阅读说明：本技术 一种用纳米磁珠提取核酸的试剂盒及提取方法 (Kit for extracting nucleic acid by using nano magnetic beads and extraction method ) 是由 崔大祥 刘泽熙 王丹 刘关 于 2021-08-25 设计创作，主要内容包括：本发明涉及一种用生物纳米磁珠法提取DNA的试剂盒及其提取方法,该试剂盒包括了裂解液,去抑制剂,洗涤液,洗脱液及磁珠。利用磁珠法操作简便且适用范围广的优点,用处理过的生物纳米磁珠在特定溶液环境中对核酸进行高特异性的吸附与结合,以便于裂解和分离蛋白质、多糖等生物大分子结构的杂质,从而提升结果核酸产物的产出及纯度。本发明的试剂盒适用于短片段DNA,且具有简易,安全,成本廉价等优点。(The invention relates to a kit for extracting DNA by a biological nanometer magnetic bead method and an extraction method thereof. The magnetic bead method is utilized to have the advantages of simple and convenient operation and wide application range, and the treated biological nano magnetic beads are used for carrying out high-specificity adsorption and combination on nucleic acid in a specific solution environment so as to facilitate the cracking and separation of impurities with biological macromolecular structures such as protein, polysaccharide and the like, thereby improving the yield and purity of the nucleic acid product. The kit is suitable for short-fragment DNA, and has the advantages of simplicity, safety, low cost and the like.)

1. The utility model provides a kit of using nanometer magnetic bead method to extract DNA which characterized in that, has included the lysate, has removed inhibitor, washing liquid, eluant and biological nanometer magnetic bead, wherein:

solution I: consists of 10 mM-100 mM Tris-Cl buffer solution, 5 mM-20 mM Ethylene Diamine Tetraacetic Acid (EDTA) solvent, 0.5% -3.5% detergent and 200 mM-300 mM guanidine salt;

solution II: consists of 10 mM-100 mM Tris-Cl and 5 mM-10 mM EDTA;

solution III: consists of high-purity ethanol with the mass concentration of not less than 75 percent;

solution IV: ultrapure water/deionized water;

the biological nanometer magnetic beads are silicon hydroxyl nanometer magnetic bead suspension stored in a NaCl buffer solution and ferroferric oxide magnetic cores.

2. The kit for extracting DNA by using nanobagnetic bead method according to claim 1, wherein the solvent Tris-Cl and EDTA has pH value of about 7.4.

3. The kit for extracting DNA by using a nano magnetic bead method according to claim 1, wherein the hydroxyl nano magnetic bead suspension is a silicon hydroxyl superparamagnetic nano magnetic bead, a ferroferric oxide magnetic core with an average particle size of 1 μm, and is stored in a NaCl buffer solution.

4. The kit for extracting DNA using nanobead according to claim 1 or 3, wherein the concentration of the nanobead suspension is 25 mg/ml.

5. The kit for extracting DNA by using the nanobagnetic bead method according to claim 1, wherein in the solution I, the guanidinium salt is guanidinium hydrochloride, the de-inhibitor is proteinase k, and the detergent is Sodium Dodecyl Sulfate (SDS) and polyethylene glycol octyl phenyl ether (Triton x-100).

6. The kit for extracting DNA by using the nano magnetic bead method as claimed in claim 1, wherein the ethanol with a mass concentration of not less than 75% in the solution III is absolute ethanol, and the volume ratio of the ethanol to the mixed solution of the magnetic bead suspension is 3: 1.

7. A method for extracting DNA using the kit according to any one of claims 1 to 6, comprising the steps of:

1) reacting the solution I with the inhibitor, ribonucleic acid Carrier RNA, biological nano magnetic bead suspension and a sample to be extracted, heating and fully cracking to enable the magnetic beads to be adsorbed and combined with nucleic acid;

2) cleaning the mixed adsorption solution by using the solutions II and III, and separating protein and polysaccharide impurities to be eluted;

3) and washing and desorbing the nucleic acid by using the solution IV to obtain a purified sample.

8. The extraction method according to claim 7, wherein a magnetic device is used in combination with the bio-nano magnetic beads to promote the magnetic beads to be suspended in the liquid.

9. The extraction method according to claim 8, wherein the magnetic device is a simple magnetic rack for holding the buffer solution of the sample and the magnetic beads to assist the adsorption separation washing process.

10. The extraction method according to any one of claims 7 to 9, characterized by comprising the steps of:

adding 20-500 mu L of sample, 100-500 mu L of solution I, 20-100 mu L of inhibitor, 1-5 mu L of Carrier RNA and 10-30 mu L of silicon hydroxyl nano magnetic bead suspension into a 1.5ml centrifuge tube, slightly shaking and uniformly mixing, and placing in a warm bath at 55-60 ℃ for 10-30 minutes;

step two, adding 250-; and/or the like, and/or,

adding 150-; and/or the like, and/or,

step four: adding the solution III and repeating the third step;

step five: adding 50-150 μ L of solution IV, mixing, centrifuging, bathing at 55-60 deg.C for 10-20 min, placing on magnetic frame, standing for 2-3 min, sucking solution, removing magnetic beads to obtain final product, and storing properly.

Technical Field

The invention belongs to the field of molecular biology, and particularly relates to a kit for extracting nucleic acid by using nano magnetic beads and an extraction method, in particular to a simple and convenient nucleic acid extraction method for manually extracting DNA by using nano magnetic beads.

Background

Nucleic acid is divided into DNA and RNA, is an essential basic unit in the composition of life forms, and widely exists in animal and plant cells and microorganisms. Among them, DNA carries necessary genetic information for synthesizing RNA and protein, and is the most important macromolecular substance in the mechanism of life development and normal operation.

The nucleic acid extraction technology is the most basic and key link in the field of molecular biology, brings the possibility of obtaining target genes, providing a PCR replication template and being used for establishing a library for subsequent downstream experiments, and provides a foundation for better understanding of a biomolecule structure and development and functional application of people.

Nucleic acid is an acidic macromolecular substance, participates in a plurality of biological reactions such as genetic information expression, protein reaction synthesis and the like, and is an indispensable basic substance in the detection field. The existing nucleic acid purification and separation mainly aims to separate nucleic acid (DNA/RNA) and other impurities such as protein, polysaccharide, salt and the like through test steps to obtain purified nucleic acid molecules. On the premise of ensuring that the integrity of the internal structure is not damaged as much as possible, other pollution is eliminated as much as possible, and meanwhile, the output is amplified. Common methods include chloroform extraction, centrifugation column method, magnetic bead method, etc. Most of them need to be subjected to various steps such as cracking, separation, purification and washing.

The magnetic bead method is a relatively newly developed nucleic acid extraction method, and the surfaces of nano microbead particles are optimized and modified to prepare the silicon hydroxyl nano magnetic beads with superparamagnetism. Through the function of the surface chemical functional groups, the nucleic acid molecules can form specific adsorption and combination under different conditions (solution salinity, pH value change control and the like), and pure nucleic acid products to be obtained are reserved and separated again in a way of releasing adsorption. The method can avoid using toxic organic solvents such as chloroform and the like, simplifies partial manual operation, relatively simplifies the implementation method and reagent preparation, and provides new possibility for high-flux full-automatic flow of instruments.

Disclosure of Invention

The invention aims to provide a kit for extracting nucleic acid by using nano magnetic beads.

Yet another object of the present invention is to: provides a method for extracting nucleic acid by the kit, which achieves the aim of applying the method to the follow-up research such as downstream amplification sequencing and the like by extracting nucleic acid.

The purpose of the invention is realized by the following scheme: a kit for extracting DNA by a nano magnetic bead method comprises lysis solution, inhibitor removal, washing solution, eluent and biological nano magnetic beads, wherein:

solution I: consists of 10 mM-100 mM Tris-Cl buffer solution, 5 mM-20 mM Ethylene Diamine Tetraacetic Acid (EDTA) solvent, 0.5% -3.5% detergent and 200 mM-300 mM guanidine salt;

solution II: consists of 10 mM-100 mM Tris-Cl and 5 mM-10 mM EDTA;

solution III: consists of high-purity ethanol with the mass concentration of not less than 75 percent;

solution IV: ultrapure water/deionized water;

the biological nano magnetic beads are silicon hydroxyl nano magnetic bead suspension stored in NaCl buffer solution.

Wherein, the preferable pH value of Tris-Cl and EDTA solvent is approximately equal to 7.4.

Preferably, the hydroxyl nano magnetic bead suspension is silicon hydroxyl superparamagnetic nano magnetic beads and ferroferric oxide magnetic cores, the average particle size is 1 mu m, and the silicon hydroxyl superparamagnetic nano magnetic beads and the ferroferric oxide magnetic cores are stored in a NaCl buffer solution.

Further, the concentration of the nano magnetic bead suspension is 25 mg/ml.

In the solution I, the guanidine salt is guanidine hydrochloride, the de-inhibitor is proteinase k, and the detergent is Sodium Dodecyl Sulfate (SDS) and polyethylene glycol octyl phenyl ether (Triton x-100).

In the solution III, ethanol with the mass concentration not lower than 75% can be replaced by absolute ethanol, and the volume ratio of the ethanol to the magnetic bead suspension mixed solution is 3: 1.

The invention provides a method for extracting DNA by using the kit, which comprises the following steps:

1) reacting the solution I with the inhibitor, ribonucleic acid Carrier RNA, biological nano magnetic bead suspension and a sample to be extracted, heating and fully cracking to enable the magnetic beads to be adsorbed and combined with nucleic acid;

2) cleaning the mixed adsorption solution by using the solutions II and III, and separating protein and polysaccharide impurities to be eluted;

3) and washing and desorbing the nucleic acid by using the solution IV to obtain a purified sample.

Furthermore, in the extraction method of the invention, a magnetic device is used in combination with the biological nano magnetic beads to promote the magnetic beads and the liquid to be adsorbed and suspended.

Preferably, the magnetic device is a simple magnetic frame for holding a buffer solution for combining the sample and the magnetic beads and assisting the adsorption separation cleaning process.

The kit is used for extracting DNA, and the matched solution comprises a solution I, a solution II, a solution III, a solution IV, a silicon hydroxyl nano magnetic bead suspension and a magnetic device matched with the magnetic beads, and the method comprises the following steps:

adding 20-500 mu L of sample, 100-500 mu L of solution I, 20-100 mu L of inhibitor, 1-5 mu L of Carrier RNA and 10-30 mu L of silicon hydroxyl nano magnetic bead suspension into a 1.5ml centrifuge tube, slightly shaking and uniformly mixing, and placing in a warm bath at 55-60 ℃ for 10-30 minutes;

step two, adding 250-;

adding 150-; and/or the like, and/or,

step four: adding the solution III and repeating the third step;

step five: adding 50-150 μ L of solution IV, mixing, centrifuging, bathing at 55-60 deg.C for 10-20 min, placing on magnetic frame, standing for 2-3 min, sucking solution, removing magnetic beads to obtain the final product, and storing the final product nucleic acid.

The principle of the invention is as follows: in solution i, the lysate contained: the Tris-Cl buffer system can provide a stable solution environment for a fragile nucleic acid basic structure after membrane rupture so as to ensure the purity and integrity of finished nucleic acid; EDTA is a divalent ion chelating agent, chelating divalent metal cations such as Mg²⁺、Ca²⁺Etc. to prevent the excitation or promotion of rnase activity, by inhibiting these lytic enzymes to maximize the protection of nucleic acids; triton X-100 and SDS, these common non-ionic and anionic detergents, guanidine hydrochloride, in combination with proteinase K, can effectively and strongly destroy protein structureThe method is characterized in that the nucleic acid is denatured, the nucleic acid is fully separated from the nucleic acid, the nucleic acid is released after being separated from the protein structure, and then the nucleic acid is adsorbed and retained by matching with the biological nano magnetic beads with silicon-based oxidation on the surface in a concentrated salt environment, and other redundant impurities such as polysaccharide, lipid and the like are precipitated and eluted step by step.

The reagents contained in the invention are all chemical reagents which can be purchased, prepared and stored under the conventional conditions, have no violent toxicity, can cause harm to human bodies or contain potential leakage risk, are easy to transport including nano magnetic beads and are stored in the environment of 2-8 ℃; the method can extract a large amount of DNA in cells/bacteria through simple steps, can be directly applied to downstream multiple subsequent experiments including but not limited to PCR/q-RTPCR/LAMP/sequencing/library construction and the like, and provides a new idea for simplifying nucleic acid extraction steps.

The invention utilizes the advantages of simple operation and wide application range of the magnetic bead method, and the treated biological nano magnetic beads are used for carrying out high-specificity adsorption and combination on nucleic acid in a specific solution environment so as to facilitate cracking and separation of impurities with biomacromolecule structures such as protein, polysaccharide and the like, thereby improving the yield and purity of the nucleic acid product. The kit is suitable for short-fragment DNA, and has the advantages of simplicity, safety, low cost and the like.

Drawings

FIG. 1 shows the finished product extracted from the kit of the present invention;

FIG. 2 is a graph showing the result of dsDNA concentration of finished products extracted by Magpure kit;

FIG. 3 is a graph showing the results of dsDNA concentration of finished products extracted by using magenta magnetic beads in combination with the solution of the kit of the present invention;

FIG. 4 is a graph showing the dsDNA concentration of finished products extracted by using magenta solution in combination with the magnetic beads of the kit;

FIG. 5 shows agarose gel electrophoresis based on the samples obtained, and samples were sequentially added in the order shown in the above table, and the first two compartments were defined as lambda DNA 250bp and DNA ladder control groups.

Detailed Description

In order to facilitate further elucidation and understanding of the invention, embodiments of the invention will now be described in more detail with reference to the examples. The following examples are merely illustrative of the present invention and the scope of the invention described should not be limited to the examples.

Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. The terminology used in the description of the invention herein is for the purpose of describing particular embodiments only and is not intended to be limiting of the invention. Those who do not specify specific conditions in the examples of the present invention follow conventional conditions or conditions recommended by the manufacturer. The reagents or instruments used are not indicated by manufacturers, and are all conventional products available on the market.

This example was obtained by using self-made magnetic beads and preparing the above-mentioned series of solutions I-IV according to the comparative MagPure Circulating DNA Maxi Kit experiment, and the results are shown in the attached drawing.

A comparative commercial kit is available from magenta Tiangen Bio Inc., Cat No.12917PC-50(50 preps).

Taking a part of escherichia coli sample, sucking and removing supernatant, and mixing precipitate with pure water for mixing;

step one, adding 500 mu L of escherichia coli sample and 500 mu L of solution I (containing 100mM Tris-Cl,10mM EDTA,1% Triton X-100,0.5% SDS and 200mM guanidine hydrochloride), 20-100 mu L of inhibitor-removed protease K, 1 mu L of Carrier RNA and 10 mu L of silicon hydroxyl nano magnetic bead suspension into a 1.5ml centrifuge tube, slightly shaking and uniformly mixing, and then placing the mixture in a 58 ℃ warm bath for 10 minutes;

adding 250 mu L of solution II (containing 10mM Tris-Cl and 5mM EDTA) into the centrifuge tube, slightly shaking and uniformly mixing, standing the centrifuge tube on a magnetic frame, standing for 2 minutes, and sucking and removing supernatant after magnetic beads are adsorbed with nucleic acid;

adding 150 mu L of the solution III into a centrifuge tube, slightly shaking and uniformly mixing, opening a bottle cap in a sterile enzyme-free super clean bench environment, standing on a magnetic frame for 2 minutes, and sucking and removing supernatant;

step four: adding 150 mu L of the solution IV into a centrifuge tube, uniformly mixing, simply centrifuging, then carrying out warm bath at 58 ℃ for 10 minutes, then placing on a magnetic frame, standing for 2-3 minutes, absorbing the solution, discarding magnetic beads to obtain a finished product, and properly storing the finished product of nucleic acid.

In the results of this example, the concentration of the extracted finished product was measured by using qubit4.0, and after the finished product was diluted by 50 times, the diluted finished product was diluted by 20 times at a ratio of 10:190, and the results obtained are shown in fig. 1 to 4, and are listed in the result list 1:

from the obtained samples, agarose gel electrophoresis was performed to prepare samples, the samples were sequentially loaded in the order of table 1, and the first two cells were set as lambda DNA 250bp and DNA ladder control groups, and the results are shown in fig. 5:

FIG. 5 is a graph showing the effect of gel in the UV mode of a gel electrophoresis apparatus, wherein GelRed is used as a staining agent, the lambda DNA gene fragment of the second well is too large to run through the gel field, and the strong contrast of the last 4 groups is perfect, and the difference is similar according to the data in Table 1.

The invention provides a practical idea which is feasible, safe and convenient for the extraction of nucleic acid by a magnetic bead method; it is to be understood that this invention is not limited to the particular methodology, protocols, and materials described herein as such may, within the scope of sound, vary and equivalents thereof may be substituted without departing from the scope of the invention.