CN1970752A - Method for recovery of DNA by using magnetic nanocomposite and reagent kit therefor - Google Patents

Abstract

The method and kit for recovering DNA by using magnetic nanocomposite materials of the present invention relate to the field of biotechnology. Recovering DNA in a recovered sample such as a DNA-containing gel sample, the steps include: (a) mixing the magnetic nanocomposite material with the recovered sample to form a solid-liquid homogeneous dispersion suspension; (b) using a magnetic field to absorb the DNA The magnetic nanocomposite material is separated from the solid-liquid homogeneous dispersion suspension to obtain the magnetic nanocomposite material adsorbed with DNA; (c) after the impurities are washed away with the washing solution, the DNA is combined with the eluent from the magnetic nanocomposite analyzed from the material. The kit for recovering DNA by using magnetic nanocomposite material contains: magnetic nanocomposite material solution, dissolving solution, washing solution I, washing solution II and eluent. The invention can realize recovery and purification of DNA in one step; the total extraction efficiency is above 80 percent; the method is simple and fast; and the extraction quantity of DNA can be estimated.

Description

Method and kit for recovering DNA using magnetic nanocomposite material

technical field

The present invention relates to the field of biotechnology. More specifically, the present invention relates to a method for recovering DNA using magnetic nanoparticles and a kit for recovering DNA.

Background technique

The recovery of DNA has a wide range of applications, such as recovery of high-purity DNA from DNA polymerase chain reaction (PCR) amplification products, polyacrylamide gel, agarose gel, digested biological samples for genetic fingerprint identification, gene Sequencing, library construction, PCR testing, etc. DNA recovery technology is a key technology in molecular biology experiments. The efficiency and purity of recovered DNA will directly affect the success of subsequent experiments. Therefore, developing fast, efficient, simple and low-cost DNA recovery methods has always One of the goals of academics.

At present, the main DNA recovery methods are chemical method, glass bead method and column separation method. The steps are relatively cumbersome and the purity of the recovered DNA is not high.

The kits produced by some foreign companies, such as those produced by Promega, Qiagen, and Gene, are expensive and cannot be effectively promoted.

With the improvement and progress of nanotechnology, the synthesis technology of magnetic nanocomposite materials and its application prospect in the biological field have attracted more and more attention. In particular, it has the advantages of simple operation of the separation method, cheap equipment required, and fast separation speed, making it gradually become an indispensable experimental method for molecular biology research, and its application is becoming more and more extensive.

Magnetic composite materials used in biotechnology are generally required to have the following characteristics: (1) have paramagnetism, and are easy to adsorb and elute. (2) It has a larger magnetization intensity to ensure separation efficiency and improve sensitivity. (3) The surface of composite materials should be easy to be chemically modified to connect with different biological and drug molecules. (4) Good biocompatibility is required when used in vivo. Composite materials designed for this purpose usually have a sandwich structure. The inner core is generally superparamagnetic magnetic nanoparticles, and the middle is a coating layer. Most of the existing products currently use polymer materials as the coating layer, and the outermost layer is modified with Biological tissues have functional groups with specific effects to meet the requirements of biological separation.

Commercial magnetic composite materials mostly use polymer microspheres obtained by emulsion polymerization, which are coated with nano-sized magnetic particles. However, the structure of polymer spheres is usually relatively loose, and the magnetic particles contained in them are easy to detach from the microspheres during long-term storage, thereby affecting the performance of the product. Generally, the polymer microspheres obtained by emulsion polymerization are large in size, mostly in the micron range, which cannot meet the requirements of some application fields that require smaller size particles. Another type of magnetic particle coating material commonly used is polysaccharide molecules, such as dextran, chitosan, etc., but the coating layer formed by such molecules is more unstable, and its interaction with magnetic particles is weak, and the same There is the disadvantage that the magnetic particles are easy to fall off, resulting in product failure. Therefore, there is an urgent need in this field to develop new techniques for recovering DNA easily and efficiently.

The existing technology similar to the present invention is Chinese patent ZL03111351.6, the title of the invention is "magnetic core-shell particles with strong magnetic field corresponding ability and its preparation method", which discloses a magnetic nanocomposite material with silicon dioxide as a coating layer The synthetic method, the synthesized magnetic nanocomposite material is suitable for the separation and purification of biological materials and pharmaceutical products, and can be used as a magnetic control directional delivery carrier for biological molecules and drug molecules.

Currently, there is no technology for recovering DNA using magnetic nanocomposites.

Contents of the invention

The technical problem to be solved by the present invention is to recover DNA by using the magnetic nanocomposite material with silicon dioxide as the coating layer, so as to achieve the purpose of simplicity, speed, high efficiency and low cost.

The inventors of the present invention, on the basis of preparing a magnetic nanocomposite material coated with silicon dioxide, found that the magnetic nanocomposite material has a high affinity with DNA molecules and can be used as an effective medium for recovering DNA. One object of the present invention is to disclose a method for recovering DNA simply and efficiently using magnetic nanocomposite materials.

Another object of the present invention is to provide a kit for recovering DNA simply and effectively by utilizing magnetic nanocomposite materials.

In the first aspect of the present invention, a kind of method utilizing magnetic nanocomposite material to reclaim DNA is provided, and said reclaiming DNA is from the PCR amplification product that has been mixed with lysate, uses lysate to make DNA from gel sample DNA is recovered from recovered samples such as the released adsorbate sample or the biological sample in which the DNA mixed with the lysate has been released; the recovery steps include:

(a) mixing the magnetic nanocomposite material with the recovered sample to form a solid-liquid homogeneous dispersion suspension, wherein the diameter of the magnetic nanocomposite material is 0.2-10 μm;

(b) separating the magnetic nanocomposite material with adsorbed DNA from the solid-liquid homogeneous dispersion suspension with a magnetic field, thereby obtaining the magnetic nanocomposite material with adsorbed DNA;

(c) After the impurities are washed away with the washing solution, the DNA is resolved from the magnetic nanocomposite material with the washing solution, so as to obtain a pure DNA solution; the washing solution has a component of 1 to 8 mol/l Salt solution, 0.5mol/l Tris-HCl hydrochloride (Tris-HCl), pH=6.7, 5-50mmol/l ethylenediaminetetraacetic acid (EDTA) or 0-2mol/l acetic acid Sodium; said eluent is deionized water or 10±2mmol/l Tris-HCl, pH=8.0.

The method of the invention can simply and efficiently recover DNA from DNA-containing samples such as gels.

In another preferred example, the gel samples are polyacrylamide gels, agarose gels, dextran gels, etc. and other samples requiring DNA recovery.

In another preferred example, the DNA is released from the gel sample with the lysate to obtain a DNA-containing adsorption liquid sample, the lysate has a composition of 1 to 8 mol/l salt solution, a pH=0.5 mol/l 6.7 Tris-HCl hydrochloride (Tris-HCl), 5-50 mmol/l ethylenediaminetetraacetic acid (EDTA) and 0.5-5% polyethylene glycol octylphenyl ether (Triton).

In another preferred embodiment, the sample of the adsorption solution is prepared by the following steps: mixing the gel sample containing DNA with the solution, and incubating at 35° C. to 80° C. for 10 to 40 minutes.

In another preferred example, the core of the magnetic nanocomposite material is magnetic ferrite nanoparticles, including Fe ₃ O ₄ nanoparticles, γ-Fe ₂ O ₃ nanoparticles or those doped with transition metal elements and their compounds. Superparamagnetic ferrite nanoparticles with a silica shell.

In another preferred example, step (c) further includes the step of washing with washing liquid I or washing liquid II successively; the number of washings is 1-5 times, preferably 2-3 times. The composition of washing liquid I is 1-8mol/l salt solution, 0.5mol/l Tris-HCl, pH=6.7, 5-50mmol/l EDTA; the composition of washing liquid II is 0-2mol/l sodium acetate.

The DNA-adsorbed magnetic nanocomposite obtained from step (b) of the method of the present invention can be directly used, for example, as a template for PCR reaction. Of course, DNA can also be desorbed from the magnetic nanocomposite to form a DNA solution. Preferably, step (c) is performed again, adding an eluent containing Tris-HCl to dissolve the DNA from the magnetic nanocomposite material, thereby obtaining a DNA solution.

In the second aspect of the present invention, a DNA recovery kit is adopted, which contains a magnetic nanocomposite material, the magnetic nanocomposite material has a magnetic inner core and a silicon-oxygen structure coating layer, and its diameter is 0.2-10 μm.

Described test kit also contains:

Dissolving liquid, its composition is 1～8mol/l salt solution, 0.5mol/l Tris-HCl, pH=6.7, 5～50mmol/l EDTA, 0.5～5% Triton;

Washing solution I, whose components are 1-8 mol/l salt solution, 0.5 mol/l Tris-HCl, pH=6.7, 5-50 mmol/l EDTA;

Washing liquid II, its component is the sodium acetate of 0～2mol/l (can be water);

The eluent is 10±2mmol/l Tris-HCl, pH=8.0 or deionized water.

In another preferred example, in the dissolving solution, the salt solution is NaCl or NaClO ₄ solution or NaI solution, and the concentration of the salt solution is 5 ± 1mol/l; the concentration of the EDTA is 15 ± 5mmol/l ;

In the washing solution I, the salt solution is NaCl or NaClO ₄ solution or NaI solution, and the concentration of the salt solution is 4 ± 1mol/l; the EDTA concentration is 20 ± 5mmol/l;

In the washing liquid II, the concentration of the sodium acetate is 1.5±0.5 mol/l.

The dissolving liquid, washing liquid, and eluent used in the present invention are not particularly limited, and various dissolving liquids, washing liquids, and eluting liquids used in the art can be used. In addition, the dissolving solution, washing solution and eluting solution in the kit can be concentrated or diluent.

The size of the novel composite magnetic material prepared by the method of the invention can be adjusted between 0.2-10 μm, and the magnetic core is covered by a dense silicon-oxygen structure layer, which can completely avoid the shedding of magnetic particles. At the same time, it can slow down the oxidation rate of the core and prolong its service life. The silicon-oxygen structure layer covered by the outer layer has good biocompatibility. After surface modification with different silylating reagents, the composite magnetic material can be combined with different drugs or biomolecules to meet special purposes such as separation and purification of biological samples in vitro, detection, and directional transport of drugs or other biomolecules in vivo.

Main advantage of the present invention is:

1. Using superparamagnetic magnetic nanocomposite materials, one-step recovery and purification of DNA can be achieved.

2. The method is simple and the extraction is fast, usually the operation can be completed within 1 hour.

3. Can effectively remove inhibitors and pollutants, suitable for molecular biology laboratories.

4. According to the design of the kit, the amount of DNA extracted can be estimated, which can serve as an indicator for future operations.

5. Using bionano technology for product development, the efficiency of DNA extraction is greatly improved, and the total extraction efficiency is above 80%.

6. It can be used in conjunction with a variety of biological technologies, such as PCR technology, enzyme cutting technology, cloning technology, etc.

7. It can realize the automation of DNA recovery operation.

Detailed ways

Below in conjunction with specific embodiment, further illustrate the present invention. It should be understood that these examples are only used to illustrate the present invention and are not intended to limit the scope of the present invention. The experimental method that does not indicate specific conditions in the following examples is usually according to conventional conditions, such as Sambrook et al., Molecular cloning: the conditions described in the laboratory manual (New York: Cold Spring Harbor Laboratory Press, 1989), or according to the conditions of manufacture conditions recommended by the manufacturer.

Example 1

Preparation of Magnetic Nanocomposites

54.90 g of FeCl ₃ ·6H ₂ O and 20.20 g of FeCl ₂ ·4H ₂ O were weighed and dissolved in 200 ml of water that had been purged with nitrogen and deoxygenated to obtain a mixed solution. Take 800ml of water in a 1L round-bottomed flask to pass through nitrogen to remove oxygen, add 50ml of concentrated ammonia water with a concentration of 25-28% by mass, and quickly pour the above-mentioned iron salt mixed solution into it under vigorous stirring. Reaction 1h. After the reaction is completed, a 0.1T permanent magnet is used to separate a black solid from the reaction solution, and the obtained solid is washed with high-purity water for 3 to 5 times to obtain Fe ₃ O ₄ nanoparticles with a particle diameter of 6 to 10 nm.

Weigh 5.0g Na ₂ SiO ₃ (SiO ₂ content is 45%) and dissolve it in 1000ml water, adjust the pH of the solution to 9.6 with a cationic resin, and add 2g of Fe ₃ O ₄ nanoparticles synthesized above to it. At room temperature, the reaction was carried out in a 1L round bottom flask with a stirring speed of 150rpm for 12h. After the reaction is completed, use a 0.1T permanent magnet to separate the black solid from the reaction solution, and wash the obtained solid with high-purity water for 3 to 5 times to obtain Fe ₃ O ₄ nanoparticles coated with silica with a particle size of 7 to 12 nm. .

900ml of ethanol, 100ml of water, 40ml of concentrated ammonia water with a concentration of 25-28% by mass, and 0.10g of Fe ₃ O ₄ nanoparticles synthesized by the first silicon dioxide coating were sequentially added into a 2L round bottom flask. At room temperature, 0.6 ml of ethyl orthosilicate was added dropwise at a stirring speed of 200 rpm, after 3 hours of reaction, 0.6 ml of ethyl orthosilicate was added dropwise, and the reaction was continued for 12 hours. A 0.1T permanent magnet was used to separate the black solid from the reaction solution, and the obtained solid was washed 3-5 times with high-purity water to obtain multi-core Fe ₃ O ₄ -SiO ₂ core-shell particles with an average size of 0.4 μm.

Example 2

DNA Recovery Kit

Preparation of DNA recovery kit using magnetic composite material, which includes:

0.5ml concentration is the magnetic nanocomposite material solution prepared in the embodiment 1 of 20mg/ml

100ml solution, its composition is 6mol/LNaI solution, 0.5ml Tris-HCl, pH=6.4, 25mmol/l EDTA;

100ml washing liquid II, its component is 1.5mol/l sodium acetate;

10ml eluent, deionized water.

Example 3

DNA Recovery Kit

Preparation utilizes the DNA recovery kit of magnetic composite material, and the magnetic nanocomposite material solution in it, washing liquid II are the same as embodiment 2, also include:

100ml solution, its components are 4mol/L NaCl solution, 0.5ml Tris-HCl, pH=6.4, 45mmol/l EDTA, 2.5% Triton;

100ml washing liquid I, its component is the _{NaClO4solution} of 4mol/l, the Tris-HCl of 0.5mol/l, the EDTA of pH=6.7, 25mmol/l;

10ml eluent, its composition is 10mmol/l Tris-HCl.

Example 4

DNA Recovery from Agarose Gels Using Magnetic Nanocomposites

1. Cut out the agarose gel (100mg～500mg) containing the DNA fragment, mash it and add it to the dissolving solution at a weight ratio of 1:2 to 1:4 (gel fragment:solution volume to weight ratio), and the components of the dissolving solution are the same as in the examples 2.

2. Dissolve at 50°C until the gel is completely dissolved.

3. Add magnetic nanocomposite material, and adsorb at room temperature for 20 minutes.

4. After mixing by vortexing, immediately put it on the magnetic rack attached to the kit, and let it rest for 30 seconds until the magnetic composite nanomaterial is adsorbed to the tube wall.

5. After the solution was sucked away, it was washed 3 times with the washing liquid II in Example 2.

6. After drying at room temperature for 10 minutes, add the eluent and incubate at 65°C for 15 minutes.

7. Immediately put the centrifuge tube on the magnetic rack, stand still for 30s until the magnetic nanocomposite material is adsorbed to the tube wall, suck out the eluent and transfer it to another centrifuge tube for later use.

Example 5

Recovery of DNA in Solution Using Magnetic Nanocomposites

1. Add 3 times the volume of dissolving solution to the DNA solution and mix well.

The remaining steps are followed by Steps 3-7 of Example 4.

Example 6

Recovery of DNA in Polyacrylamide Gel Using Magnetic Nanocomposite

1. Cut out the polyacrylamide gel containing DNA fragments, mash it, soak it with 2 times the volume of the solution of Example 2, and warm it at 37°C for 2 hours.

2. Centrifuge at 10,000 rpm for 3 minutes, and transfer the supernatant solution to a new centrifuge tube.

3. The rest of the steps are the same as those in 3-7 of Example 4.

Example 7

Recovery of DNA in Polyacrylamide Gel Using Magnetic Nanocomposite

1. Cut out the polyacrylamide gel containing DNA fragments, mash it, soak it with 2 times the volume of TNE, and warm it at 37°C for 2 hours.

2. Centrifuge at 10,000 rpm for 3 minutes, and transfer the supernatant solution to a new centrifuge tube.

3. Add 3 times the volume of dissolving solution to obtain the recovered sample.

4. Mixing the magnetic nanocomposite material with the recovered sample, and then using a magnet to separate the magnetic nanocomposite material with adsorbed DNA from the suspension, thereby obtaining the magnetic nanocomposite material with adsorbed DNA.

5. Wash the magnetic nanocomposite with DNA adsorbed twice with washing solution I, then wash twice with washing solution II, and finally use the eluent to resolve the DNA from the magnetic nanocomposite to obtain a pure DNA solution .

Example 8

Recovery of DNA from PCR Products Using Magnetic Nanocomposite

Add 3 times the volume of dissolving solution to the PCR amplification product solution, and mix well.

The remaining steps are according to steps 3-7 of Example 4.

Example 9

Quality Assay of Recovered DNA

The DNA recovered in Examples 4, 7, and 8 was directly subjected to electrophoresis. One of the swimming lanes is L is the DNA standard ψ×174/HaeIII (allele ladder of the PCR amplified fragment), so as to compare the DNA recovery effect of the recovered samples.

The results showed that ideal recovery results were obtained for all samples. However, the DNA recovery efficiency in agarose and polyacrylamide gels is slightly lower than that of DNA fragments recovered from PCR amplification products.

Claims (6)

1, a kind of method that reclaims DNA with magnetic nanometer composite material, it is characterized in that, said recovery DNA, be from lysate blended pcr amplification product, with lysate make adsorption liquid sample that DNA discharges from gelled specimen or the recovery samples such as biological specimen that discharged with lysate blended DNA reclaim DNA; Recycling step comprises:

(a) magnetic nanometer composite material is mixed with recovery sample, form solid-liquid homodisperse suspension liquid, the diameter of wherein said magnetic nanometer composite material is 0.2～10 μ m;

(b) magnetic nanometer composite material that will adsorb DNA with magnetic field is separated from solid-liquid homodisperse suspension liquid, thereby obtains being adsorbed with the magnetic nanometer composite material of DNA;

(c) with washings with the impurity flush away after, with elutriant DNA is parsed from magnetic nanometer composite material, thereby obtains pure dna solution; Said washings, its component are the salts solution of 1～8mol/l, the Tri(Hydroxymethyl) Amino Methane Hydrochloride of 0.5mol/l, pH=6.7, the ethylenediamine tetraacetic acid (EDTA) of 5～50mmol/l or be the sodium acetate of 0～2mol/l; Said elutriant is deionized water or 10 ± 2mmol/l Tris-HCl, pH=8.0.

2, according to the described method that reclaims DNA with magnetic nanometer composite material of claim 1, it is characterized in that, said adsorption liquid sample prepares by following steps: the gelled specimen that will contain DNA is mixed with lysate, and 35 ℃～80 ℃ temperature were bathed 10～40 minutes; Said gelled specimen is polyacrylamide gel, sepharose, dextrane gel.

According to claim 1 or 2 described methods, it is characterized in that 3, in (c) step, said washing divides and successively uses washings I, cleaning solution II washing with magnetic nanometer composite material recovery DNA; Washing times is respectively 1～5 time; The component of washings I is the salts solution of 1～8mol/l, the Tri(Hydroxymethyl) Amino Methane Hydrochloride of 0.5mol/l, pH=6.7, the ethylenediamine tetraacetic acid (EDTA) of 5～50mmol/l; The component of cleaning solution II is the sodium acetate of 0～2mol/l.

4, according to claim 1 or 2 described methods, it is characterized in that the kernel of said magnetic nanometer composite material is the magnetic ferrite nanoparticle, comprises Fe with magnetic nanometer composite material recovery DNA ₃O ₄Nanoparticle, γ-Fe ₂O ₃Nanoparticle or mix transition metal and the ferrite nano particles with superparamagnetism of compound, its shell is a silicon-dioxide.

5, a kind ofly reclaim the test kit of the method for DNA, it is characterized in that, contain in the test kit with magnetic nanometer composite material:

Magnetic nanometer composite material, said magnetic nanometer composite material have magnetic kernel and silica structure coating layer, and its diameter is 0.2～10 μ m;

Lysate, its component are the salts solution of 1～8mol/l, the Tri(Hydroxymethyl) Amino Methane Hydrochloride of 0.5mol/l, pH=6.7, the ethylenediamine tetraacetic acid (EDTA) of 5～50mmol/l, 0.5～5% Triton X-100;

Washings I, its component is the salts solution of 1～8mol/l, the Tri(Hydroxymethyl) Amino Methane Hydrochloride of 0.5mol/l, pH=6.7, the ethylenediamine tetraacetic acid (EDTA) of 5～50mmol/l;

Cleaning solution II, its component are the sodium acetate of 0～2mol/l;

Elutriant is 10 ± 2 mmol/l Tris-HCl, pH=8.0 or deionized waters.

According to the described test kit of claim 5, it is characterized in that 6, said salts solution is NaCl or NaClO ₄Solution or NaI solution, and concentration of salt solution is 5 ± 1mol/l; The concentration of the ethylenediamine tetraacetic acid (EDTA) of being washed is 15 ± 5mmol/l; In washings I, said salts solution is NaCl or NaClO ₄Solution or NaI solution, and the concentration of salts solution is 4 ± 1mol/l; Said ethylenediamine tetraacetic acid (EDTA) concentration is 20 ± 5mmol/l; In cleaning solution II, the concentration of said sodium acetate is 1.5 ± 0.5mol/l.