CN111961663A - Genome magnetic bead extraction kit and extraction method - Google Patents
Genome magnetic bead extraction kit and extraction method Download PDFInfo
- Publication number
- CN111961663A CN111961663A CN202010922941.6A CN202010922941A CN111961663A CN 111961663 A CN111961663 A CN 111961663A CN 202010922941 A CN202010922941 A CN 202010922941A CN 111961663 A CN111961663 A CN 111961663A
- Authority
- CN
- China
- Prior art keywords
- solution
- nucleic acid
- magnetic beads
- kit
- magnetic
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
- Pending
Links
Images
Classifications
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
- C12N15/09—Recombinant DNA-technology
- C12N15/10—Processes for the isolation, preparation or purification of DNA or RNA
- C12N15/1003—Extracting or separating nucleic acids from biological samples, e.g. pure separation or isolation methods; Conditions, buffers or apparatuses therefor
- C12N15/1006—Extracting or separating nucleic acids from biological samples, e.g. pure separation or isolation methods; Conditions, buffers or apparatuses therefor by means of a solid support carrier, e.g. particles, polymers
- C12N15/1013—Extracting or separating nucleic acids from biological samples, e.g. pure separation or isolation methods; Conditions, buffers or apparatuses therefor by means of a solid support carrier, e.g. particles, polymers by using magnetic beads
Landscapes
- Chemical & Material Sciences (AREA)
- Engineering & Computer Science (AREA)
- Health & Medical Sciences (AREA)
- Biomedical Technology (AREA)
- Life Sciences & Earth Sciences (AREA)
- Genetics & Genomics (AREA)
- Bioinformatics & Cheminformatics (AREA)
- Organic Chemistry (AREA)
- Zoology (AREA)
- Wood Science & Technology (AREA)
- Biotechnology (AREA)
- General Engineering & Computer Science (AREA)
- Crystallography & Structural Chemistry (AREA)
- Plant Pathology (AREA)
- Molecular Biology (AREA)
- Microbiology (AREA)
- Biophysics (AREA)
- Physics & Mathematics (AREA)
- Biochemistry (AREA)
- General Health & Medical Sciences (AREA)
- Analytical Chemistry (AREA)
- Measuring Or Testing Involving Enzymes Or Micro-Organisms (AREA)
Abstract
The invention discloses a genome magnetic bead extraction kit, which comprises: lysis solution, proteinase K, isopropanol, hydroxyl magnetic beads, washing solution and eluent; the formula of the lysis solution is as follows: 25-75 mM Tris-HCl with pH value of 7.5-8.5, 3-5M guanidinium isothiocyanate, 5-15% polidocanol, 5-15% PEG 8000; the washing liquid comprises a liquid A and a liquid B, wherein the liquid A: 120-130 mmol/l NaCl, and absolute ethyl alcohol with volume concentration of 80-90%; and the liquid B: 25-35 mmol/l Tris, 80-90% volume concentration absolute ethyl alcohol; the formula of the eluent comprises: 9.5 to 10.5mM Tris-HCl; 0.8 to 1.2mM EDTA. The concentration of the nucleic acid extracted by the kit is higher, the phenomenon of impurity carrying and dragging of nucleic acid agarose gel electrophoresis is avoided, and the purity of the extracted nucleic acid is better.
Description
Technical Field
The invention relates to the technical field of biology, in particular to a genome magnetic bead extraction kit and an extraction method.
Background
The nucleic acid extraction method includes phenol chloroform method, salting-out method, column adsorption method, etc. The traditional method for extracting nucleic acid has the advantages and disadvantages, but the extraction effect is not good enough, and the traditional method is only suitable for manual extraction and is not suitable for automation. The magnetic bead method utilizes the specific adsorption of magnetic beads on nucleic acid, separates and obtains the nucleic acid under the action of a magnetic field, and is suitable for automatic extraction.
The magnetic beads have good surface effect and volume effect, good selective magnetic responsiveness, stable physicochemical properties and the like, so that the nucleic acid extraction by the bead method is widely applied. However, different magnetic bead method nucleic acid extraction methods have different final nucleic acid extraction efficiencies due to different components and contents of selected lysis solution, binding solution, washing solution and the like. On the other hand, based on the diversity of the samples to be detected, including cells, bacteria, serum, plasma, semen, hair, sleeping liquid, urine or hydrothorax and ascites, etc., the extraction effect and the extraction efficiency of different samples to be detected adopting the same magnetic bead method nucleic acid extraction method are also greatly different.
The saliva is a nucleic acid which is easy to obtain, a sample to be detected is taken, the components of the saliva are greatly different from those in serum and plasma, and the saliva is colorless and tasteless liquid, is acidic and has high viscosity. Contains amylase, lysozyme, peroxidase, mucin, mucopolysaccharide, phospholipid, etc. Mucin and mucopolysaccharide are main substances for thickening saliva, so that the application of a magnetic bead method in extracting nucleic acid in the saliva is limited.
The existing magnetic bead extraction kit has the following defects: low extraction efficiency, low concentration of extracted nucleic acid and poor purity.
Therefore, how to develop a magnetic bead extraction kit and an extraction method with high extraction efficiency, high nucleic acid concentration and high purity is a technical problem to be solved urgently.
Disclosure of Invention
The invention aims to provide a genome magnetic bead extraction kit, the whole extraction process is carried out at normal temperature, nucleic acid with higher concentration and better purity is extracted, and convenience is provided for subsequent molecular diagnosis, target gene detection and the like.
In order to achieve the above object, the present invention provides a genomic magnetic bead extraction kit, comprising: lysis solution, proteinase K, isopropanol, hydroxyl magnetic beads, washing solution and eluent;
the formula of the lysis solution is as follows: 25-75 mM Tris-HCl with the pH value of 7.5-8.5, 3-5M guanidinium isothiocyanate, 5-15% polidocanol and 5-15% PEG 8000;
the washing liquid comprises a liquid A and a liquid B, wherein the liquid A is as follows: 120-130 mmol/l NaCl, and absolute ethyl alcohol with volume concentration of 80-90%; the solution B is as follows: 25-35 mmol/l Tris, 80-90% volume concentration absolute ethyl alcohol;
the formula of the eluent is as follows: 9.5 to 10.5mM Tris-HCl; 0.8 to 1.2mM EDTA.
Further, the formula of the lysis solution is as follows: 50mM Tris-HCl, pH8.0, 4M guanidinium isothiocyanate, 10% polidocanol, 10% PEG 8000.
Further, the washing liquid comprises a liquid A and a liquid B, wherein the liquid A is: 125mmol/l NaCl, absolute ethyl alcohol with volume concentration of 85 percent; the solution B is as follows: 30mmol/l Tris, 85% by volume absolute ethanol.
Further, the formula of the eluent is as follows: 10mM Tris-HCl; 1mM EDTA.
Further, the concentration of the proteinase K is 20-60 mg/ml.
Further, the concentration of the isopropanol is 200-400 ul/ml.
The invention also provides a method for extracting a genome by using the kit, which comprises the following steps:
adding protease K, lysis solution and isopropanol into a sample to be detected, uniformly mixing, adding hydroxyl magnetic beads, and uniformly mixing to obtain a mixed solution;
performing magnetic separation on the mixed solution, and taking a precipitate to obtain first magnetic beads;
adding the first magnetic beads into a washing solution A, pouring out supernatant after magnetic separation, adding a washing solution B, and pouring out supernatant after magnetic separation to obtain second magnetic beads;
and drying the second magnetic beads, adding an eluent for elution, and carrying out magnetic separation to obtain a supernatant, namely the extracted genome.
Further, the volume ratio of the proteinase K, the lysis solution, the isopropanol and the hydroxyl magnetic beads is 2: (48-52): (33-37): 2.
Further, adding the hydroxyl magnetic beads, and uniformly mixing by vortex oscillation.
Further, heating for 5-10 min at 60-70 ℃ during elution; during the magnetic separation, the centrifugal tube is placed on a magnetic separator until the solution is clear.
One or more technical solutions in the embodiments of the present invention have at least the following technical effects or advantages:
the concentration of nucleic acid extracted by the genome magnetic bead extraction kit and the extraction method provided by the invention is higher, and the nucleic acid agarose gel electrophoresis has no impurity and dragging phenomena, which indicates that the purity of the extracted nucleic acid is better; the lysate does not need to be heated when in use, can ensure the complete lysis of cells and release nucleic acid at normal temperature, and is favorable for the combination of the nucleic acid and magnetic beads; the washing solution can remove protein and other impurities adsorbed on the fiber membrane, such as polysaccharide, cellulose, lipid, etc. Compared with a nutria kit, the reagent has the advantages of high concentration purity of extracted nucleic acid and bright target gene band. When the washing time is shortened to 1min, the washing liquid has better extraction effect than the beaver kit for extracting nucleic acid; the air drying time is shortened to 5min, and the elution time is shortened to 7min, so that the reagent has better extraction effect than the beaver kit. Therefore, the final nucleic acid extraction time is shortened to 3min for cracking and binding, 1min for washing A, 1min for washing B, 5min for air drying and 7min for elution, and the extraction time is greatly shortened.
Drawings
In order to more clearly illustrate the technical solutions in the embodiments of the present invention, the drawings needed to be used in the description of the embodiments are briefly introduced below, and it is obvious that the drawings in the following description are some embodiments of the present invention, and it is obvious for those skilled in the art to obtain other drawings based on the drawings without creative efforts.
FIG. 1 is a drawing showing the detection of a gene of interest and the agarose gel electrophoresis of nucleic acid in Experimental example 1 of the present invention; wherein nos. 1 to 7 represent comparative example 1 to comparative example 7, respectively, No. 8 represents example 1, No. 9 to 11 represent comparative example 8 to comparative example 10, respectively;
FIG. 2 is a drawing showing the detection of a gene of interest and the agarose gel electrophoresis of nucleic acid in Experimental example 2 of the present invention; wherein nos. 1 to 7 represent comparative example 11 to comparative example 17, respectively, No. 8 represents example 1, and No. 9 represents comparative example 18; the radix asparagi represents a detection kit adopting radix asparagi, and NC represents a negative control group;
FIG. 3 is a drawing showing detection of a gene of interest and nucleic acid agarose gel electrophoresis in Experimental example 3 of the present invention; wherein, No. 1 represents comparative example 19, respectively, No. 2 represents examples 1, and No. 4 to 8 represent comparative examples 21 to 25, respectively;
FIG. 4 is a diagram showing the detection of a target gene and the agarose gel electrophoresis of nucleic acid in Experimental example 4 of the present invention. (A) The figure is the nucleic acid target gene extracted by three kinds of magnetic beads and the nucleic acid agarose gel electrophoresis picture; (B) the figure shows the concentration detection result of nucleic acid extracted by three magnetic beads;
FIG. 5 is a drawing showing the detection of a target gene with a shortened nucleic acid extraction time and the nucleic acid agarose gel electrophoresis in Experimental example 5; (A) FIG. shows the lysis and binding steps combined with the present invention in comparison to a beaver kit; (B) the figure shows that the invention is compared with a beaver kit for shortening the washing time; (C) the figure shows that the invention is compared with a beaver kit for shortening the airing time; (D) the figure shows that the elution time is shortened by comparing the invention with a beaver kit;
FIG. 6 is a diagram showing the detection of a target gene and the agarose gel electrophoresis of nucleic acid in example 1;
FIG. 7 is a diagram showing the detection of a target gene and the agarose gel electrophoresis of nucleic acid in example 2;
FIG. 8 is a diagram showing the detection of a target gene and the agarose gel electrophoresis of nucleic acid in example 3.
Detailed Description
The present invention will be described in detail below with reference to specific embodiments and examples, and the advantages and various effects of the present invention will be more clearly apparent therefrom. It will be understood by those skilled in the art that these specific embodiments and examples are for the purpose of illustrating the invention and are not to be construed as limiting the invention.
Throughout the specification, unless otherwise specifically noted, terms used herein should be understood as having meanings as commonly used in the art. Accordingly, 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. If there is a conflict, the present specification will control.
Unless otherwise specifically stated, various raw materials, reagents, instruments, equipment and the like used in the present invention are commercially available or can be obtained by an existing method. The terms "first", "second", and the like in the present invention do not denote any order, and they may be understood as nouns.
In order to solve the technical problems, the general idea of the embodiment of the application is as follows:
according to an exemplary embodiment of the present invention, there is provided a genome magnetic bead extraction kit, including: lysis solution, proteinase K, isopropanol, hydroxyl magnetic beads, washing solution and eluent;
the formula of the lysis solution is as follows: 25-75 mM Tris-HCl with the pH value of 7.5-8.5, 3-5M guanidinium isothiocyanate, 5-15% polidocanol and 5-15% PEG 8000;
the washing liquid comprises a liquid A and a liquid B, wherein the liquid A is as follows: 120-130 mmol/l NaCl, and absolute ethyl alcohol with volume concentration of 80-90%; the solution B is as follows: 25-35 mmol/l Tris, 80-90% volume concentration absolute ethyl alcohol;
the formula of the eluent is as follows: 9.5 to 10.5mM Tris-HCl; 0.8 to 1.2mM EDTA.
The invention provides a genome magnetic bead extraction kit, which has higher nucleic acid concentration, and has no impurity and drag phenomena in nucleic acid agarose gel electrophoresis, thus indicating that the purity of extracted nucleic acid is better; the lysate does not need to be heated when in use, can ensure the complete lysis of cells and release nucleic acid at normal temperature, and is favorable for the combination of the nucleic acid and magnetic beads; the washing solution can remove protein and other impurities adsorbed on the fiber membrane, such as polysaccharide, cellulose, lipid, etc.
The invention provides a genome magnetic bead extraction kit which can extract all genomes including DNA and RNA; in the present example, saliva genomic DNA was extracted.
As a preferred embodiment, the formulation of the lysis solution is: 50mM Tris-HCl, pH8.0, 4M guanidinium isothiocyanate, 10% polidocanol, 10% PEG 8000.
In a preferred embodiment, the washing solution comprises solution a and solution B, wherein solution a is: 125mmol/l NaCl, absolute ethyl alcohol with volume concentration of 85 percent; the solution B is as follows: 30mmol/l Tris, 85% by volume absolute ethanol.
As a preferred embodiment, the formulation of the eluent is: 10mM Tris-HCl; 1mM EDTA.
In a preferred embodiment, the hydroxyl magnetic bead is a hydroxyl magnetic bead 70304-5(OH) from beaver nanotechnology, Inc.
In a preferred embodiment, the concentration of proteinase K is 20-60 mg/ml. Can extract nucleic acid with higher concentration and better purity
In a preferred embodiment, the concentration of the isopropanol is 200 to 400 ul/ml. Nucleic acid with higher concentration and purity can be extracted.
According to an exemplary embodiment of the present invention, there is provided a method for extracting a genome using the kit, the method including:
adding protease K, lysis solution and isopropanol into a sample to be detected, uniformly mixing, adding hydroxyl magnetic beads, and uniformly mixing to obtain a mixed solution;
performing magnetic separation on the mixed solution, and taking a precipitate to obtain first magnetic beads;
adding the first magnetic beads into a washing solution A, pouring out supernatant after magnetic separation, adding a washing solution B, and pouring out supernatant after magnetic separation to obtain second magnetic beads;
and drying the second magnetic beads, adding an eluent for elution, and carrying out magnetic separation to obtain a supernatant, namely the extracted genome.
The volume ratio of the protease K to the lysis solution to the isopropanol to the hydroxyl magnetic beads is 2: (48-52): (33-37): 2.
and adding the hydroxyl magnetic beads, and uniformly mixing by adopting a vortex oscillation uniform mixing mode.
During elution, heating at 60-70 ℃ for 5-10 min; during the magnetic separation, the centrifugal tube is placed on a magnetic separator until the solution is clear.
The detection principle of the genome magnetic bead extraction kit provided by the invention is as follows:
the function of the lysis solution is to cause the cell to be lysed, and the genomic DNA in the cell is released; the invention improves the components and the content of the cracking liquid, the combination does not need to be heated, the cells can be ensured to be completely cracked and the nucleic acid can be released at normal temperature, and the combination of the nucleic acid and the magnetic beads is facilitated. The lysate can be used for extracting nucleic acid in cells, bacteria, serum, plasma, saliva, urine or pleural effusion, preferably serum or plasma, and more preferably extracting nucleic acid in cells, bacteria, serum, plasma, saliva, urine or pleural effusion and saliva of a sample to be detected in PCR detection. Preferably, the PCR method comprises:
detecting a target gene: action
Primer: Action-F: GTTTGAGACCTTCAACACCCCA
Action-R：CAGGAAGGAAGGCTGGAAGAGT
Adding protease K, lysate and isopropanol into a PCR reaction tube, uniformly mixing, adding hydroxyl magnetic beads, uniformly mixing, adding a sample to be detected, adding an amplification primer pair and other PCR reaction system components, uniformly mixing, standing, performing magnetic attraction and then sucking to obtain a mixed solution; and then washing, eluting and carrying out magnetic separation to obtain supernatant, namely the extracted genome.
The proteinase K is used for degrading membrane protein and degrading protein combined with DNA so as to fully dissociate the DNA;
isopropyl alcohol: hydrophobic, can better precipitate nucleic acid, when isopropanol precipitates nucleic acid, high concentration salt exists to enable a large amount of polysaccharide to exist in solution, thereby achieving the effect of removing polysaccharide;
washing liquid: can remove protein and other impurities adsorbed on the fiber membrane, such as polysaccharide, cellulose, lipid, etc. The washing liquid has good extraction effect.
Eluent: is a nuclease-free liquid, and is treated with diethyl pyrophosphate (DEPC) to elute the nucleic acids adsorbed on the magnetic beads.
The extraction by the magnetic bead method is based on the same principle as that of an adsorption membrane centrifugal column, and the superparamagnetic silicon oxide nanometer magnetic bead is prepared by improving and modifying the surfaces of superparamagnetic nanometer particles by using a nanometer technology. The magnetic beads can be specifically identified and efficiently combined with nucleic acid molecules on a microscopic interface. The superparamagnetism of the nanometer magnetic microsphere coated by the silicon dioxide can separate DNA and RNA from samples such as blood, animal tissues, food, pathogenic microorganisms and the like under the action of salt (guanidine hydrochloride, guanidine isothiocyanate and the like) and an external magnetic field, and can be applied to various fields such as clinical disease diagnosis, transfusion safety, forensic medicine identification, environmental microorganism detection, food safety detection, molecular biology research and the like.
The following will describe the preparation method of the genomic magnetic bead extraction kit of the present application in detail with reference to examples, comparative examples and experimental data.
Example 1
1. The genome magnetic bead extraction kit provided by the embodiment of the invention comprises: lysis solution, proteinase K, isopropanol, washing solution, eluent and hydroxyl magnetic beads;
the formula of the lysis solution is as follows: : 50mM Tris-HCl, pH8.0, 4M guanidinium isothiocyanate, 10% polidocanol, 10% PEG 8000;
the washing liquid comprises a liquid A and a liquid B, wherein the liquid A is as follows: 125mmol/l NaCl, absolute ethyl alcohol with volume concentration of 85 percent; the solution B is as follows: 30mmol/l Tris, 85% volume concentration absolute ethyl alcohol;
the formula of the eluent is as follows: 8mM Tris-HCl; 0.5mM EDTA.
2. Application method of genome magnetic bead extraction kit
(1) Sample lysis binding:
adding 125 mu L of sample, 20 mu L of proteinase K solution and 500 mu L of lysis solution into a 1.5mL centrifuge tube, adding 350 mu L of isopropanol into the centrifuge tube, adding 20 mu L of magnetic bead suspension, uniformly mixing by vortex oscillation (the speed is between 9 and 10) for 3min, and standing for 2 min.
(2) Magnetic separation
Placing the centrifugal tube on a magnetic frame and standing for 20s until the magnetic beads are completely adsorbed; if the magnetic beads are covered in the centrifuge tube, the EP tube can be kept on the magnetic frame, and the whole body is turned upside down for 2-3 times until the magnetic beads are completely adsorbed. The EP tube was kept fixed on the magnetic stand and the supernatant was aspirated off with a pipette, during which time contact with the magnetic beads was avoided.
(3) Cleaning solution A
Add 800. mu.L of Wash I to 1.5mL centrifuge tube, remove 1.5mL centrifuge tube from magnetic rack, blow away magnetic beads with pipette, vortex (between 9-10) for 1min, magnetically separate (see step 2), repeat this operation 1 time.
(4) Cleaning solution B
Using 800. mu.L of washing solution II (see procedure 3)
(5) Drying
Keeping the centrifuge tube on the magnetic separator, opening the cover at room temperature, standing for 5min, and taking down the centrifuge tube.
Note: if liquid residue is found in the reaction tube in the drying process, a small-range liquid transfer device can be used for sucking and discarding the liquid.
(6) Elution is carried out
Adding 100 mu L of eluent, carrying out vortex oscillation for 1min to fully suspend magnetic beads, heating at 65 ℃ for 7min, placing the centrifugal tube on a magnetic separator until the solution is clarified, transferring the supernatant into a new centrifugal tube of 1.5mL to obtain purified saliva DNA, and storing at-20 ℃. FIG. 6 is a diagram showing the detection of a target gene and the agarose gel electrophoresis of nucleic acid in example 1; the target gene detection strip is bright, and nucleic acid is found to have no dragging and dispersion phenomena by running the nucleic acid gel.
Example 2
1. The genome magnetic bead extraction kit provided by the embodiment of the invention comprises: lysis solution, proteinase K, isopropanol, washing solution, eluent and hydroxyl magnetic beads;
the formula of the lysis solution is as follows: Tris-HCl 25mM and pH7.5, guanidinium isothiocyanate 3M, polidocanol 5%, PEG8000 5%;
the washing liquid comprises a liquid A and a liquid B, wherein the liquid A is as follows: 120mmol/l NaCl, 80% volume concentration absolute ethyl alcohol; the solution B is as follows: 25mmol/l Tris, 80% volume concentration absolute ethyl alcohol;
the formula of the eluent is as follows: 9.5mM Tris-HCl; 0.8mM EDTA.
2. The method of using the kit for extracting genomic magnetic beads was the same as in example 1. FIG. 7 is a diagram showing the detection of a target gene and the agarose gel electrophoresis of nucleic acid in example 1; the target gene detection strip is bright, and nucleic acid is found to have no dragging and dispersion phenomena by running the nucleic acid gel.
Example 3
1. The genome magnetic bead extraction kit provided by the embodiment of the invention comprises: lysis solution, proteinase K, isopropanol, washing solution, eluent and hydroxyl magnetic beads;
the formula of the lysis solution is as follows: Tris-HCl 75mM and pH8.5, guanidinium isothiocyanate 5M, polidocanol 15%, PEG8000 15%;
the washing liquid comprises a liquid A and a liquid B, wherein the liquid A is as follows: 130mmol/l NaCl, absolute ethyl alcohol with volume concentration of 90%; the solution B is as follows: 35mmol/l Tris, absolute ethyl alcohol with volume concentration of 90%;
the formula of the eluent is as follows: 10.5mM Tris-HCl; 1.2mM EDTA.
2. The method of using the kit for extracting genomic magnetic beads was the same as in example 1. FIG. 8 is a photograph of the detection of a gene of interest and the agarose gel electrophoresis of nucleic acid in example 1; the target gene detection strip is bright, and nucleic acid is found to have no dragging and dispersion phenomena by running the nucleic acid gel.
Comparative examples 1 to 10
The formulations of the lysates in comparative examples 1-10 are different from those in example 1, and the other components are the same as those in example 1, as shown in Table 1.
TABLE 1 lysate formulations
Comparative examples 11 to 18
The formulations of the cleaning solutions of comparative examples 11 to 18 were different from those of example 1, and the other components were the same as those of example 1, as shown in Table 2.
TABLE 2 detergent formulations
Comparative examples 19 to 25
The eluent formulations in comparative examples 19-25 differ from example 1, and the other components are the same as in example 1, as shown in Table 3.
TABLE 3 eluent formulations
| Group of | Number of eluent | Eluent |
| Comparative example 19 | 1 | 20mM Tris-HCl |
| Example 1 | 2 | 10mM Tris-HCl；1mM EDTA |
| Comparative example 20 | 3 | 8mM Tris-HCl；0.5mM EDTA |
| Comparative example 21 | 4 | 30mM Tris-HCl；2mM EDTA |
| Comparative example 22 | 5 | 10mM Tris-HCl；0.1mM EDTA |
| Comparative example 23 | 6 | 0.25mM EDTANa2 |
| Comparative example 24 | 7 | 0.75mM EDTANa2 |
| Comparative example 25 | 8 | DEPC water |
Comparative examples 26 to 27
The magnetic beads in comparative examples 26 to 27 are different from those in example 1, and the other components are the same as those in example 1, and are specifically shown in Table 4.
TABLE 4
Experimental example 1
Under different cracking temperatures, the kits of each example and comparative example are respectively adopted to extract nucleic acid from the same number of human cells, the nucleic acid concentration, purity and target gene expression level are detected, and compared with the beaver saliva nucleic acid extraction kit, the results are shown in figure 1 and table 5.
TABLE 5 concentration and purity of nucleic acids extracted from lysate
| Experiment number | Cracking temperature (. degree.C.) | Nucleic acid concentration (ng/ul) | Purity of nucleic acid (260/280) |
| Comparative example 1 | 56 | 284 | 1.88 |
| Comparative example 2 | 56 | 715 | 1.86 |
| Comparative example 3 | 56 | 705 | 1.95 |
| Comparative example 4 | 56 | 250 | 1.99 |
| Comparative example 5 | 56 | 478 | 1.9 |
| Comparative example 6 | 56 | 953 | 1.93 |
| Comparative example 7 | 56 | 443 | 1.89 |
| Example 1 | 56 | 954 | 1.9 |
| Comparative example 8 | 56 | 66 | 2 |
| Comparative example 9 | 56 | 911 | 2 |
| Comparative example 10 | 56 | 786 | 1.97 |
| Comparative example 4 | At normal temperature | 394 | 1.99 |
| Comparative example 6 | At normal temperature | 954 | 1.98 |
| Comparative example 7 | At normal temperature | 935 | 2 |
| Example 1 | At normal temperature | 1136 | 2.04 |
| Comparative example 9 | At normal temperature | 995 | 1.99 |
| Comparative example 10 | At normal temperature | 946 | 2 |
| Beaver | 56 | 751 | 2.03 |
As can be seen from the data results in Table 5, the concentration of the nucleic acid extracted by the kit of example 1 was the highest without heating (1136 ng/ul was reached), and as can be seen from FIG. 1, the band of the target gene extracted by the kit of example 1 was brighter, and the nucleic acid was found to be free from the phenomenon of smearing and diffusion by running the nucleic acid gel, indicating that the nucleic acid was not degraded.
Experimental example 2
The kit of each example and comparative example is used for extracting nucleic acid from the same number of human cells under the same conditions, the concentration, purity and target gene expression level of the nucleic acid are detected, and the results are shown in FIG. 2 and Table 6 by comparing with the washing solution of the beaver saliva nucleic acid extraction kit.
TABLE 6 washing solution extraction nucleic acid concentration and purity
| Experiment number | Nucleic acid concentration (ng/ul) | Purity of nucleic acid (260/280) |
| Comparative example 11 | 197 | 1.86 |
| Comparative example 12 | 838 | 1.82 |
| Comparative example 13 | 904 | 1.83 |
| Comparative example 14 | 157 | 1.89 |
| Comparative example 15 | 760 | 1.86 |
| Comparative example 16 | 259 | 1.83 |
| Comparative example 17 | 858 | 1.81 |
| Example 1 | 1234 | 1.83 |
| Comparative example 18 | 811 | 1.8 |
| Beaver | 1113 | 1.88 |
As can be seen from the data results in Table 6, the concentration of the nucleic acid extracted by the kit of example 1 was the highest (reached to 1234ng/ul), and as can be seen from FIG. 2, the target gene detection band extracted by the kit of example 1 was brighter, and nucleic acid was found to be free of streaking and diffusion phenomena by running nucleic acid, indicating that the nucleic acid was not degraded.
Experimental example 3
The kit of each example and comparative example was used to extract nucleic acid from the same number of human cells under the same conditions, and the results of the nucleic acid concentration, purity and expression level of the target gene were determined and compared with the washing solution of the beaver saliva nucleic acid extraction kit, and are shown in FIG. 3 and Table 7.
TABLE 7 elution extraction of nucleic acid concentration and purity
| Experiment number | Nucleic acid concentration (ng/ul) | Purity of nucleic acid (260/280) |
| Comparative example 19 | 1121 | 1.84 |
| Example 1 | 1246 | 1.86 |
| Comparative example 20 | 828 | 1.84 |
| Comparative example 21 | 1063 | 1.84 |
| Comparative example 22 | 714 | 1.87 |
| Comparative example 23 | 389 | 1.83 |
| Comparative example 24 | 688 | 1.87 |
| Comparative example 25 | 1003 | 1.86 |
| Beaver | 971 | 1.82 |
As can be seen from the data results in Table 7, the concentration of the nucleic acid extracted by the kit of example 1 was the highest (reached 1246ng/ul), and as can be seen from FIG. 3, the target gene detection band extracted by the kit of example 1 was brighter, and nucleic acid was found to be free of streaking and diffusion phenomena by running gel on nucleic acid, indicating that the nucleic acid was not degraded.
Experimental example 4
Nucleic acid extraction was performed on the same number of human cells under the same conditions using the kits of comparative examples 26 to 27 and example 1, respectively, and the nucleic acid concentration, purity and expression level of the target gene were measured, and the results are shown in FIG. 4.
Hydroxymagnetic bead 70304-5(OH) from beaver nanotechnology Co., Ltd, carboxymagnetic bead 70107-5(COOH) from beaver nanotechnology Co., Ltd, and beaver saliva extraction kit as control (OH). Three times of repeated experiments are compared to find that the concentration of the nucleic acid extracted by the hydroxyl magnetic beads is highest, and a target gene detection strip is brighter.
Experimental example 5
The extraction kit of example 1 of the present invention was compared with a beaver kit:
(1) the cleavage was combined with the binding step: adding lysis solution, binding solution, proteinase K and magnetic beads into the sample, performing vortex oscillation for 3min, and performing the same treatment by using a beaver kit. The comparison of the extracted nucleic acids shows that the concentration and purity of the nucleic acid extracted by the reagent are better, and the target gene band is brighter (FIG. 5A).
(2) When the washing time of the extraction kit of the embodiment 1 and the washing time of the beaver kit are both shortened to 1min, the washing liquid has better extraction effect than the beaver kit in extracting nucleic acid purity (fig. 5B).
(3) The extraction effect of the reagent of the invention is better than that of the beaver kit when the air-drying time of the extraction kit and the beaver kit of the invention embodiment 1 are both shortened to 5min and the elution time is both shortened to 7min (fig. 5C and D).
Therefore, finally, the nucleic acid extraction time of the kit of the embodiment 1 is shortened to 3min for cracking and binding, washing A is carried out for 1min, washing B is carried out for 1min, air drying is carried out for 5min, and elution is carried out for 7min, so that the extraction time is greatly shortened.
In conclusion, the comparison of 11 formulas in the lysis process proves that the formula of the example 1 can well lyse cells and release nucleic acid at normal temperature. Simplifies the operation steps, shortens the operation time and ensures the concentration and the purity of the nucleic acid extraction. The target gene detection also proves that the PCR reagent has no influence on the subsequent PCR experiment and has a bright target band.
The invention also optimizes the washing liquid. By comparing the extraction effects of the 9 kinds of washing solutions, the invention proves that the concentration of the nucleic acid extracted by the formula of the washing solution in the embodiment 1 is higher, and the nucleic acid agarose gel electrophoresis has no impurity and dragging phenomena, thereby indicating that the purity of the extracted nucleic acid is better.
Also for the elution process, the formulations of the present invention work better than other formulations.
The cracking process is carried out at normal temperature, so that the experimental steps are simplified, the automatic extraction is more favorably realized, the formula and experimental conditions of the whole extraction process are optimized, the extraction of nucleic acid with higher concentration is realized, and the time of the whole extraction process is shortened.
Finally, it should also be noted that the terms "comprises," "comprising," or any other variation thereof, are intended to cover a non-exclusive inclusion, such that a process, method, article, or apparatus that comprises a list of elements does not include only those elements but may include other elements not expressly listed or inherent to such process, method, article, or apparatus.
While preferred embodiments of the present invention have been described, additional variations and modifications in those embodiments may occur to those skilled in the art once they learn of the basic inventive concepts. Therefore, it is intended that the appended claims be interpreted as including preferred embodiments and all such alterations and modifications as fall within the scope of the invention.
It will be apparent to those skilled in the art that various changes and modifications may be made in the present invention without departing from the spirit and scope of the invention. Thus, if such modifications and variations of the present invention fall within the scope of the claims of the present invention and their equivalents, the present invention is also intended to include such modifications and variations.
Claims (10)
1. A genome magnetic bead extraction kit is characterized by comprising: lysis solution, proteinase K, isopropanol, hydroxyl magnetic beads, washing solution and eluent;
the formula of the lysis solution is as follows: 25-75 mM Tris-HCl with pH value of 7.5-8.5, 3-5M guanidinium isothiocyanate, 5-15% polidocanol, 5-15% PEG 8000;
the washing liquid comprises a liquid A and a liquid B, wherein the liquid A is as follows: 120-130 mmol/l NaCl, and absolute ethyl alcohol with volume concentration of 80-90%; the solution B is as follows: 25-35 mmol/l Tris, 80-90% volume concentration absolute ethyl alcohol;
the formula of the eluent is as follows: 9.5 to 10.5mM Tris-HCl; 0.8 to 1.2mM EDTA.
2. The kit for extracting genome magnetic beads according to claim 1, wherein the formula of the lysis solution is as follows: 50mM Tris-HCl, pH8.0, 4M guanidinium isothiocyanate, 10% polidocanol, 10% PEG 8000.
3. The kit for extracting genomic magnetic beads according to claim 1, wherein the washing solution comprises a solution A and a solution B, wherein the solution A is: 125mmol/l NaCl, absolute ethyl alcohol with volume concentration of 85 percent; the solution B is as follows: 30mmol/l Tris, 85% by volume absolute ethanol.
4. The genome magnetic bead extraction kit of claim 1, wherein the eluent is prepared from the following formula: 10mM Tris-HCl; 1mM EDTA.
5. The kit for extracting genomic magnetic beads according to claim 1, wherein the concentration of proteinase K is 20-60 mg/ml.
6. The kit for extracting genomic magnetic beads according to claim 1, wherein the concentration of isopropanol is 200-400 ul/ml.
7. A method for extracting a genome using the kit of any one of claims 1 to 6, the method comprising:
adding protease K, lysis solution and isopropanol into a sample to be detected, uniformly mixing, adding hydroxyl magnetic beads, and uniformly mixing to obtain a mixed solution;
performing magnetic separation on the mixed solution, and taking a precipitate to obtain first magnetic beads;
adding the first magnetic beads into a washing solution A, pouring out supernatant after magnetic separation, adding a washing solution B, and pouring out supernatant after magnetic separation to obtain second magnetic beads;
and drying the second magnetic beads, adding an eluent for elution, and carrying out magnetic separation to obtain a supernatant, namely the extracted genome.
8. The method of claim 7, wherein the volume ratio of proteinase K, lysis solution, isopropanol, and hydroxyl magnetic beads is 2: (48-52): (33-37): 2.
9. the method of claim 7, wherein the hydroxyl-added magnetic beads are mixed by vortexing and shaking.
10. The method according to claim 7, wherein the elution is carried out by heating at 60 to 70 ℃ for 5 to 10 min; during the magnetic separation, the centrifugal tube is placed on a magnetic separator until the solution is clear.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN202010922941.6A CN111961663A (en) | 2020-09-04 | 2020-09-04 | Genome magnetic bead extraction kit and extraction method |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN202010922941.6A CN111961663A (en) | 2020-09-04 | 2020-09-04 | Genome magnetic bead extraction kit and extraction method |
Publications (1)
| Publication Number | Publication Date |
|---|---|
| CN111961663A true CN111961663A (en) | 2020-11-20 |
Family
ID=73392205
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN202010922941.6A Pending CN111961663A (en) | 2020-09-04 | 2020-09-04 | Genome magnetic bead extraction kit and extraction method |
Country Status (1)
| Country | Link |
|---|---|
| CN (1) | CN111961663A (en) |
Cited By (5)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN113322253A (en) * | 2021-04-09 | 2021-08-31 | 长沙理工大学 | Kit for extracting genome DNA based on magnetic material and application method |
| CN114164206A (en) * | 2021-12-16 | 2022-03-11 | 苏州海狸生物医学工程有限公司 | Soybean genome DNA extraction reagent and application thereof |
| CN114317523A (en) * | 2021-12-24 | 2022-04-12 | 深圳市华晨阳科技有限公司 | Extraction reagent and extraction kit for high-purity nucleic acid |
| CN114934041A (en) * | 2022-04-27 | 2022-08-23 | 北京金豪制药股份有限公司 | Reagent and method for extracting nucleic acid |
| CN116949036A (en) * | 2023-09-11 | 2023-10-27 | 成都斯马特科技有限公司 | Kit for rapidly extracting nucleic acid from ascites and extraction method |
Citations (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN101935645A (en) * | 2010-09-13 | 2011-01-05 | 原平皓(天津)生物技术有限公司 | Kit for extracting DNA from histiocytes and method thereof |
| CN105368820A (en) * | 2015-12-22 | 2016-03-02 | 南京先进激光技术研究院 | Whole blood DNA (deoxyribonucleic acid) extraction kit based on paramagnetic particle method and application of extraction kit |
| CN109722431A (en) * | 2019-01-21 | 2019-05-07 | 上海科华生物工程股份有限公司 | It is a kind of based on paramagnetic particle method without alcohol Viral nucleic acid extraction reagent box |
| CN110938624A (en) * | 2019-12-27 | 2020-03-31 | 深圳市海普洛斯生物科技有限公司 | Kit for extracting genome DNA and application thereof |
-
2020
- 2020-09-04 CN CN202010922941.6A patent/CN111961663A/en active Pending
Patent Citations (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN101935645A (en) * | 2010-09-13 | 2011-01-05 | 原平皓(天津)生物技术有限公司 | Kit for extracting DNA from histiocytes and method thereof |
| CN105368820A (en) * | 2015-12-22 | 2016-03-02 | 南京先进激光技术研究院 | Whole blood DNA (deoxyribonucleic acid) extraction kit based on paramagnetic particle method and application of extraction kit |
| CN109722431A (en) * | 2019-01-21 | 2019-05-07 | 上海科华生物工程股份有限公司 | It is a kind of based on paramagnetic particle method without alcohol Viral nucleic acid extraction reagent box |
| CN110938624A (en) * | 2019-12-27 | 2020-03-31 | 深圳市海普洛斯生物科技有限公司 | Kit for extracting genome DNA and application thereof |
Non-Patent Citations (3)
| Title |
|---|
| ZHANG, M等: "Adsorption of DNA by using polydopamine modified magnetic nanoparticles based on solid-phase extraction", 《ANALYTICAL BIOCHEMISTRY》 * |
| 朴英实等: "《分子病理生物学实验技术指南》", 31 May 2015 * |
| 李民: "《法医物证DNA检验技术的探索性研究》", 30 June 2017 * |
Cited By (9)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN113322253A (en) * | 2021-04-09 | 2021-08-31 | 长沙理工大学 | Kit for extracting genome DNA based on magnetic material and application method |
| CN114164206A (en) * | 2021-12-16 | 2022-03-11 | 苏州海狸生物医学工程有限公司 | Soybean genome DNA extraction reagent and application thereof |
| CN114164206B (en) * | 2021-12-16 | 2023-08-04 | 苏州海狸生物医学工程有限公司 | Soybean genome DNA extraction reagent and application thereof |
| CN114317523A (en) * | 2021-12-24 | 2022-04-12 | 深圳市华晨阳科技有限公司 | Extraction reagent and extraction kit for high-purity nucleic acid |
| CN114317523B (en) * | 2021-12-24 | 2024-04-02 | 深圳市华晨阳科技有限公司 | Extraction reagent and extraction kit for high-purity nucleic acid |
| CN114934041A (en) * | 2022-04-27 | 2022-08-23 | 北京金豪制药股份有限公司 | Reagent and method for extracting nucleic acid |
| CN114934041B (en) * | 2022-04-27 | 2023-12-15 | 北京金豪制药股份有限公司 | Reagent and method for extracting nucleic acid |
| CN116949036A (en) * | 2023-09-11 | 2023-10-27 | 成都斯马特科技有限公司 | Kit for rapidly extracting nucleic acid from ascites and extraction method |
| CN116949036B (en) * | 2023-09-11 | 2023-12-15 | 成都斯马特科技有限公司 | Kit for rapidly extracting nucleic acid from ascites and extraction method |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| CN111961663A (en) | Genome magnetic bead extraction kit and extraction method | |
| US8691969B2 (en) | Isolation of nucleic acid | |
| CN112980830B (en) | Kit for extracting nucleic acid by magnetic bead method, magnetic bead and preparation method thereof | |
| CN106574265B (en) | Method for isolating RNA with high yield | |
| WO2014063651A1 (en) | Method for purifying nucleic acid and kit | |
| CN111057705B (en) | Kit for extracting free nucleic acid and use method | |
| AU6789500A (en) | Simultaneous isolation and quantitation of dna | |
| JP2006517225A (en) | Chemical treatment of biological samples for nucleic acid extraction and kits for the treatment | |
| US9051563B2 (en) | Nucleic acid purification | |
| CN106754884B (en) | Kit and application thereof | |
| CN112481254B (en) | Method and kit for removing host DNA and enriching microorganisms by one-step method | |
| CN112941067A (en) | Lysis binding solution for whole blood nucleic acid extraction and kit and application thereof | |
| WO2001062976A1 (en) | Rapid nucleic acid separation, isolation and purification methods | |
| CN110863037A (en) | Microarray chip capable of detecting various fungal genome DNAs (deoxyribonucleic acids) and preparation method thereof | |
| CN110923338A (en) | Microarray chip capable of detecting various bacterial genome DNAs and preparation method thereof | |
| CN108866042B (en) | Extraction method of trace RNA | |
| JP2002212198A (en) | Method for separating nucleic acid and carrier for separating nucleic acid | |
| JP2002209580A (en) | Method for isolating nucleic acid and globular carrier | |
| CN114395614A (en) | Method for rapidly detecting body fluid metagenome pathogenic microorganisms in high flux | |
| CN117587004A (en) | Lysis binding solution for extracting fungus DNA, kit and extraction method and application thereof | |
| JP3811767B6 (en) | Method for purifying nucleic acids from a homogeneous mixture | |
| CN116135975A (en) | Nucleic acid extraction and purification kit and use method thereof | |
| JP3811767B2 (en) | Method for purifying nucleic acids from a homogeneous mixture | |
| CN116286798A (en) | Kit and method suitable for bacterial DNA extraction and purification | |
| EP1510578A2 (en) | Simultaneous isolation and quantitation of dna |
Legal Events
| Date | Code | Title | Description |
|---|---|---|---|
| PB01 | Publication | ||
| PB01 | Publication | ||
| SE01 | Entry into force of request for substantive examination | ||
| SE01 | Entry into force of request for substantive examination | ||
| RJ01 | Rejection of invention patent application after publication |
Application publication date: 20201120 |
|
| RJ01 | Rejection of invention patent application after publication |