CN110938624A - Kit for extracting genome DNA and application thereof - Google Patents
Kit for extracting genome DNA and application thereof Download PDFInfo
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- CN110938624A CN110938624A CN201911374467.1A CN201911374467A CN110938624A CN 110938624 A CN110938624 A CN 110938624A CN 201911374467 A CN201911374467 A CN 201911374467A CN 110938624 A CN110938624 A CN 110938624A
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- 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
Abstract
The application discloses a kit for extracting genome DNA and application thereof. The kit comprises lysate and erythrocyte lysate; the lysate contains 20-300mM Tris-HCl, 20-100mM EDTA, 0.3-2M NaCl, 0.5-5% PEG8000, 0.5-10% Triton X-100, SDS or Tween 20; the erythrocyte lysate contains 10-100mM Tris-HCl, 1-50mM EDTA, 10-300mM NaCl and 0.01-0.2% Triton X-100. This application kit optimizes through lysate and red blood cell lysate, can effectively get rid of the polysaccharide, combines phenol, prevents that phenol and DNA from combining, solves and glues thick and muddy problem, is applicable to multiple different samples, and the genome DNA purity and the volume of extracting can both satisfy various low reaches experiment user demands.
Description
Technical Field
The application relates to the technical field of genome DNA extraction, in particular to a kit for extracting genome DNA and application thereof.
Background
Nucleic acid is a very important biological macromolecule, which plays an irreplaceable role in a series of life processes of reproduction, growth, development, aging, death and the like of organisms, is called the 'leading part' of the biological world, and is now an important research subject in a plurality of life science fields of biochemistry, molecular biology, genetics, biomedicine and the like. Therefore, it is an essential first step to develop other studies to extract high-quality and high-purity nucleic acid from many complex biological macromolecules.
The principles of DNA extraction include: the integrity of the primary structure of the DNA is ensured; organic solvents having an inhibitory effect on enzymes and metal ions at too high a concentration should not be present; contamination of other biological macromolecules, such as proteins, polysaccharides and lipid molecules, should be minimized; excluding contamination with other nucleic acid molecules, such as RNA.
Currently, DNA extraction methods are mainly divided into three types: phenol chloroform extraction method, centrifugal column method, and magnetic bead method. The phenol chloroform extraction method is to denature and extract protein by phenol and chloroform, and then obtain DNA by precipitation of an organic reagent; the method has the advantages of low cost and easy realization; the defects are that the DNA purity of a special sample is low, the phenol chloroform reagent is toxic, a centrifuge is required to continuously centrifuge, and the efficiency is low when a large amount of samples are extracted. The main principle of the centrifugal column method is that functional groups with adsorption effect on nucleic acid are fixed on a centrifugal column membrane, and different cracking reagents and washing reagents are added, and repeated centrifugation is carried out, so that the purpose of separating nucleic acid from impurities is achieved, and purified nucleic acid is obtained. The centrifugal column method has the advantages of high DNA purity and quality, convenient operation and capability of micro-extraction; the disadvantage is that repeated centrifugation is required, which is not suitable for large-scale extraction. The principle of the magnetic bead method is that a specific active functional group which has an adsorption effect on nucleic acid is modified on the surface of a magnetic bead, washing liquid of different lysate binding solutions can be specifically bound with a target substance under a specific condition, and meanwhile, the magnetic property of the magnetic bead can be utilized to conveniently realize directional movement and enrichment under the action of an external magnetic field, so that the purpose of separating the nucleic acid from impurities is achieved, and then the separation and purification of the target substance are realized to obtain purified nucleic acid. The magnetic bead method has the advantages of convenient extraction, safety, no toxicity, no need of centrifugation and suitability for high-flux extraction; the defects are that the purity of a special sample is poor, and the phenomena of magnetic bead residue, eluent with color and the like are easy to occur. At present, most of commercially available DNA extraction kits employ a centrifugal column method or a magnetic bead method.
Human tissues, blood cells and saliva are the most common genomic DNA extraction and detection samples in the field of medical examination. The problems of viscous eluent, low purity and the like easily occur in the extraction of genome DNA in blood cells. The extraction of genome DNA in saliva is also prone to problems of low yield, viscous and turbid eluent and the like.
In general, most of the currently marketed genomic DNA extraction kits can only extract genomic DNA from one sample, such as the genomic DNA extraction kit in blood cells or the genomic DNA extraction kit in saliva, and as mentioned above, the existing kits generally have the problems of viscous eluent, low purity, low yield, and the like.
Disclosure of Invention
The purpose of the application is to provide an improved kit for extracting genomic DNA and application thereof.
In order to achieve the purpose, the following technical scheme is adopted in the application:
the first aspect of the application discloses a kit for extracting genome DNA, which adopts a magnetic bead method to extract the genome DNA of an isolated sample, wherein the isolated sample is a human tissue sample, blood cells or saliva, and the kit comprises magnetic beads, lysis solution, erythrocyte lysis solution, binding solution, cleaning solution 1 and cleaning solution 2; the lysis solution contains 20-300mmol/L Tris-HCl, 20-100mmol/L Ethylene Diamine Tetraacetic Acid (EDTA), 0.3-2mol/L NaCl, 0.5-5% PEG8000, and 0.5-10% at least one of Triton X-100, SDS and Tween-20; the erythrocyte lysate contains 10-100mmol/L Tris-HCl, 1-50mmol/L EDTA, 10-300mmol/L NaCl and 0.01-0.2% TritonX-100.
It should be noted that the kit of the present application provides two solutions for lysis, namely, a lysate and a red blood cell lysate; the lysis solution is independently used for extracting the genome DNA of a human tissue sample and saliva, and the erythrocyte lysis solution and the lysis solution are used in combination and are mainly used for extracting the genome DNA of blood cells; the other components, including the magnetic beads, the binding solution, the cleaning solution 1 and the cleaning solution 2, are all universal, i.e., can be used for human tissue samples, blood cells and saliva. The reagent kit of the application especially develops two kinds of solutions for cracking, namely, the lysate and the erythrocyte lysate, aiming at saliva and erythrocytes, and through optimization and improvement of the two kinds of lysis solutions, the reagent kit can effectively remove polysaccharide and combine phenol to prevent the combination of phenol and DNA during cracking, thereby solving the problems of viscosity, turbidity and the like in the extraction process and improving the purity and yield of extraction of human tissue samples, blood cells and saliva genome DNA. The two optimized lysis solutions in the kit can denature proteins, isolate chromosomes and release nucleic acids during lysis, and are suitable for extracting genomic DNA of various samples such as tissues, blood, cells, bacteria and the like. The genomic DNA extracted by the kit can meet the requirements of various downstream experiments in quality and quantity, such as molecular marker detection, gene sequencing and the like; in addition, the kit is adopted for extracting the genome DNA, the total time of sample cracking, combination, cleaning and elution is only about 1h, and compared with the commercially available kit of the same type, the kit has the advantages of simple flow, high extraction quality and high extraction efficiency. It can be understood that the kit of the present application can be applied to the extraction of genomic DNA of various relatively complex clinical samples, such as tissues, blood cells, saliva, etc.; of course, it can also be used for extraction of genomic DNA from bacteria and the like.
It should be further noted that, one of the keys of the present application, namely, the optimization and improvement of the lysis solution and the erythrocyte lysis solution, as for other components, such as magnetic beads, binding solution, cleaning solution 1 and cleaning solution 2, reference may be made to the existing magnetic bead method reagent or kit, for example, the binding solution employs isopropanol or absolute ethanol; however, in order to obtain a better genomic DNA extraction effect, the washing solution 1 and the washing solution 2 are defined in the preferred embodiment of the present invention, and the details are described in the following technical solutions.
In addition, it is understood that the kit of the present application may further include other reagents conventionally used in the magnetic bead method, such as proteinase K, an eluent, and the like, in addition to the magnetic beads, the lysis solution, the binding solution, the washing solution 1, and the washing solution 2. Among them, proteinase K may be incorporated into the kit of the present application or may be commercially available. As for the eluent, the TE solution used for DNA extraction is generally a TE solution or directly sterilized deionized water without nuclease, wherein the TE solution can be prepared by self or obtained commercially, and the sterilized deionized water is a solvent which is used conventionally in laboratories. Therefore, proteinase K and the eluent can be selectively combined in the kit of the present application as needed, and are not particularly limited herein.
Preferably, the kit of the application can only be used, and the lysis solution consists of 100mmol/L Tris-HCl, 20mmol/L EDTA, 1.4mol/L NaCl, 1% PEG8000 and 1% Tween-20; the erythrocyte lysate is composed of 30mmol/L Tris-HCl, 5mmol/L EDTA, 60mmol/L NaCl and 0.015% Triton X-100.
It should be noted that the above components and specific concentrations are specifically adopted in one implementation manner of the present application, and the formula with better genomic DNA extraction effect is proved, which does not exclude that allowable adjustment of chemical dosage can be performed on the basis of the preferable formula of the present application, for example, deviation of plus or minus 5% of dosage of the present application can basically achieve the effect of the present application.
Preferably, the cleaning solution 1 contains 1-4mol/L of guanidine isothiocyanate or guanidine hydrochloride and 40% -70% of absolute ethyl alcohol.
Preferably, the cleaning solution 1 consists of 2mol/L guanidine hydrochloride and 50% absolute ethyl alcohol.
Preferably, the cleaning solution 2 contains 10-100mmol/L NaCl and 70% -80% absolute ethyl alcohol.
Preferably, the cleaning solution 2 is composed of 50mmol/L NaCl and 75% absolute ethanol.
It should be noted that, the kit of the present application actually improves two lysis solutions, cleaning solution 1 and cleaning solution 2, respectively, so that, under the condition of low requirement or some special use conditions, the two lysis solutions, cleaning solution 1 and cleaning solution 2 of the present application can be used separately and separately in combination with the existing magnetic bead method reagent, thereby having corresponding effects. For example, the lysis solution of the application can be better suitable for extracting genomic DNA of saliva or human tissue samples by combining with the existing magnetic bead method reagent, and particularly, the problems of low yield, viscous eluent, turbidity and the like of saliva genomic DNA extraction are solved; the erythrocyte lysate is combined with the existing magnetic bead method reagent, and the lysate combination comprises the existing magnetic bead method lysate, so that the problems of viscosity, low purity and the like of an eluent extracted from the genome DNA of the hemocyte can be solved to a certain extent; the cleaning solution 1 and the cleaning solution 2 are combined with the existing magnetic bead method reagent, so that the magnetic beads can be more effectively combined with the genome DNA, and impurities such as protein, salt ions and the like can be effectively removed.
Therefore, the second aspect of the present application discloses a lysate for magnetic bead method to extract genomic DNA, which is the lysate in the kit of the present application, wherein the lysate contains 20-300mmol/L Tris-HCl, 20-100mmol/L EDTA, 0.3-2mol/L NaCl, 0.5-5% PEG8000, and 0.5-10% at least one of Triton X-100, SDS and Tween-20.
Preferably, the lysis solution of the present application consists of 100mmol/L Tris-HCl, 20mmol/L EDTA, 1.4mol/L NaCl, 1% PEG8000 and 1% Tween-20.
The third aspect of the application discloses a red blood cell lysate for extracting blood cell genome DNA by a magnetic bead method, wherein the red blood cell lysate is also the red blood cell lysate in the kit, and the red blood cell lysate contains 10-100mmol/L Tris-HCl, 1-50mmol/L EDTA, 10-300mmol/L NaCl and 0.01-0.2% Triton X-100.
Preferably, the erythrocyte lysate of the present application consists of 30mmol/L Tris-HCl, 5mmol/L EDTA, 60mmol/L NaCl and 0.015% Triton X-100.
The fourth aspect of the application discloses a cleaning solution for extracting genomic DNA by a magnetic bead method, which comprises a cleaning solution 1 and a cleaning solution 2, wherein the cleaning solution 1 and the cleaning solution 2 are the cleaning solution 1 and the cleaning solution 2 in the kit of the application.
In a fifth aspect, the present application discloses the use of the kit of the present application, the lysate of the present application, or the wash of the present application for the extraction of genomic DNA from a human tissue sample or saliva.
The sixth aspect of the present application discloses the use of the kit of the present application, the lysate of the present application, the red blood cell lysate of the present application, or the wash solution of the present application for the extraction of genomic DNA of blood cells.
It can be understood that the kit of the present application is developed mainly for the problems existing in the extraction of genomic DNA of blood cells and saliva, and therefore, the reagents therein, such as lysate, erythrocyte lysate, cleaning solution, etc., can be used for the extraction of genomic DNA of blood cells and saliva accordingly.
Due to the adoption of the technical scheme, the beneficial effects of the application are as follows:
according to the kit for extracting the genome DNA, the lysis solution and the erythrocyte lysis solution are optimized, so that the kit can effectively remove polysaccharide, combine phenol and prevent phenol from being combined with DNA during lysis, and the problems of viscosity, turbidity and the like in the extraction process are solved; the kit is suitable for extracting genomic DNA of various samples such as tissues, blood, cells, bacteria and the like, and the extracted genomic DNA can meet the use requirements of various downstream experiments in terms of quality and quantity; moreover, the kit has simple and convenient flow, and improves the quality and efficiency of extracting the genome DNA.
Drawings
FIG. 1 is a graph showing the results of Agilent 4200 analysis of genomic DNA extracted from human tissue sample 1 in the example of the present application;
FIG. 2 is a graph showing the results of Agilent 4200 analysis of genomic DNA extracted from human tissue sample 2 in the example of the present application;
FIG. 3 is a graph showing the results of Agilent 4200 analysis of genomic DNA extracted from the blood cell sample 1 in the example of the present application;
FIG. 4 is a graph showing the results of Agilent 4200 analysis of genomic DNA extracted from the blood cell sample 2 in the example of the present application;
FIG. 5 is a graph showing the results of Agilent 4200 analysis of genomic DNA extracted from saliva sample 1 in the example of the present application;
FIG. 6 is a graph showing the results of Agilent 4200 analysis of genomic DNA extracted from saliva sample 2 in the example of the present application.
Detailed Description
The existing magnetic bead method reagent or kit has poor genome DNA extraction effect on clinical samples such as human tissues, blood cells, saliva and the like, and has low purity and yield, and the genome DNA extraction kit of the blood cells cannot be used for extracting the genome DNA of the saliva or tissues, on the contrary, the saliva genome DNA extraction kit cannot be used for extracting the genome DNA of the blood cells or tissues, so that a plurality of extraction kits are usually adopted when the genomic DNA of the clinical samples is extracted. Although there are also related studies on universal genomic DNA extraction kits, for example, patent application 201610022017 has developed a universal kit suitable for easily lysed liquid samples such as plasma, serum, cells, urine, saliva, etc.; however, it is not clearly applicable to tissue samples and blood cell samples.
The application develops a new universal genome DNA extraction kit applicable to three clinical samples, namely human tissues, blood cells, saliva and the like, and the kit comprises magnetic beads, lysate, erythrocyte lysate, binding solution, cleaning solution 1 and cleaning solution 2; the lysate contains 20-300mmol/L Tris-HCl, 20-100mmol/L EDTA, 0.3-2mol/L NaCl, 0.5-5% PEG8000, and at least one of 0.5-10% Triton X-100, SDS and Tween-20; the erythrocyte lysate contains 10-100mmol/L Tris-HCl, 1-50mmol/L EDTA, 10-300mmol/L NaCl and 0.01-0.2% Triton X-100.
In the lysate, a Tris-HCl buffer system plays a role in stabilizing the pH of a reaction system; EDTA is a metal ion chelating agent, and can inhibit DNase activity; NaCl provides a high-salt environment, so that DNA is fully dissolved in a liquid phase and separated from protein precipitate, and DNA sodium salt precipitate is more easily formed in an ethanol environment; PEG8000 is an organic polymer with strong hydrophilicity, can remove polysaccharide, is combined with phenol, and prevents phenol from being combined with DNA; triton X-100, SDS, Tween-20 and the like are nonionic surfactants, can dissolve cell membrane proteins and are beneficial to separating the proteins from DNA. This application is through optimizing the improvement to the lysate, can effectively solve the thick and muddy scheduling problem that appears among the extraction process to, not only can be applicable to saliva genome DNA and draw, can be applicable to the genome DNA of multiple samples such as human tissue sample, bacterium moreover and draw.
In the erythrocyte lysate, a Tris-HCl buffer system plays a role in stabilizing the pH of a reaction system; EDTA is a metal ion chelating agent, and can inhibit DNase activity; NaCl provides a high-salt environment, so that DNA is fully dissolved in a liquid phase and separated from protein precipitate, and DNA sodium salt precipitate is more easily formed in an ethanol environment; triton X-100 is a nonionic surfactant, can dissolve cell membrane protein, and is beneficial to separation of protein and DNA. When the erythrocyte lysate is used, the erythrocyte lysate is combined with the lysate, complex blood samples such as cryopreserved blood, blood clots and the like can be processed, the extraction of multi-sample genome DNA can be better adapted, and the problems of viscosity and low purity of eluate in the extraction of the erythrocyte genome DNA are solved.
In a further improvement scheme, the cleaning solution 1 and the cleaning solution 2 are optimized, and the cleaning solution 1 contains 1-4mol/L of guanidine isothiocyanate or guanidine hydrochloride and 40-70% of absolute ethyl alcohol; the cleaning solution 2 contains 10-100mmol/L NaCl and 70% -80% absolute ethyl alcohol. In the cleaning solution 1, guanidinium isothiocyanate or guanidinium hydrochloride is high chaotropic salt and can destroy cell structures to denature proteins; the absolute ethyl alcohol is used for removing impurities such as salt ions and the like. In the cleaning solution 2, NaCl is used for providing a high-salt environment, so that DNA is fully dissolved in a liquid phase and separated from protein precipitate; the absolute ethyl alcohol further removes impurities such as salt ions and the like.
The kit can be suitable for various forms of clinical samples, including complex blood samples such as cryopreserved blood, blood clots and the like, human tissue samples, blood cells, saliva and the like, and other animal samples and bacteria, solves the problems of low viscosity and low purity of eluent extracted from blood cell genome DNA, low extraction yield of saliva genome DNA, viscosity and turbidity of eluent and the like, and can meet the use requirements of various downstream experiments on the quality and yield of the genome DNA extracted from different samples; moreover, the kit is simple in operation process, low in required sample initial amount and suitable for an automatic workstation, namely the kit can be transplanted into the automatic workstation, and genomic DNA extraction is realized in an automatic mode; moreover, the cost of extracting 1 sample by the kit is about 1.7 yuan through accounting, and the kit is only about 1/4 of the same type of commercial products, so that the extraction cost of the genome DNA is greatly reduced.
The present application is described in further detail below with reference to specific embodiments and the attached drawings. The following examples are intended to be illustrative of the present application only and should not be construed as limiting the present application.
Example one
The kit specifically comprises lysis solution, proteinase K, magnetic bead suspension, erythrocyte lysis solution, binding solution, cleaning solution 1, cleaning solution 2 and eluent.
Wherein, the lysis solution consists of 100mmol/L Tris-HCl, 20mmol/L EDTA, 1.4mol/L NaCl, 1% PEG8000 and 1% Tween-20.
Proteinase K was purchased from Tiangen Biotechnology and magnetic bead suspensions were purchased from Meiji Biotechnology.
The erythrocyte lysate is composed of 30mmol/L Tris-HCl, 5mmol/L EDTA, 60mmol/L NaCl and 0.015% TritonX-100.
The binding solution is isopropanol.
The cleaning solution 1 consists of 2mol/L guanidine hydrochloride and 50% absolute ethyl alcohol.
The eluent is sterilized deionized water without nuclease.
The kit of this example was used as follows:
(1) for human tissue samples, 30mg of tissue was placed in 2.0mL centrifuge tubes, 300. mu.L of lysis buffer and 20. mu.L of proteinase K were added, 1 example of 4mm grinding beads were added, ground on a grinder at 60Hz for 60s, and then incubated in a 56 ℃ metal bath for 30 min;
(2) for a blood cell sample, 500 mu L of blood cells are taken and put into a 2.0mL centrifuge tube, 1mL of erythrocyte lysate is added, after vortex mixing, the mixture is centrifuged at 12000rpm for 1min, the supernatant is discarded, 1mL of erythrocyte lysate is added again, after vortex mixing, the mixture is centrifuged at 12000rpm for 1min, the supernatant is discarded, 300 mu L of lysate and 20 mu L of proteinase K are added, the mixture is shaken and mixed evenly, and the mixture is incubated in a 56 ℃ metal bath for 30 min;
(3) for the saliva sample, 300 μ L of saliva is taken and put into a 1.5mL centrifuge tube, 300 μ L of lysis solution and 20 μ L of proteinase K are added, the mixture is evenly mixed by shaking and is incubated in a 56 ℃ metal bath for 30 min;
(4) adding 0.6-1 times volume of binding solution and 10 μ L of magnetic bead suspension, and shaking for 10 min;
(5) after the instantaneous separation, the magnetic absorption is carried out for 1min on a rack, and the supernatant is discarded; adding 500 μ L of cleaning solution 1, vortex shaking for 30s, placing on shelf, magnetically attracting for 1min, and discarding supernatant;
(6) adding 500 μ L of cleaning solution 2, vortex shaking for 30s, magnetically attracting on the upper rack for 1min, and discarding the supernatant; cleaning with the cleaning solution 2 for 1 time;
(7) air drying at room temperature for 3-5min, adding 60 μ L of eluent, vortex vibrating magnetic beads, and incubating in 56 deg.C metal bath for 5 min;
(8) after the centrifugal separation, the supernatant is magnetically sucked on a rack, and is transferred to a new centrifugal tube, so that the extracted genomic DNA is obtained and stored at the temperature of minus 20 ℃.
In this example, two human tissue samples, two blood cell samples and two saliva samples were subjected to extraction of genomic DNA and quality detection of the extracted genomic DNA according to the above kit and method. Specifically, this example examined all extracted genomic DNAs, a260, a280 and a 230; and calculating A260/280 and A260/230 to characterize the purity of the genomic DNA. The concentration of the extracted genomic DNA was determined using QUBIT, and the size and content of the main fragment of the extracted genomic DNA were analyzed using agilent 4200. The detection results are shown in table 1; fig. 1 to 6 show the analysis results of agilent 4200, fig. 1 is a graph showing the analysis results of human tissue sample 1, fig. 2 is a graph showing the analysis results of human tissue sample 2, fig. 3 is a graph showing the analysis results of blood cell sample 1, fig. 4 is a graph showing the analysis results of blood cell sample 2, fig. 5 is a graph showing the analysis results of saliva sample 1, and fig. 6 is a graph showing the analysis results of saliva sample 2.
Table 1 quality test results of extracted genomic DNA
The results of table 1 and fig. 1 to 6 show that the kit of the present example can effectively extract genomic DNA of blood cells, saliva and human tissues, and the purity and concentration of the extracted genomic DNA are high, so that the kit can well meet the requirements of subsequent use.
Example two
In this example, the components and the concentrations of the components of the lysis solution, the erythrocyte lysis solution, the cleaning solution 1 and the cleaning solution 2 were tested on the basis of the first example, and the details are as follows:
1. formulation test of lysate
In this example, different concentrations of each component in the cracking liquid were tested, as shown in table 2.
TABLE 2 lysate formulation test
In this example, seven formulations of lysates of experiments 1 to 7 were prepared according to table 2, and the remaining reagents, e.g., proteinase K, erythrocyte lysate, binding solution, washing solution 1, washing solution 2, etc., were the same as in example one, and genomic DNA extraction was performed on two human tissue samples, two blood cell samples, and two saliva samples of example one by the same method as in example one. And the concentration and purity of the extracted genomic DNA were measured according to the method of example one. The results show that the lysis solutions of the seven formulations of the present example can finally extract genomic DNA meeting the use requirements, and especially the tests 6 and 7 have relatively good effects, which are close to the first example.
2. Erythrocyte lysate formulation test
In this example, different concentrations of each component in the red blood cell lysate were tested, as shown in Table 3.
TABLE 3 erythrocyte lysate formulation test
In this example, four formulations of erythrocyte lysates of tests 1 to 4 were prepared according to Table 3, and the remaining reagents, e.g., proteinase K, lysate, binding solution, cleaning solution 1, cleaning solution 2, etc., were the same as in example one, and genomic DNA extraction was performed on the two blood cell samples of example one by the same method as in example one. And the concentration and purity of the extracted genomic DNA were measured according to the method of example one. The results show that the four formulations of red blood cell lysate liquid of the present example can finally extract genomic DNA meeting the use requirements, especially the effect of experiment 2 is relatively good.
3. Cleaning solution formulation test
In this example, the different concentrations of the components in cleaning solution 1 and cleaning solution 2 were tested, as shown in Table 4.
Table 4 cleaning solution formulation test
In this example, cleaning solution 1 and cleaning solution 2 of five formulations of tests 1 to 5 were prepared according to table 4, and the remaining reagents, such as proteinase K, lysis solution, erythrocyte lysis solution, binding solution, etc., were the same as in example one, and genomic DNA extraction was performed on two human tissue samples, two blood cell samples, and two saliva samples of example one by the same method as in example one. And the concentration and purity of the extracted genomic DNA were measured according to the method of example one. The results show that the cleaning solution 1 and the cleaning solution 2 of the five formulas of the present example can finally extract and obtain genomic DNA meeting the use requirements, and especially the tests 2 and 5 have relatively good effects.
The above test results show that the lysis solution in the kit of this example contains 20-300mmol/L Tris-HCl, 20-100mmol/L EDTA, 0.3-2mol/L NaCl, 0.5-5% PEG8000, and 0.5-10% of at least one of Triton X-100, SDS and Tween-20; the erythrocyte lysate contains 10-100mmol/L Tris-HCl, 1-50mmol/L EDTA, 10-300mmol/L NaCl and 0.01-0.2% Triton X-100; the cleaning solution 1 contains 1-4mol/L of guanidinium isothiocyanate or guanidinium hydrochloride and 40-70% of absolute ethyl alcohol; the cleaning solution 2 contains 10-100mmol/L NaCl and 70% -80% absolute ethyl alcohol; can basically meet the requirement of extracting genome DNA of human tissues, blood cells and saliva. Combining the results of the first embodiment, the lysis solution consists of 100mmol/L Tris-HCl, 20mmol/L EDTA, 1.4mol/L NaCl, 1% PEG8000 and 1% Tween-20; the erythrocyte lysate is composed of 30mmol/L Tris-HCl, 5mmol/L EDTA, 60mmol/L NaCl and 0.015% Triton X-100; the cleaning solution 1 consists of 2mol/L guanidine hydrochloride and 50% absolute ethyl alcohol; the cleaning solution 2 consists of 50mmol/L NaCl and 75% absolute ethyl alcohol; the preferred kit embodiment of this example is optimal.
The foregoing is a more detailed description of the present application in connection with specific embodiments thereof, and it is not intended that the present application be limited to the specific embodiments thereof. It will be apparent to those skilled in the art from this disclosure that many more simple derivations or substitutions can be made without departing from the spirit of the disclosure.
Claims (10)
1. A kit for extracting genome DNA, wherein the kit adopts a magnetic bead method to extract the genome DNA of an isolated sample, and the isolated sample is a human tissue sample, blood cells or saliva, and is characterized in that: the kit comprises magnetic beads, lysis solution, erythrocyte lysis solution, binding solution, cleaning solution 1 and cleaning solution 2;
the lysis solution contains 20-300mmol/L Tris-HCl, 20-100mmol/L EDTA, 0.3-2mol/L NaCl, 0.5-5% PEG8000,
and 0.5% -10% of at least one of Triton X-100, SDS and Tween-20;
the erythrocyte lysate contains 10-100mmol/L Tris-HCl, 1-50mmol/L EDTA, 10-300mmol/L NaCl and 0.01-0.2% Triton X-100.
2. The kit of claim 1, wherein: the lysis solution consists of 100mmol/L Tris-HCl, 20mmol/L EDTA, 1.4mol/L NaCl, 1% PEG8000 and 1% Tween-20;
the erythrocyte lysate is composed of 30mmol/L Tris-HCl, 5mmol/L EDTA, 60mmol/L NaCl and 0.015% Triton X-100.
3. The kit according to claim 1 or 2, characterized in that: the cleaning solution 1 contains 1-4mol/L of guanidine isothiocyanate or guanidine hydrochloride and 40% -70% of absolute ethyl alcohol.
4. The kit of claim 3, wherein: the cleaning solution 1 consists of 2mol/L guanidine hydrochloride and 50% absolute ethyl alcohol.
5. The kit according to claim 1 or 2, characterized in that: the cleaning solution 2 contains 10-100mmol/L NaCl and 70% -80% absolute ethyl alcohol;
preferably, the cleaning solution 2 consists of 50mmol/L NaCl and 75% absolute ethyl alcohol.
6. A lysis solution for extracting genome DNA by a magnetic bead method is characterized in that: the lysis solution contains 20-300mmol/L Tris-HCl, 20-100mmol/L EDTA, 0.3-2mol/L NaCl, 0.5-5% PEG8000,
and 0.5% -10% of at least one of Triton X-100, SDS and Tween-20;
preferably, the lysis solution consists of 100mmol/L Tris-HCl, 20mmol/L EDTA, 1.4mol/L NaCl, 1% PEG8000 and 1% Tween-20.
7. A erythrocyte lysate used for extracting genome DNA of blood cells by a magnetic bead method is characterized in that: the erythrocyte lysate contains 10-100mmol/L Tris-HCl, 1-50mmol/L EDTA, 10-300mmol/L NaCl and 0.01-0.2% Triton X-100;
preferably, the erythrocyte lysate consists of 30mmol/L Tris-HCl, 5mmol/L EDTA, 60mmol/L NaCl and 0.015% Triton X-100.
8. A cleaning solution for extracting genomic DNA by a magnetic bead method comprises a cleaning solution 1 and a cleaning solution 2, and is characterized in that: the cleaning solution 1 contains 1-4mol/L of guanidine isothiocyanate or guanidine hydrochloride and 40% -70% of absolute ethyl alcohol, and the cleaning solution 2 contains 10-100mmol/L of NaCl and 70% -80% of absolute ethyl alcohol;
preferably, the cleaning solution 1 consists of 2mol/L guanidine hydrochloride and 50% absolute ethyl alcohol, and the cleaning solution 2 consists of 50mmol/L NaCl and 75% absolute ethyl alcohol.
9. Use of a kit according to any one of claims 1-5, a lysate according to claim 6 or a wash solution according to claim 8 for the extraction of genomic DNA from a human tissue sample or saliva.
10. Use of the kit according to any one of claims 1 to 5, the lysate according to claim 6, the erythrocyte lysate according to claim 7 or the wash solution according to claim 8 for the extraction of genomic DNA from blood cells.
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Cited By (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN111961663A (en) * | 2020-09-04 | 2020-11-20 | 华芯生物科技(武汉)有限公司 | Genome magnetic bead extraction kit and extraction method |
| CN113736772A (en) * | 2021-09-02 | 2021-12-03 | 北京艾迪康医学检验实验室有限公司 | Saliva DNA extraction method |
| CN114317524A (en) * | 2021-12-28 | 2022-04-12 | 国家粮食和物资储备局科学研究院 | Reagent, kit and extraction method for extracting DNA of grain kernel attaching fungi |
Citations (10)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN101613697A (en) * | 2009-08-05 | 2009-12-30 | 公安部物证鉴定中心 | A kind of method of extracting purify DNA |
| WO2011083429A1 (en) * | 2010-01-07 | 2011-07-14 | Bigtec Private Limited | A method for isolation of nucleic acids and a kit 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 |
| CN107663521A (en) * | 2016-07-28 | 2018-02-06 | 深圳华大基因股份有限公司 | Plasma free nucleic acid extraction kit and its application |
| CN107779451A (en) * | 2017-11-20 | 2018-03-09 | 广州海思医疗科技有限公司 | A kind of mankind's dissociative DNA extracting method and its kit |
| CN107922940A (en) * | 2015-03-12 | 2018-04-17 | 财团法人峩山社会福祉财团 | The method of seperated nuclear acid from FFPE tissues |
| CN108192891A (en) * | 2018-02-09 | 2018-06-22 | 湖南优品司生物科技有限公司 | A kind of nucleic acid purification reagent based on paramagnetic particle method |
| CN109402113A (en) * | 2018-11-26 | 2019-03-01 | 广东腾飞基因科技股份有限公司 | Dried blood spot genome extraction kit and extracting method based on polystyrene magnetic beads |
| CN109852610A (en) * | 2019-03-19 | 2019-06-07 | 宁波艾捷康宁生物科技有限公司 | One step washs paramagnetic particle method saliva DNA extraction kit |
| CN109913445A (en) * | 2019-03-19 | 2019-06-21 | 宁波艾捷康宁生物科技有限公司 | One step washs paramagnetic particle method blood DNA extracts kit |
-
2019
- 2019-12-27 CN CN201911374467.1A patent/CN110938624A/en active Pending
Patent Citations (10)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN101613697A (en) * | 2009-08-05 | 2009-12-30 | 公安部物证鉴定中心 | A kind of method of extracting purify DNA |
| WO2011083429A1 (en) * | 2010-01-07 | 2011-07-14 | Bigtec Private Limited | A method for isolation of nucleic acids and a kit thereof |
| CN107922940A (en) * | 2015-03-12 | 2018-04-17 | 财团法人峩山社会福祉财团 | The method of seperated nuclear acid from FFPE tissues |
| 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 |
| CN107663521A (en) * | 2016-07-28 | 2018-02-06 | 深圳华大基因股份有限公司 | Plasma free nucleic acid extraction kit and its application |
| CN107779451A (en) * | 2017-11-20 | 2018-03-09 | 广州海思医疗科技有限公司 | A kind of mankind's dissociative DNA extracting method and its kit |
| CN108192891A (en) * | 2018-02-09 | 2018-06-22 | 湖南优品司生物科技有限公司 | A kind of nucleic acid purification reagent based on paramagnetic particle method |
| CN109402113A (en) * | 2018-11-26 | 2019-03-01 | 广东腾飞基因科技股份有限公司 | Dried blood spot genome extraction kit and extracting method based on polystyrene magnetic beads |
| CN109852610A (en) * | 2019-03-19 | 2019-06-07 | 宁波艾捷康宁生物科技有限公司 | One step washs paramagnetic particle method saliva DNA extraction kit |
| CN109913445A (en) * | 2019-03-19 | 2019-06-21 | 宁波艾捷康宁生物科技有限公司 | One step washs paramagnetic particle method blood DNA extracts kit |
Non-Patent Citations (1)
| Title |
|---|
| 苏燕 等: "《医学生物化学与分子生物学实验技术双语教程》", 30 September 2015, 人民军医出版社 * |
Cited By (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN111961663A (en) * | 2020-09-04 | 2020-11-20 | 华芯生物科技(武汉)有限公司 | Genome magnetic bead extraction kit and extraction method |
| CN113736772A (en) * | 2021-09-02 | 2021-12-03 | 北京艾迪康医学检验实验室有限公司 | Saliva DNA extraction method |
| CN114317524A (en) * | 2021-12-28 | 2022-04-12 | 国家粮食和物资储备局科学研究院 | Reagent, kit and extraction method for extracting DNA of grain kernel attaching fungi |
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