CN121136972A - A lysis buffer and precipitation buffer for DNA extraction using magnetic beads - Google Patents
A lysis buffer and precipitation buffer for DNA extraction using magnetic beadsInfo
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- CN121136972A CN121136972A CN202511548165.7A CN202511548165A CN121136972A CN 121136972 A CN121136972 A CN 121136972A CN 202511548165 A CN202511548165 A CN 202511548165A CN 121136972 A CN121136972 A CN 121136972A
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Abstract
The invention discloses a lysate and a precipitation solution for extracting DNA by a magnetic bead method. The lysate comprises SDS, EDTA, DTT and NaCl, the precipitation solution comprises Tris-HCl, liCl, naCl and DTT, and a kit and a method for extracting DNA by a magnetic bead method are provided based on the lysate. The lysate, the precipitation solution and the kit provided by the invention are suitable for rapid extraction of most sample DNA, and have the advantages of low cost, simplicity in operation, high recovery rate, no toxicity and harm, and the like, and have a wide application prospect.
Description
Technical Field
The invention belongs to the technical field of biological medicine, and particularly relates to a lysate and a precipitation solution for extracting DNA by a magnetic bead method.
Background
The performance of life studies (sequence detection, gene expression and identification, abundance analysis, mutation library construction, etc.) often requires starting from genomic DNA or total RNA. High quality extraction of nucleic acids is also one of the most fundamental, necessary and important procedures in experimental techniques of molecular biology. DNA is the most important bioinformatic molecule as a carrier of genetic information, and is the main object of molecular biology research.
Methods of nucleic acid extraction have evolved over the years. The extraction method of DNA mainly comprises a boiling cleavage method, a phenol chloroform extraction method, an anionic detergent method, a CTAB method, a centrifugal column method and the like, and the methods have advantages and disadvantages. At present, the DNA separation technology is developing towards simplicity, convenience, high quality, high purity and high flux so as to meet the experimental requirements of large samples and low cost. The magnetic bead method nucleic acid extraction is a superparamagnetism nanometer magnetic bead technology based on surface modification, belongs to a high and new technology product crossing bioscience and nanomaterial science, is widely applied to the fields of gene diagnosis, forensic identification, clinical medicine, environmental monitoring and the like, and can realize automatic and high-flux operation. However, the existing magnetic bead method still has problems in the extraction process, such as low binding efficiency of magnetic beads and DNA, more DNA loss in the washing process, and unsatisfactory purity and concentration of extracted DNA, which limit the wide application of the magnetic bead method to a certain extent.
Therefore, it is important to provide a novel method for extracting genomic DNA with high DNA purity, high anti-interference capability and high compatibility of amplification reagents.
Disclosure of Invention
Based on the shortcomings of the prior art, the invention aims to provide a magnetic bead method for extracting lysate, precipitation solution and kit for extracting different sample types.
In order to achieve the above purpose, the present invention adopts the following technical scheme:
In a first aspect, the invention provides a lysis conjugate for DNA extraction.
Further, the lysis-binding fluid comprises SDS, EDTA, DTT and NaCl.
Further, the concentration of SDS is 0.1% -0.5%.
Further, the EDTA concentration is 0.1-1mM.
Further, the concentration of DTT is 50-500mM.
Further, the concentration of NaCl is 0.1-1mM.
Further, the concentration of SDS was 0.33%.
Further, the concentration of EDTA was 0.33mM.
Further, the concentration of DTT is 166.67mM.
Further, the concentration of NaCl was 0.5M.
In the present invention, "M" is a shorthand for mol/L and "mM" is mmol/L.
In the application, the applicant proves through experiments that the lysis binding solution disclosed by the application is obviously superior to the commercially available lysis solution, can more effectively lyse biological samples, release DNA and improve the binding efficiency of subsequent magnetic beads and DNA.
In a second aspect, the present invention provides a precipitation solution for DNA extraction.
Further, the precipitation solution includes Tris-HCl, liCl, naCl and DTT.
Further, the concentration of Tris-HCl is 10-100mM.
Further, the pH value of the Tris-HCl is 6.0-9.0.
Further, the concentration of LiCl is 100-500mM.
Further, the concentration of NaCl is 1-5M.
Further, the concentration of DTT is 2-20mM.
Further, the concentration of Tris-HCl was 100mM.
Further, the pH of Tris-HCl was 7.0.
Further, the concentration of LiCl was 400mM.
Further, the concentration of NaCl was 2M.
Further, the concentration of DTT was 10mM.
In the invention, the applicant searches out the optimal concentration and proportion of each component of the DNA precipitation liquid by optimizing the formula of the DNA precipitation liquid, enhances the interaction between the magnetic beads and DNA, and further improves the binding efficiency, thereby improving the extraction yield of the DNA.
The third aspect of the invention provides a kit for extracting DNA by a magnetic bead method.
Further, the kit comprises the lysis binding solution according to the first aspect of the invention and the precipitation solution according to the second aspect of the invention.
Further, the kit further comprises proteinase K, isopropanol, 90% ethanol and 100% absolute ethanol.
Further, the concentration of isopropyl alcohol is 100%.
Further, the kit further comprises magnetic beads.
Further, the magnetic beads are superparamagnetic beads with carboxyl groups or amino groups modified on the surfaces.
Further, the particle size of the magnetic beads is 100-500nm.
Further, the particle diameter of the magnetic beads was 500nm.
Further, the kit further comprises a DNA elution buffer.
Further, the DNA eluting buffer is a mixed solution of Tris-HCl and EDTA or deionized water.
Further, the concentration of Tris-HCl is 1-10mM.
Further, the concentration of Tris-HCl was 10mM.
Further, the EDTA concentration is 0.1-1mM.
Further, the concentration of EDTA was 1mM.
Further, the pH of the Tris-HCl and EDTA mixture was 8.0.
Further, the kit also includes instructions.
In some embodiments, the instructions state that the kit is used to extract DNA from a biological sample.
In some embodiments, the kit further contains one or more sterile containers, which may be in the form of a box, ampoule, bottle, vial, tube, pouch, blister pack, or other suitable container known in the art. Such containers may be made of plastic, glass, laminated paper, metal foil, or other materials suitable for containing medicaments.
In a fourth aspect, the present invention provides a method for extracting DNA by a magnetic bead method.
Further, the method comprises the step of performing DNA extraction using the kit according to the third aspect of the present invention.
Further, the method comprises the following steps:
(1) Pretreatment, namely mixing a sample with the lysis binding solution and proteinase K according to the first aspect of the invention, and incubating;
(2) Magnetic binding, namely mixing the mixed solution obtained in the step (1) with the lysis binding solution, 100% isopropanol and magnetic beads, and incubating at room temperature;
(3) Magnetic separation, namely magnetic separation, and discarding supernatant;
(4) Adding the precipitation solution according to the second aspect of the invention and 90% ethanol into the precipitate obtained in the step (3), uniformly mixing, magnetically separating, and discarding the supernatant;
(5) Washing, namely adding 100% absolute ethyl alcohol into the precipitate obtained in the step (4), uniformly mixing, magnetically separating, and discarding the supernatant;
(6) Eluting, namely drying the precipitate obtained in the step (5), adding an eluting buffer solution, incubating, magnetically separating, and collecting supernatant to obtain the separated and purified DNA.
Further, the concentration of proteinase K is 1-10mg/mL, preferably 10mg/mL.
Further, the incubation time in the step (1) is 10-20min, preferably 20min.
Further, the incubation temperature in step (1) is 50-55 ℃, preferably 50 ℃.
Further, the incubation time in the step (2) is 10-20min, preferably 20min.
Further, the magnetic separation time is 1-2min, preferably 2min.
Further, the drying time is 2-3min.
Further, the incubation time in the step (6) is 3-5min, preferably 5min.
Further, the incubation temperature in step (6) is 70-75 ℃, preferably 70 ℃.
The method for extracting nucleic acid by using magnetic bead method involves four steps of cracking, combining, rinsing and eluting, the combining step involves mixing DNA precipitation solution and magnetic beads, most of the combining solution in the prior art contains guanidine salt, the guanidine salt can promote precipitation of nucleic acid and combination with magnetic beads, but most of magnetic beads cannot be preserved for a long time with guanidine salt.
In the invention, magnetic separation refers to a process of using a magnetic field generated by a magnetic frame to enable magnetic substances (such as magnetic beads) in a centrifuge tube to rapidly move and be adsorbed to the wall (one side close to the magnetic pole of the magnetic frame) of the centrifuge tube under the action of the magnetic field, so that the magnetic substances are rapidly separated from other non-magnetic components in the solution.
In a fifth aspect, the invention provides the use of the lysis binding solution according to the first aspect of the invention, the precipitation solution according to the second aspect of the invention, and the kit according to the third aspect of the invention for extracting DNA from a biological sample.
Further, the biological sample includes whole blood, blood spots, cell cultures, bacterial cultures, tissues, semen, sperm spots, hair, or saliva.
When a range of values is disclosed herein, the range is considered to be continuous and includes both the minimum and maximum values for the range, as well as each value between such minimum and maximum values. Further, when a range refers to an integer, each integer between the minimum and maximum values of the range is included. Further, when multiple range description features or characteristics are provided, the ranges may be combined. In other words, unless otherwise indicated, all ranges disclosed herein are to be understood to include any and all subranges subsumed therein.
The words "preferably," "more preferably," and the like in the present invention refer to embodiments of the invention that may provide certain benefits in some instances. However, other embodiments may be preferred under the same or other circumstances. Furthermore, the recitation of one or more preferred embodiments does not imply that other embodiments are not useful, nor is it intended to exclude other embodiments from the scope of the invention.
In the description of the present invention, the terms "one embodiment," "some embodiments," "illustrative embodiments," "examples," "specific examples," or "some examples," etc. describe mean that a particular feature, structure, material, or characteristic described in connection with the embodiment or example is included in at least one embodiment or example of the present invention. In this specification, schematic representations of the above terms do not necessarily refer to the same embodiments or examples. Furthermore, the particular features, structures, materials, or characteristics described may be combined in any suitable manner in any one or more embodiments or examples.
The invention has the advantages and beneficial effects that:
1. The invention can more effectively crack biological samples to release DNA by optimizing the formula of the pretreatment lysate, and improves the subsequent combination efficiency of magnetic beads and DNA.
2. The interaction between the magnetic beads and the DNA is enhanced by adopting the optimized DNA precipitation solution, and the combination efficiency is further improved, so that the extraction yield of the DNA is improved.
3. The designed washing buffer solution can effectively remove impurities, reduce the loss of DNA in the washing process, and improve the purity of the extracted DNA.
4. The whole extraction process is simple and quick to operate, the whole process can be completed within 1 hour, the laboratory efficiency is obviously improved, complicated instruments and equipment are not needed, the method is suitable for processing large-scale samples, and the risk of cross contamination of the samples is avoided.
5. The extracted DNA has high purity and high concentration, and can meet the requirements of various subsequent molecular biology experiments.
6. The invention has the advantages of less used reagents, safe and nontoxic raw materials, and no influence on human body because the operation is carried out in the microcentrifuge tube.
Drawings
FIG. 1 is a graph of qPCR for amplifying an E.coli bacterial liquid sample; curve 1, cracking high concentration sample with the cracking liquid; curve 2. Concentration sample in the pyrolysis of the pyrolysis liquid of the invention; curve 3 for the low concentration sample of the inventive lysate, curve 4 for the high concentration sample of the comparative example 1, curve 5 for the medium concentration sample of the comparative example 1, curve 6 for the low concentration sample of the comparative example 1, curve 7 for the high concentration sample of the comparative example 2, curve 8 for the medium concentration sample of the comparative example 2, and curve 9 for the low concentration sample of the comparative example 2;
FIG. 2 is a graph of qPCR for amplifying CHO cell samples, curve 1 is a high concentration sample of the inventive lysate, curve 2 is a medium concentration sample of the inventive lysate, curve 3 is a low concentration sample of the inventive lysate, curve 4 is a high concentration sample of the inventive lysate, curve 1 is a medium concentration sample of the comparative example, curve 5 is a medium concentration sample of the comparative example 1, curve 6 is a low concentration sample of the comparative example 1, curve 7 is a high concentration sample of the comparative example 2, curve 8 is a medium concentration sample of the comparative example 2, and curve 9 is a low concentration sample of the comparative example 2;
FIG. 3 is a graph of qPCR of amplified JEV inactivated porcine serum samples; curve 1, cracking high concentration sample with the cracking liquid; curve 2. Concentration sample in the pyrolysis of the pyrolysis liquid of the invention; curve 3 for the low concentration sample of the inventive lysate, curve 4 for the high concentration sample of the comparative example 1, curve 5 for the medium concentration sample of the comparative example 1, curve 6 for the low concentration sample of the comparative example 1, curve 7 for the high concentration sample of the comparative example 2, curve 8 for the medium concentration sample of the comparative example 2, and curve 9 for the low concentration sample of the comparative example 2;
FIG. 4 is a graph showing qPCR amplification of a sample nucleic acid of HEK293 cells, curve 1 showing qPCR amplification of a sample nucleic acid of high concentration extracted by a magnetic bead kit of the present invention, curve 2 showing qPCR amplification of a sample nucleic acid of low concentration extracted by a magnetic bead kit of the present invention, curve 3 showing qPCR amplification of a sample nucleic acid of low concentration extracted by a magnetic bead kit of the present invention, curve 4 showing qPCR amplification of a sample nucleic acid of high concentration extracted by a commercial magnetic bead kit, curve 5 showing qPCR amplification of a sample nucleic acid of low concentration extracted by a commercial magnetic bead kit, and curve 6 showing qPCR amplification of a sample nucleic acid of low concentration extracted by a commercial magnetic bead kit;
FIG. 5 is a qPCR profile and a standard profile for an amplified E.coli DNA standard.
Detailed Description
The present invention will be further described with reference to specific embodiments, but the scope of the present invention is not limited thereto, and any person skilled in the art, who is within the scope of the present invention, should make equivalent substitutions or modifications according to the technical scheme of the present invention and the inventive concept thereof, and should be covered by the scope of the present invention.
The specific conditions are not noted in the examples below, and are carried out according to conventional conditions or conditions recommended by the manufacturer. The reagents or apparatus used were conventional products commercially available without the manufacturer's attention.
Example 1 the lysis binding fluid of the invention is suitable for lysis of different types of samples
The lysis binding solution for extracting genome DNA by the magnetic bead method consists of the following components of 0.33 percent SDS,166.67mM DTT,0.33mM EDTA,0.5M NaCl and water as a solvent.
In order to verify the effect of the lysis solution of the invention, two commercially available conventional lysis solutions of the magnetic bead method were selected as control groups, and a comparative experiment was performed with the lysis-binding solution of the embodiment of the invention. The comparative example 1 is a pretreatment kit (magnetic bead method) for universal host DNA residual samples of Ekesai ResiQuant, which comprises ：Lysis/Binding Buffer、Wash Buffer、Elution Buffer、Proteinase K Buffer、Magnetic Bead Solution、Proteinase K、Yeast tRNA and Glycogen with the product number of CRB00-0011, and the comparative example 2-day universal genome DNA extraction kit (DP 705) comprises tissue digestion solution, lysate GHT, buffer GDZ, proteinase K, magnetic bead suspension GH, elution buffer TB with the product number of DP705-01.
The experimental sample is colibacillus liquid, CHO cells and whole pig blood. Samples of E.coli bacterial liquid, CHO cells and whole pig blood were taken, respectively, and subjected to 25-fold gradient dilution with 1xTE Buffer, and labeled as high-, medium-and low-concentration samples diluted 5 2 -fold, 5 4 -fold and 5-fold 6 -fold, respectively.
The genomic DNA was extracted according to the conventional procedure for magnetic bead extraction using the control and the lysis conjugate of the invention, respectively. 2. Mu.L of the DNA extract was used as a template, and fluorescent quantitative PCR was performed.
The results of the detection are shown in Table 1 and FIGS. 1 to 3. As can be seen from the analysis results in Table 1, the kit of the present invention has significantly better nucleic acid extraction or purification effect, and under the same sample verification, the Ct value is smaller (smaller Ct value indicates more initial template amount) for both the high concentration sample, the medium concentration sample and the low concentration sample, which indicates that the cleavage binding solution of the present invention releases higher nucleic acid when used for nucleic acid cleavage recovery than the control group. The results of FIGS. 1-3 also show that the lysis-binding fluid of the present invention exhibits excellent extraction (Ct values are smaller) for different concentrations and different types of samples.
TABLE 1 Ct value results for the inventive and control lysis-binding solutions
Example 2 optimization of the formulation of the precipitation solution II according to the invention
1. In order to explore the influence of each component in the kit on the extraction result of nucleic acid in a sample, the following formula of the precipitation solution II is obtained by adjusting the concentration and the pH value of the components of the precipitation solution II.
The concentration of the precipitate II-1:Tris-HCl pH7.0 was 100mM, the concentration of LiCl was 400mM, the concentration of NaCl was 2M, and the concentration of DTT was 10mM.
The concentration of Tris-HCl pH6.0, liCl 400mM, naCl 2M and DTT 10mM was used as the precipitation solution II-2.
The concentration of Tris-HCl pH8.0, liCl 400mM, naCl 2M and DTT 10mM was used as the precipitation solution II-3.
The concentration of Tris-HCl pH9.0 was 100mM, the concentration of LiCl was 400mM, the concentration of NaCl was 2M, and the concentration of DTT was 10mM.
The concentration of the precipitate II-5 was 100mM, the concentration of LiCl was 40mM, the concentration of NaCl was 2M, and the concentration of DTT was 10mM.
The concentration of the precipitate II-6:Tris-HCl pH7.0 was 100mM, the concentration of NaCl was 2M, and the concentration of DTT was 10mM.
The concentration of the precipitate II-7:Tris-HCl pH7.0 was 100mM, the concentration of LiCl was 400mM, the concentration of NaCl was 200mM, and the concentration of DTT was 10mM.
The concentration of the precipitate II-8:Tris-HCl pH7.0 was 100mM, the concentration of LiCl was 400mM, the concentration of NaCl was 4M, and the concentration of DTT was 10mM.
The concentration of the precipitate II-9:Tris-HCl pH7.0 was 100mM, the concentration of LiCl was 400mM, and the concentration of NaCl was 2M.
The concentration of Tris-HCl pH7.0 was 100mM, the concentration of LiCl was 400mM, the concentration of NaCl was 2M, and the concentration of DTT was 50mM.
2. Sample treatment mice liver tissue was minced or ground to a powder with liquid nitrogen, then 0.01g was weighed and added to 100 μl of 1xTE Buffer.
(1) Pretreatment, namely taking 100 mu L of a sample, sequentially adding 18 mu L of a lysis binding solution (the lysis binding solution of the invention described in example 1) and 20 mu L of a 10mg/mL proteinase K solution, mixing uniformly upside down, centrifuging, and standing at 50 ℃ for 20min;
(2) Magnetic combination, namely, after the reaction is finished, sequentially adding 30 mu L of cracking combination liquid, 400 mu L of precipitation liquid I and 10 mu L of magnetic beads, uniformly mixing the mixture upside down, and standing the mixture at room temperature for 20 minutes, wherein the precipitation liquid I comprises isopropanol with the concentration of 100%, the concentration of the magnetic beads in the magnetic beads is 20-50mg/mL, preferably 50mg/mL, the magnetic beads are superparamagnetic nano particles with carboxyl or amino modified on the surfaces, and the particle size is 100-500 nm, preferably 500nm;
(3) Magnetic separation, namely placing the centrifuge tube on a magnetic rack after instantaneous centrifugation, standing for 2min, and absorbing and discarding supernatant;
(4) Adding 300 mu L of precipitation liquid II into a centrifuge tube, adding 300 mu L of wash buffer I into the centrifuge tube, uniformly mixing, standing for 2min, centrifuging the centrifuge tube, then placing the centrifuge tube on a magnetic rack, magnetically separating for 2min, and absorbing and discarding supernatant, wherein the precipitation liquid II is respectively the precipitation liquid II-1, the precipitation liquid II-2, the precipitation liquid II-3, the precipitation liquid II-4, the precipitation liquid II-5, the precipitation liquid II-6, the precipitation liquid II-7, the precipitation liquid II-8, the precipitation liquid II-9 and the precipitation liquid II-10, and the wash buffer I comprises ethanol with the concentration of 90%;
(5) Washing, namely adding 500 mu L of wash buffer II into a centrifuge tube, uniformly mixing and placing the mixture for 2min, centrifuging the centrifuge tube, placing the centrifuge tube on a magnetic rack, magnetically separating the centrifuge tube for 2min, and absorbing and discarding supernatant, wherein the wash buffer II comprises 100% absolute ethyl alcohol;
(6) Drying, namely centrifuging the centrifuge tube, then placing the centrifuge tube on a magnetic rack, magnetically separating for 2min, sucking residual liquid, and air-drying for 2-3min (without excessive drying) to remove residual ethanol;
(7) And (3) eluting, namely adding 100 mu L of elution buffer into the dried magnetic beads, incubating for 5min at 70 ℃, then placing in a magnetic field for magnetic separation, and collecting supernatant to obtain the extracted DNA, wherein the elution buffer is 10mM Tris-HCl,1mM EDTA,pH 8.0.
3. After the DNA extracts were diluted 100-fold with 1xTE Buffer, 2. Mu.L of the DNA extract was used as a template, and fluorescent quantitative PCR was performed. As a result of the detection, as shown in Table 2, the precipitation solution II-1 was found to have the best amount of nucleic acid (Ct value smaller) extracted from the nucleic acid sample of the mouse liver tissue sample.
TABLE 2 extraction results of different precipitants
Example 3 comparing the extraction Effect of the magnetic bead extraction kit of the present invention with the commercially available magnetic bead extraction kit
In this example, HEK293 cells collected by the applicant were used as samples, and 10-fold gradient dilutions were performed with 1xTE Buffer, and the samples were labeled as high-, medium-and low-concentration samples diluted 10 1 -fold, 10 2 -fold, and 10 3 -fold, respectively. The invention extracts nucleic acid in a sample, and the specific extraction process is as follows:
(1) Pretreatment, namely taking 100 mu L of a sample, sequentially adding 18 mu L of lysis binding solution (example 1) and 20 mu L of 10mg/mL proteinase K solution, uniformly mixing the solution upside down, and standing for 20min at 50 ℃ by short centrifugation;
(2) Magnetic combination, namely sequentially adding 30 mu L of lysis binding solution (example 1), 400 mu L of precipitation solution I and 10 mu L of magnetic beads into the centrifuge tube, uniformly mixing the solution with the solution upside down, standing the solution at room temperature for 20min, wherein the precipitation solution I is 100% isopropanol, the concentration of the magnetic beads in the magnetic beads is 20-50mg/mL, preferably 50mg/mL, and the magnetic beads in the magnetic beads are superparamagnetic nano particles with carboxyl or amino modified on the surfaces, and the particle size is 100-500 nm, preferably 500nm;
(3) Magnetic separation, namely placing the centrifuge tube on a magnetic rack after instantaneous centrifugation, standing for 2min, and absorbing and discarding supernatant;
(4) Adding 300 mu L of precipitation liquid II into a centrifuge tube, adding 300 mu L of wash buffer I into the centrifuge tube, uniformly mixing and placing the mixture for 2min, centrifuging the centrifuge tube, placing the centrifuge tube on a magnetic rack, magnetically separating the centrifuge tube for 2min, absorbing supernatant, wherein the precipitation liquid II comprises 0.1% -0.5% SDS, 10-100 mM Tris-HCl, 50-500 mM LiCl, 1-10 mM NaCl, 2-20 mM DTT, preferably 100mM Tris-HCl pH7.0, 400mM LiCl, 2M NaCl and 10mM DTT, and the wash buffer I is 90% ethanol.
(5) Washing, namely adding 500 mu L of wash buffer II into a centrifuge tube, uniformly mixing and placing the mixture for 2min, centrifuging the centrifuge tube, placing the centrifuge tube on a magnetic rack, magnetically separating the centrifuge tube for 2min, and absorbing and discarding supernatant, wherein the wash buffer II comprises 100% absolute ethyl alcohol;
(6) Drying, namely centrifuging the centrifuge tube, then placing the centrifuge tube on a magnetic rack, magnetically separating for 2min, sucking residual liquid, and air-drying for 2-3min (without excessive drying) to remove residual ethanol;
(7) Eluting, namely adding an eluting buffer solution into the dried magnetic beads, incubating for 5min at 70 ℃, then performing magnetic separation in a magnetic field, and collecting supernatant, namely the extracted DNA, wherein the eluting buffer solution is 10mM Tris-HCl,1mM EDTA,pH 8.0 buffer solution or deionized water.
Comparative example nucleic acid in a sample was extracted as follows:
(1) 100. Mu.L of the sample was added to a 1.5mL centrifuge tube, and then 20. Mu.L of protease K, 100. Mu. L Proteinase K Buffer and 20. Mu.L of 5M NaCl were added, inverted or vortexed well and briefly centrifuged. Incubating at 70 ℃ for 15min, and standing at room temperature for 5min;
(2) Adding freshly prepared Lysis/Binding solution (200. Mu.L of Lysis/Binding buffer+9. Mu.L of glycogen+0.2. Mu. L YEAST TRNA) to the above tube, vortexing, briefly centrifuging, further adding 200. Mu.L of isopropanol, vortexing, briefly centrifuging;
(3) Mixing the magnetic bead suspension Magnetic Bead Solution for 30 seconds or reversely for 10 times, adding 5 mu L of each sample, and mixing for 10 minutes at room temperature;
(4) Placing the centrifuge tube on a magnetic rack after instantaneous centrifugation, standing for 2min, and removing the supernatant;
(5) Adding 700 mu L of Wash Buffer into the sample tube, and carrying out vortex oscillation for 30 seconds or reversely and uniformly mixing for 10 times to resuspend the magnetic beads;
(6) Placing the centrifuge tube on a magnetic rack after instantaneous centrifugation, standing for 2min, and removing the supernatant;
(7) Transferring the centrifuge tube to a centrifuge tube rack, adding 700 mu L of 70% ethanol, and carrying out vortex oscillation for 30 seconds or reversely and uniformly mixing for 10 times to resuspend the magnetic beads;
(8) Placing the centrifuge tube on a magnetic rack after instantaneous centrifugation, standing for 2min, and removing the supernatant;
(9) Centrifuging the centrifuge tube, placing on a magnetic rack, magnetically separating for 2min, sucking residual liquid, air drying for 2-3min, and removing residual ethanol;
(10) Eluting, namely adding 100 mu L of the solution Buffer into the dried magnetic beads, incubating for 15min at 70 ℃, then placing the magnetic beads in a magnetic field for magnetic separation, and collecting supernatant, namely the extracted DNA.
The nucleic acid measurement results extracted by the invention and the nucleic acid measurement results extracted by the comparative example are shown in Table 3 and FIG. 4, and the results show that the nucleic acid amounts of HEK293 cell samples extracted by the magnetic bead method kit are higher than those of the magnetic bead method kit sold in the market.
TABLE 3 determination of nucleic acid extracted by different extraction methods
Example 4 addition of the standard recovery
(1) The standard curve was prepared using a commercially available E.coli DNA standard substance (Jinbo Co., ltd., GB 1001107) as a standard substance. E.coli DNA was diluted to 3 ng/. Mu.L with 1xTE Buffer between templates, and E.coli DNA (3 ng/. Mu.L) was further subjected to 10-fold gradient dilution at a concentration of 300 pg/. Mu.L, 30 pg/. Mu.L, 3 pg/. Mu.L, 300 fg/. Mu.L, 30 fg/. Mu.L, 3 fg/. Mu.L, 0.3 fg/. Mu.L in this order. The specific procedure for dilution of the E.coli DNA standard is as follows:
1) E, placing the coli DNA (30 ng/. Mu.L) and 1xTE Buffer on ice for melting, slightly oscillating and uniformly mixing after the complete melting, and instantaneously separating for 10s;
2) Taking 9 clean 1.5ml centrifuge tubes, respectively marking St1, st2, st3, st4, st5, st6, st7 and St8, and adding 90 mu L of 1xTE Buffer into the St1, st2, st3, st4, st5, st6, st7 and St8 centrifuge tubes;
3) Then 10 mu L of E.coli DNA (30 ng/. Mu.l) was taken out of the tube and added to the St1 tube, and the mixture was immediately separated for 20s after being mixed by a vortex machine;
4) Taking 10 mu L from the St1 pipe, adding into the St2 pipe, and immediately separating for 20s after the vortex meter is uniformly mixed;
5) Sequentially taking 10 mu L from the front pipe, adding into the rear pipe, uniformly mixing by a vortex meter, and instantaneously separating for 20s;
6) All samples were capped and placed in a 4 ℃ refrigerator for use.
(2) Preparing a sample, namely preparing 10mg/mL BSA, accurately weighing 100mg of BSA powder, fixing the volume to 10mL by using 1xTE Buffer, uniformly vortex mixing the sample to be tested until the sample is fully melted, and reserving the sample in a refrigerator at 4 ℃.
(3) The preparation of the labeled sample (ERC) the high concentration labeled sample (ERC-1) was prepared by adding 90. Mu.L of the test sample (10 mg/mL BSA) to a centrifuge tube, then adding 10. Mu.L of St1 (3 ng/. Mu.L), mixing well by a vortex machine, and then immediately separating for 20s, and adding a theoretical labeled amount of 30ng.
Medium concentration labeled sample (ERC-2) 90. Mu.L of test sample (10 mg/mL BSA) was added to the centrifuge tube, 10. Mu.L of St1 (3 ng/. Mu.L) was added, and the mixture was vortexed and then was separated for 20 seconds, followed by a theoretical 30ng addition.
The low concentration labeled sample (ERC-3) was prepared by adding 90. Mu.L of the test sample (10 mg/mL BSA) to a centrifuge tube, adding 10. Mu.L of St1 (3 ng/. Mu.L), mixing the mixture with a vortex, and then immediately separating the mixture for 20s, and adding 30ng of the theoretical labeled sample.
(4) DNA recovery the DNA recovery procedure of the present invention in example 3 was carried out by taking 100. Mu.L of 10mg/mL BSA, 100. Mu.L of ERC-1 sample, 100. Mu.L of ERC-2 sample, 100. Mu.L of ERC-3 sample, and 100. Mu.L of 1xTE Buffer.
(5) DNA detection, namely taking 2 mu L of standard products with different gradients and DNA extracting solution as templates respectively, and carrying out fluorescent quantitative PCR detection. The detection results are shown in tables 4-5 and fig. 5, and the labeling recovery rate of the magnetic bead method kit is 80% -130%.
In conclusion, the research is carried out through a large number of experiments and process optimization of the extraction and recovery processes, and finally the reagent formula and the reaction system are determined. The reaction system completes the extraction, purification and identification process of the genome DNA under the advantages of low cost, simple operation, high recovery rate and no toxicity or harm, and is suitable for the rapid extraction of most sample DNA.
The above description of the embodiments is only for the understanding of the method of the present invention and its core ideas. It should be noted that it will be apparent to those skilled in the art that several improvements and modifications can be made to the present invention without departing from the principle of the invention, and these improvements and modifications will fall within the scope of the claims of the invention.
Claims (10)
1. A lysis conjugate for DNA extraction, the lysis conjugate comprising SDS, EDTA, DTT and NaCl;
Preferably, the concentration of SDS is 0.1% -0.5%;
Preferably, the EDTA is present at a concentration of 0.1 to 1mM;
preferably, the concentration of DTT is 50-500mM;
preferably, the concentration of NaCl is 0.1-1mM.
2. The lysis conjugate of claim 1, wherein the concentration of SDS is 0.33%;
Preferably, the EDTA is present at a concentration of 0.33mM;
preferably, the concentration of DTT is 166.67mM;
preferably, the concentration of NaCl is 0.5M.
3. A precipitation solution for DNA extraction, characterized in that the precipitation solution comprises Tris-HCl, liCl, naCl and DTT;
preferably, the concentration of Tris-HCl is 10-100mM;
Preferably, the pH value of the Tris-HCl is 6.0-9.0;
preferably, the concentration of LiCl is 100-500mM;
preferably, the concentration of NaCl is 1-5M;
preferably, the concentration of DTT is 2-20mM.
4. A precipitation solution according to claim 3, wherein the Tris-HCl concentration is 100mM;
Preferably, the pH of Tris-HCl is 7.0;
Preferably, the concentration of LiCl is 400mM;
Preferably, the concentration of NaCl is 2M;
preferably, the concentration of DTT is 10mM.
5. A kit for extracting DNA by a magnetic bead method, wherein the kit comprises the lysis conjugate solution according to claim 1 or 2 and the precipitation solution according to claim 3 or 4.
6. The kit of claim 5, further comprising proteinase K, isopropanol, 90% ethanol, and 100% absolute ethanol;
Preferably, the concentration of isopropyl alcohol is 100%;
preferably, the kit further comprises magnetic beads;
preferably, the magnetic beads are superparamagnetic beads with carboxyl groups or amino groups modified on the surfaces;
preferably, the particle size of the magnetic beads is 100-500nm;
preferably, the particle size of the magnetic beads is 500nm.
7. The kit of claim 5, further comprising a DNA elution buffer;
preferably, the DNA eluting buffer is a mixed solution of Tris-HCl and EDTA or deionized water;
preferably, the concentration of Tris-HCl is 1-10mM;
preferably, the concentration of Tris-HCl is 10mM;
Preferably, the EDTA is present at a concentration of 0.1 to 1mM;
Preferably, the EDTA is present at a concentration of 1mM;
preferably, the pH of the Tris-HCl and EDTA mixture is 8.0;
preferably, the kit further comprises instructions.
8. A method for extracting DNA by a magnetic bead method, characterized in that the method comprises a step of extracting DNA using the kit of any one of claims 5 to 7.
9. The method according to claim 8, characterized in that it comprises the steps of:
(1) Pretreatment, namely mixing a sample with the lysis binding solution and proteinase K according to claim 1 or 2, and incubating;
(2) Magnetic binding, namely mixing the mixed solution obtained in the step (1) with the lysis binding solution, 100% isopropanol and magnetic beads according to the claim 1 or 2, and incubating at room temperature;
(3) Magnetic separation, namely magnetic separation, and discarding supernatant;
(4) Adding the precipitation solution according to claim 3 or 4 and 90% ethanol into the precipitate obtained in the step (3), uniformly mixing, magnetically separating, and discarding the supernatant;
(5) Washing, namely adding 100% absolute ethyl alcohol into the precipitate obtained in the step (4), uniformly mixing, magnetically separating, and discarding the supernatant;
(6) Eluting, namely drying the precipitate obtained in the step (5), adding an eluting buffer solution, incubating, magnetically separating, and collecting supernatant to obtain separated and purified DNA;
preferably, the concentration of proteinase K is 1-10mg/mL, preferably 10mg/mL;
preferably, the incubation time in step (1) is 10-20min, preferably 20min;
preferably, the incubation temperature of step (1) is 50-55 ℃, preferably 50 ℃;
Preferably, the incubation time in step (2) is 10-20min, preferably 20min;
Preferably, the magnetic separation time is 1-2min, preferably 2min;
preferably, the drying time is 2-3min;
preferably, the incubation time in step (6) is 3-5min, preferably 5min;
preferably, the incubation temperature of step (6) is 70-75 ℃, preferably 70 ℃.
10. Use of the lysis binding fluid of claim 1 or 2, the precipitation fluid of claim 3 or 4, the kit of any one of claims 5-7 for extracting DNA from a biological sample;
preferably, the biological sample comprises whole blood, blood spots, cell cultures, bacterial cultures, tissues, semen, sperm spots, hair or saliva.
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| CN202511548165.7A CN121136972A (en) | 2025-10-28 | 2025-10-28 | A lysis buffer and precipitation buffer for DNA extraction using magnetic beads |
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