Kit and method for extracting fruit DNA

Abstract

The invention discloses a kit and a method for extracting fruit DNA, and belongs to the technical field of biology. The kit comprises a lysate, wherein the lysate comprises the following components: 1 to 3 M of inorganic salt, 30 to 80 mM of EDTA (Ethylene Diamine Tetraacetic Acid), 0.05 to 0.12 M of Tris-HCl with the pH (Potential of Hydrogen) being 8.0, 1.3 to 2.8% m / v of CTAB (Cetyltrimethyl Ammonium Bromide), 7 to 16% v / v of Tween 20 and 3 to 4% v / v of phenol. By utilizing the kit and the method disclosed by the invention, DNA in fruits such as waxberries and grapes can be completely extracted, the yield is high, and the purity is high.

Description

Technical Field

[0001] The present application relates to the field of biotechnology, and in particular, to a kit and method for extracting fruit DNA. Background Art

[0002] Bayberry and grape are special fruit resources in my country. They are rich in anthocyanins and other flavonoids and have strong antioxidant activity and significant blood sugar regulating activity. The preparation of the main hypoglycemic components of bayberry fruit extract by column chromatography and countercurrent chromatography is simple, efficient, and has a high recovery rate, which can meet the needs of product development such as pilot preparation. The application of key technologies for the study of the hypoglycemic activity of bayberry fruit extract and the preparation of main effective components has important social and economic value for the research and development of more special medical foods or hypoglycemic products suitable for diabetic patients.

[0003] Conventional genomic DNA extraction methods or DNA extraction kits cannot obtain high-quality bayberry and grape pulp DNA, resulting in difficulty in releasing bayberry and grape pulp DNA during digestion, low extraction yield, and low purity, which is difficult to meet the high requirements of molecular biology. Summary of the invention

[0004] In order to solve at least one of the above technical problems, the technical solution adopted in this application is as follows.

[0005] The first aspect of the present application provides a kit for extracting fruit DNA, the kit comprising a lysis solution, the components of which are: 1~3M inorganic salt, 30~80mM EDTA, 0.05~0.12M Tris-HCl pH 8.0, 1.3~2.8%m / v CTAB, 7~16% v / v Tween 20, and phenol 20~30:1.

[0006] In some specific embodiments of the present application, the components of the lysate are: 1.5 M inorganic salt, 50 mM EDTA, 0.1 M Tris-HCl pH 8.0, 2.0% m / v CTAB, 10% v / v Tween 20, phenol 25:1.

[0007] In some embodiments of the present application, the kit further comprises mercaptoethanol and NaOH.

[0008] In some embodiments of the present application, the inorganic salt is NaCl, KCl, MgCl2 and / or ZnCl2.

[0009] In the present application, the fruit is bayberry or grape fruit, and further, the pulp of the fruit is used for DNA extraction.

[0010] The second aspect of the present application provides a method for extracting fruit DNA using any kit described in the first aspect of the present application, comprising the following steps: S1, mixing the lysate with the pulp of the fruit, and incubating at 60-75° C. for 20-35 min; S2, centrifuge and take the supernatant, add equal volumes of Tris-saturated phenol and chloroform:isoamyl alcohol (24:1), vortex to mix, and let stand for 3-10 min; S3, centrifuge and take the supernatant, add an equal volume of chloroform:isoamyl alcohol (24:1), and vortex to mix; S4, centrifuge and take the supernatant, add an equal volume of isopropanol and 1 / 10 volume of 3M NaAC, mix by inversion, and let stand at -20°C for 1 h; S5, centrifuge and discard the supernatant, wash the precipitate with 75% ethanol, and obtain the DNA of the fruit.

[0011] In some embodiments of the present application, before mixing with the lysis solution, the pulp of the fruit is first mixed with mercaptoethanol and 0.1M NaOH, allowed to stand on ice for 8 to 15 minutes, and then centrifuged to discard the supernatant, wherein the ratio of mercaptoethanol to 0.1M NaOH is 3:5.

[0012] In some embodiments of the present application, in step S1, after the lysate is mixed with the pulp of the fruit, one steel ball with a diameter of 6 mm and one steel ball with a diameter of 3 mm are added, and the mixture is ground at 50 Hz for 3 to 10 minutes.

[0013] In some embodiments of the present application, the step of removing RNA is further included: S6. Dissolve the precipitate in step S5 in 20-50 μL ddH2O, add 1 μL RNase, and incubate at 37°C.

[0014] In some embodiments of the present application, the step of purifying the DNA using magnetic beads is further included. Specifically: (1) Add 0.6 times the volume of supernatant and mix well with magnetic beads, gently pipette and mix for more than 10 times, and let stand at room temperature for 5 minutes; (2) Place on the magnetic stand for 5 minutes until the solution becomes clear, then carefully aspirate and discard the supernatant; (3) Add 200 μL of freshly prepared 80% ethanol, let stand at room temperature for 30 seconds, and discard the supernatant; Preferably, repeat steps (1) to (3) once; (4) Centrifuge briefly, discard the ethanol, and dry the magnetic beads at room temperature for 5 minutes; (5) Add 70-100 μL of 10 mM Tris-HCl eluent, mix gently by pipetting, and let stand at room temperature for 5 min. (6) Let the solution stand at room temperature for 5 min until it becomes clear. Carefully transfer the supernatant to the corresponding new sample storage tube and store it at -20°C.

[0015] Compared with the prior art, this application has the following beneficial effects: By using the kit and method of the present application, DNA in fruits such as bayberry and grape can be completely extracted with high yield and purity.

[0016] It should be understood that the content described in this section is not intended to identify the key or important features of the embodiments of the present application, nor is it intended to limit the scope of the present application. Other features of the present application will become easily understood through the following description. BRIEF DESCRIPTION OF THE DRAWINGS

[0017] By reading the detailed description below with reference to the accompanying drawings, the above and other purposes, features and advantages of the exemplary embodiments of the present application will become readily understood. In the accompanying drawings, several embodiments of the present application are shown in an exemplary and non-limiting manner, wherein: Figure 1 The electrophoresis diagram of extracting bayberry pulp DNA using different lysis solutions in Example 1 of the present application is shown; Figure 2 The electrophoresis diagram of extracting bayberry pulp DNA in comparative examples 1 to 3 of the present application is shown; Figure 3 The electrophoresis diagram of extracting grape pulp DNA using different lysis solutions in Example 2 of the present application is shown; Figure 4 The electrophoresis diagram of DNA extracted from grape pulp using the reagents and methods of Comparative Examples 1 to 3 in Example 2 of the present application is shown. DETAILED DESCRIPTION

[0018] Unless otherwise indicated, implied from the context, or customary in the prior art, all parts and percentages in this application are based on weight, and the tests and characterization methods used are all synchronized with the filing date of this application. Where applicable, the contents of any patent, patent application or disclosure involved in this application are fully incorporated herein by reference, and their equivalent patent families are also introduced as references, especially the definitions of relevant terms in the art disclosed in these documents. If the definition of a specific term disclosed in the prior art is inconsistent with any definition provided in this application, the definition of the term provided in this application shall prevail.

[0019] Numerical ranges in this application are approximate values, so unless otherwise specified, they may include values ​​outside the range. Numerical ranges include all values ​​from the lower limit to the upper limit in increments of 1 unit, provided that there is an interval of at least 2 units between any lower value and any higher value. For a range containing a value less than 1 or containing a fraction greater than 1 (e.g., 1.1, 1.5, etc.), 1 unit is appropriately considered to be 0.0001, 0.001, 0.01 or 0.1. For a range containing a single digit less than 10 (e.g., 1 to 5), 1 unit is generally considered to be 0.1. These are only specific examples of what is intended to be expressed, and all possible combinations of the values ​​between the lowest and highest values ​​listed are considered to be clearly recorded in this application.

[0020] The terms "comprising", "including", "having" and their derivatives do not exclude the presence of any other components, steps or processes, and are irrelevant to whether these other components, steps or processes are disclosed in this application. To eliminate any doubt, unless explicitly stated, all compositions using the terms "comprising", "including", or "having" in this application may include any additional additives, adjuvants or compounds. On the contrary, except for those necessary for operating performance, the term "essentially consisting of..." excludes any other components, steps or processes from the scope of any description of the term below. The term "consisting of..." does not include any components, steps or processes that are not specifically described or listed. Unless explicitly stated, the term "or" refers to the listed individual members or any combination thereof.

[0021] In order to make the technical problems, technical solutions and beneficial effects solved by the present application more clearly understood, the present application is further described in detail below in conjunction with embodiments.

[0022] The following examples are used to demonstrate the preferred embodiments of the present application. It will be understood by those skilled in the art that the techniques disclosed in the following examples represent techniques discovered by the inventors that can be used to implement the present application, and therefore can be considered as preferred embodiments of the present application. However, it should be understood by those skilled in the art based on this specification that many modifications can be made to the specific embodiments disclosed herein, and still the same or similar results can be obtained without departing from the spirit or scope of the present application.

[0023] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this application belongs, and the disclosures and materials cited therein are hereby incorporated by reference.

[0024] Those skilled in the art will recognize, or be able to ascertain using no more than routine experimentation, many technical equivalents to the specific embodiments of the invention described herein. Such equivalents are intended to be encompassed by the claims.

[0025] The experimental methods in the following examples are conventional methods unless otherwise specified. The instruments and equipment used in the following examples are conventional laboratory instruments and equipment unless otherwise specified; the experimental materials used in the following examples are purchased from conventional biochemical reagent stores unless otherwise specified.

[0026] Example 1 Reagents and methods for extracting DNA from bayberry pulp 1. Preparation of reagents and instruments (1) Preparation of lysate Lysis buffer A (lysate disclosed in CA103266107A): 1.4 M NaCl, 20 mM ethylenediaminetetraacetate (EDTA), 0.1 M Tris-HCl pH 8.0, 2% m / v cetyltrimethylammonium bromide (CTAB), 8% m / v β-mercaptoethanol; Lysis solution B (lysate disclosed in CN110592072A): 2M NaCl, 25mM EDTA, 0.1M Tris-HCl pH 8.0, 2% m / v CTAB, 2% m / v PVP40, 0.35M D-Sorbitol, 0.1M anhydrous sodium sulfite, 2% m / v β-mercaptoethanol.

[0027] Lysis Buffer C: 1.5 M NaCl, 50 mM EDTA, 0.1 M Tris-HCl pH 8.0, 2.0% m / v CTAB, 10% v / v Tween 20, 4% v / v phenol.

[0028] (2) Instrument Setting 1: Turn on the water bath in advance and set the temperature to 65°C.

[0029] (3) Instrument Setting 2: Turn on the metal bath, set the temperature to 37°C, and set the time to 1 h.

[0030] (4) Preparation of rinsing solution: 80% ethanol.

[0031] (5) Preparation of eluent: 10 mM Tris-HCl.

[0032] 2. Methods for DNA extraction from bayberry pulp (1) Prepare three 2 mL centrifuge tubes numbered A, B, and C, and take equal portions of frozen bayberry pulp samples and place them in the centrifuge tubes to fill half of the tubes; (2) Add 30 μL of mercaptoethanol and 50 μL of 0.1 M NaOH to each of the three centrifuge tubes, vortex to mix, centrifuge gently, and place on ice for 10 min. (3) Centrifuge at 10,000 rpm for 10 min at room temperature and discard the supernatant; (4) Add lysis buffer A, lysis buffer B, and lysis buffer C to three centrifuge tubes respectively; then add one 6 mm and one 3 mm diameter steel ball respectively, and grind at 50 Hz for 5 min; (5) Place the ground centrifuge tube in a float and incubate in a 65°C water bath for 30 min, inverting and mixing 5 times; (6) After water bath, centrifuge at 8000 rpm for 10 min at room temperature and collect the supernatant; (7) Add 450 μL Tris-saturated phenol (source leaf bio, pH>7.8) and 450 μL chloroform:isoamyl alcohol = 24:1, vortex to mix, let stand for 5 min, and centrifuge at 15°C, 12,000 rpm for 10 min; (8) Take the supernatant, add an equal volume of chloroform:isoamyl alcohol (24:1), vortex to mix, and centrifuge at 12,000 rpm for 10 min at 4°C; (9) Take the supernatant, add an equal volume of isopropanol and 1 / 10 volume of NaAC (3 M), mix by inversion, and let stand at -20 °C for 1 h. (10) Centrifuge at 12,000 rpm for 20 min at 4°C and remove the supernatant. Wash the precipitate with 75% ethanol; (11) Repeat the above steps twice; (12) Open the lid and wait until the residual alcohol evaporates. Then dissolve the precipitate in an appropriate volume (20-50 μL) of ddH2O, add 1 μL RNase, and incubate at 37°C for 1 h. (13) Add 0.6 times the volume of supernatant to the three centrifuge tubes in the previous step. Mix the magnetic beads evenly. Pipette gently for more than 10 times and let stand at room temperature for 5 min. (15) Place on the magnetic stand for 5 min until the solution becomes clear. Carefully aspirate and discard the supernatant, taking care not to disturb the magnetic beads. (16) Keep the above three centrifuge tubes fixed on the magnetic rack, add 200 μL of freshly prepared 80% ethanol to the tubes, let stand at room temperature for 30 seconds, and then discard the supernatant, being careful not to disturb the magnetic beads; (17) Repeat the above steps once; (18) Centrifuge briefly, then keep the three centrifuge tubes fixed on the magnetic rack, discard the ethanol, and dry the magnetic beads at room temperature for 5 minutes; (19) Remove the three centrifuge tubes from the magnetic rack, add 70-100 μL of 10 mM Tris-HCl elution buffer, gently pipette to mix, and let stand at room temperature for 5 min; (20) Place three centrifuge tubes on a magnetic rack and let them stand at room temperature for 5 min until the solution becomes clear. Carefully transfer the supernatant to the corresponding new sample storage tubes and store them at -20°C.

[0033] 3. Agarose quality inspection The extracted DNA was subjected to 1% agarose gel quality test and the total amount of DNA and OD260 / OD280 values ​​were detected. Figure 1 And as shown in Table 1.

[0034] Table 1 Results of DNA extraction from bayberry pulp using different lysis buffers

[0035] Depend on Figure 1 As shown in Table 1, the total amount of DNA obtained by using lysis buffer A to extract DNA from bayberry pulp is very small, and the main DNA is degraded ( Figure 1 The purity is low in lane A. When DNA was extracted from bayberry pulp using lysis buffer B, the total amount of DNA obtained was also small, and even no bands could be observed ( Figure 1 Lane B), the purity is also not high; while using lysis buffer C to extract bayberry pulp DNA, the total amount of DNA obtained is large, and the main band is intact without degradation ( Figure 1 The purity of the obtained DNA is also very high.

[0036] Comparative Example 1 This comparative example provides a solution for extracting DNA from bayberry pulp, using lysate C1 to replace lysate C in Example 1. The difference between lysate C1 and lysate C in Example 1 is that PVP is used to replace Tween 20, the concentration remains unchanged, and other components remain unchanged. The method for extracting DNA from bayberry pulp is the same as in Example 1.

[0037] Comparative Example 2 This comparative example provides a solution for extracting DNA from bayberry pulp, using lysate C2 to replace lysate C in Example 1. The difference between lysate C2 and lysate C in Example 1 is that 4% v / v phenol is omitted, and other components remain unchanged. The method for extracting DNA from bayberry pulp is the same as in Example 1.

[0038] Comparative Example 3 This comparative example provides a solution for extracting DNA from waxberry pulp, using lysis solution C for lysis, and the other reagents are the same. In the extraction step, step (2) is omitted, that is, mercaptoethanol and NaOH are not added.

[0039] The test results of the red bayberry pulp DNA extracted from Comparative Examples 1 to 3 are as follows: Figure 2 And as shown in Table 2.

[0040] Table 2 Results of DNA extraction from bayberry pulp using different comparative lysis buffers

[0041] Depend on Figure 2 As shown in Table 2, when Tween 20 was replaced with PVP (Comparative Example 1), the total amount of DNA obtained decreased significantly and the purity decreased; when 4% v / v phenol was omitted (Comparative Example 2), the total amount of DNA obtained also decreased and was severely degraded (such as Figure 2 Lane D2 in the middle); if mercaptoethanol and NaOH are not added during sample pretreatment (Comparative Example 3), the total amount of DNA extracted is seriously reduced, and even the main band cannot be detected in agarose gel electrophoresis (such as Figure 2 Lane D3 in the figure shows that adding mercaptoethanol and NaOH in the pretreatment can cause repeated lysis of the sample, thereby greatly improving the DNA.

[0042] Example 2 Grape pulp DNA extraction reagent Lysis solution A, lysate B and lysate C in Example 1 were used respectively, and the extraction steps and quality inspection steps were the same as those in Example 1. The test results of the extracted grape pulp DNA are as follows: Figure 3 And as shown in Table 3.

[0043] Table 3 Results of DNA extraction from grape pulp using different lysis buffers

[0044] Depend on Figure 3 As shown in Table 3, the total amount of DNA obtained by using lysis buffer A to extract DNA from bayberry pulp was very small (only 0.36 μg), and the main band was degraded ( Figure 3 The purity is low when using lysis buffer B to extract DNA from bayberry pulp. The total amount of DNA obtained is also small (1.08 μg), and the band is weak ( Figure 3 Lane B), the purity is also not high; while using lysis buffer C to extract bayberry pulp DNA, the total amount of DNA obtained is large (2.34μg), and the main band is intact without degradation ( Figure 3 The purity of the obtained DNA is also very high.

[0045] The same reagents and methods as in Comparative Examples 1 to 3 were used to extract DNA from grape pulp. The test results are as follows: Figure 4 And as shown in Table 4.

[0046] Table 4 Results of DNA extraction from grape pulp using different comparative lysis buffers

[0047] Depend on Figure 4As shown in Table 4, when Tween 20 was replaced with PVP (Comparative Example 1), the total amount of DNA obtained was significantly reduced (only 0.76 μg), and the purity decreased; when 4% v / v phenol was omitted (Comparative Example 2), the total amount of DNA obtained also decreased and was severely degraded (such as Figure 4 If mercaptoethanol and NaOH are not added during sample pretreatment (Comparative Example 3), the total amount of DNA extracted is severely reduced (only 0.33 μg), and even the main band cannot be detected in agarose gel electrophoresis (such as Figure 4 D3 lane in the middle). It shows that adding mercaptoethanol and NaOH in the pretreatment can make the sample repeatedly lyse, so that the DNA is greatly improved. All documents mentioned in this application are cited as references in this application, just as each document is cited as reference separately. In addition, it should be understood that after reading the above teaching content of this application, those skilled in the art can make various changes or modifications to this application, and these equivalent forms also fall within the scope defined by the claims attached to this application.

Claims

1. A kit for extracting fruit DNA, characterized in that: The kit includes a lysis solution, and the components of the lysis solution are: 1-3M inorganic salt, 30-80mM EDTA, 0.05-0.12M Tris-HCl pH 8.0, 1.3-2.8% m / v CTAB, 7-16% v / v Tween 20, and 3-4% v / v phenol.

2. The kit according to claim 1, characterized in that The components of the lysate are: 1.5 M inorganic salt, 50 mM EDTA, 0.1 M Tris-HCl pH 8.0, 2.0% m / v CTAB, 10% v / v Tween 20, 4% v / v phenol.

3. The kit according to claim 1 or 2, characterized in that The kit also includes mercaptoethanol and NaOH.

4. The kit according to claim 1 or 2, characterized in that The inorganic salt is NaCl, KCl, MgCl2 and / or ZnCl2.

5. The kit according to claim 1 or 2, characterized in that The fruit is bayberry or grape fruit.

6. A method for extracting fruit DNA using the kit according to claim 1 or 2, characterized in that: The following steps are involved: S1, mixing the lysate with the pulp of the fruit, and incubating at 60-75° C. for 20-35 min; S2, centrifuge and take the supernatant, add equal volumes of Tris-saturated phenol and chloroform:isoamyl alcohol (24:1), vortex to mix, and let stand for 3-10 min; S3, centrifuge and take the supernatant, add an equal volume of chloroform:isoamyl alcohol (24:1), and vortex to mix; S4, centrifuge and take the supernatant, add an equal volume of isopropanol and 1 / 10 volume of 3M NaAC, mix by inversion, and let stand at -20°C for 1 h; S5, centrifuge and discard the supernatant, wash the precipitate with 75% ethanol, and obtain the DNA of the fruit.

7. The method according to claim 6, characterized in that Before mixing with the lysis solution, the fruit pulp is first mixed with mercaptoethanol and 0.1M NaOH, and then placed on ice for 8 to 15 minutes and centrifuged to discard the supernatant, wherein the usage ratio of mercaptoethanol to 0.1M NaOH is 3:

5.

8. The method according to claim 6 or 7, characterized in that: In step S1, after the lysate is mixed with the fruit pulp, one steel ball with a diameter of 6 mm and one steel ball with a diameter of 3 mm are added, and the mixture is ground at 50 Hz for 3 to 10 minutes.

9. The method according to claim 6 or 7, characterized in that: The RNA removal step is further included: S6. Dissolve the precipitate in step S5 in 20-50 μL ddH2O, add 1 μL RNase, and incubate at 37°C.

10. The method according to claim 9, characterized in that The method further comprises the step of purifying the DNA using magnetic beads.

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