Plant genome DNA rapid crude extract and extraction method based on crude extract

Abstract

The invention discloses a plant genome DNA rapid crude extract and an extraction method based on the crude extract. The DNA crude extract is prepared from the following components: 60 to 150 mM of Tris-HCl and 5 to 12 mM of EDTA (Ethylene Diamine Tetraacetic Acid), and the pH (Potential of Hydrogen) of the DNA crude extract is 7.5 to 8.5. The extraction method based on the crude extract comprises the following steps: adding a plant tissue sample and a DNA extracting solution into a centrifugal tube or a deep-hole plate, grinding the sample, and carrying out warm bath on the sample to obtain the crude extract DNA. The coarsely extracted DNA can be directly used for PCR amplification without dilution. According to the DNA crude extraction method disclosed by the invention, the crude extraction DNA can be obtained only by adding liquid once, high-temperature treatment is not needed, sample pollution possibly caused by multiple times of liquid adding and high-temperature treatment is avoided, and the operation is simple, convenient, rapid and efficient.

Description

Technical Field

[0001] This invention relates to the field of DNA extraction technology, specifically to a rapid crude extract of plant genomic DNA and an extraction method based on the crude extract. Background Technology

[0002] PCR-based molecular detection technologies have been widely applied in marker-assisted selection breeding, gene mapping, transgenic detection, and variety purity identification. The preparation of plant DNA templates is a necessary condition for successful PCR molecular detection.

[0003] Traditional DNA extraction methods include SDS and CTAB methods. These methods all require steps such as high-temperature water baths, extraction with toxic organic reagents like chloroform and isopropanol, and precipitation, making them complex and time-consuming, especially for large-scale plant sample gene detection. Therefore, establishing a simple, rapid, non-toxic, and low-cost DNA extraction method, with the template directly used for PCR amplification, can improve the efficiency of plant gene detection and promote the sustainable development of the plant gene detection field.

[0004] Currently, there are several methods for crude extraction of plant DNA. Sung-Ryul Kim et al., in Plant Breeding and Biotechnology, Vol. 4(1), pp. 99-106, 2016, mentioned a method for crude extraction of rice DNA: TPE buffer (100 mM Tris-HCl pH 9.5, 1 M KCl, 10 mM EDTA pH 8.0) was added to the ground tissue sample, incubated at 65℃ for 20-90 min, then the sample was diluted 10-fold with H2O, centrifuged, and the supernatant could be used directly as a PCR template. The drawback of this method is that high concentrations of KCl inhibit PCR, affecting the amplification efficiency.

[0005] Victor I. Klimyuk et al., in The Plant Journal, Vol. 3(3), pp. 493-494, 1993, mentioned another method for crude extraction of plant DNA: Plant sample and 40 μl of 0.25 M NaOH are added to a centrifuge tube, boiled for 30 seconds, followed by the addition of 40 μl of 0.25 M HCl and 20 μl of 0.5 M Tris-HCl (pH 8.0), and boiled for 2 minutes. The sample can then be used directly for PCR. This method involves two boiling steps and two liquid addition steps, which can easily lead to sample contamination.

[0006] Application No. 201710605160.2 discloses a method for extracting plant DNA: a small amount of leaves are cut, rapidly ground into a homogenate, and an extraction solution (20-50 mM Tris-HCl and 13-25 mM EDTA, pH 8.0) is added. The mixture is heated at 70-90℃ for 10-15 min and centrifuged at high speed for 1 min to obtain a DNA extract that can be directly used for PCR. Summary of the Invention

[0007] The purpose of this invention is to overcome the shortcomings of the above-mentioned crude plant genomic DNA extraction methods and provide a more efficient, simple, and less sample-contamination-prone crude plant genomic DNA extraction method.

[0008] The objective of this invention is achieved through the following technical solution: A rapid crude extract of plant genomic DNA comprises the following components: 60–150 mM Tris-HCl and 5–12 mM EDTA. The pH of the crude extract is 7.5–8.5.

[0009] The extraction method based on the above-mentioned rapid crude extract of plant genomic DNA involves the following steps: 1) Prepare a rapid crude extract of plant genomic DNA and store it at room temperature; 2) Take 10-100 mg of plant tissue, put it into a 2.0 mL centrifuge tube, and add 2 steel balls with a diameter of 4 mm and 100-200 μL of extraction solution at the same time; 3) Homogenize the tissue using a homogenizer until the extract changes color; 4) After homogenization, transfer the sample to a constant temperature incubator at 60~65℃ for 20~40 minutes; 5) Samples that have been incubated in the warm water can be directly used for PCR amplification after cooling to room temperature.

[0010] The steel balls in step 2) and the homogenizer in step 3) can be replaced by other methods, such as crushing the tissue with a grinding rod.

[0011] The crude extract sample after removing the steel balls can be stored at room temperature for more than 5 weeks without any change in quality.

[0012] Compared with the prior art, the beneficial effects of the present invention are: less inhibition of subsequent PCR, only one addition of extraction solution is needed during sample preparation, no high-temperature heating is required, greatly reducing contamination between samples and significantly improving the success rate of subsequent PCR identification. Attached Figure Description

[0013] To more clearly illustrate the specific embodiments of the present invention or the technical solutions in the prior art, the drawings used in the specific embodiments will be described in detail below. Obviously, the drawings described below are some embodiments of the present invention. For those skilled in the art, other drawings can be obtained from these drawings without creative effort.

[0014] Figure 1 Electrophoretic images of PCR products (transgenic identification) after storage at room temperature for 1 week and 5 weeks using extracts of different concentrations and crude extracts. M: DL2000 Marker, 250bp and 500bp were labeled; 1-12: different individual plants from the same transgenic rice line. Agarose gel concentration was 1.5%, and the PCR product size of the T1 primer pair was 338bp. Figure 1 The results showed that different concentrations of Tris-HCl and EDTA combinations had varying effects on the crude extraction of sample DNA. The combination of 100 mM Tris-HCl and 10 mM EDTA was superior to that of 50 mM Tris-HCl and 5 mM EDTA, while the low-concentration combination of 10 mM Tris-HCl and 1 mM EDTA showed the worst results. Even after the crude samples were stored at room temperature for 5 weeks (from 1 week), the crude samples extracted with the 100 mM Tris-HCl and 10 mM EDTA combination could still be successfully detected, while the detection success rate decreased with lower concentration combinations.

[0015] Figure 2 Electrophoretic images of PCR products (co-dominant markers) after storage at room temperature for 1 week and 5 weeks using extracts of different concentrations and crude extracts. 1–12: Segregating populations of progeny from backcross heterozygous single plants of rice varieties. Agarose gel concentration was 5%, and the PCR products amplified from parental DNA using the InDel3 primer pair were 213 and 184 bp, respectively. Figure 2 The results showed that different concentrations of Tris-HCl and EDTA combinations had varying effects on crude DNA extraction. The combinations of 100 mM Tris-HCl and 10 mM EDTA and 50 mM Tris-HCl and 5 mM EDTA were superior to the low concentration combination of 10 mM Tris-HCl and 1 mM EDTA. Even after the crude samples were stored at room temperature for 5 weeks (from 1 week), the crude samples extracted with the 100 mM Tris-HCl and 10 mM EDTA combination could still be successfully detected, while the detection success rate decreased with lower concentration combinations.

[0016] Figure 3This invention demonstrates the application of the crude DNA extraction method in molecular breeding for population screening of target genotypes. Lanes 1-72: Results of dual PCR products from segregating populations of backcross heterozygous single plants of rice varieties. The InDel1 primer pair amplified PCR products of parental DNA at 142 and 126 bp, respectively, while the InDel2 primer pair amplified PCR products of parental DNA at 183 and 207 bp, respectively. At a 5% agarose gel concentration, the four PCR products were clearly distinguishable. Analysis revealed that plants 54 and 65 were plants with genotype exchange. Figure 3 This invention demonstrates that the crude DNA extraction method can be used for dual PCR, which can significantly improve the efficiency of genotype screening in molecular breeding and has important application value. Detailed Implementation

[0017] The embodiments of the present invention will be described in detail below with reference to examples. However, those skilled in the art will understand that the following examples are for illustrative purposes only and should not be considered as limiting the scope of the invention. Unless otherwise specified in the examples, conventional conditions or conditions recommended by the manufacturer are followed. Reagents or instruments whose manufacturers are not specified are all commercially available conventional products.

[0018] Example 1: The effect of different concentrations of extract and the storage time of crude extract samples at room temperature on subsequent PCR.

[0019] Experimental Design: Tris-HCl and EDTA are considered standard reagents for nucleic acid extraction. This invention uses 60-150 mM Tris-HCl and 5-12 mM EDTA as crude DNA extraction reagents. However, some technical descriptions (e.g., application number 201710605160.2) suggest that 20-50 mM Tris-HCl can be used for crude DNA extraction. Therefore, it is unknown whether different concentrations of Tris-HCl have the same extraction effect. This experiment designs extraction solutions with different concentrations of Tris-HCl and EDTA and compares the PCR results of samples after crude DNA extraction to demonstrate whether the concentration combination of this invention can significantly improve the efficiency of crude DNA extraction. Simultaneously, the effects of storing the corresponding crude extracted samples at room temperature for 1 week and 5 weeks on subsequent PCR are compared. Two different samples were selected for verification. Sample A (experimental materials provided by Researcher Li Yunhai of the Institute of Genetics and Developmental Biology, Chinese Academy of Sciences) consisted of mature leaves of transgenic rice plants (to verify the presence of transgenic T1 gene). Sample B (experimental materials provided by Researcher Li Yunhai of the Institute of Genetics and Developmental Biology, Chinese Academy of Sciences) consisted of young leaves of segregating populations of backcross heterozygous single plants of rice varieties (to verify the genotypic status of InDel3 in this population).

[0020] Experimental steps:

[0021] 1. Preparation of crude DNA

[0022] (1) Prepare a rapid crude extract of plant genomic DNA (100 mM Tris-HCl, 10 mM EDTA, pH 8.0). Then, dilute the crude extract by 2 times and 10 times to obtain crude extracts with lower concentrations (50 mM Tris-HCl, 5 mM EDTA, pH 8.0) and (10 mM Tris-HCl, 1 mM EDTA, pH 8.0), respectively.

[0023] (2) Take 10-100 mg of leaves from 12 individual plants of each sample A and sample B and put them into 2.0 ml centrifuge tubes. Add 2 steel balls with a diameter of 4 mm to each centrifuge tube. Take 3 replicates for each individual plant and add 100 μl of crude extract of different concentrations between different replicates.

[0024] (3) Homogenize the mixture for 1 min using a homogenizer.

[0025] (4) Transfer the sample to a constant temperature incubator and incubate at 65℃ for 30 min.

[0026] (5) After the sample is cooled to room temperature, in order to avoid the steel ball affecting the crude extract components in the aqueous solution and thus affecting the subsequent PCR results, open the centrifuge tube cap and quickly pour out the steel ball, and then immediately put the centrifuge tube upright. At this time, although there is some loss of crude extract in the centrifuge tube, the remaining part is enough to complete the subsequent experiments.

[0027] (6) Tightly seal the centrifuge tube, remove the steel ball, and store the crude extract at room temperature for more than 5 weeks. Select samples stored for 1 week and 5 weeks for PCR comparison and verification.

[0028] 2. DNA PCR reaction system and reaction procedure

[0029] PCR amplification was performed using 2×Rapid Taq Master Mix (P222) purchased from Nanjing Novizan Biotechnology Co., Ltd. Sample A was amplified using the T1 primer pair, and sample B was amplified using the InDel3 primer pair. The reaction mixture consisted of 9 μl of 2×Rapid Taq Master Mix, 0.4 μl each of forward and reverse primers (10 μM), 2 μl of crude extract, and ddH2O to a total reaction volume of 15 μl. The reaction program was as follows: 95℃ pre-denaturation for 5 min, 95℃ denaturation for 20 sec, annealing at 55℃ (T1 primer pair) or 52℃ (InDel3 primer pair) for 20 sec, extension at 72℃ for 20 sec, denaturation to extension for 35 (T1 primer pair) or 37 (InDel3 primer pair) cycles, and a final extension at 72℃ for 5 min. The primer sequences used are shown in Table 1. Table 1 Primer sequences used in this invention

[0030] 3. Agarose gel electrophoresis and gel imaging of PCR products

[0031] Prepare 1.5% and 5% agarose gels. For sample A, use 1.5% agarose gel electrophoresis (voltage 5V / cm) for PCR products; for sample B, use 5% agarose gel electrophoresis (voltage 5V / cm). Stop electrophoresis when the PCR products can be distinguished by size, and take pictures with a gel imaging device for storage.

[0032] Results analysis:

[0033] Figure 1 and Figure 2 Electrophoretic images show PCR products from different concentrations of extract and their crude extracts, Sample A and Sample B, stored at room temperature for 1 week and 5 weeks, respectively. Figure 1 The results showed that different concentrations of Tris-HCl and EDTA combinations had varying effects on the crude extraction of DNA from sample A. The combination of 100 mM Tris-HCl and 10 mM EDTA was superior to that of 50 mM Tris-HCl and 5 mM EDTA, while the low-concentration combination of 10 mM Tris-HCl and 1 mM EDTA showed the worst results. Even after the crude extracted samples were stored at room temperature for 5 weeks (from 1 week), the crude extract with the 100 mM Tris-HCl and 10 mM EDTA combination could still be successfully detected, while the detection success rate decreased with lower concentration combinations. Figure 2 The results showed that different concentrations of Tris-HCl and EDTA combinations had varying effects on the crude extraction of B DNA from samples. The combinations of 100 mM Tris-HCl and 10 mM EDTA and 50 mM Tris-HCl and 5 mM EDTA were superior to the low concentration combination of 10 mM Tris-HCl and 1 mM EDTA. Even after the crude samples were stored at room temperature for 5 weeks (from 1 week), the crude samples extracted with the 100 mM Tris-HCl and 10 mM EDTA combination could still be successfully detected, while the detection success rate decreased with lower concentration combinations. These results indicate that the 100 mM Tris-HCl and 10 mM EDTA combination is more advantageous than lower concentration combinations for crude extraction of plant DNA.

[0034] Example 2: Application of the crude DNA extraction method of the present invention in population screening of target genotypes in molecular breeding. Experimental Design: In applications such as QTL mapping and marker-assisted selection breeding, identifying and screening target genotypes from large genetic populations is a tedious but essential step. If DNA is extracted first using the traditional CTAB method followed by PCR identification, it inevitably increases the workload of genomic DNA extraction, which is time-consuming and labor-intensive. Therefore, it is necessary to find a rapid, efficient, and cost-effective crude DNA extraction method for subsequent genotype screening. The purpose of this experiment is to use the crude DNA extraction method of this invention to crudely extract segregating population C (experimental materials provided by Researcher Li Yunhai of the Institute of Genetics and Developmental Biology, Chinese Academy of Sciences) from the progeny of backcross heterozygous single plants of rice varieties, and to screen single plants from which recombination exchange between InDel1 and InDel2 markers occurs.

[0035] Experimental steps: The preparation of crude DNA, the DNA PCR reaction procedure, and the agarose gel electrophoresis and gel imaging method of the PCR products are the same as those for sample B in Example 1. The difference is that the DNA PCR reaction system has two primer pairs (10 μM), InDel1 and InDel2 (Table 1), with 0.4 μl of each primer.

[0036] Results analysis: Figure 3 The DNA extraction method of this invention, after crude extraction of rice variety backcross heterozygous progeny segregating population C, was used for PCR identification using two primer pairs, InDel1 and InDel2. InDel1 and InDel2 are approximately 700 kb apart on rice chromosome 3. The InDel1 primer pair amplified the PCR products of both parents' DNA, yielding 142 bp (recurrent parent) and 126 bp (donor), respectively. The InDel2 primer pair amplified the PCR products of both parents' DNA, yielding 183 bp (recurrent parent) and 207 bp (donor), respectively. Figure 3 It can be seen that the two markers are linked in most individual plants, while plants 54 and 65 are plants where the genotype has been exchanged. Figure 3 This invention demonstrates that the crude DNA extraction method can be used for dual PCR, which can significantly improve the efficiency of genotype screening in molecular breeding and has important application value.

Claims

1. A DNA extraction solution, characterized in that, It includes the following components: 60~150 mM Tris-HCl and 5~12 mM EDTA, with a pH of 7.5~8.

5.

2. A reagent kit, characterized in that, Includes the DNA extraction solution as described in claim 1.

3. The application of the DNA extraction solution of claim 1 or the kit of claim 2 in the extraction of plant DNA.

4. A method for extracting plant DNA, characterized in that, Includes the following steps: Plant tissue samples and the DNA extraction solution described in claim 1 are added to centrifuge tubes or deep-well plates, the samples are ground, and the samples are incubated in a warm water bath to obtain crude DNA.

5. The method according to claim 4, wherein the plant tissue sample includes parts of the plant such as leaves, inflorescences, stems, roots, and seeds.

6. The method according to claim 4, wherein the plants include monocotyledons and dicotyledons.

7. According to the method described in claim 4, the grinding sample can be homogenized by combining steel balls and a homogenizer, or it can be replaced by other methods, such as crushing with a grinding rod.

8. The method according to claim 4, wherein the bath temperature is 60~65℃ and the bath time is 20~40 min.

9. In the method according to claim 4, the mass-to-volume ratio of the plant sample and the DNA extraction solution according to claim 1 is preferably 1:2 to 1:

40.

10. A method for verifying whether plant DNA has been successfully extracted, characterized in that, The process includes the following steps: using the crude DNA extract obtained by the method described in claim 4, performing PCR amplification and gel electrophoresis detection; when an electrophoretic band appears, it indicates that the method described in claim 4 has successfully crudely extracted plant DNA; If no electrophoretic bands appear, it indicates that the method described in claim 4 has failed to successfully extract crude plant DNA.

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