CN111808848A

CN111808848A - Large-scale extraction method of wheat seed DNA high-salt magnetic beads

Info

Publication number: CN111808848A
Application number: CN202010858282.4A
Authority: CN
Inventors: 庞斌双; 张明明; 刘丽华; 李宏博; 刘阳娜; 张风廷; 张立平
Original assignee: Beijing Academy of Agriculture and Forestry Sciences
Current assignee: Beijing Academy of Agriculture and Forestry Sciences
Priority date: 2020-08-24
Filing date: 2020-08-24
Publication date: 2020-10-23

Abstract

The invention belongs to the technical field of agricultural biology, and particularly relates to a large-scale extraction method of wheat seed DNA high-salt magnetic beads. The invention takes a plurality of DNA extraction methods as reference, and develops a DNA formula and a method suitable for large-scale rapid extraction of wheat seeds by adjusting a formula and an extraction procedure for extracting DNA with high salt.

Description

Large-scale extraction method of wheat seed DNA high-salt magnetic beads

Technical Field

The invention belongs to the technical field of agricultural biology, and particularly relates to a large-scale extraction method of wheat seed DNA high-salt magnetic beads.

Background

Wheat is an important food crop in China, and the technical bottleneck of high-purity large-scale DNA extraction of wheat needs to be solved urgently in the fields of molecular biology, molecular genetics and the like, such as wheat variety identification, gene mining, correlation analysis and the like. The starch content in wheat seeds is about 57-75%, and the starch and DNA are coprecipitated during DNA extraction, so that the yield and quality of the DNA and the stability of later-stage PCR amplification are influenced. At present, various extraction methods are available to obtain high-quality and high-yield DNA, but most methods use leaves as materials. With the completion of wheat whole genome sequencing and the rapid and rapid development of molecular biology, molecular genetics and bioinformatics, the demand for extracting DNA in a large scale by using seeds as materials is increasing day by day.

At present, the most common DNA extraction method for wheat is a CTAB method. With the continuous progress of research methods, DNA extraction methods newly proposed on wheat crops include a high-salt low-pH method, a TENR method and the like, which can also extract DNA with higher quality without extraction of organic reagents such as phenol, chloroform and the like, and the appearance of various kits enables the DNA extraction to be faster, simpler and more environment-friendly. However, the kit method has high cost and complex operation, and cannot realize large-scale extraction, and the DNA extracted by the CTAB method has relatively poor quality and purity.

The internationally universal wheat DNA extraction method mostly takes wheat seedlings as materials, the time for sprouting is more than 7 days, the time is long, and the timeliness requirement of quick extraction and quick detection can be guaranteed by extracting DNA from wheat seed powder; the conventional CTAB method needs phenol chloroform or chloroform reagents which are main components for removing impurities such as polysaccharide, starch, protein and the like, have extremely strong corrosivity, can generate phosgene toxic gas and cause damage to organs such as respiratory tract and the like.

Disclosure of Invention

The invention aims to provide a large-scale extraction method of wheat seed DNA high-salt magnetic beads.

The method for extracting the DNA from the wheat seeds on a large scale by using the high-salt magnetic beads comprises the following steps:

(1) adding 700 μ LDNA extractive solution into 100mg wheat powder, mixing, incubating at 65 deg.C for 30min, adding 2.5mol/L KAc solution (1/3 volume of DNA extractive solution), shaking, standing at room temperature for 10min, centrifuging, and collecting supernatant;

(2) adding isopropanol with the volume 0.5 times that of the supernatant and 5 mu L of magnetic beads into the supernatant, uniformly mixing the magnetic beads and the supernatant, standing at room temperature for 3-5 min, and enriching the magnetic beads at the bottom of the extraction tube by magnetic force;

(3) keeping the extraction tube under the action of magnetic force, pouring out liquid, and sucking to dry;

(4) adding 700 mu L of 70% ethanol, fully rinsing the magnetic beads and the tube wall, and repeating the step (3);

(5) adding 200. mu.L of 0.5 × TE buffer, dissolving and mixing uniformly, dissolving DNA from the magnetic beads into the eluent, enriching the magnetic beads at the bottom of the extraction tube by magnetic force, sucking out the DNA solution and storing at low temperature.

According to the invention, the scale extraction method of the wheat seed DNA high-salt magnetic beads comprises the following steps: 14g SDS powder, 100mL of 1mol/L NaAc solution, 100mL of 0.5mol/L EDTA solution and 100mL of 5mol/L NaCl solution, and water is added to the mixture to make the volume of the mixture to be 1000 mL.

According to the large-scale extraction method of the wheat seed DNA high-salt magnetic beads, 41.02g of anhydrous NaAc is weighed and dissolved in a proper amount of water, water is added to the water to a constant volume of 500mL, and the mixture is autoclaved at 121 ℃ for 20min to prepare a 1mol/L NaAc solution.

The method for extracting the DNA from the wheat seeds in the high-salt magnetic beads on a large scale comprises the step of weighing 186.1g of Na₂EDTA·2H₂Dissolving O in 800mL of water, adjusting the pH value to 8.0 by using NaOH, adding water to a constant volume of 1000mL, and carrying out autoclaving at 121 ℃ for 20min to prepare 0.5mol/L EDTA solution.

According to the large-scale extraction method of the wheat seed DNA high-salt magnetic beads, 146.1g of solid NaCl is weighed, water is added to the solid NaCl, the volume is adjusted to 500mL, the mixture is sterilized at 121 ℃ for 20min under high pressure, and a 5mol/L NaCl solution is prepared.

According to the method for extracting the DNA from the wheat seeds in the large scale by the high-salt magnetic beads, 112.68g of KAc are weighed and dissolved in a proper amount of water, water is added to the water to be constant volume to 500mL, and the solution is sterilized at 121 ℃ for 20min under high pressure to prepare 2.5mol/L of KAc solution.

According to the large-scale extraction method of the wheat seed DNA high-salt magnetic beads, 121.1g of Tris is weighed and dissolved in 800mL of water, the pH value is adjusted to 8.0 by concentrated HCl, the water is added to a constant volume of 1000mL, and the mixture is autoclaved at 121 ℃ for 20min to prepare 1 mol/LTris-HCl.

According to the method for extracting the DNA from the wheat seeds on a large scale by using the high-salt magnetic beads, 5mL of 1mol/L LTris-HCl solution and 1mL of 0.5mol/L EDTA solution are measured, water is added to the solution to be constant volume of 1000mL, and a 0.5 × TE buffer solution is prepared.

According to the specific embodiment of the invention, the formula of the reagent for rapidly extracting the DNA of the wheat seeds in large scale is as follows:

the method for extracting the DNA from the wheat seeds in the large scale by the high-salt magnetic beads comprises the following steps:

(1) grinding a mixed seed sample or single seed of the wheat variety to be detected into flour by using a grinding instrument, and uniformly mixing;

(2) weighing 40-100mg of flour from the seed flour ground in the step (1), placing the flour at the bottom of a 96-hole deep-hole plate, adding 700 mu LDNA extracting solution into each hole, covering a silica gel pad, fully and uniformly mixing, placing the mixture in an oven, incubating for 30min at 65 ℃, turning upside down and uniformly mixing for 2 times in the period, adding 230 mu L of 2.5mol/L KAc solution, fully shaking, and standing for 10min at room temperature; centrifuging at 10000rpm/min for 10min, and transferring the supernatant into another 96-well deep-well plate by sucking 600 μ L with workstation or row gun.

(3) Adding 300 mu L of isopropanol and 5 mu L of magnetic beads into each hole of the new 96-hole deep-hole plate in the step (2), tightly covering by using a silica gel pad, uniformly mixing the magnetic beads and the supernatant, standing for 3-5 min, and uniformly mixing for 2 times in the period; and placing the deep-hole plate on a magnetic frame, standing for 1-2 min to enable the magnetic beads to be uniformly enriched at the bottom of the deep-hole plate, and turning the deep-hole plate for 2 times in the period to enable the magnetic beads attached to other positions in the deep-hole plate to be enriched at the bottom of the deep-hole plate.

(4) Keeping the deep hole plate on the magnetic frame, uncovering the silica gel pad, pouring out the liquid, placing the deep hole plate on the absorbent paper with the deep hole plate downward, standing for 2min, and allowing the liquid to flow out of the deep hole plate sufficiently.

(5) Adding 700 μ L70% ethanol into each hole of the deep hole plate, covering with silica gel pad, keeping the deep hole plate on the magnetic frame, turning over, rinsing the magnetic beads and the tube wall, and repeating step (4)

(6) And (5) repeating the steps (5) and (4) once again, thoroughly removing the liquid in the deep hole plate, and airing for 10min at room temperature.

(7) And (3) adding 100-200 mu L of 0.5 × TE buffer dissolving solution into each hole of the deep-hole plate obtained in the step (6), dissolving and uniformly mixing DNA, dissolving nucleic acid into eluent from magnetic beads, placing the deep-hole plate on a magnetic frame, sucking out the DNA solution from a 96-hole DNA collecting plate by using a liquid transfer workstation or a discharging gun until the magnetic beads are enriched at the bottom of the deep-hole plate, and storing at-20 ℃ for later use.

While the pH of the extract solution of the conventional high-salt low-pH method needs to be adjusted, the present inventors have discovered that the amount of extraction, quality, purity and stability of the amplification product are excellent, although the pH is not adjusted. Further optimization is carried out on the formula and the workflow in order to meet the requirement of batch DNA extraction in the detection center.

The method is improved in comparison with a high-salt low-pH method as follows: 1) the extraction amount of DNA is large, and the requirement of preparing a PCR reaction system for hundreds of times can be met; 2) after the magnetic bead method is introduced, the quality and purity of DNA are improved, and an amplification product is stable; 3) the reagent preparation is time-saving, simple and quick, the pH value is not required to be adjusted, the pH value of the high-salt low-pH formula needs to be adjusted in the process of preparing sodium acetate, potassium acetate and all extracting solutions with the pH value of 4.8, and the sodium acetate and the potassium acetate are not easy to dissolve in the process of adjusting the pH value; 4) after the extract is warm-bathed, 230 μ L of 2.5mol/L KAc solution is added, and 250 μ L of 2.5mol/L KAc solution is added for high-salt low-pH method; 5) after the magnetic frame is added, the centrifugation step is reduced, a plurality of 96-hole deep-hole plates can be processed simultaneously, the extraction flux and efficiency are greatly improved, and the seed powder is used for quickly realizing large-scale extraction, so that the detection time can be greatly shortened compared with a germination extraction method, and the reported timeliness is improved.

The invention has the beneficial effects that:

the method of the invention uses all conventional cheap inorganic reagents, the price is cheap, wherein SDS reagent is the effective component for removing impurities such as protein, etc., and auxiliary equipment such as magnetic beads and magnetic racks is an effective technical means for ensuring the removal of impurities such as polysaccharide, protein, etc., and the magnetic beads only combine DNA and do not combine organic matters such as polysaccharide, protein, etc., and inorganic matters, etc., so the purity and quality of extracted DNA are ensured after the magnetic beads are cleaned.

According to the method, the reagent is prepared in a time-saving, simple and quick manner without adjusting the pH value, the pH value of the high-salt low-pH formula needs to be adjusted in the process of preparing sodium acetate, potassium acetate and all extracting solutions with the pH value of 4.8, and the sodium acetate and the potassium acetate are not easy to dissolve in the process of adjusting the pH value.

According to the method, after the magnetic beads and the magnetic frame are added, the centrifugation steps are reduced, a plurality of 96-hole deep-hole plates can be processed at the same time, the extraction flux and the extraction efficiency are greatly improved, the application of the magnetic beads and the magnetic frame changes the traditional complicated procedures of independent absorption, sample addition and centrifugation of a single centrifugal tube, the problem can be solved by a mode of sample addition or sample absorption of a workstation at one time, the whole plate is centrifuged during centrifugation, and the whole plate can be toppled in the process of cleaning DNA impurities, so that a large amount of manpower is saved.

The method is suitable for large-scale extraction, the quality and yield of the extracted DNA are high, the purity and quality of the DNA are stable, and the technical support is provided for the fields of large-scale gene typing, correlation analysis, variety identification and the like of wheat in the future.

Drawings

FIG. 1 shows the results of SSR primer amplification for different DNA extraction methods;

FIG. 2 shows the SNP genotyping results of DNA extracted by an example of the present invention;

FIG. 3 shows the effect of DNA extracted by the example of the present invention in identifying the photoperiod genes Ppd-D1a (a) and Ppd-D1b (b).

Detailed Description

According to the specific embodiment of the invention, the method for extracting the DNA from the wheat seeds in a large scale by using the high-salt magnetic beads comprises the following steps:

(2) weighing 40-100mg of flour from the seed flour ground in the step (1), placing the flour at the bottom of a 96-hole deep-hole plate, adding 700 mu LDNA extracting solution into each hole by using a liquid transfer workstation or a row gun, covering a silica gel pad, fully and uniformly mixing, placing the mixture in an oven, incubating the mixture for 30min at 65 ℃, turning upside down and uniformly mixing the mixture for 2 times, adding 230 mu L of 2.5mol/L KAc solution, fully shaking the mixture uniformly, and standing the mixture for 10min at room temperature; centrifuging at 10000rpm/min for 10min, and transferring the supernatant into another 96-well deep-well plate by sucking 600 μ L with workstation or row gun.

(3) Adding 300 mu L of isopropanol and 5 mu L of magnetic beads into each hole of the new 96-hole deep-hole plate in the step (2) by using a liquid transfer workstation or a discharging gun, tightly covering by using a silica gel pad, uniformly mixing the magnetic beads and the supernatant, standing for 3-5 min, and uniformly mixing for 2 times; and placing the deep-hole plate on a magnetic frame, standing for 1-2 min to enable the magnetic beads to be uniformly enriched at the bottom of the deep-hole plate, and turning the deep-hole plate for 2 times in the period to enable the magnetic beads attached to other positions in the deep-hole plate to be enriched at the bottom of the deep-hole plate.

(5) Adding 700 μ L70% ethanol into each hole of the deep-hole plate with a liquid transfer workstation or a row gun, covering with silica gel pad, keeping the deep-hole plate on a magnetic frame, turning over, rinsing the magnetic beads and the tube wall, and repeating the step (4)

(7) And (3) adding 100 and 200 mu L of 0.5 multiplied by TEbuffer dissolving solution into each hole of the deep-hole plate obtained in the step (6) by using a liquid transfer workstation or a discharging gun, dissolving the nucleic acid from the magnetic beads into the eluent after the DNA is dissolved and uniformly mixed, placing the deep-hole plate on a magnetic frame, sucking the DNA solution out of a 96-hole DNA collecting plate by using the liquid transfer workstation or the discharging gun when the magnetic beads are enriched at the bottom of the deep-hole plate, and storing the DNA solution at the temperature of minus 20 ℃ for later use.

EXAMPLE 1 Effect of setting of different extraction parameters on DNA extraction Effect

Chinese spring and Zhongyou 9507 of wheat as research material and high-salt and low-pH value method as contrast (Zhangming et al 2020) to respectively adjust sodium acetate and potassium acetateThe pH value is 4.8, and the two reagents are not adjusted to be pH groups, wherein room temperature cracking and cracking at 65 ℃ are respectively set in each group of experiments, and the amount of potassium acetate added after cracking is 1/3 of the original volume and 2/3 of the original volume respectively set in the two groups of experiments; two groups of control experiments of room temperature isopropanol and frozen isopropanol are respectively set in two groups of experiments during precipitation, and respectively optimized experimental schemes are determined. The experimental results show that different materials have certain difference in the yield of extracted DNA due to the difference in the starch content. The results of combining the two materials show that the DNA yield of the formula is higher than that of the pH value adjustment (tables 1 and 2) when the pH values of sodium acetate and potassium acetate are not adjusted and the pH value of the DNA extraction working solution is not adjusted, wherein the DNA yield of the formula is 581ng/mg of flour, the DNA yield of the formula is 528ng/mg of flour, and the DNA yield of each mg of flour is 53ng/mg of flour. The yield of DNA cracked under the constant temperature environment of 65 ℃ in the formula is 107ng/mg higher than that of DNA cracked at room temperature; the results of the treatment with isopropanol when the DNA was precipitated in the cold and the treatment with isopropanol at ordinary temperature are shown in Table 1, and the DNA purity (OD) in the treatment at ordinary temperature_260/2302.11) higher than the freezing process environment (OD)_260/2301.96), but the DNA yield difference is not large, which indicates that the DNA extraction adopts 0.5 volume of isopropanol at normal temperature to be more beneficial to the high-quality DNA extraction. As can be seen from Table 1, the optimal amount of potassium acetate was 581ng/mg for 1/3 volumes of the extract, which is much higher than 415ng/mg for 2/3 volumes, and the results in Table 2 are similar. The optimum amount of potassium acetate in the extraction reagent is 1/3% by volume of the extract. As can be seen from tables 1 and 2, the lysis conditions at 65 ℃ are on average 100ng/mg higher than the DNA yield under room temperature lysis conditions, regardless of whether the pH is adjusted, and if the DNA yield is high, the 65 ℃ lysis should be selected preferentially.

In conclusion, it was finally determined that the optimum amount of potassium acetate was 1/3% by volume of the extract; the pH value is not regulated in the preparation process of the potassium acetate and sodium acetate solution, so that the high yield and high quality of DNA are facilitated; when DNA is precipitated, isopropanol with the volume 0.5 times that of the supernatant at room temperature is adopted, so that the yield and DNA quality are better; the 65 ℃ cracking condition is higher than the room temperature cracking yield, and if the requirement on the DNA yield is not high, the room temperature cracking condition can be adopted for extraction.

TABLE 1 DNA quality and yield under various control conditions without pH adjustment of potassium acetate and sodium acetate solutions

TABLE 2 DNA quality and yield under various control conditions when pH4.8 was adjusted with potassium acetate and sodium acetate solutions

2. Determination of optimal amount of magnetic beads

Selecting 4 varieties of Jingdong 17, Jingdong 22, Jingdong 23, Jingdong 24 and the like, wherein the corresponding numbers are 1613, 1614, 1615 and 1616 respectively, the magnetic bead dosage is set to be two gradients of 5uL and 10uL, each variety of each gradient is set to be repeated for 4 times, each gradient is repeated for 100mg of flour, performing an experiment according to the experimental steps by using a 96-hole deep-hole plate of the same plate, and finally dissolving DNA in 200uL of 0.5 × TE. The average result of extracting DNA from each sample in each magnetic bead gradient shows (table 3-table 4), the different DNA yields of the samples are different, but from the average value of 4 varieties to be tested, 5uL magnetic beads can be saturated and adhered with the extracted DNA amount, the yield of the DNA can be reduced by increasing the magnetic bead amount, and the cost cannot be increased, so that the 5uL magnetic beads for extracting 100mg of flour DNA are finally determined.

TABLE 3 DNA extraction results with 5uL magnetic beads for each sample

TABLE 4 DNA extraction results with 10uL magnetic beads for each sample

Example 2 comparison of the effectiveness of the extraction of DNA according to the invention with other conventional methods

Taking 5 wheat varieties, setting each variety in each group of experiments for 3 times of repetition, multiplying 100mg of seed flour each time, extracting DNA of dry seed powder by using a kit, CTAB and a high-salt magnetic bead method respectively, and finally dissolving the DNA by using 100 microliters of solution, wherein the results show that (table 5-table 7)) the DNA yield of different varieties has obvious difference but consistent general trend, wherein the average yield of DNA extracted by using the high-salt magnetic bead method is 487ng/mg, OD260/280 is 1.9, and OD260/230 is 2.0; the average yield of DNA extracted from seeds by the kit method is 400ng/mg, OD260/280 is 2.0, and OD260/230 is 2.6; the average yield of DNA extracted from seeds by the CTAB method is 401ng/mg, OD260/280 is 2.1, and OD260/230 is 1.0. The method has the advantages that the yield of the DNA extracted from the seeds by the high-salt magnetic bead method is highest, the purity and the quality of the DNA are good, the operation is simple, the cost is low, the large-area popularization is facilitated, the yield of the DNA extracted by the kit method is equivalent to that of the CTAB method, the yield of the DNA is close to that of the CTAB method, the cost of the kit method is high, the operation is complex, the large-scale extraction cannot be realized, the quality and the purity of the DNA extracted by the CTAB method are relatively poor, and.

TABLE 5 concentration and quality of DNA extracted by the kit method

TABLE 6 concentration and quality of DNA extracted by high CTAB method

TABLE 7 concentration and quality of DNA extracted by high-salt magnetic bead method

Example 3 application of DNA extracted according to the invention in SSR marker identification

The SSR primers are amplified by using special 42 for wheat authenticity identification for varieties of DNA extracted by a high-salt magnetic bead method, a kit method, a high-salt low-pH method and a tender bud CTAB method of seeds, the detection result of products by a capillary fluorescence electrophoresis method shows (figure 1), the amplification peak value effect of the DNA extracted by the high-salt magnetic bead method is obviously better than that of the seed DNA extracted by the high-salt low-pH method and the kit method and that of the tender bud DNA extracted by the CTAB method, and the DNA extracted by the high-salt magnetic bead method can meet the basic requirement of PCR amplification.

Example 4 application of DNA extracted according to the present invention to SNP marker genotyping

The DNA of 94 wheat seed samples is extracted by adopting a high-salt magnetic bead method, partial results of amplification product genotyping of the 94 samples are shown in figure 2 by using the DNA as a template and using KASP primers with 96 SNP loci to amplify by using a KASP SNpline technology, and obviously, the quality of the DNA extracted by the method can meet the requirement of SNP genotyping, the clustering effect is very good, and the DNA extraction method is good in universality.

Example 4 application of DNA extracted according to the present invention to Gene identification

As shown in FIG. 3, 4 seed DNAs of Jimai 19, Chinese spring, Rugen 502, Xiaoyan 22 and the like are extracted by the method, target fragments are amplified by taking the DNAs and specific primers of the photoperiod genes Ppd-D1a (a) and Ppd-D1b (b) as templates and primers, and the results of two times of repetition of agarose electrophoresis of amplified products show that the Jimai 19, the Rugen 502 and the Xiaoyan 22 all carry the target genes Ppd-D1a, while the Chinese spring does not carry the genes; only Chinese spring in the 4 wheat varieties carries the Ppd-D1b gene, but only Chinese spring is spring wheat, which indicates that the method can meet the basic requirements of conventional molecular biology and gene cloning.

Claims

1. A large-scale extraction method of wheat seed DNA high-salt magnetic beads is characterized by comprising the following steps:

2. The method for extracting the wheat seed DNA from the magnetic beads with high salt content on a large scale according to claim 1, wherein the formula of the DNA extracting solution is as follows: 14g of SDS powder, 100mL of 1mol/LNaAc solution, 100mL of 0.5mol/L EDTA solution and 100mL of 5mol/L LNaCl solution, and adding water to a constant volume of 1000 mL.

3. The large-scale extraction method of the wheat seed DNA high-salt magnetic beads according to claim 2, characterized in that 41.02g of anhydrous NaAc is weighed and dissolved in a proper amount of water, water is added to the solution to a constant volume of 500mL, and the solution is sterilized at 121 ℃ for 20min under high pressure to obtain a 1mol/LNaAc solution.

4. The method for large-scale extraction of the wheat seed DNA high-salt magnetic beads according to claim 2, wherein 186.1g of Na is weighed₂EDTA·2H₂Dissolving O in 800mL of water, adjusting the pH value to 8.0 by using NaOH, adding water to a constant volume of 1000mL to prepare 0.5mol/L EDTA solution, and autoclaving at 121 ℃ for 20 min.

5. The large-scale extraction method of the wheat seed DNA high-salt magnetic beads as claimed in claim 2, wherein 146.1g of solid NaCl is weighed, water is added to a constant volume of 500mL, a 5mol/L NaCl solution is prepared, and autoclaving is carried out at 121 ℃ for 20 min.

6. The large-scale extraction method of the wheat seed DNA high-salt magnetic beads as claimed in claim 1, wherein 112.68g of KAc are weighed and dissolved in a proper amount of water, water is added to the water to a constant volume of 500mL, and the mixture is autoclaved at 121 ℃ for 20min to prepare a 2.5mol/LKAC solution.

7. The large-scale extraction method of the wheat seed DNA high-salt magnetic beads as claimed in claim 1, wherein 121.1g of Tris is weighed and dissolved in 800mL of water, the pH value is adjusted to 8.0 by concentrated HCl, the water is added to the volume to be 1000mL, and the solution is autoclaved at 121 ℃ for 20min (the pH value is adjusted after the solution is cooled to room temperature).

8. The method for extracting the DNA from the wheat seeds on a large scale by the high-salt magnetic beads according to claim 1, wherein 5mL of a 1mol/L LTris-HCl solution and 1mL of a 0.5mol/L EDTA solution are measured, and water is added to the solution to reach a volume of 1000 mL.