Lysate for extracting plant DNA containing polysaccharide and polyphenol and kit

Abstract

The invention discloses a lysate for extracting plant DNA (deoxyribonucleic acid) containing polysaccharide and polyphenol. The lysate comprises the following components in percentage by weight: 50 to 500 mmol / L of buffer solution, 1 to 2 mol / L of chloride, 2 to 5 percent of surfactant, 10 to 50 mmol / L of metal ion complex and 10 to 100 mmol / L of antioxidant. The invention provides a method for rapidly, simply and conveniently extracting high-quality and high-yield DNA from various plant materials by utilizing the specific adsorption effect of a purification column on DNA. The whole operation process of the method is only 30-40 min, the process is simple, convenient and rapid, and a reference mode is provided for workers in the field of related plant DNA.

Description

Technical Field

[0001] The invention relates to the technical field of molecular biology, and in particular to a lysate and a kit for extracting plant DNA containing polysaccharides and polyphenols. Background Art

[0002] DNA is generally considered to be the main genetic material in organisms. Extracting nucleic acids from plant tissues is a necessary prerequisite for conducting research in plant molecular biology. Extracting nucleic acids with high purity and good integrity from plant tissues is the key to many studies. Traditional methods for isolating nucleic acids mainly include boiling, repeated freezing and thawing, chemical methods, and extraction methods. The most common extraction steps are lysis, extraction, centrifugation, precipitation, and washing. Although most samples can obtain DNA with relatively good quality and yield using traditional techniques, RNA enzymes are required, which is time-consuming, labor-intensive, and costly. In addition, for plant tissues rich in polysaccharides and polyphenols, it is difficult to effectively separate DNA from viscous polysaccharide solutions, resulting in DNA loss and affecting downstream experiments. Therefore, how to quickly and efficiently obtain high-quality DNA from plants is still a difficult problem. Summary of the invention

[0003] The object of the present invention is to overcome at least one disadvantage of the prior art and to provide a lysis solution and a kit for extracting plant DNA containing polysaccharides and polyphenols.

[0004] The technical solution adopted by the present invention is: In a first aspect, the present invention provides a lysis solution for extracting DNA from plants containing polysaccharides and polyphenols, wherein the lysis solution contains the following components: a buffer, lithium chloride, a surfactant, a metal ion complex and an antioxidant.

[0005] In some examples, the contents of the components of the lysate are: 50-500 mmol / L buffer, 1-2 mol / L chloride, 2%-5% surfactant, 10-50 mmol / L metal ion complex, and 10-100 mmol / L antioxidant.

[0006] In a second aspect, the present invention provides a rapid DNA extraction kit for extracting plant DNA containing polysaccharides and polyphenols, characterized in that it comprises the lysis solution, column membrane adsorption solution, rinsing solution 1, and rinsing solution 2 described in the first aspect.

[0007] In some examples, the column membrane adsorption solution is composed of: 2-5 mol / L chaotropic salt and 10-50% volume fraction of alcohol solution.

[0008] In some examples, the chaotropic salt is selected from guanidine hydrochloride or guanidine isothiocyanate.

[0009] In some examples, the alcohol solution is an isopropanol solution or an ethanol solution.

[0010] In some examples, the rinse solution 1 is composed of: 10-50 mmol / L buffer, 1-5 mol / L chaotropic salt, and 40-60% volume fraction of ethanol solution.

[0011] In some examples, the rinse solution 2 is composed of: 50-90% by volume ethanol solution.

[0012] In some examples, the surfactant is cetyltrimethylammonium bromide, the metal ion complex is EDTA, and the antioxidant is sodium thiosulfate.

[0013] In a third aspect, the present invention provides a method for extracting plant DNA containing polysaccharides and polyphenols, using the DNA rapid extraction kit provided in the second aspect for extraction, comprising the following steps: 1) Add lysis buffer to the sample to be extracted and mix well. After thermal lysis, extract with chloroform and collect the supernatant. 2) mixing the supernatant with the column membrane adsorption solution and applying the mixture to the column for purification; 3) Wash with rinse solution 1 and rinse solution 2 for 1 to 2 times respectively, and discard the waste liquid; 4) Add sterile water or elution buffer and let stand for 3 to 5 minutes to obtain DNA solution.

[0014] The beneficial effects of the present invention are: The present invention utilizes the specific adsorption of DNA by the purification column to provide a method for extracting high-quality and high-yield DNA from various plant materials in a quick and easy manner. The entire operation process of the method only takes 30 to 40 minutes, and the process is simple and rapid, providing a reference method for workers in the field of plant DNA. BRIEF DESCRIPTION OF THE DRAWINGS

[0015] Figure 1 The figure is a diagram of extraction results of different plant leaf samples, in which: lane M represents marker; lanes 1-4 represent the extraction results of Examples 1-4.

[0016] Figure 2 This is a diagram of the extraction results of the optimized process using green radish leaves as a reference, in which: lane M represents a marker; lanes 1-8 represent the extraction results of comparative examples 1-8; and lane 9 represents the extraction results of Example 1 of the present invention. DETAILED DESCRIPTION

[0017] The following will be combined with the embodiments of the present invention to clearly and completely describe the technical solutions in the embodiments of the present invention. Obviously, the described embodiments are only part of the embodiments of the present invention, not all of the embodiments. In the examples of the present invention, if there is no additional description, all solutions involving percentage content are volume concentrations.

[0018] Example 1 The specific extraction steps of this embodiment are as follows: 1) Grind 100 mg of freshly picked green radish leaves in liquid nitrogen, add 700 μL of lysis buffer, shake, and incubate at 65°C for 10 min; 2) Add 700 μL of chloroform, vortex for 10 s, centrifuge, and collect the supernatant; 3) Add 700 μL of column membrane adsorption solution to the above supernatant, load onto the column, centrifuge, discard the waste liquid, load the remaining mixed solution onto the column, centrifuge, and discard the waste liquid; 4) Add 500 μL of rinse solution 1, centrifuge, and discard the waste liquid; 5) Add 650 μL of rinse solution 2, centrifuge, and discard the waste liquid; 6) Add 650 μL of rinse solution 2, centrifuge, and discard the waste liquid; 7) Dry at room temperature for 5 min, add 100 μL of sterile water or elution buffer preheated to 65°C, let stand for 5 min, and centrifuge. The resulting liquid is a high-concentration, high-purity DNA solution.

[0019] The composition of the lysis buffer was 100 mmol / L Tris-HCl (pH=8.0), 20 mmol / L EDTA, 1.4 mol / L lithium chloride, and 50 mmol / L sodium thiosulfate.

[0020] The column membrane adsorption liquid consists of 4 mol / L guanidine hydrochloride and 35% isopropanol.

[0021] The composition of the rinse solution 1 is 10 mmol / L Tris-HCl, 1 mol / L guanidine hydrochloride, and 60% anhydrous ethanol.

[0022] The composition of rinse solution 2 is 80% anhydrous ethanol.

[0023] Example 2 The specific extraction steps of this embodiment are as follows: 1) Grind 100 mg of freshly picked Ageratum cochinchinensis in liquid nitrogen, add 700 μL of lysis buffer, shake, and incubate at 65°C for 10 min; 2) Add 700 μL of chloroform, vortex for 10 s, centrifuge, and collect the supernatant; 3) Add 700 μL of column membrane adsorption solution to the above supernatant, load onto the column, centrifuge, discard the waste liquid, load the remaining mixed solution onto the column, centrifuge, and discard the waste liquid; 4) Add 500 μL of rinse solution 1, centrifuge, and discard the waste liquid; 5) Add 650 μL of rinse solution 2, centrifuge, and discard the waste liquid; 6) Add 650 μL of rinse solution 2, centrifuge, and discard the waste liquid; 7) Dry at room temperature for 5 min, add 100 μL of sterile water or elution buffer preheated to 65°C, let stand for 5 min, and centrifuge. The resulting liquid is a high-concentration, high-purity DNA solution.

[0024] The composition of the lysis buffer was 100 mmol / L Tris-HCl (pH=8.0), 20 mmol / L EDTA, 1.4 mol / L lithium chloride, and 50 mmol / L sodium thiosulfate.

[0025] The column membrane adsorption liquid consists of 4 mol / L guanidine hydrochloride and 35% isopropanol.

[0026] The composition of the rinse solution 1 is 10 mmol / L Tris-HCl, 1 mol / L guanidine hydrochloride, and 60% anhydrous ethanol.

[0027] The composition of rinse solution 2 is 80% anhydrous ethanol.

[0028] Example 3 The specific extraction steps of this embodiment are as follows: 1) Grind 100 mg of freshly picked Sonchus endive leaves in liquid nitrogen, add 700 μL of lysis buffer, shake, and incubate at 65°C for 10 min; 2) Add 700 μL of chloroform, vortex for 10 s, centrifuge, and collect the supernatant; 3) Add 700 μL of column membrane adsorption solution to the above supernatant, load onto the column, centrifuge, discard the waste liquid, load the remaining mixed solution onto the column, centrifuge, and discard the waste liquid; 4) Add 500 μL of rinse solution 1, centrifuge, and discard the waste liquid; 5) Add 650 μL of rinse solution 2, centrifuge, and discard the waste liquid; 6) Add 650 μL of rinse solution 2, centrifuge, and discard the waste liquid; 7) Dry at room temperature for 5 min, add 100 μL of sterile water or elution buffer preheated to 65°C, let stand for 5 min, and centrifuge. The resulting liquid is a high-concentration, high-purity DNA solution.

[0029] The composition of the lysis buffer was 100 mmol / L Tris-HCl (pH=8.0), 20 mmol / L EDTA, 1.4 mol / L lithium chloride, and 50 mmol / L sodium thiosulfate.

[0030] The column membrane adsorption liquid consists of 4 mol / L guanidine hydrochloride and 35% isopropanol.

[0031] The composition of the rinse solution 1 is 10 mmol / L Tris-HCl, 1 mol / L guanidine hydrochloride, and 60% anhydrous ethanol.

[0032] The composition of rinse solution 2 is 80% anhydrous ethanol.

[0033] Example 4 The specific extraction steps of this embodiment are as follows: 1) Grind 100 mg of freshly picked Bidens pilosa in liquid nitrogen, add 700 μL of lysis buffer, shake, and incubate at 65°C for 10 min; 2) Add 700 μL of chloroform, vortex for 10 s, centrifuge, and collect the supernatant; 3) Add 700 μL of column membrane adsorption solution to the above supernatant, load onto the column, centrifuge, discard the waste liquid, load the remaining mixed solution onto the column, centrifuge, and discard the waste liquid; 4) Add 500 μL of rinse solution 1, centrifuge, and discard the waste liquid; 5) Add 650 μL of rinse solution 2, centrifuge, and discard the waste liquid; 6) Add 650 μL of rinse solution 2, centrifuge, and discard the waste liquid; 7) Dry at room temperature for 5 min, add 100 μL of sterile water or elution buffer preheated to 65°C, let stand for 5 min, and centrifuge. The resulting liquid is a high-concentration, high-purity DNA solution.

[0034] The composition of the lysis buffer was 100 mmol / L Tris-HCl (pH=8.0), 20 mmol / L EDTA, 1.4 mol / L lithium chloride, and 50 mmol / L sodium thiosulfate.

[0035] The column membrane adsorption liquid consists of 4 mol / L guanidine hydrochloride and 35% isopropanol.

[0036] The composition of the rinse solution 1 is 10 mmol / L Tris-HCl, 1 mol / L guanidine hydrochloride, and 60% anhydrous ethanol.

[0037] The composition of rinse solution 2 is 80% anhydrous ethanol.

[0038] Comparative Example 1 The only difference from Example 1 is that the composition of the lysate is 100 mmol / L Tris-HCl (pH=7.4), 20 mmol / L EDTA, 1.4 mol / L lithium chloride, and 50 mmol / L sodium thiosulfate.

[0039] Comparative Example 2 The only difference from Example 1 is that the lysis solution consists of 200 mmol / L Tris-HCl (pH=8.0), 20 mmol / L EDTA, 1.4 mol / L lithium chloride, and 50 mmol / L sodium thiosulfate.

[0040] Comparative Example 3 The only difference from Example 1 is that the lysis solution consists of 200 mmol / L Tris-HCl (pH=8.0), 25 mmol / L EDTA, 1.4 mol / L lithium chloride, and 50 mmol / L sodium thiosulfate.

[0041] Comparative Example 4 The only difference from Example 1 is that the lysis solution consists of 100 mmol / L Tris-HCl (pH=8.0), 25 mmol / L EDTA, 1.4 mol / L lithium chloride, and 0.5% PVPP.

[0042] Comparative Example 5 The only difference from Example 1 is that the lysis solution consists of 100 mmol / L Tris-HCl (pH=8.0), 25 mmol / L EDTA, 1.4 mol / L sodium chloride, and 50 mmol / L sodium thiosulfate.

[0043] Comparative Example 6 The only difference from Example 1 is that the incubation time of the lysate is shortened to 5 min.

[0044] Comparative Example 7 The only difference from Example 1 is that the composition of the column membrane adsorption liquid is 225 mmol / L sodium acetate-acetic acid buffer (pH=5.2), 2 mol / L guanidine isothiocyanate, and 3 mol / L guanidine hydrochloride.

[0045] Comparative Example 8 The only difference from Example 1 is that the composition of the column membrane adsorption liquid is 35% isopropanol.

[0046] like Figure 1 and Figure 2 As shown, the concentration and purity of the DNA extracted from Examples 1 to 4 and Comparative Examples 1 to 5 were measured using a nucleic acid quantifier, and the measurement results are recorded in Table 1: Table 1

[0047] Note: OD260 / OD280 is an important parameter for evaluating the purity of DNA samples. The ratio is usually between 1.7 and 1.9. A ratio below 1.7 indicates protein contamination, and a ratio above 1.9 indicates RNA contamination. OD260 / OD230 is another important parameter for evaluating the purity of DNA samples. The ratio is usually greater than 2.0. If it is lower than 2.0, there may be contamination by impurities such as ethanol, carbohydrates, peptides, and phenol.

[0048] The above is a further detailed description of the present invention, which should not be regarded as a limitation on the specific implementation of the present invention. For ordinary technicians in the technical field to which the present invention belongs, simple deduction or replacement without departing from the concept of the present invention is within the protection scope of the present invention.

Claims

1. A lysate for extracting plant DNA containing polysaccharides and polyphenols, characterized in that: The lysate contains the following components: buffer, lithium chloride, surfactant, metal ion complex and antioxidant.

2. The lysate according to claim 1, characterized in that The contents of the components of the lysate are as follows: 50-500 mmol / L buffer, 1-2 mol / L chloride, 2%-5% surfactant, 10-50 mmol / L metal ion complex, and 10-100 mmol / L antioxidant.

3. A rapid DNA extraction kit for extracting plant DNA containing polysaccharides and polyphenols, characterized in that: It comprises the lysis solution, column membrane adsorption solution, rinsing solution 1 and rinsing solution 2 as described in claim 1.

4. The rapid DNA extraction kit according to claim 3, characterized in that: The column membrane adsorption liquid is composed of: 2-5 mol / L chaotropic salt and 10-50% volume fraction of alcohol solution.

5. The rapid DNA extraction kit according to claim 4, characterized in that: The chaotropic salt is selected from guanidine hydrochloride or guanidine isothiocyanate.

6. The rapid DNA extraction kit according to claim 4, characterized in that: The alcohol solution is an isopropanol solution or an ethanol solution.

7. The rapid DNA extraction kit according to claim 3, characterized in that: The rinse solution 1 is composed of: 10-50 mmol / L buffer, 1-5 mol / L chaotropic salt, and 40-60% volume fraction of ethanol solution.

8. The rapid DNA extraction kit according to claim 3, characterized in that: The rinse solution 2 is composed of: 50-90% by volume of an ethanol solution.

9. The lysate according to claim 1, characterized in that The surfactant is hexadecyltrimethylammonium bromide, the metal ion complex is EDTA, and the antioxidant is sodium thiosulfate.

10. A method for extracting plant DNA containing polysaccharides and polyphenols, characterized in that: The rapid DNA extraction kit according to claim 3 is used for extraction, comprising the following steps: 1) Add lysis buffer to the sample to be extracted and mix well. After thermal lysis, extract with chloroform and collect the supernatant. 2) mixing the supernatant with the column membrane adsorption solution and applying the mixture to the column for purification; 3) Wash with rinse solution 1 and rinse solution 2 for 1 to 2 times respectively, and discard the waste liquid; 4) Add sterile water or elution buffer and let stand for 3 to 5 minutes to obtain DNA solution.

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