specifically adsorb plasmid DNA and then elutes DNA in a low-salt or aqueous state, which takes a short time to complete The entire process in 90 minutes compared to chromatography or cesium chloride, and 10-15. mu.g DNA can be obtained from 1.5mL of bacterial solution (OD600 is 3.5) although it is simpler than cesium chloride and chromatography, but requires multiple centrifugation during separation and purification, which is complicated in magnetic separation operation compared to magnetic nano-micro material separation techniques, it has unique advantages over other methods: 1) the size is small, the specific surface area is large, DNA can be efficiently extracted, and the requirement of extracting the DNA of a trace biological sample is met; 2) the surface of the material can be chemically modified so as to carry out specific adsorption with DNA; 3) the quantity of functional groups on the surface of the material is rich, and the requirement of quantitative DNA can be realized by controlling the quantity of the functional groups; 4) the superparamagnetic nano material has good resuspension property after the magnetic field is removed, so that the requirements of an automatic operation platform can be met, a DNA sample can be extracted in a large scale, and the influence of human factors is reduced; 5) the method is rapid, simple and convenient in separation operation, is suitable for most biological samples, and can obtain satisfactory DNA extraction results for beginners with less experience.

Disclosure of Invention

Therefore, the technical problem to be solved by the invention is to overcome the defects of complicated plasmid DNA extraction steps and low efficiency in the prior art, and provide a novel method for purifying plasmid DNA which is rapid, simple, convenient, high-efficiency, high-quality, high in cost performance and harmless.

In order to solve the technical problems, the invention provides a kit for extracting plasmid DNA, which comprises a magnetic separation medium and at least one plasmid extraction reagent, wherein the magnetic separation medium is a magnetic nano microsphere with the particle size of 50-1000 nm.

In the invention, the magnetic nano-microsphere is modified by organic molecules, comprises at least one silicon hydroxyl group, has high magnetic component (> 50%) and superparamagnetism, and has no porous matrix inside.

In one embodiment of the invention, the plasmid extraction reagent comprises a bacterial suspension R1, thallus lysates R2 and R3, an adsorption buffer R4, a washing solution R5 and an eluent R6.

In one embodiment of the invention, the composition of the bacterial suspension R1 is: 10mmol/L EDTA, 50mmol/L Tris-HCl (pH 7.5), 30-50. mu.g/mL RNase A.

In one embodiment of the present invention, the lysate R2 of the cells is 0.4mol/L NaOH solution, and the lysate R3 of the cells is 2% SDS solution.

In one embodiment of the present invention, the composition of the adsorption buffer R4 is: 2.5-4 mol/L guanidine hydrochloride (pH 5.0), 200-300 mmol/L sodium chloride, 15-50 mmol/L potassium acetate, 1-3% glacial acetic acid, 12-18% isopropanol.

In one embodiment of the invention, the wash solution is 70% ethanol.

In one embodiment of the invention, the composition of the elution buffer is: 10mmol/L Tris-HCl, 1mmol/L EDTA.

The invention also provides a method for extracting plasmid DNA by a magnetic bead method, a kit for extracting the plasmid DNA is used, and the method comprises the following steps,

(1) contacting a liquid containing plasmid DNA with the magnetic separation medium such that plasmid DNA is adsorbed onto the outer surface of the magnetic separation medium;

(2) and eluting the plasmid DNA from the surface of the magnetic separation medium by means of elution, thereby recovering the plasmid DNA.

In an embodiment of the present invention, the method specifically includes:

(1) taking 1.5mL of bacterial liquid, centrifuging at the rotating speed of 12000rpm for 30 seconds at room temperature, and then removing supernatant;

(2) adding the bacterial suspension R1 into the precipitate, and performing vortex oscillation to uniformly mix the bacterial suspension R1;

(3) adding a freshly prepared thallus lysate into the mixed solution, and turning the test tube upside down until the solution is clear;

(4) adding adsorption buffer solution R4 and magnetic separation medium, reversing the tube from top to bottom for 5-10 times, and standing at room temperature for 5min to adsorb plasmid DNA;

(5) magnetically separating and recovering the DNA-medium complex;

(6) adding a washing solution R5, and washing the DNA-medium complex twice;

(7) eluent R6 was added and left at room temperature for 3min to recover the elution buffer containing plasmid DNA.

In one embodiment of the present invention, the lysate is obtained by mixing lysate R2 and lysate R3 at a ratio of 1: 1.

Compared with the prior art, the technical scheme of the invention has the following advantages:

1) the operation is simple and convenient, and expensive instruments and facilities are not needed;

2) the method has the advantages of no need of centrifugation, high separation speed, short time consumption, and capability of completing the whole process within 12 minutes;

3) the yield is high, for 1.5mL of bacterial liquid, the high-copy plasmid with the bacterial growth density OD600 of about 0.09 is cultured for 16 hours conventionally, and 10-14 mu g of DNA can be captured by 1mg of magnetic nano-microspheres;

4) the purity is high, protein and RNA pollution are avoided, and the purified plasmid DNA can be used for molecular biology experiments such as enzyme digestion, PCR and eukaryotic cell transfection;

5) the price is low, and the cost is low because the magnetic nano-microspheres synthesized by the invention and the buffer solution prepared by the invention are mostly domestic organic and inorganic reagents;

6) no toxic reagent is used, the operation is safe, and the worry of environmental pollution is avoided;

7) the operation is carried out at room temperature and the automation is easy.

Drawings

In order that the present disclosure may be more readily and clearly understood, reference is now made to the following detailed description of the present disclosure taken in conjunction with the accompanying drawings, in which:

FIG. 1 shows the electrophoretogram of the plasmid DNA obtained by purification and the cleavage and PCR products of example 2.

Detailed Description

The present invention is further described below in conjunction with the following figures and specific examples so that those skilled in the art may better understand the present invention and practice it, but the examples are not intended to limit the present invention.

The term "plasmid" as used herein is interchangeable with the term "plasmid DNA" and includes various plasmid forms, namely open circular (oc, also known as nicked plasmid DNA) and supercoiled (ccc) plasmid DNA.

The term "separation medium" refers to a solid phase material in magnetic separation technology. By this is meant a magnetic nanomaterial that has been surface functionalized.

As used herein, the "surface" of the separation medium means the outer surface.

The term "purification" means herein the separation of the desired component from other components.

Example 1: separation and purification of pETBlue plasmid DNA

1.5ml of the bacterial pellet containing pETBlue plasmid, which was cultured overnight, was suspended in 100. mu. L R1 solution, 150. mu.L of freshly prepared lysate (R2 and R3 mixed at 1: 1) was added, and the solution was inverted several times upside down until it was clear. Then 300 mul of adsorption solution R4 and 100 mul of 3mg/mL magnetic microspheres are added, the tube is inverted from top to bottom for 5-10 times, and then the mixture is placed at room temperature for 5 min. And (4) placing the centrifugal tube on a magnetic separation frame for about 5-10 seconds, and discarding the supernatant. Plasmid DNA is selectively adsorbed on the surface of the microspheres. The microsphere-DNA complex was washed twice with 500. mu.L 70% ethanol.

After ethanol was completely evaporated at room temperature, 100. mu.L of Tris-EDTA (pH 8.0) solution was added to the complex, and the precipitate was blown up and down to fully suspend the microspheres, and then left at room temperature for 3 minutes to elute DNA. The centrifuge tube was then placed on a separation rack, the eluate was recovered, and its purity was checked by agarose gel electrophoresis and uv spectroscopy and quantified.

The pEGFP-N1 plasmid DNA purified by this method had a yield of 14. mu.g and a high purity, and was free from contamination with genomic DNA, RNA and proteins.

Example 2: isolation of pEGFP-N1 plasmid DNA

1) The sample was 1.5mL of bacteria of pEGFP-N1 plasmid, and the isolation method and the procedure were carried out as described in reference to example 1.

2) The sample was 1.5mL of pEGFP-N1 plasmid bacteria, and plasmid DNA was extracted using the QIAprep Spin Miniprep Kit (Qiagen, Inc., available from commercial Inc.) according to the instructions. Detecting the DNA obtained by separation and purification by agarose gel electrophoresis;

3) taking 1 mu g of DNA purified by the method and 1 mu L of DNA purified by a Qiagen company kit, adding 1 mu L of EcoR I enzyme and 2 mu L of 10x buffer, complementing the reaction system to be 20 mu L by using sterilized ultrapure water, carrying out water bath constant temperature reaction at 37 ℃ for 3 hours, sampling and carrying out gel electrophoresis;

4) PCR experiment: taking 10ng of each plasmid DNA extracted by the two methods as a template, adding EcoRI and Not I enzyme cutting sites into an upstream primer and a downstream primer 5 ', wherein the upstream primer is 5'-TAGAATT CATGGTGAGCAAGGGC-3'; the downstream primers were 5'-AATGCGGCCGCTTACTTGTA-3' (500pmol/L) each at 1. mu.L. 10 XExTaq buffer (5. mu.L), dNTP mix (2.5 mmol/L each) (4. mu.L), Takara ExTaq (5U/. mu.L) (0.5. mu.L) were added thereto, and the reaction system was finally made up to 50. mu.L with sterilized ultrapure water. The reaction conditions are divided into three stages: firstly, denaturation is carried out for 5min at 94 ℃; then, denaturation at 94 ℃ for 30s, annealing at 60 ℃ for 30s, and extension at 72 ℃ for 1min were carried out, and this portion was cycled 30 times. And finally, extending for 10min at 72 ℃, and taking the PCR product to carry out gel electrophoresis for detection after the extension is finished.

FIG. 1 shows the electrophoretogram of the purified plasmid DNA and its digestion and PCR products. In FIG. 1, 1-6 are plasmid DNA extracted by Qiagen kit and its products of restriction enzyme digestion and PCR, and 7-12 are plasmid DNA purified by the method of the present invention and its products of restriction enzyme digestion and PCR. The lane samples are as follows: 1 and 2: plasmid DNA; 3 and 4: performing enzyme digestion on the product; 5 and 6: PCR products; 7 and 8: plasmid DNA; 9 and 10: performing enzyme digestion on the product; 11 and 12: PCR products; m: mu.L of Wide range DNA marker (500-12000bp), 2. mu.L of each sample was added to lanes 1-12.

The results in FIG. 1 show that the plasmid DNA extracted by the present invention has purity and yield not inferior to plasmid purification kits of foreign first-class enterprises, and can be applied to downstream molecular biology experiments.

It should be understood that the above examples are only for clarity of illustration and are not intended to limit the embodiments. Other variations and modifications will be apparent to persons skilled in the art in light of the above description. And are neither required nor exhaustive of all embodiments. And obvious variations or modifications therefrom are within the scope of the invention.

Claims (10)

1. A kit for extracting plasmid DNA, which is characterized in that: the method comprises a magnetic separation medium and at least one plasmid extraction reagent, wherein the magnetic separation medium is a magnetic nano microsphere with the particle size of 50-1000 nm.

2. The kit for extracting plasmid DNA according to claim 1, wherein: the plasmid extraction reagent comprises a bacterial suspension R1, thallus lysate R2 and R3, an adsorption buffer R4, a washing solution R5 and an eluent R6.

3. The kit for extracting plasmid DNA according to claim 2, wherein: the composition of the bacterial suspension R1 was: 10mmol/L EDTA, 50mmol/L Tris-HCl (pH 7.5), 30-50. mu.g/mL RNase A.

4. The kit for extracting plasmid DNA according to claim 2, wherein: the cell lysate R2 is 0.4mol/L NaOH solution, and the cell lysate R3 is 2% SDS solution.

5. The kit for extracting plasmid DNA according to claim 2, wherein: the adsorption buffer R4 comprises the following components: 2.5-4 mol/L guanidine hydrochloride (pH 5.0), 200-300 mmol/L sodium chloride, 15-50 mmol/L potassium acetate, 1-3% glacial acetic acid, 12-18% isopropanol.

6. The kit for extracting plasmid DNA according to claim 2, wherein: the washing solution was 70% ethanol.

7. The kit for extracting plasmid DNA according to claim 2, wherein: the composition of the elution buffer was: 10mmol/L Tris-HCl, 1mmol/L EDTA.

8. A method for extracting plasmid DNA by a magnetic bead method is characterized in that: the kit for extracting plasmid DNA according to any one of claims 1 to 7, comprising the steps of,

(1) contacting a liquid containing plasmid DNA with the magnetic separation medium such that plasmid DNA is adsorbed onto the outer surface of the magnetic separation medium;

(2) and eluting the plasmid DNA from the surface of the magnetic separation medium by means of elution, thereby recovering the plasmid DNA.

9. The method for extracting plasmid DNA by magnetic bead method according to claim 8, wherein: the method specifically comprises the following steps:

(1) taking 1.5mL of bacterial liquid, centrifuging at the rotating speed of 12000rpm for 30 seconds at room temperature, and then removing supernatant;

(2) adding the bacterial suspension R1 into the precipitate, and performing vortex oscillation to uniformly mix the bacterial suspension R1;

(3) adding a freshly prepared thallus lysate into the mixed solution obtained in the step (2), and turning the test tube upside down until the solution is clear;

(4) adding adsorption buffer solution R4 and magnetic separation medium, reversing the tube from top to bottom for 5-10 times, and standing at room temperature for 5min to adsorb plasmid DNA;

(5) magnetically separating and recovering the DNA-medium complex;

(6) adding a washing solution R5, and washing the DNA-medium complex twice;

(7) eluent R6 was added and left at room temperature for 3min to recover the elution buffer containing plasmid DNA.

10. The method for extracting plasmid DNA by magnetic bead method according to claim 9, wherein: the cell lysate is obtained by mixing cell lysate R2 and R3 according to the ratio of 1: 1.

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