CN112048503A

CN112048503A - Kit for extracting plant genome DNA by high-throughput rapid magnetic bead method and extraction method

Info

Publication number: CN112048503A
Application number: CN202010940039.7A
Authority: CN
Inventors: 刘君; 程溪柳; 高英; 王欢; 孙莹璐; 柳华峰; 石添添
Original assignee: Biotechnology Research Institute of CAAS; Institute of Crop Sciences of Chinese Academy of Agricultural Sciences
Current assignee: Biotechnology Research Institute of CAAS; Institute of Crop Sciences of Chinese Academy of Agricultural Sciences
Priority date: 2020-09-09
Filing date: 2020-09-09
Publication date: 2020-12-08

Abstract

The invention discloses a kit for extracting plant genome DNA by a high-throughput rapid magnetic bead method and extraction. Compared with the existing magnetic bead genome DNA separating and purifying kit, the kit and the corresponding method are more efficient and simpler. The kit is matched with a 96-well kit, 96 samples can be completed in each operation, and each person can simultaneously operate 4-8 sets of 96-well kits through testing, namely each person can operate 400 samples and 800 samples in the same time, so that the efficiency is greatly improved.

Description

Kit for extracting plant genome DNA by high-throughput rapid magnetic bead method and extraction method

Technical Field

The invention belongs to the field of molecular biology, and particularly relates to a kit for extracting plant tissue genome DNA by using magnetic beads and an extraction method thereof.

Background

The molecular biological detection technology of nucleic acid is one of the most important means for the development foundation and routine application of modern medicine and plant biology. The extracted and purified genome DNA can be directly used for subsequent PCR detection, indicator paper detection, NGS second-generation sequencing library preparation and other works. To the extent of current technological development, obtaining high quality, high purity, high integrity plant genomic DNA in the laboratory remains a time consuming and labor intensive process that is not completely stable. Plants may be planted at 2-3 crops per year, a large number of plant samples, thousands to tens of thousands of samples, are often required to be identified within a short time between 2 planting, and the traditional method consumes a large amount of manpower and time and is often difficult to complete within a required time. A new simple, efficient and rapid plant genome DNA extraction method is developed to obtain a high-quality sample, and the method has very important application value.

The traditional nucleic acid separation method mainly comprises the processes of cell wall breaking, solvent extraction, precipitation, high-speed centrifugation and the like, active nuclease and other protein components in a liquid phase are removed by phenol or chloroform, then ethanol is used for removing salt, and the nucleic acid DNA can be obtained after washing. Some membrane adsorption can also be used to assist in DNA extraction and purification, such as silica gel membrane, polysaccharide membrane. The purification method has the advantages of complicated steps, long time consumption and low recovery rate, and phenol and chloroform are used in the extraction process, so that the respiratory tract of a human is damaged, and the health of the human is influenced.

The magnetic bead purification technology is a new method developed in recent years, and is characterized by being simple and effective, and the main principle is that silicon dioxide is uniformly coated on a Fe magnetic core with superparamagnetism, and the connection of the silicon dioxide can specifically adsorb negative charge nucleic acid. Compared with the traditional method, the magnetic bead method has the advantages that no virulent reagent is required to participate in the whole process, multi-step high-rotation-speed centrifugation is avoided, the steps are fewer, and the method is simple and easy to implement. However, the existing magnetic bead separation method has the problems of low yield or low efficiency, most of the existing magnetic bead separation methods are suitable for single channels or 8 channels, and high-flux DNA extraction cannot be really realized; or a full-automatic motorized 96-channel, which is imported to a large-scale instrument and is not beneficial to popularization application.

Disclosure of Invention

The invention aims to provide a kit for extracting plant tissue genome DNA by a high-throughput and high-efficiency magnetic bead method and an extraction method thereof aiming at the defects of the prior art.

The invention provides a DNA extracting agent which comprises a magnetic bead suspension and a lysis solution 1, wherein solutes in the lysis solution 1 are benzalkonium bromide, sodium dodecyl sulfate, sodium chloride, ethylenediamine tetraacetic acid, polyvinylpyrrolidone, polyethylene glycol octyl phenyl ether, tween20 and a Tris-HCL buffer solution of sodium azide.

Wherein the volume fraction of the ammonium bromide (CTAB) is 2%, the volume fraction of the Sodium Dodecyl Sulfate (SDS) is 1% -2%, the volume fraction of the polyethylene glycol octyl phenyl ether (TritonX) is 0.1%, and the volume fraction of the Tween20 (Tween20) is 1%; the volume fraction of sodium azide (SodiumAzide) is 1%, the content of sodium chloride is 0.2mol/L, the content of Ethylene Diamine Tetraacetic Acid (EDTA) is 10mmol/L, the content of polyvinylpyrrolidone (PVP) is 10mg/L, and the concentration of the Tris-HCL buffer solution is 100mmol/L, pH and is 8.5.

The DNA extracting agent comprises a binding solution, a rinsing solution and an eluent, wherein the binding solution is 100% isopropanol and 2M GITC (isopropyl guanidine sulfate); the rinsing liquid is 80% ethanol; the eluent is deionized water.

The content of the magnetic beads in the magnetic bead suspension is 200 mg/ml.

The diameter of the magnetic bead is 80-1000 nm.

The invention also provides a DNA extraction kit, which comprises any one of the DNA extraction reagents.

The DNA extraction kit also comprises a 96-hole deep-hole centrifugal plate, a 96-hole magnetic stirring rod and a 5 mm-diameter steel ball, and is matched with a 96-hole semi-automatic magnetic frame for use.

The invention also provides a method for extracting plant genome DNA by a paramagnetic particle method, which uses the DNA extracting agent andor the DNA extracting kit and comprises the following steps:

1) crushing plant tissues, adding lysis solution 1, and standing at 65 ℃ for 40 minutes;

2) centrifuging the mixed solution in the step 1), taking supernatant, adding the supernatant into the binding solution and the magnetic bead suspension, uniformly mixing, and standing for 3-10 minutes;

3) immersing the magnetic beads in the step 2 in a rinsing liquid, standing for 20 seconds, and cleaning for the 1 st time; this step was repeated once more.

4) And standing the washed magnetic beads for 2 minutes, airing, and eluting with an eluent to obtain the purified DNA.

Wherein the standing temperature in the step l) is 65 ℃, and the standing time is 40-60 minutes; the centrifugation condition of the step 2) is 4000rpm centrifugation for 5 min; the volume ratio of the lysis solution 1 to the binding solution to the magnetic bead suspension to the eluent is 600: 5-20: 100.

wherein, in the step l), the mass-to-volume ratio of the sample to the lysate is 150mg:600 mu 1; in the step 2), the volume ratio of the supernatant to the combined liquid is 1: 1; in the step (3), the volume ratio of the magnetic beads participating in nucleic acid extraction to the rinsing liquid is 1: 60.

Wherein the plant tissue is one of fresh plant leaves, dry plant leaves and plant seeds.

The invention has the beneficial effects that: the invention provides a set of high-throughput DNA extraction kit suitable for a semi-automatic method. Compared with the existing magnetic bead genome DNA separation and purification kit, the DNA extraction reagent (kit) and the corresponding method are more efficient and simple, and the operation is more convenient, and on the premise of using the same raw materials, the DNA extraction reagent (kit) can obtain DNA with higher concentration, thereby greatly improving the DNA extraction effect. In addition, the kit is matched with a 96-well kit, 96 samples can be completed in each operation, and each person can simultaneously operate 4-8 sets of 96-well kits through testing, namely each person can operate 400 samples and 800 samples in the same time, so that the efficiency is greatly improved.

Drawings

FIG. 1 is a diagram showing the results of DNA detection of rice, wheat and maize genomes extracted by different methods, wherein 1, 4 and 7 are rice; 2. 5 and 8 are wheat; 3. 6 and 9 are corn. 1-3, a reagent kit for extracting DNA from plant tissues by a Luoyang Edison magnetic bead method is used; 4-6, the kit 1 of the present invention; 7-9, the Solebao plant DNA extraction kit was used.

FIG. 2 is a result chart of the

reagent kit

1, 2 for extracting corn, wheat and rice; among them, 1 to 3 used is the kit 1 of the present invention; 4-6, the kit 2 of the present invention is used. 1 and 4 for maize, 2 and 5 for rice; 3 and 6 are wheat.

FIG. 3 is a graph showing the results of extracting corn, millet, rice and wheat with the kit 2 of the present invention, wherein 1234 corresponds to corn, millet, rice and wheat, respectively.

FIG. 4 is a graph showing the concentration of DNA extracted from corn, rice and wheat using different kits according to the present invention.

Detailed Description

The present invention is described in further detail below with reference to specific embodiments, which are given for the purpose of illustration only and are not intended to limit the scope of the invention. The examples provided below serve as a guide for further modifications by a person skilled in the art and do not constitute a limitation of the invention in any way.

The experimental procedures in the following examples are conventional unless otherwise specified. Materials, reagents and the like used in the following examples are commercially available unless otherwise specified.

In the following examples, the wheat was spring of China, the corn was Zhengdan 58, the rice was 93-11, and the millet was Zhonggu 2.

CTAB (ammonium bromide, sigma, Cat No. H9151-250G), Sodium chloride (Chinese medicine, Cat No.10019318), EDTA (ethylene diamine tetraacetic acid, West Long, Cat No.1-32), Tris-HCL (amresco, Cat No.0234), TritonX (polyethylene glycol octyl phenyl ether, sigma, Cat No. V900502), Tween20 (Tween20, sigma, Cat No. P1379), Sodium Azide (Sodium Azide, Merck Millipore, Cat No.01-0032-00), RNase A (ribonuclease A, Thermo, Cat No. R1253), isopropyl alcohol (Chinese medicine, 80109218), GITC (isothio gua, ma, V900474), Sodium SDS (dodecyl, sigma, L6026), PVP (polyvinylpyrrolidone, sigma 004 234257) and ethanol (Western XL 1) in the following examples.

EXAMPLE 1 preparation of the kit

1. Preparation of kit 1

Preparing a DNA extracting agent 1, wherein the DNA extracting agent 1 comprises a lysis solution 1, a binding solution, a rinsing solution and an eluent, wherein the lysis solution 1 is a mixed solution obtained by adding CTAB, SDS, Sodium chloride, EDTA, TritonX, Tween20 and Sodium Azide into a Tris-HCL buffer solution, the Tris-HCL buffer solution is 100mmol/L, and the pH value is 8.5; the contents of each component in the mixed solution are respectively 1% CTAB, 1% SDS,0.1mol/L Sodium chloride, 10mmol/L EDTA,100mmol/L Tris-HCL, pH 8.5, 0.1% TritonX, 1% Tween20, 1% Sodium Azide and 10mg/L PVP; the magnetic bead suspension is 100mg/ml, and the percentage contents are volume contents; the binding solution is 100% isopropanol, 1MGITC (isopropyl guanidine sulfate); the rinsing liquid is 80% ethanol; the eluent is deionized water. The lysis solution 1, the binding solution, the rinsing solution and the eluent are all independently packaged and can be used independently.

The reagent can be matched with a 96-hole deep-hole centrifugal plate, a 96-hole magnetic stirring rod and a 5 mm-diameter steel ball to be used with a 96-hole semi-automatic magnetic frame to finish the extraction of DNA.

2. Preparation of kit 2

Preparing a DNA extracting agent 2, wherein the DNA extracting agent 2 comprises a lysis solution 1, a binding solution, a rinsing solution and an eluent, wherein the lysis solution 1 is a mixed solution obtained by adding CTAB, SDS, Sodium chloride, EDTA, TritonX, Tween20 and Sodium Azide into a Tris-HCL buffer solution, the Tris-HCL buffer solution is 100mmol/L, and the pH value is 8.5; the contents of each component in the mixed solution are respectively 2% CTAB, 2% SDS,0.2mol/L sodium chloride, 10mmol/L EDTA,100mmol/L Tris-HCL, pH 8.5, 10mM PVP, 0.1% TritonX and 1% Tween 20; 1% of Sodium Azide, wherein the percentage contents are volume contents; the magnetic bead suspension is 200 mg/ml; the binding solution is 100% isopropanol, 1M GITC (isopropyl guanidine sulfate); the rinsing liquid is 80% ethanol; the eluent is dd water. The lysis solution 1, the binding solution, the rinsing solution and the eluent are all independently packaged and can be used independently.

Example 2 extraction of genomic DNA from maize, rice and wheat Using different kits

1. Extraction of corn genomic DNA Using kit 1

The extraction of the genome DNA of the corn leaf tissue by using the kit 1 (including the DNA extractant 1 in the kit 1) comprises the following steps:

1) 150mg of plant tissues (fresh plant leaves) are taken, samples are placed in a 96-hole deep-hole plate matched with a semi-automatic magnetic bead adsorption magnetic frame, and the samples are fully ground in a plant homogenizer after being frozen.

2) The ground and pulverized plant tissues were added to 600. mu.l of lysis solution and incubated at 65 ℃ for 40 minutes to obtain a lysis solution mixture.

3) The lysate mixture was centrifuged at 4000rpm for l0 minutes at ambient temperature, the sample was observed to be sedimented completely, and the supernatant was aspirated for use.

4.) transfer the supernatant to a new 96-well deep-well plate, add equal volume of isopropanol and 10. mu.l of magnetic bead suspension, mix well, and stand for 10 minutes.

5) Pressing the adsorption sleeve on a semi-automatic magnetic bead adsorption magnetic frame, and probing the magnetic rod of the magnetic frame into a 96-hole deep-hole plate for adsorption for 5 min.

6) And (4) putting the magnetic bar adsorbed with the magnetic beads into the rinsing liquid, and rinsing twice for 30 seconds.

7) After the magnetic beads were dried, they were eluted in deionized water.

8) Using a Qubit (Thermo)^TM) The concentration was measured and the results were recorded in 3 replicates, averaged and the results are recorded in table 1.

9) The results of the electrophoresis detection are shown in lane 6 of FIG. 1 and lane 1 of FIG. 2.

2. Extraction of wheat genomic DNA Using kit 1

And (3) replacing the corn leaf tissue in the step (1) with wheat leaf tissue, extracting wheat genome DNA without changing other steps, and carrying out electrophoresis detection on an experimental result shown in a lane 5 of a figure 1 and a lane 3 of a figure 2.

3. Extraction of Rice genomic DNA Using kit 1

And (3) replacing the corn leaf tissue in the step (1) with rice leaf tissue, extracting rice genome DNA (deoxyribonucleic acid) in the unchanged other steps, and carrying out electrophoresis detection on an experimental result shown in a lane 4 of a figure 1 and a lane 2 of a figure 2.

4. Extraction of corn genomic DNA Using kit 2

Replacing the kit 1 in the step 1 with the kit 1 (containing the DNA extractant 2), extracting the corn genomic DNA without changing other steps, and carrying out electrophoresis detection on the experimental result shown in a lane 4 of a figure 2 and a lane 1 of a figure 3.

5. Extraction of wheat genomic DNA Using kit 2

And (3) replacing the corn leaf tissue in the step (4) with wheat leaf tissue, extracting wheat genome DNA without changing other steps, and carrying out electrophoresis detection on an experimental result shown in a lane 6 of a figure 2 and a lane 4 of the figure 2.

6. Extraction of Rice genomic DNA Using kit 2

And (3) replacing the corn leaf tissue in the step (4) with the rice leaf tissue, and extracting the rice genome DNA without changing other steps, wherein the results are shown in table 1. The results of the electrophoresis detection are shown in lane 5 of FIG. 2 and lane 3 of FIG. 3.

7. Extraction of millet genomic DNA Using kit 2

And (3) replacing the corn leaf tissue in the step (4) with rice leaf tissue, extracting rice genome DNA (deoxyribonucleic acid) without changing other steps, and carrying out electrophoresis detection on an experimental result shown in a lane 2 of a figure 3.

8. Extraction of corn genomic DNA using Solebao plant DNA extraction kit

Extracting genome DNA of a corn leaf tissue by using a Solebao plant DNA extraction kit, wherein the method comprises the following steps:

1) 100mg of plant tissue (fresh plant leaves) was taken and placed in a 2ml centrifuge tube, frozen and ground thoroughly in a plant homogenizer.

2) Adding the ground and pulverized plant tissues into 400 ul of the solution A, 20ul of RNase A (10mg/ml) and 5ul of beta-mercaptoethanol in a centrifuge tube, fully reversing and uniformly mixing, and standing at room temperature for 10 min.

3) 140ul of solution B was added, mixed well by inversion, centrifuged at 12000rpm for 10min and the supernatant transferred to a fresh centrifuge tube.

4) Adding solution C with the same volume as the supernatant, fully inverting and mixing, adding absolute ethyl alcohol with the same volume as the solution C, and centrifuging at 12000rpm for 5 min.

5) 600ul of the rinse solution was added to the adsorption column, centrifuged at 12000rpm for 1min, and the waste solution was discarded.

6) And (5) repeating the step.

7) Placing the adsorption column into a clean centrifuge tube, adding 50-200ul of eluent dropwise, standing at room temperature for 1-5min, and centrifuging at 12000rpm for 2 min.

The results of the electrophoresis detection experiment are shown in lane 3 of FIG. 1.

9. Wheat genome DNA extraction by using Solebao plant DNA extraction kit

And (3) replacing the corn leaf tissue in the step (7) with the wheat leaf tissue, and extracting the wheat genome DNA electrophoresis detection experiment result without changing other steps as shown in a lane 2 of a figure 1.

10. Extraction of rice genome DNA by using Solebao plant DNA extraction kit

And (3) replacing the corn leaf tissue in the step (7) with rice leaf tissue, extracting rice genome DNA (deoxyribonucleic acid) without changing other steps, and carrying out electrophoresis detection on an experimental result shown in a lane 1 of a figure 1.

11. Corn genome DNA (deoxyribonucleic acid) extracted by using Loyang Edison paramagnetic particle method plant tissue DNA extraction kit

The method comprises the following steps of extracting the genome DNA of the maize leaf tissue by using a Loyang Edison magnetic bead method plant tissue DNA extraction kit:

1) taking 0.2 g of fresh plant sample and/or 0.1 g of dried plant sample, putting the fresh plant sample and/or the dried plant sample into a centrifugal tube of 1.5ml or 2ml, simultaneously adding 3-6 ceramic beads, completely freezing by liquid nitrogen, and transferring to a freezing and grinding instrument (MM400) for high-speed homogenization for 2-3 minutes.

2) 0.54ml Buffer SPL was added to the sample tube, vortexed for 60 seconds and then water-washed for 15 minutes at 65 ℃.

3) 0.18ml Buffer PS was added to the sample tube and vortexed for 60 seconds and then allowed to stand on ice for 20 minutes.

4) Centrifuging at 4000-5000 g for 20 minutes at room temperature.

5) 400 μ l of the supernatant was transferred to a 2ml96 deep well plate.

6) Add 80. mu.L of the suspension of magnetic beads and 550. mu.L of binding solution (with isopropanol added) and mix well by inversion. And (3) vortexing the EP tube at a high speed for 6min to fully combine the magnetic beads with the nucleic acid, and standing for 2min after the vortexing is finished.

7) Placing the EP tube on a magnetic frame and standing for 20s until the magnetic beads are completely adsorbed; if the magnetic beads are covered in the EP tube, the EP tube can be kept on the magnetic frame, and the whole body is turned upside down for 2-3 times until the magnetic beads are completely adsorbed.

8) Adding 600 μ L of cleaning solution into the EP tube, taking off the EP tube from the magnetic frame, blowing away the magnetic beads with a pipette, vortexing and shaking for 2min, and performing magnetic separation.

9) Add 100 u L eluent (or deionized water) to the tube bottom, transfer the magnetic beads after resuspension to another clean EP tube, 65 ℃ warm bath for 5min, then vortex elution for 2min, ensure the magnetic beads and nucleic acid complete elution dissociation.

The results of the electrophoresis detection experiment are shown in lane 9 of FIG. 1.

12. Wheat genome DNA extraction kit using Loyang Edison paramagnetic particle method plant tissue DNA extraction method

And (3) replacing the corn leaf tissue in the step (10) with the wheat leaf tissue, and extracting the wheat genome DNA without changing other steps, wherein the results are shown in table 1. The results of the electrophoresis detection experiment are shown in lane 8 of FIG. 1.

13. Extraction of rice genome DNA by using Loyang Edison paramagnetic particle method plant tissue DNA extraction kit

The maize leaf tissue in step 10 was replaced with rice leaf tissue, and the rice genome DNA was extracted without changing the other steps, and the results are shown in table 1. The results of the electrophoresis detection experiment are shown in lane 7 of FIG. 1.

The results of statistics of the concentrations of the DNAs extracted from corn, rice and wheat by the different kits are shown in Table 1 and FIG. 3, and it can be seen from the data in Table 1 that the extraction yields of the kit 1 and the kit 2 of the present invention are significantly improved in each crop species compared with the extraction yields of other DNA extraction kits (according to the results of more than 3 repeated experiments).

TABLE 1 DNA concentrations extracted from corn, rice and wheat using different kits

The present invention has been described in detail above. It will be apparent to those skilled in the art that the invention can be practiced in a wide range of equivalent parameters, concentrations, and conditions without departing from the spirit and scope of the invention and without undue experimentation. While the invention has been described with reference to specific embodiments, it will be appreciated that the invention can be further modified. In general, this application is intended to cover any variations, uses, or adaptations of the invention following, in general, the principles of the invention and including such departures from the present disclosure as come within known or customary practice within the art to which the invention pertains. The use of some of the essential features is possible within the scope of the claims attached below.

Claims

1. The DNA extracting agent is characterized by comprising a magnetic bead suspension and a lysate 1, wherein the lysate 1 is a Tris-HCL buffer solution containing ammonium bromide, sodium dodecyl sulfate, sodium chloride, ethylenediamine tetraacetic acid, polyvinylpyrrolidone, polyethylene glycol octyl phenyl ether, Tween20 and sodium azide.

2. The DNA extractant of claim 1, wherein the lysate 1 contains 2% by volume of ammonium bromate, 1% to 2% by volume of sodium dodecyl sulfate, 0.1% by volume of octyl phenyl ether of polyethylene glycol, 1% by volume of Tween20, 1% by volume of sodium azide, 0.2mol/L of sodium chloride, 10mmol/L of ethylenediaminetetraacetic acid, and 10mg/L of polyvinylpyrrolidone; the concentration of the Tris-HCL buffer solution is 100mmol/L, pH to be 8.5.

3. The DNA extractant of claim 1 or comprising a binding solution, a rinsing solution and an eluent, wherein the binding solution is 100% isopropanol and 2M GITC (isopropyl guanidine); the rinsing liquid is 80% ethanol; the eluent is deionized water.

4. The DNA extractant of any one of claims 1 to 3, wherein the magnetic bead content in the magnetic bead suspension is 200 mg/ml; the diameter of the magnetic bead is 80-1000 nm.

5. A DNA extraction kit comprising the DNA extraction reagent according to any one of claims 1 to 4.

6. The DNA extraction kit of claim 5, further comprising a 96-well deep well centrifuge plate, a 96-well magnetic stir bar and 5mm diameter steel balls, for use in cooperation with a 96-well semi-automatic magnetic stand.

7. A method for extracting plant genomic DNA by a magnetic bead method, which comprises using the DNA extracting agent according to any one of claims 1 to 4 and the DNA extracting kit according to claim 5 or 6, and which comprises:

1) crushing plant tissues, adding lysis solution 1, and standing;

2) centrifuging the mixed solution in the step 1), taking supernatant, adding the supernatant into the binding solution and the magnetic bead suspension, uniformly mixing, and standing;

3) washing the magnetic beads in the step 2 in a rinsing liquid for 2 times;

4) and drying the washed magnetic beads, and eluting with an eluent to obtain the purified DNA.

8. The method for extracting plant genomic DNA by the magnetic bead method according to claim 7, wherein the standing temperature of the step l) is 65 ℃, the standing time is 40-50min, and the centrifugation condition is 4000rpm for 5 min; the centrifugation condition in the step 2) is 4000rpm centrifugation for 20min, and the standing time is 10 min; the volume ratio of the lysis solution 1 to the binding solution to the magnetic bead suspension to the eluent is 600: 5-10:600.

9. The method for extracting plant genomic DNA by the magnetic bead method according to claim 7, wherein in the step l), the mass-to-volume ratio of the sample to the lysate is 150mg:600 μ 1; in the step 2), the volume ratio of the supernatant to the combined liquid is 1: 1; in the step (3), the volume ratio of the magnetic beads participating in nucleic acid extraction to the rinsing liquid is 1: 60.

10. The method for extracting plant genomic DNA by the magnetic bead method according to claim 7, wherein the plant tissue is one of fresh plant leaves, dry plant leaves and plant seeds.