CN112391378A

CN112391378A - Kit and preparation method thereof

Info

Publication number: CN112391378A
Application number: CN201910737825.4A
Authority: CN
Inventors: 张辉
Original assignee: Xinkaiyuan Huicheng Wuhan Medical Technology Co ltd
Current assignee: Xinkaiyuan Huicheng Wuhan Medical Technology Co ltd
Priority date: 2019-08-12
Filing date: 2019-08-12
Publication date: 2021-02-23

Abstract

The invention discloses a kit and a manufacturing method thereof, the kit is used for extracting genome DNA, and the kit comprises cell lysate, nano magnetic beads and cleaning fluid, wherein the nano magnetic beads are Fe of silicon dioxide₃O₄The diameter is 200-500 nm, and the cell lysate comprises erythrocyte lysate and leukocyte lysate. The kit and the preparation method thereof provided by the invention have the advantages of simple and easy operation, safety, no toxicity, few components, low use cost, high DNA yield and very wide application prospect, and can be suitable for extracting various animal tissues.

Description

Kit and preparation method thereof

Technical Field

The invention relates to a kit and a manufacturing method thereof, belonging to the field of biological medical treatment.

Background

With the development of molecular biology techniques, research on the genome level has become a hot spot. In molecular genetics and molecular epidemiology, whether whole genome association analysis (GWAS), candidate SNP site genotyping, or genomic library establishment, it is necessary to extract genomic DNA from various types of samples. Because blood samples are convenient to obtain and contain abundant and reliable genomic DNA, many of the above researches have taken blood as a sample to extract complete genomic DNA. The concentration, purity and integrity of the primary structure of the extracted and purified genomic DNA can affect the subsequent research, so that an efficient and reliable genomic DNA extraction method is urgently needed for basic research of molecular genetics.

At present, the commonly used DNA extraction methods include phenol chloroform extraction, centrifugal column method, magnetic bead method and the like, and each of these methods has advantages and disadvantages. The phenol chloroform extraction method needs frequent use of toxic reagents, is easy to bring safety problems, and has complicated extraction process and low extraction efficiency. The column centrifugation method adopts silicon substrate materials to efficiently and specifically adsorb DNA under a certain high-salt buffer system, needs to use toxic reagents such as phenol, chloroform and the like, consumes more time in the extraction process of the centrifugal column method, and influences the speed of overall analysis and detection. The magnetic bead method is a novel extraction method for adsorbing DNA by a new nano material, has the advantages of less safe reagent dosage, complete DNA molecular structure and the like, but the quality of the extracted DNA is closely related to the quality of the magnetic bead, the quality of the magnetic bead is good or bad, and the quality of the extracted DNA is determined to be high or low to a great extent. Therefore, the application develops a nucleic acid extraction method and an extraction kit which are simple to operate, low in requirements on extraction instruments and personnel, non-toxic and harmless, capable of effectively removing impurities and free of influencing DNA structures.

Disclosure of Invention

Aiming at the problems in the prior art, the invention aims to provide a kit which is simple and convenient to operate and has good safety and a manufacturing method thereof.

The first invention of the invention aims to provide a kit, wherein the kit adopts magnetic nano microspheres as markers to separate genome DNA, is used for extracting the genome DNA, and comprises cell lysis solution, nano magnetic beads and cleaning solution, wherein the nano magnetic beads are Fe of silicon dioxide₃O₄The diameter is 200-500 nm, and the cell lysate comprises erythrocyte lysate and leukocyte lysate.

Since the cellular composition in blood is different from that of tissue cells, the method for extracting genomic DNA according to the present invention is to extract genomic DNA from leukocytes or tissue cells. Any animal tissue containing DNA may be used as the DNA extraction target in the present invention. As a preferred embodiment, the tissue is a buccal swab.

Preferably, the concentration of the nano magnetic beads is 50-200 mg/ml.

Preferably, the erythrocyte lysate comprises 5mol/L NaCl, 1M Tris-HCl and 0.5M EDTA.

The erythrocyte lysate is a mixed solution of 0.2mL of 5mol/L NaCl, 2mL of 1M Tris-HCl and 0.02mL of 0.5M EDTA. Wherein, the removal of the red blood cells is performed until the red blood cells are completely lysed, and if the operation is not complete, the operation can be repeated for a plurality of times until the red blood cells are completely lysed.

Preferably, the leukocyte lysate comprises SDS, 1M Tris-HCl with pH 7-9, and 0.5M EDTA with pH 8.0.

More preferably, the leukocyte lysate contains 1g SDS, 1M Tris-HCl 1mL, NaCl 0.585g and 0.5M EDTA 1mL per 100mL of leukocyte lysate, and the pH of the solution is 8.0.

Preferably, the wash solution comprises wash solution I and wash solution II.

More preferably, the washing solution I comprises 2mM of guanidinium isothiocyanate, 10-40 mM of Tris-HCl with the pH value of 7-9 and 20-50% of absolute ethyl alcohol. In a preferred embodiment, wash solution I comprises guanidinium isothiocyanate at a concentration of 2mM, 30mM Tris-HCl (pH 8.0), and 30% absolute ethanol.

More preferably, the washing solution II comprises 1-5 mM of Tris-HCl with pH 7-9, 20-50 mM of NaCl and 50-80% of absolute ethyl alcohol. In a preferred embodiment, washing solution II comprises Tris-HCl (pH 8.0) at a concentration of 3mM, NaCl at a concentration of 30mM, and 65% absolute ethanol.

A second object of the present invention is to provide a method for producing the above kit, the method comprising:

a) preparing magnetic nano microspheres: the prepared nano Fe₃O₄Dispersing particles (with the particle size of 200-500 nm) in the silicon dioxide microspheres, ultrasonically stirring for 1-3 h at the temperature of not higher than 20 ℃, and sealing and cleaning after magnetic particles are formed;

b) red blood cell lysate, white blood cell lysate, washing liquid I and washing liquid II are respectively prepared according to the component proportion, and the components are uniformly mixed and then sterilized by high pressure or a filter membrane.

Preferably, the silica microspheres are subjected to ultrasonic stirring for 3-10 hours at the temperature of not less than 50 ℃.

The above silica-coated Fe₃O₄The preparation method of the nano magnetic beads can be carried out by referring to the preparation method in the prior art, and is not described herein again.

The experimental data show that: according to the kit provided by the invention, the DNA extraction purity is 1.6-2.1 in the result of extracting more than 500 human samples; the yield of genomic DNA from 200uL of whole blood was not less than 1 ug.

The above-mentioned components are preferred embodiments of the present invention, and any components modified or substituted based on the above-mentioned disclosed components should be considered as falling within the scope of the present invention as long as they can exert corresponding effects.

Compared with the prior art, the kit and the preparation method thereof provided by the invention have the following technical effects:

1) the components are simple, the raw materials are safe and reliable, and the environment is friendly; the kit can effectively remove impurities, particularly protein impurities, in an animal sample to be extracted, and ensures the purity and concentration of the extracted DNA;

2) the extraction method is simple and convenient to operate, the steps are simple, and the extraction time of the kit is only about 1 h;

3) the DNA extraction by using the kit of the extraction method can be carried out only by using conventional centrifugal equipment in a laboratory, and the steps are obviously simplified;

4) the DNA extracted by the kit has good concentration and purity and stable extraction effect.

In a word, the extraction method has simple operation steps, few components, lower requirements on operators, high efficiency and accuracy in extracting the genomic DNA, and higher DNA purity.

Detailed Description

The present invention will be described more fully hereinafter with reference to the following examples.

The DNA extraction technology based on the nano magnetic beads specifically adsorbs DNA molecules of a sample solution by using hydrophilic nano magnetic beads, the adsorbed DNA-magnetic bead compound is separated from the sample solution under the action of an external magnetic field, protein and salt ions in the solution are removed by washing through a washing solution, and finally, eluent is added into the magnetic beads only remaining with the adsorbed DNA, and the DNA is released into the eluent.

The reagents adopted by the DNA extraction kit in the embodiment are self-prepared in the laboratory of the company if no special description exists, and the details are as follows:

erythrocyte lysate (component a): 0.2mL of 5mol/L NaCl, 2mL of 1M Tris-HCl and 0.02mL of 0.5M EDTA, and uniformly mixing the components and then sterilizing the components by using a high pressure or filter membrane;

leukocyte lysate (component B): each 100mL of the mixture contained 1g of SDS, 1mL of Tris-HCl (1M) (pH 8.0), 0.585g of NaCl, and 1mL of EDTA (0.5M) (pH 8.0), and the mixture was mixed well and sterilized by autoclaving or filtration;

magnetic nanobead (component C): the magnetic beads are Fe wrapped with silicon dioxide₃O₄The diameter is 200-500 nm, and the concentration is 50-200 mg/ml;

washing solution I (component D): guanidinium isothiocyanate with concentration of 2mM, Tris-HCl with concentration of 30mM (pH 8.0), and 30% anhydrous ethanol, mixing, and sterilizing under high pressure or with filter membrane;

wash II (component E): Tris-HCl (pH 8.0) at a concentration of 3mM, NaCl at a concentration of 30mM, and 65% absolute ethanol, mixed well and then sterilized by autoclaving or filtration.

The preparation method of the nano magnetic bead (component C) comprises the following steps: the prepared nano Fe₃O₄Dispersing particles (with the particle size of 200-500 nm) in the treated silicon dioxide microspheres, ultrasonically stirring for 1-3 hours at the temperature of not higher than 20 ℃, and sealing and cleaning after obtaining magnetic particles to obtain the nano magnetic beads.

Example 1

In this embodiment, taking a human blood sample as an example, genomic DNA in leukocytes is extracted, and the specific extraction process includes the following steps:

before the start of the experiment it was necessary to know: all experimental operations are carried out at room temperature, a centrifugal tube and a chromatographic column need to be marked in the experimental process, and covers need to be covered in the centrifugation and oscillation steps.

a. Removing red blood cells

Taking 200 mu L of the uniformly mixed human blood sample, adding 800 mu LA solution, reversing and uniformly mixing, and centrifuging at 3000rpm for 5 min;

discard the supernatant, add 800 μ LA solution, reverse mix, centrifuge at 3000rpm for 5 min. If the precipitate after centrifugation is still red, the solution A can be repeatedly added and then centrifugation is carried out until the precipitate is white, and the supernatant is discarded to retain the white precipitate.

b. Lysing leukocytes

And (B) taking the white precipitate in the step a, adding 200 mu L of the solution B, uniformly mixing, carrying out water bath at 56 ℃ for 10min, and blowing and uniformly mixing by using a pipette when carrying out water bath for 5 min.

c. Adsorption of nano magnetic beads

And C, adding 20 mu L of solution C into the reaction system in the step b, reversing, uniformly mixing, and standing at room temperature for 10 min.

Separating and cleaning DNA-magnetic bead compound

And c, placing the centrifuge tube containing the reaction system in the step c on a magnetic frame for 30s, and after the nano magnetic beads are completely absorbed to the tube wall, absorbing the supernatant solution by using a sterile pipette tip. Adding 500 mu l of D liquid into the nano magnetic beads on the tube wall, shaking and uniformly mixing for 1min, placing on a magnetic frame for 30s, and sucking and removing the supernatant solution by using a sterile pipette tip.

Adding 500 mu l of the E liquid into the nano magnetic beads on the tube wall, shaking and uniformly mixing for 1min, placing on a magnetic frame for 30s, using a sterile pipette tip to suck and remove the supernatant solution, and uncapping and drying at room temperature for 10-20min until no liquid remains in the tube.

The comparative example used the blood samples of examples 2 to 3 of Chinese patent application CN 106868000A, wherein examples 2 and 3 were performed. According to the embodiment disclosed in this application, a solution was prepared and a comparative experiment was performed to extract DNA using the sample of example 1 in this application according to the procedure. The extraction results were as follows:

comparative example 1 comparison of conventional DNA extraction method and reference extraction method for anticoagulant fluid sample

As can be seen from the above table, the concentration of DNA extracted by the DNA extraction method of the present invention is much higher than that of DNA extracted by other methods from the same sample, and the purity of DNA is maintained at a higher level.

According to statistics of results of DNA extraction of over 500 human blood and oral swab extraction, the DNA extraction purity is 1.6-2.1 of OD value; and the yield of the genomic DNA obtained from 200ul of the whole blood sample is not less than 1 ug.

Finally, it must be said here that: the above embodiments are only used for further detailed description of the technical solutions of the present invention, and should not be understood as limiting the scope of the present invention, and the insubstantial modifications and adaptations made by those skilled in the art according to the above descriptions of the present invention are within the scope of the present invention.

Claims

1. A kit, characterized in that: the kit is used for extracting genome DNA and comprises cell lysate, nano magnetic beads and cleaning solution, wherein the nano magnetic beads are Fe of silicon dioxide₃O₄The diameter is 200-500 nm, and the cell lysate comprises erythrocyte lysate and leukocyte lysate.

2. The kit of claim 1, wherein: the concentration of the nano magnetic beads is 50-200 mg/ml.

3. The kit of claim 1, wherein: the genomic DNA sample extracted by the kit is derived from blood or oral swab.

4. The kit of claim 1, wherein: the erythrocyte lysate comprises 5mol/L NaCl, 1M Tris-HCl and 0.5M EDTA.

5. The kit of claim 1, wherein: the leukocyte lysate comprises SDS, 1M Tris-HCl with the pH value of 7-9 and 0.5M EDTA with the pH value of 8.0.

6. The kit of claim 1, wherein: the washing solution comprises washing solution I and washing solution II.

7. The kit of claim 6, wherein: the washing solution I comprises 2mM of guanidinium isothiocyanate, 10-40 mM of Tris-HCl with the pH value of 7-9 and 20-50% of absolute ethyl alcohol.

8. The kit of claim 6, wherein: the washing solution II comprises 1-5 mM Tris-HCl with pH 7-9, 20-50 mM NaCl and 50-80% absolute ethyl alcohol.

9. A method for manufacturing the kit according to claim 1, wherein the method for manufacturing the kit comprises:

a) preparing magnetic nano microspheres: the prepared nano Fe₃O₄Dispersing the particles in silicon dioxide microspheres, ultrasonically stirring for 1-3 h at the temperature of not higher than 20 ℃, and sealing and cleaning the magnetic particles;

10. The method for preparing the kit according to claim 9, wherein the silica microspheres are previously subjected to ultrasonic stirring at a temperature of not less than 50 ℃ for 3-10 hours.