CN113913421A - Reagent and method for extracting DNA from whole blood - Google Patents
Reagent and method for extracting DNA from whole blood Download PDFInfo
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Abstract
The invention relates to the technical field of biology, in particular to a whole blood DNA extraction reagent and an extraction method. The whole blood DNA extraction reagent provided by the invention consists of lysis solution, isopropanol, magnetic bead suspension, washing solution I, washing solution II, eluent and proteinase K. Wherein the cracking liquid consists of guanidine compounds, tris (hydroxymethyl) aminomethane, a stabilizer and a surfactant. The reagent is simple and convenient to operate, sample cracking and nucleic acid and magnetic bead combination can be realized on an automatic nucleic acid extractor by one step, the obtained genome DNA is high in content, good in integrity and high in purity, 1-96 samples can be extracted simultaneously, and the rapid, high-flux and fully-automatic extraction and purification of nucleic acid are realized.
Description
Technical Field
The invention relates to the technical field of biology, in particular to a whole blood DNA extraction reagent and an extraction method.
Background
With the rapid development of molecular biology technology, research on the genome level has become a hot point of research. Many conventional experiments of molecular biology are premised on extraction of genomic DNA, whether PCR amplification, gene sequencing or construction of a genomic library, the genomic DNA needs to be extracted, and the quality of the extracted genomic DNA directly affects downstream molecular biology experiments. With the rapid development of modern disease diagnosis technology and precise medical treatment, the amount of samples to be processed is increasing, so that an efficient, reliable and high-throughput DNA extraction method becomes an urgent need in related research fields.
There are various methods for extracting and purifying genome DNA, such as phenol chloroform extraction, SDS method, centrifugal column method and magnetic bead method, and these methods have advantages and disadvantages. The whole blood DNA extraction kit researched based on the method has various types, but the current whole blood DNA extraction kit has various defects in extraction, such as the need of pretreatment of samples, long treatment time, complex extraction process, long extraction process time, easy cross contamination among samples and the like. When the sample size is large, errors are prone to occur, and a large amount of time, manpower and material resources are wasted.
The automatic nucleic acid extractor is an instrument which automatically finishes the extraction work of sample nucleic acid by using matched nucleic acid extraction reagents and is divided into two types: one type is a large-scale automatic instrument, generally called an automatic liquid workstation, which is expensive in price and high in operation cost and is suitable for extracting thousands of samples of the same type at a time. The other type is a small-sized automatic extraction instrument which utilizes a packaged matched reagent to automatically complete the extraction and purification process, and has the advantages of low equipment price, low running cost and convenient operation, thereby being increasingly applied.
The matched reagent of the small-sized automatic extraction instrument has obvious influence on the extraction effect, only a few of current kits can realize high-flux and automatic extraction, and the steps of cell lysis and combination with magnetic beads are required to be separately carried out, so that the complete automation is not realized. Therefore, it is imperative to provide a high-throughput, fully automated whole blood DNA extraction reagent in order to more efficiently incorporate the use of the magnetic bead method for nucleic acid extraction.
Disclosure of Invention
In view of the above, the technical problem to be solved by the present invention is to provide a high-throughput, fully automated whole blood DNA extraction reagent and extraction method
The whole blood DNA extraction reagent provided by the invention comprises lysis solution, magnetic beads, isopropanol, washing solution I, washing solution II, eluent and proteinase K;
the lysis solution comprises: guanidine compounds, tris (hydroxymethyl) aminomethane, stabilizers and surfactants;
the washing solution I comprises: guanidine compounds, tris (hydroxymethyl) aminomethane, stabilizers, surfactants and isopropanol;
the washing liquid II is an ethanol water solution;
the eluent comprises tris.
The whole blood DNA extraction reagent provided by the invention is simple and convenient to operate, can realize sample cracking and nucleic acid and magnetic bead combination on an automatic nucleic acid extractor by one step, has high content of genomic DNA, good integrity and high purity, can simultaneously complete extraction of 1-96 samples, and realizes rapid, high-throughput and fully-automatic extraction and purification of nucleic acid.
In the invention, the lysis solution consists of water and 1M-10M guanidine compounds, 10 mM-50 mM trihydroxymethyl aminomethane, 5 mM-50 mM stabilizer and 1 vol% -10 vol% surfactant.
In the invention, the washing solution I consists of water, 0.5M-5M guanidine compound, 0.1M-1M sodium chloride, 1 mM-10 mM stabilizer, 5 mM-20 mM tris (hydroxymethyl) aminomethane, 0.5 vol% -10 vol% surfactant and 40 vol% -80 vol% isopropanol.
In some embodiments, the guanidine compound is at least one of guanidine hydrochloride or guanidine cyanamide sulfate. The surfactant is at least one selected from Tween-20, Triton X-100 and NP-40. The stabilizer is selected from EDTA or sodium citrate.
In some embodiments, the lysis solution comprises water and 1M to 10M guanidine hydrochloride, 10mM to 50mM tris, 5mM to 50mM EDTA, and 1 vol% to 10 vol% surfactant.
In some embodiments, the washing solution I is composed of water and 0.5M to 5M guanidine hydrochloride, 0.1M to 1M sodium chloride, 1mM to 10mM EDTA, 5mM to 20mM tris, 0.5 vol% to 10 vol% surfactant, and 40 vol% to 80 vol% isopropyl alcohol.
In the invention, the magnetic beads are silicon oxide hydroxyl nano magnetic beads. The diameter of the superparamagnetic silicon oxide hydroxyl nano magnetic microsphere is 100-2000 nm. The magnetic beads of the present invention are present in a suspension of magnetic beads. Wherein the buffer solution for suspending the magnetic beads is 10-80% ethanol water solution. In the adopted magnetic bead suspension, the concentration of the magnetic beads is 5-50 mg/mL. Preferably, the concentration of the magnetic beads is 10 mg/mL.
In the invention, the washing liquid II is ethanol water solution with the volume fraction of 60-100%. Preferably, the washing solution II is ethanol water solution with the volume fraction of 70%.
In the invention, the pH value of the eluent is 7.0-9.0, wherein the concentration of the trihydroxymethyl aminomethane is 0.1 mM-50 mM. Preferably, the concentration of tris in the eluate is 10 mM.
In the present invention, the concentration of proteinase K is 20 mg/mL.
The reagent provided by the invention can be used for preparing a kit for extracting DNA from whole blood.
The whole blood DNA extraction kit comprises the extraction reagent provided by the invention. Other tools or reagents required for extraction may also be included in the kit.
The invention also provides a method for extracting the DNA of the whole blood, which comprises the following steps:
step 1: mixing whole blood, proteinase K, lysis solution, isopropanol and magnetic beads, incubating and reacting, and separating the magnetic beads;
step 2: and (2) washing the magnetic beads separated in the step (1) by a washing solution I and a washing solution II in sequence, eluting by using an eluent, separating the magnetic beads, and taking supernate to obtain a whole blood DNA solution.
In the present invention, the whole blood is fresh whole blood or anticoagulated whole blood. Wherein the anticoagulant has no effect on the extraction result.
In the invention, the volume ratio of the whole blood, the proteinase K, the lysis solution, the isopropanol and the magnetic beads is 200:10:300:300: 10.
In the invention, the incubation reaction conditions in the step 1 comprise incubation at 55 ℃ for 5min and then a binding reaction for 5 min; the elution conditions described in step 2 included incubation at 55 ℃ for 5 min.
The whole blood DNA extraction reagent provided by the invention consists of lysis solution, isopropanol, magnetic bead suspension, washing solution I, washing solution II, eluent and proteinase K. Wherein the cracking liquid consists of guanidine compounds, tris (hydroxymethyl) aminomethane, a stabilizer and a surfactant. The reagent is simple and convenient to operate, sample cracking and nucleic acid and magnetic bead combination can be realized on an automatic nucleic acid extractor by one step, the obtained genome DNA is high in content, good in integrity and high in purity, 1-96 samples can be extracted simultaneously, and the rapid, high-flux and fully-automatic extraction and purification of nucleic acid are realized.
Drawings
FIG. 1 is a diagram showing the comparison of the integrity of the genome in the method of the present invention and the extraction method of the Omega magnetic bead blood extraction kit in example 1;
FIG. 2 is a schematic representation of the addition of reagents to each column of a 96-well plate using the method of the present invention in example 2.
Detailed Description
The invention provides a whole blood DNA extraction reagent and an extraction method, and a person skilled in the art can realize the whole blood DNA extraction reagent by appropriately improving process parameters according to the content in the text. It is expressly intended that all such similar substitutes and modifications which would be obvious to one skilled in the art are deemed to be included in the invention. While the methods and applications of this invention have been described in terms of preferred embodiments, it will be apparent to those of ordinary skill in the art that variations and modifications in the methods and applications described herein, as well as other suitable variations and combinations, may be made to implement and use the techniques of this invention without departing from the spirit and scope of the invention.
The invention provides a whole blood DNA extraction kit based on nano magnetic beads, which consists of lysis solution, isopropanol, magnetic bead suspension, washing solution I, washing solution II, eluent and proteinase K.
The cracking liquid contains guanidine compounds, trihydroxymethyl aminomethane, a stabilizer and a surfactant; the guanidine compound is selected from guanidine hydrochloride, guanidine isocyanate or a combination thereof; the magnetic beads are silicon oxide hydroxyl nano magnetic beads; the washing solution I comprises guanidine compounds, trihydroxymethyl aminomethane, a stabilizer, a surfactant and isopropanol; the surfactant is selected from Tween-20, Triton X-100, NP-40 or the combination thereof; the stabilizer is EDTA or sodium citrate. The washing liquid II is an ethanol solution; the eluent comprises tris.
In the magnetic bead suspension, the magnetic beads are silicon oxide hydroxyl nano magnetic beads, specifically superparamagnetic silicon oxide hydroxyl nano magnetic microspheres, the diameter of which is 100-2000 nm, and the concentration of which is 5-50 mg/mL.
The pH value of the lysis solution is 7.0-10.0, and the lysis solution comprises 1-10M guanidine compounds, 10-50 mM tris (hydroxymethyl) aminomethane, 5-50 mM stabilizer and 1-10% of surfactant. The guanidine compound is selected from guanidine hydrochloride, guanidine isocyanate or a combination thereof.
The washing liquid comprises a washing liquid I and a washing liquid II; the washing solution I contains 0.5-5M guanidine hydrochloride, 0.1-1M sodium chloride, 1-10 mM stabilizer, 5-20 mM tris (hydroxymethyl) aminomethane, 0.5-10% by volume of surfactant and 40-80% by volume of isopropanol. The surfactant is selected from Tween-20, Triton X-100, NP-40 or their combination. The washing liquid II contains ethanol with the volume ratio of 60-100%.
The eluent comprises 0.1-50 mM trihydroxymethyl aminomethane, and the pH value is 7.0-9.0.
The invention provides an application method of the kit for extracting whole blood DNA by a one-step method, which comprises the following steps:
(1) adding frozen anticoagulated whole blood or fresh whole blood, proteinase K, lysate, isopropanol and nano magnetic beads into a new centrifugal tube, uniformly mixing by vortex, incubating for 5 minutes at 55 ℃, reacting for 5 minutes in a combined manner, placing the centrifugal tube on a magnetic separator, and discarding all liquid in an EP tube;
(2) adding the washing solution I into the centrifuge tube, oscillating and uniformly mixing, placing on a magnetic separator, discarding all liquid in the centrifuge tube, and repeating the step once;
(3) adding a washing solution II into the centrifugal tube, oscillating and uniformly mixing, placing on a magnetic separator, and removing all liquid in the centrifugal tube;
(4) adding the eluent into the centrifuge tube, incubating for 5min at 55 deg.C, placing on a magnetic separator, discarding all liquid in the centrifuge tube, transferring the eluent into a new centrifuge tube, and storing at-20 deg.C for use.
Wherein, the lysate added in the step (1) is 200-400 μ L, the isopropanol is 200-400 μ L, the whole blood is 100-300 μ L, and the nano magnetic beads are 50-200 μ L; the washing liquid I and the washing liquid II added in the steps (2) and (3) are 400-1000 mu L; the nucleic acid eluent added in the step (4) is 50-200 mu L.
The invention also provides an application method of the kit for one-step nucleic acid extraction, which comprises the following steps:
(1) sequentially adding frozen anticoagulated whole blood or fresh whole blood, proteinase K, lysate and isopropanol into the 1 st column and the 7 th column of the 96 deep-well plate;
(2) adding nano magnetic beads into the 96 deep-well plate in the 2 nd column and the 8 th column;
(3) adding washing solution I into the 96 deep-hole plate in the 3 rd column and the 9 th column;
(4) adding washing solution I into the 96 deep-hole plate in the 4 th column and the 10 th column;
(5) washing liquid II is added into the 5 th row and the 11 th row of the 96 deep-hole plate;
(6) eluent is added into the 6 th column and the 12 th column of the 96 deep-well plate;
(7) the nucleic acid extractor was run and after completion the eluates from column 6 and column 12 of the 96-well plate were transferred to a new EP tube and stored at-20 ℃ until use.
Wherein, the lysate added in the step (1) is 200-400 mu L, the isopropanol is 200-400 mu L, and the whole blood is 100-300 mu L; adding 50-200 mu L of nano magnetic beads in the step (2); the washing liquid I and the washing liquid II added in the steps (3), (4) and (5) are 400-1000 mu L; the nucleic acid eluent added in the step (6) is 50-200 mu L.
The test materials adopted by the invention are all common commercial products and can be purchased in the market. The invention is further illustrated by the following examples:
example 1
The kit comprises the following seven reagents:
cracking solution: pH 8.5, containing 7MGuanidine hydrochloride50mM tris, 50mM EDTA, 10% Tween-20.
② isopropanol.
③ nanometer magnetic bead suspension: the superparamagnetic silica hydroxyl nano magnetic microsphere has a solution of 30% ethanol, a diameter of 100nm and a concentration of 10 mg/mL.
Fourthly, washing liquid I: 4M guanidine hydrochloride, 1M sodium chloride, 10mM EDTA, 20mM tris (hydroxymethyl) aminomethane, 10% by volume of Tween-20, and 60% by volume of isopropanol.
Washing liquid II: 70% by volume of ethanol
Sixthly, eluent: 10mM Tris, pH 8.0
(iii) protease K: the concentration was 20 mg/mL.
Example 2
The kit comprises the following seven reagents:
cracking solution: pH 9.0, containing 4MCyaniguanidine isosulfate50mM tris, 50mM MEDTA, 1% Tween-20.
② isopropanol.
③ nanometer magnetic bead suspension: the superparamagnetic silica hydroxyl nano magnetic microsphere has a solution of 30% ethanol, a diameter of 100nm and a concentration of 10 mg/mL.
Fourthly, washing liquid I: 4M guanidine hydrochloride, 1M sodium chloride, 10mM EDTA, 20mM tris (hydroxymethyl) aminomethane, 10% by volume of Tween-20, and 60% by volume of isopropanol.
Washing liquid II: 70% by volume of ethanol
Sixthly, eluent: 10mM Tris, pH 8.0
(iii) protease K: the concentration was 20 mg/mL.
Example 3:
the kit comprises the following seven reagents:
cracking solution: the pH was 8.5 and contained 2M guanidine hydrochloride, 50mM Tris, 50mM EDTA, 3% Tween-20.
② isopropanol.
③ nanometer magnetic bead suspension: the superparamagnetic silica hydroxyl nano magnetic microsphere has a solution of 30% ethanol, a diameter of 100nm and a concentration of 10 mg/mL.
Fourthly, washing liquid I: 4M guanidine hydrochloride, 1M sodium chloride, 10mM EDTA, 20mM tris (hydroxymethyl) aminomethane, 10% by volume of Tween-20, and 60% by volume of isopropanol.
Washing liquid II: 70% by volume of ethanol
Sixthly, eluent: 10mM Tris, pH 8.0
(iii) protease K: the concentration was 20 mg/mL.
Example 4
The kit comprises the following seven reagents:
cracking solution: pH 8.5, containing 9M guanidine hydrochloride, 50mM Tris, 50mM EDTA, 3% Tween-20.
② isopropanol.
③ nanometer magnetic bead suspension: the superparamagnetic silica hydroxyl nano magnetic microsphere has a solution of 30% ethanol, a diameter of 100nm and a concentration of 10 mg/mL.
Fourthly, washing liquid I: 4M guanidine hydrochloride, 1M sodium chloride, 10mM EDTA, 20mM tris (hydroxymethyl) aminomethane, 10% by volume of Tween-20, and 60% by volume of isopropanol.
Washing liquid II: 70% by volume of ethanol
Sixthly, eluent: 10mM Tris, pH 8.0
(iii) protease K: the concentration was 20 mg/mL.
Effect verification
The whole blood samples were extracted using the kits of the examples using different methods, respectively:
firstly, a one-step method for extracting DNA from whole blood comprises the following steps:
(1) taking a new centrifuge tube, adding 200 mu L of anticoagulated whole blood or fresh whole blood, 10 mu L of proteinase K, 300 mu L of lysate, 300 mu L of isopropanol and 10 mu L of nano magnetic beads into the centrifuge tube, uniformly mixing by vortex, incubating for 5 minutes at 55 ℃, then carrying out combined reaction for 5 minutes, placing the centrifuge tube on a magnetic separator, and discarding all liquid in an EP tube;
(2) adding 500 mu L of washing solution I into a centrifuge tube, oscillating and uniformly mixing, placing on a magnetic separator, discarding all liquid in the centrifuge tube, and repeating the step once;
(3) adding 500 mu L of washing liquid II into the centrifugal tube, oscillating and uniformly mixing, placing on a magnetic separator, and discarding all liquid in the centrifugal tube;
(4) adding 100 μ L of eluent into the centrifuge tube, incubating at 55 deg.C for 5min, placing on a magnetic separator, discarding all liquid in the centrifuge tube, transferring the eluent into a new centrifuge tube, and storing at-20 deg.C for use.
While the above experiment was completed, the same whole blood sample was extracted using the control reagent Omega magnetic bead method blood genomic DNA extraction kit, and the elution volume was 100. mu.L.
And (3) agarose gel electrophoresis detection: the extracted 6 parts of whole blood DNA were subjected to agarose gel electrophoresis. FIG. 1 shows that the extracted DNA band is single, clear and complete, and has no degradation, and the brightness of the extracted DNA band is equivalent to the effect of the Omega magnetic bead method blood genome DNA extraction kit.
After the kit and the control reagent (Omega magnetic bead method blood genome DNA extraction kit) in the embodiment 1 of the invention are extracted, the concentration and purity of DNA are detected by using an OneDrop 1000 ultra-micro ultraviolet spectrophotometer, and the result is shown in figure 1 and table 1.
TABLE 1
As can be seen from FIG. 1 and Table 1, the kit of the present invention was extracted at the same level of concentration and purity as the Omega magnetic bead method blood genomic DNA extraction kit.
Secondly, an automatic extraction instrument is utilized, the kit of the embodiment 1-4 is adopted to carry out high-flux nucleic acid extraction on the whole blood sample, and the method comprises the following steps:
(1) adding 200 mu L of whole blood, 10 mu L of proteinase K, 300 mu L of lysate and 300 mu L of isopropanol into the 96 deep-well plate in the 1 st column and the 7 th column in sequence;
(2) adding 200 mu L of nano magnetic beads into the 2 nd column and the 8 th column of the 96 deep-well plate;
(3) adding 500 mu L of washing solution I into the 3 rd column and the 9 th column of the 96 deep-well plate;
(4) adding 500 mu L of washing solution I into the 4 th column and the 10 th column of the 96 deep-well plate;
(5) adding 500 mu L of washing solution II into the 5 th row and the 11 th row of the 96 deep-well plate;
(6) adding 100 μ L of eluent into the 6 th column and the 12 th column of the 96 deep-well plate;
(7) operating the nucleic acid extractor, transferring the eluent of the 6 th column and the 12 th column of the 96-deep-hole plate into a new EP tube after the operation is finished,
storing at-20 deg.C for use.
The nucleic acid autosampler program settings are as in table 2.
TABLE 2 nucleic acid auto-extractor program settings
FIG. 2 is a schematic diagram showing a side view of a 96-well plate in which 16 whole blood samples can be processed at a time, in columns 1 to 12, after reagents of the kit of the present invention are added.
After extraction, the DNA concentration and purity were measured with a OneDrop 1000 ultra-micro UV spectrophotometer, the results are shown in tables 3-6.
TABLE 3 detection results of DNA products extracted by the kit of example 1 through the automatic nucleic acid extractor
TABLE 4 detection results of DNA products extracted by the kit of example 2 through the automatic nucleic acid extractor
| Sample (I) | A260nm | A280nm | 230nm | 260/280 | 260/230 | Concentration (ng/. mu.L) |
| 1 | 0.692 | 0.364 | 0.991 | 1.91 | 0.7 | 34.60 |
| 2 | 0.656 | 0.348 | 0.834 | 1.89 | 0.79 | 32.78 |
| 3 | 0.932 | 0.514 | 1.113 | 1.81 | 0.84 | 46.57 |
| 4 | 0.729 | 0.399 | 0.807 | 1.83 | 0.91 | 36.43 |
| 5 | 1.132 | 0.645 | 2.136 | 1.76 | 0.53 | 56.59 |
| 6 | 0.969 | 0.546 | 1.936 | 1.78 | 0.5 | 48.44 |
| 7 | 0.931 | 0.529 | 1.622 | 1.76 | 0.57 | 46.56 |
| 8 | 1.028 | 0.586 | 1.826 | 1.76 | 0.56 | 51.40 |
TABLE 5 detection results of DNA products extracted by the kit from example 3 by the automatic nucleic acid extractor
TABLE 6 detection results of DNA products extracted by the kit of example 4 through the automatic nucleic acid extractor
| Sample (I) | A260nm | A280nm | A230nm | 260/280 | 260/230 | Concentration (ng/. mu.L) |
| 1 | 1.018 | 0.563 | 0.539 | 1.81 | 1.89 | 50.89 |
| 2 | 1.06 | 0.591 | 0.607 | 1.79 | 1.74 | 52.99 |
| 3 | 1.169 | 0.648 | 0.633 | 1.8 | 1.85 | 58.43 |
| 4 | 1.266 | 0.705 | 0.659 | 1.8 | 1.92 | 63.28 |
| 5 | 0.888 | 0.493 | 0.505 | 1.8 | 1.76 | 44.42 |
| 6 | 1.023 | 0.575 | 0.546 | 1.78 | 1.87 | 51.13 |
| 7 | 0.928 | 0.519 | 0.523 | 1.79 | 1.77 | 46.38 |
| 8 | 1.061 | 0.602 | 0.675 | 1.76 | 1.57 | 53.07 |
As can be seen from the results in the table, the DNA obtained by extraction was successfully obtained in each example, wherein the product obtained by extracting the whole blood DNA using the kit of example 1 was high in content and good in purity. Compared with the product obtained by extracting the whole blood DNA with the kit in the embodiment 1, the product obtained by extracting the whole blood DNA with the kit in the embodiment 2-4 has lower content and poorer purity 260/230, which shows that the lysis binding capacity of the whole blood DNA extracted with the lysate is poorer.
The foregoing is only a preferred embodiment of the present invention, and it should be noted that it is obvious to those skilled in the art that various modifications and improvements can be made without departing from the principle of the present invention, and these modifications and improvements should also be considered as the protection scope of the present invention.
Claims (10)
1. The whole blood DNA extraction reagent is characterized by comprising lysis solution, magnetic beads, isopropanol, washing solution I, washing solution II, eluent and proteinase K;
the lysis solution comprises: guanidine compounds, tris (hydroxymethyl) aminomethane, stabilizers and surfactants;
the washing solution I comprises: guanidine compounds, tris (hydroxymethyl) aminomethane, stabilizers, surfactants and isopropanol;
the washing liquid II is an ethanol water solution;
the eluent comprises tris.
2. The whole blood DNA extraction reagent according to claim 1, wherein the lysis solution is composed of water and 1M to 10M guanidine compound, 10mM to 50mM tris, 5mM to 50mM stabilizer, and 1 vol% to 10 vol% surfactant.
3. The whole blood DNA extraction reagent according to claim 1, wherein the washing solution I is composed of water and 0.5M to 5M guanidine compound, 0.1M to 1M sodium chloride, 1mM to 10mM stabilizer, 5mM to 20mM tris (hydroxymethyl) aminomethane, 0.5 vol% to 10 vol% surfactant, and 40 vol% to 80 vol% isopropyl alcohol.
4. The whole blood DNA extraction reagent according to any one of claims 1 to 3, wherein the guanidine compound is at least one of guanidine hydrochloride and guanidine cyanamide iso-sulfate; the stabilizer is at least one of EDTA or sodium citrate; the surfactant is at least one selected from Tween-20, Triton X-100 and NP-40.
5. The whole blood DNA extraction reagent according to claim 1, wherein the magnetic beads are silica hydroxyl nanobeads.
6. The whole blood DNA extraction reagent according to claim 1, wherein the washing solution II is an aqueous ethanol solution having a volume fraction of 60% to 100%.
7. The whole blood DNA extraction reagent according to claim 1, wherein the pH of the eluate is 7.0 to 9.0, and the concentration of tris (hydroxymethyl) aminomethane is 0.1mM to 50 mM.
8. A method for extracting DNA from whole blood, comprising:
step 1: mixing whole blood, proteinase K, lysis solution, isopropanol and magnetic beads, incubating and reacting, and separating the magnetic beads;
step 2: and (2) washing the magnetic beads separated in the step (1) by a washing solution I and a washing solution II in sequence, eluting by using an eluent, separating the magnetic beads, and taking supernate to obtain a whole blood DNA solution.
9. The extraction method according to claim 8, wherein the volume ratio of the whole blood, proteinase K, the lysate, the isopropanol, and the magnetic beads is 200:10:300:300: 10.
10. The extraction method according to claim 8,
the incubation reaction conditions in step 1 include incubation at 55 ℃ for 5min, followed by a binding reaction for 5 min;
the elution conditions described in step 2 included incubation at 55 ℃ for 5 min.
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Cited By (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN116064503A (en) * | 2023-04-07 | 2023-05-05 | 深圳市梓健生物科技有限公司 | Protease-free rapid magnetic bead method nucleic acid extraction kit and nucleic acid extraction and purification method |
| CN117264945A (en) * | 2023-11-22 | 2023-12-22 | 山东新华医疗器械股份有限公司 | Nucleic acid extraction kit based on magnetic bead method, application thereof and nucleic acid extraction method |
| CN117535283A (en) * | 2024-01-08 | 2024-02-09 | 广州凯普医药科技有限公司 | Kit for extracting blood genome DNA by magnetic bead method and application thereof |
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| CN117264945A (en) * | 2023-11-22 | 2023-12-22 | 山东新华医疗器械股份有限公司 | Nucleic acid extraction kit based on magnetic bead method, application thereof and nucleic acid extraction method |
| CN117535283A (en) * | 2024-01-08 | 2024-02-09 | 广州凯普医药科技有限公司 | Kit for extracting blood genome DNA by magnetic bead method and application thereof |
| CN117535283B (en) * | 2024-01-08 | 2024-03-29 | 广州凯普医药科技有限公司 | Kit for extracting blood genome DNA by magnetic bead method and application thereof |
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