Extraction method and inspection method of plant genome DNA (Deoxyribose Nucleic Acid)

Abstract

The invention provides an extraction method and a test method of plant genome DNA, and relates to the technical field of biotechnology, genome DNA can be efficiently extracted from various different types of plant samples through specific lysate components and concentration proportions and accurately calculated precipitant dosage, the extraction success rate and the DNA yield are improved, and the method is suitable for large-scale popularization and application. The optimized centrifugal condition in the extraction method can effectively separate the precipitated genome DNA, the purity and integrity of the DNA are ensured, a high-quality sample is provided for subsequent inspection and analysis, and for the inspection method of the plant genome DNA, the specificity and efficiency of PCR amplification are improved through the accurately calculated primer annealing temperature, the accuracy of the detection result is ensured, and the method is suitable for popularization and application. The PCR amplification product can be clearly separated by adopting 1.5% agarose gel electrophoresis concentration and specific electrophoresis conditions, so that the concentration and purity of DNA can be accurately analyzed and calculated.

Description

Technical Field

[0001] The present invention belongs to the field of biotechnology, and more specifically, relates to a method for extracting and testing plant genomic DNA. Background Art

[0002] With the rapid development of biotechnology, the research on plant genome is becoming more and more in-depth. The extraction and testing of plant genomic DNA is the basis and key step for plant molecular biology research, genetic breeding, gene function analysis and other work.

[0003] In past studies, there have been a variety of plant genomic DNA extraction methods, but these methods often have some limitations. For example, some methods are complicated to operate and require a lot of time and manpower; some methods extract DNA with low purity and contain more impurities, which affects subsequent experimental analysis; and some methods are not effective for certain types of plant samples, resulting in small amounts of DNA extraction or poor quality.

[0004] For the testing methods of plant genomic DNA, traditional technologies may have problems such as low accuracy, low specificity or complicated operation. During PCR amplification, inaccurate primer annealing temperature may lead to nonspecific amplification, affecting the reliability of the test results. Non-optimal conditions of agarose gel electrophoresis may lead to unclear bands, making it difficult to accurately judge the concentration and purity of DNA.

[0005] In addition, with the continuous deepening of research and the increase in application needs, the requirements for plant genomic DNA extraction and testing methods are becoming higher and higher. Not only do the methods need to be efficient, high-purity and high-yield, but they also need to be applicable to a variety of different types of plant samples and be able to be closely combined with modern biotechnology methods such as gene sequencing to provide more accurate and comprehensive information for plant gene function research and genetic improvement.

[0006] Therefore, developing a plant genomic DNA extraction and testing method that is simple to operate, efficient, accurate, and widely applicable has important scientific significance and application value. Summary of the invention

[0007] In order to solve the above technical problems, the present invention provides a method for extracting and testing plant genomic DNA to solve the above problems.

[0008] A method for extracting plant genomic DNA comprises the following steps:

[0009] Step A: Select fresh plant samples to be tested and pre-treat them to remove impurities and surface contaminants;

[0010] Step B: Lysing the plant sample to be tested with a lysis solution, wherein the components of the lysis solution include SDS, EDTA and Tris-HCl, and the concentration ratio thereof is determined according to the formula 0.5X+0.2Y+0.3Z=1, wherein X, Y, and Z are the concentrations of SDS, EDTA, and Tris-HCl, respectively (unit: mol / L);

[0011] Step C: adding a precipitant to precipitate the DNA in the lysed solution, wherein the precipitant is ethanol, and the amount of the precipitant is calculated according to the sample mass M (unit: g) according to the formula N=2.5M, where N is the volume of ethanol (unit: mL);

[0012] Step D: The precipitated mixture was centrifuged at a speed of 12,000 rpm for 15 minutes to obtain precipitated genomic DNA.

[0013] Preferably, in step A, the pretreatment includes washing, drying and shredding operations.

[0014] Preferably, in step C, the precipitant is ethanol or isopropanol.

[0015] Preferably, in step D, the supernatant is removed after centrifugation and the precipitate is retained.

[0016] A method for testing plant genomic DNA comprises the following steps:

[0017] Step E: Using the extracted plant genomic DNA as a template, using specific primers for PCR amplification, the annealing temperature of the primers T_{a} is calculated according to the formula T_{a}=64.9+41×(G+C) / (A+T+G+C)-820 / L, where G, C, A, T are the numbers of guanine, cytosine, adenine, and thymine in the primers, respectively, and L is the primer length (unit: bp);

[0018] Step F: performing agarose gel electrophoresis on the PCR amplification product, with a gel concentration of 1.5%;

[0019] Step G: Use image analysis software to analyze the electrophoresis results and calculate the concentration and purity of the DNA. The DNA concentration C_{DNA} is calculated according to the formula C_{DNA}=50×A_{260}×dilution factor, where A_{260} is the absorbance at 260 nm.

[0020] Preferably, in step E, the PCR reaction system includes a DNA template, primers, dNTPs (at a concentration of 2.5 mmol / L), Taq enzyme (5 U / μL) and a buffer.

[0021] Preferably, in step F, the electrophoresis voltage is 120 volts and the electrophoresis time is 30 minutes.

[0022] Preferably, in step G, the concentration of DNA is calculated using a standard curve based on the brightness and clarity of the DNA bands in the electrophoresis pattern.

[0023] Preferably, after step G is completed, the PCR amplification product is sequenced, and the sequencing method is Sanger sequencing.

[0024] Preferably, the sequencing results are compared with a database of known plant genome sequences to determine the species and genetic characteristics of the plant.

[0025] Compared with the prior art, the present invention has the following beneficial effects:

[0026] 1. The present invention provides a method for extracting plant genomic DNA. Through specific lysis solution components and concentration ratios, and accurately calculated precipitant dosages, genomic DNA can be efficiently extracted from a variety of different types of plant samples, thereby improving the success rate of extraction and the yield of DNA.

[0027] 2. The optimized centrifugation conditions in the extraction method can effectively separate the precipitated genomic DNA, ensure the purity and integrity of the DNA, and provide high-quality samples for subsequent testing and analysis.

[0028] 3. For the testing method of plant genomic DNA, the specificity and efficiency of PCR amplification are improved by accurately calculating the primer annealing temperature, ensuring the accuracy of the test results.

[0029] 4. Using 1.5% agarose gel electrophoresis concentration and specific electrophoresis conditions can clearly separate PCR amplification products, facilitating accurate analysis and calculation of DNA concentration and purity.

[0030] 5. The method of calculating DNA concentration using image analysis software combined with a standard curve improves the accuracy and repeatability of concentration determination.

[0031] 6. Sequencing the PCR amplification products and comparing them with the known plant genome sequence database can accurately determine the plant species and genetic characteristics, providing a reliable technical means for plant genetic research, variety identification and gene function analysis.

[0032] 7. The extraction and testing methods of the present invention are relatively simple to operate, and the reagents and equipment are common, and are easy to promote and apply in laboratories.

[0033] 8. This method is applicable to a variety of plant samples, including vegetables, fruits, flowers and crops, which broadens its application scope.

[0034] 9. After repeatability verification, the extraction and inspection methods of the present invention have good repeatability and stability, and can provide reliable and consistent results for different studies and applications. BRIEF DESCRIPTION OF THE DRAWINGS

[0035] Figure 1 It is a schematic flow diagram of the extraction method of the present invention;

[0036] Figure 2 It is a schematic flow chart of the inspection method in the present invention. DETAILED DESCRIPTION

[0037] The following embodiments of the present invention are described in further detail in conjunction with the accompanying drawings and examples. The following examples are used to illustrate the present invention, but are not intended to limit the scope of the present invention.

[0038] The extraction and testing of plant genomic DNA is of great significance to the fields of plant genetic research, variety identification, gene function analysis, etc. This specific implementation method aims to elaborate a method for extracting and testing plant genomic DNA, and provide accurate and reliable technical support for related research and applications.

[0039] Experimental materials: Select a variety of different types of fresh plant samples, including but not limited to:

[0040] Vegetables: spinach, celery, cucumber;

[0041] Fruits: strawberries, oranges, bananas;

[0042] Flowers: roses, chrysanthemums, tulips;

[0043] Crops: wheat, corn, rice;

[0044] Woody plants: fragrant fruit tree, tulip tree, Cyclobalanopsis microphylla;

[0045] Multiple individuals were selected for each plant sample to ensure the representativeness and repeatability of the experimental results.

[0046] Experimental reagents:

[0047] Lysis solution: comprising sodium dodecyl sulfate (SDS), ethylenediaminetetraacetic acid (EDTA) and tris(hydroxymethyl)aminomethane hydrochloride (Tris-HCl), prepared according to a concentration ratio of 0.5X+0.2Y+0.3Z=1, wherein X, Y, and Z are the concentrations of SDS, EDTA, and Tris-HCl, respectively (unit: mol / L);

[0048] Precipitant: ethanol, isopropanol;

[0049] PCR amplification reagents: specific primers, dNTPs (concentration 2.5 mmol / L), Taq enzyme (5 U / μL), buffer;

[0050] Agarose gel electrophoresis reagents: agarose, electrophoresis buffer.

[0051] Experimental equipment:

[0052] High-speed centrifuge: The speed can reach more than 12,000 rpm;

[0053] Pipette: different ranges, accuracy 0.1-1000μL;

[0054] PCR instrument: can accurately control temperature and time;

[0055] Agarose gel electrophoresis apparatus: equipped with a stable power supply and electrophoresis tank;

[0056] UV spectrophotometer: used to measure the absorbance of DNA;

[0057] Image analysis software: such as ImageJ, etc.

[0058] Plant genomic DNA extraction method:

[0059] Example 1: Extraction of spinach genomic DNA:

[0060] Sample pretreatment: Select fresh spinach leaves, remove the yellowed and damaged parts, wash the impurities and dust on the surface of the leaves with distilled water, absorb the moisture on the surface of the leaves with filter paper, and cut the leaves into small pieces of about 0.5cm×0.5cm;

[0061] Sample lysis: weigh 1g of chopped spinach leaves, put them into a pre-cooled mortar, add liquid nitrogen and quickly grind them into powder, transfer the powder to a 15mL centrifuge tube, add 5mL of lysis buffer (SDS concentration is 0.5mol / L, EDTA concentration is 0.2mol / L, Tris-HCl concentration is 0.3mol / L), mix well, and place in a 65℃ water bath for 30 minutes. During this period, gently invert the centrifuge tube every 5 minutes to mix well;

[0062] DNA precipitation: Take out the centrifuge tube, cool it to room temperature, add 2.5mL ethanol (precipitant), and gently invert the centrifuge tube 20 times to fully precipitate the DNA;

[0063] Centrifugation: Place the centrifuge tube in a high-speed centrifuge and centrifuge at 12,000 rpm for 15 minutes. After centrifugation, carefully remove the supernatant and retain the precipitate.

[0064] Example 2: Extraction of Strawberry Genomic DNA:

[0065] Sample pretreatment: Select fresh and ripe strawberry fruits, remove the stems and rotten parts, rinse the fruit surface with physiological saline to remove impurities, use sterile filter paper to absorb the moisture on the fruit surface, cut the fruit into small pieces, and homogenize with a homogenizer;

[0066] Sample lysis: Weigh 2 g of homogenized strawberry tissue, put it into a 50 mL centrifuge tube, add 10 mL of lysis buffer (SDS concentration is 0.5 mol / L, EDTA concentration is 0.2 mol / L, Tris-HCl concentration is 0.3 mol / L), mix well, and place in a 55°C water bath for 45 minutes. During this period, gently invert the centrifuge tube every 10 minutes to mix well.

[0067] DNA precipitation: Take out the centrifuge tube, cool it to room temperature, add 5 mL of isopropanol (precipitant), and gently invert the centrifuge tube 30 times to fully precipitate the DNA;

[0068] Centrifugation: Place the centrifuge tube in a high-speed centrifuge and centrifuge at 12,000 rpm for 15 minutes. After centrifugation, carefully remove the supernatant and retain the precipitate.

[0069] Example 3: Extraction of Rose Genomic DNA:

[0070] Sample pretreatment: Select fresh rose petals, remove the stamens and receptacles, wash the dust on the surface of the petals with distilled water, and cut the petals into small pieces of about 0.3 cm × 0.3 cm with scissors;

[0071] Sample lysis: weigh 0.5 g of chopped rose petals, put them into a 10 mL centrifuge tube, add 2.5 mL of lysis buffer (SDS concentration is 0.5 mol / L, EDTA concentration is 0.2 mol / L, Tris-HCl concentration is 0.3 mol / L), mix thoroughly, and place in a 60°C water bath for 35 minutes. During this period, gently invert the centrifuge tube every 8 minutes to mix;

[0072] DNA precipitation: Take out the centrifuge tube, cool it to room temperature, add 1.25mL of ethanol (precipitant), and gently invert the centrifuge tube 25 times to fully precipitate the DNA;

[0073] Centrifugation: Place the centrifuge tube in a high-speed centrifuge and centrifuge at 12,000 rpm for 15 minutes. After centrifugation, carefully remove the supernatant and retain the precipitate.

[0074] Example 4: Extraction of wheat genomic DNA:

[0075] Sample pretreatment: Select fresh wheat leaves, remove the leaf tips and leaf sheaths, wash the dirt on the leaf surface with detergent water, rinse with distilled water, absorb the moisture on the leaf surface with filter paper, and cut the leaves into small pieces about 1 cm long;

[0076] Sample lysis: Weigh 1.5 g of cut wheat leaves, put them into a 25 mL centrifuge tube, add 7.5 mL of lysis buffer (SDS concentration is 0.5 mol / L, EDTA concentration is 0.2 mol / L, Tris-HCl concentration is 0.3 mol / L), mix thoroughly, and place in a 50°C water bath for 50 minutes. During this period, gently invert the centrifuge tube every 12 minutes to mix;

[0077] DNA precipitation: Take out the centrifuge tube, cool it to room temperature, add 3.75mL ethanol (precipitant), and gently invert the centrifuge tube 28 times to fully precipitate the DNA;

[0078] Centrifugation: Place the centrifuge tube in a high-speed centrifuge and centrifuge at 12,000 rpm for 15 minutes. After centrifugation, carefully remove the supernatant and retain the precipitate.

[0079] Example 5: Extraction of Genomic DNA from the Fruit Tree

[0080] Sample pretreatment: Select fresh leaves of the fragrant fruit tree, remove dust and impurities on the leaves, rinse them with distilled water, then use filter paper to absorb the surface moisture, and cut the leaves into small pieces of about 0.5cm×0.5cm.

[0081] Sample lysis: Weigh 1 g of chopped leaves of Citrus aurantium, put them into a 15 mL centrifuge tube, add 5 mL of lysis solution (SDS concentration is 0.5 mol / L, EDTA concentration is 0.2 mol / L, Tris-HCl concentration is 0.3 mol / L), mix thoroughly, and place in a 60°C water bath for 40 minutes. During this period, gently invert the centrifuge tube every 8 minutes to mix.

[0082] DNA precipitation: Take out the centrifuge tube, cool it to room temperature, add 2.5mL of isopropanol (precipitant), and gently invert the centrifuge tube 25 times to fully precipitate the DNA.

[0083] Centrifugation: Place the centrifuge tube in a high-speed centrifuge and centrifuge at 12,000 rpm for 12 minutes. After centrifugation, carefully remove the supernatant and retain the precipitate.

[0084] Comparison and analysis of embodiments:

[0085]

[0086]

[0087] By comparing and analyzing the extraction results of the above-mentioned different plant samples, it can be found that there are certain differences in the DNA yield, purity and integrity of different plant samples. The DNA yield of spinach samples is relatively high, the purity is good, and the integrity is good; the DNA yield of strawberry samples is moderate, and the purity and integrity are also relatively ideal; the DNA yield of rose samples is low, and the purity and integrity are relatively poor; the various indicators of wheat samples are at a medium level, and the DNA yield of fragrant fruit tree samples is high, and the purity and integrity are also good. These differences may be related to factors such as the tissue structure, cell composition, and operational details of the plant samples during the extraction process.

[0088] Plant genomic DNA testing methods:

[0089] Example 1: Testing of Spinach Genomic DNA:

[0090] PCR amplification: Based on the known spinach gene sequence, specific primers were designed. The annealing temperature Ta of the primers was calculated according to the formula Ta = 64.9 + 41 × (G + C) / (A + T + G + C) - 820 / L (where G, C, A, T are the number of guanine, cytosine, adenine, and thymine in the primers, respectively, and L is the primer length). Assuming that the primer length is 20 bp, and the number of G, C, A, and T is 8, 7, 5, and 0 respectively, the annealing temperature is calculated as: Ta = 64.9 + 41 × (8 + 7) / (8 + 7 + 5 + 0) - 820 / 20 ≈ 58.5 ° C, configure the PCR reaction system: 20 μL system contains 2 μL DNA template (concentration of about 50 ng / μL), 1 μL of upstream and downstream primers (concentration of 10 μmol / L), 2 μL dNTPs (concentration of 2.5 mmol / L), 0.2 μL Taq enzyme (5U / μL), 2 μL buffer, put the reaction system into the PCR instrument, and set the reaction program: 95 ° C pre-denaturation for 5 minutes; 95 ° C denaturation for 30 seconds, 58.5 ° C annealing for 30 seconds, 72 ° C extension for 30 seconds, a total of 35 cycles; 72 ° C extension for 10 minutes;

[0091] Agarose gel electrophoresis: weigh 1.5 g agarose, add 100 mL electrophoresis buffer, heat until completely dissolved, cool to about 60 ° C, pour into the gel mold, insert the comb, and remove the comb after the gel solidifies. Mix 5 μL of PCR amplification product with 1 μL of loading buffer and add it to the gel well. At the same time, add a DNA molecular weight standard as a control, connect the electrophoresis instrument, set the electrophoresis voltage to 120 volts, and the electrophoresis time to 30 minutes;

[0092] Concentration and purity analysis: After the electrophoresis is completed, the gel is placed in a UV gel imaging system for observation and photography. The electrophoresis pattern is analyzed using image analysis software. The concentration of DNA is calculated using a standard curve based on the brightness and clarity of the DNA bands. Assuming that the standard curve equation is y=0.5x+0.1 (where y is the DNA concentration and x is the grayscale value of the band), the grayscale value of the measured target band is 500, and the DNA concentration is approximately 250ng / μL. At the same time, the absorbance at 260nm and 280nm is measured, and the A260 / A280 ratio is calculated to determine the DNA purity. If the A260 / A280 ratio is between 1.8-2.0, it indicates that the DNA purity is high.

[0093] Sequencing and alignment: The PCR amplification products were subjected to Sanger sequencing, and the sequencing results were compared with the known spinach genome sequence database to determine the gene characteristics and mutation sites.

[0094] Example 2: Testing of Strawberry Genomic DNA:

[0095] PCR amplification: Design primers for specific genes in strawberry, and determine the annealing temperature according to the above annealing temperature calculation formula. Assuming that the primer length is 22bp, and the number of G, C, A, and T is 9, 8, 5, and 0 respectively, the annealing temperature is calculated as: Ta = 64.9 + 41 × (9 + 8) / (9 + 8 + 5 + 0) - 820 / 22 ≈ 60.2 ° C, configure the PCR reaction system, and the reaction procedure is the same as the test of spinach genomic DNA;

[0096] Agarose gel electrophoresis: The preparation and electrophoresis of agarose gel are the same as those for the test of spinach genomic DNA;

[0097] Concentration and purity analysis: Image analysis and concentration and purity calculation methods are the same as those for spinach genomic DNA testing;

[0098] Sequencing and alignment: The sequencing and alignment steps were the same as those for the spinach genomic DNA.

[0099] Example 3: Testing of Rose Genomic DNA:

[0100] PCR amplification: Design the primers required for the rose genomic DNA test and calculate the annealing temperature. Assuming the primer length is 18bp, and the number of G, C, A, and T is 7, 6, 5, and 0 respectively, the annealing temperature is calculated as: Ta = 64.9 + 41 × (7 + 6) / (7 + 6 + 5 + 0) - 820 / 18 ≈ 56.8 ° C, configure the PCR reaction system, and the reaction procedure is the same as the spinach genomic DNA test;

[0101] Agarose gel electrophoresis: The preparation and electrophoresis of agarose gel are the same as those for the test of spinach genomic DNA;

[0102] Concentration and purity analysis: Image analysis and concentration and purity calculation methods are the same as those for spinach genomic DNA testing;

[0103] Sequencing and alignment: The sequencing and alignment steps were the same as those for the spinach genomic DNA.

[0104] Example 4: Testing of wheat genomic DNA:

[0105] PCR amplification: Design primers for wheat genomic DNA testing and calculate the annealing temperature. Assuming the primer length is 25 bp, and the number of G, C, A, and T is 10, 9, 6, and 0, respectively, the annealing temperature is calculated as: Ta = 64.9 + 41 × (10 + 9) / (10 + 9 + 6 + 0) - 820 / 25 ≈ 62.5 ° C, configure the PCR reaction system, and the reaction procedure is the same as the spinach genomic DNA test;

[0106] Agarose gel electrophoresis: The preparation and electrophoresis of agarose gel are the same as those for the test of spinach genomic DNA;

[0107] Concentration and purity analysis: Image analysis and concentration and purity calculation methods are the same as those for spinach genomic DNA testing;

[0108] Sequencing and alignment: The sequencing and alignment steps were the same as those for the spinach genomic DNA.

[0109] Example 5: Testing of the genome DNA of the fruit tree:

[0110] PCR amplification: Design primers for the specific gene of the fragrant fruit tree, and determine the annealing temperature according to the above annealing temperature calculation formula. Assuming that the primer length is 20bp, the number of G, C, A, and T is 8, 7, 5, and 0 respectively, the annealing temperature is calculated as: Ta = 64.9 + 41 × (8 + 7) / (8 + 7 + 5 + 0) - 820 / 20 ≈ 58.5 ° C, configure the PCR reaction system, and the reaction procedure is the same as the test of spinach genomic DNA.

[0111] Agarose gel electrophoresis: The preparation of agarose gel and electrophoresis were the same as those for the spinach genomic DNA test.

[0112] Concentration and purity analysis: Image analysis and concentration and purity calculation methods are the same as those for the test of spinach genomic DNA.

[0113] Sequencing and alignment: The sequencing and alignment steps were the same as those for the spinach genomic DNA.

[0114] Comparison and analysis of embodiments:

[0115]

[0116] By comparing and analyzing the test results of different plant samples, it can be seen that the test results of spinach and wheat are better, with obvious PCR amplification, clear electrophoresis bands, high DNA concentration and purity, and a high degree of match between the gene feature comparison results and the known sequence; the test results of strawberry are acceptable, with some differences; the test results of rose are relatively poor, with weak PCR amplification, fuzzy electrophoresis bands, and many differences in gene feature comparison results; the amplification product of the fragrant fruit tree is obvious, the bands are bright, the bands are clear, there is no obvious tailing and diffusion, and the match with the known fragrant fruit tree genome sequence database is high, with only a few differences in sites. These differences may be related to factors such as the genetic characteristics of plant samples, the specificity of primer design, and the accuracy of experimental operations.

[0117] In order to further verify the reliability and accuracy of this plant genomic DNA extraction and testing method, the following verification experiments were carried out:

[0118] Repeatability experiment:

[0119] Five plant samples of spinach, strawberry, rose and wheat were selected and operated according to the above extraction and inspection methods. The DNA yield, purity, PCR amplification effect, electrophoresis band clarity and other indicators of each experiment were recorded, and the mean, standard deviation and coefficient of variation of each indicator were calculated to evaluate the repeatability of the experimental method:

[0120]

[0121] The invention provides a method for extracting plant genomic DNA. The method can efficiently extract genomic DNA from various types of plant samples through specific lysate components and concentration ratios and accurately calculated precipitant dosages, thereby improving the success rate of extraction and the yield of DNA. The optimized centrifugal conditions in the extraction method can effectively separate precipitated genomic DNA, thereby ensuring the purity and integrity of the DNA and providing high-quality samples for subsequent inspection and analysis. For the inspection method of plant genomic DNA, the specificity and efficiency of PCR amplification are improved through accurately calculated primer annealing temperature, thereby ensuring the accuracy of the detection result. The method adopts 1.5% agarose gel electrophoresis concentration and specific electrophoresis conditions, thereby clearly separating PCR amplification products, facilitating accurate analysis and calculation of DNA concentration and purity. The method for calculating DNA concentration using image analysis software in combination with a standard curve improves the accuracy and repeatability of concentration determination. The PCR amplification products are sequenced and compared with a known plant genome sequence database, thereby accurately determining the type and gene characteristics of the plant and providing reliable technical means for plant genetic research, variety identification, gene function analysis, and the like.

[0122] The extraction and inspection method of the present invention is relatively simple to operate, the reagents and equipment are common, and it is easy to promote and apply in the laboratory. The method has good applicability to a variety of plant samples, including vegetables, fruits, flowers and crops, etc., which broadens its application scope. After repeatability verification, the extraction and inspection method of the present invention has good repeatability and stability, and can provide reliable and consistent results for different research and applications.

[0123] The embodiments of the present invention are given for the purpose of illustration and description, and are not intended to be exhaustive or to limit the invention to the disclosed forms. Many modifications and variations will be apparent to those of ordinary skill in the art. The embodiments are selected and described in order to better illustrate the principles and practical applications of the present invention and to enable those of ordinary skill in the art to understand the present invention and thereby design various embodiments with various modifications suitable for specific uses.

Claims

1. A method for extracting plant genomic DNA, characterized in that: The following steps are involved: Step A: Select fresh plant samples to be tested and pre-treat them to remove impurities and surface contaminants; Step B: Lysing the plant sample to be tested using a lysis solution, wherein the components of the lysis solution include SDS, EDTA and Tris-HCl, and the concentration ratio thereof is determined according to the formula 0.5X+0.2Y+0.3Z=1, wherein X, Y, and Z are the concentrations of SDS, EDTA, and Tris-HCl, respectively; Step C: adding a precipitant to precipitate the DNA in the lysed solution, wherein the precipitant is ethanol, and the amount of the precipitant is calculated according to the sample mass M according to the formula N=2.5M, where N is the volume of ethanol; Step D: The precipitated mixture was centrifuged at a speed of 12,000 rpm for 15 minutes to obtain precipitated genomic DNA.

2. The method for extracting plant genomic DNA according to claim 1, characterized in that: In step A, pretreatment includes washing, drying and shredding operations.

3. The method for extracting plant genomic DNA according to claim 1, characterized in that: In step C, the precipitating agent is ethanol or isopropanol.

4. The method for extracting plant genomic DNA according to claim 1, characterized in that: In step D, after centrifugation, remove the supernatant and retain the pellet.

5. A method for testing plant genomic DNA, characterized in that: The following steps are involved: Step E: Using the extracted plant genomic DNA as a template, PCR amplification is performed using specific primers, and the annealing temperature of the primers T_{a} is calculated according to the formula T_{a}=64.9+41×(G+C) / (A+T+G+C)-820 / L, where G, C, A, and T are the numbers of guanine, cytosine, adenine, and thymine in the primers, respectively, and L is the primer length; Step F: performing agarose gel electrophoresis on the PCR amplification product, with a gel concentration of 1.5%; Step G: Use image analysis software to analyze the electrophoresis results and calculate the concentration and purity of the DNA. The DNA concentration C_{DNA} is calculated according to the formula C_{DNA}=50×A_{260}×dilution factor, where A_{260} is the absorbance at 260 nm.

6. The method for testing plant genomic DNA according to claim 5, characterized in that: In step E, the PCR reaction system includes a DNA template, primers, dNTPs, Taq enzyme and buffer.

7. The method for testing plant genomic DNA according to claim 5, characterized in that: In step F, the electrophoresis voltage was 120 volts and the electrophoresis time was 30 minutes.

8. The method for testing plant genomic DNA according to claim 5, characterized in that: In step G, the concentration of DNA is calculated using a standard curve based on the brightness and clarity of the DNA bands in the electropherogram.

9. The method for testing plant genomic DNA according to claim 5, characterized in that: After step G is completed, the PCR amplification product is sequenced using the Sanger sequencing method.

10. The method for testing plant genomic DNA according to claim 9, characterized in that: The sequencing results are compared with the known plant genome sequence database to determine the plant species and genetic characteristics.

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