Lysate for rapidly extracting animal and plant genomes and application method thereof

Abstract

The invention provides a lysis solution for rapidly extracting animal and plant genomes and an application method thereof.The lysis solution is prepared from, by mass, 20-25 parts of Tris-Hcl, 10-15 parts of LICI, 5-10 parts of EDTA, 1-3 parts of 3-sulfopropyl hexadecyl dimethyl betaine, 1-3 parts of (NH4) 2SO4 and 30-40 parts of ddH2O. The 3-sulfopropyl hexadecyl dimethyl betaine and (NH4) 2SO4 in the formula of the lysis solution can effectively destroy cell membranes and nuclear membranes; the lysate prepared by the method is clear in component and simple to prepare, DNA precipitation or column purification can be directly carried out after lysis, meanwhile, the lysate is suitable for various sample types including animal tissues and plant tissues, a formula does not need to be adjusted according to different sample types, and compared with a traditional lysate, the lysate has the advantages that the cost is low, and the efficiency is high. The optimized formula has the advantages of high splitting efficiency, wide application range, high DNA purity, high extraction speed and good DNA integrity.

Description

Technical Field

[0001] The invention relates to the technical field of gene extraction, in particular to a lysate for rapid extraction of animal and plant genomes and an application method thereof. Background Art

[0002] Lysis solution is a chemical reagent mainly used to break the cell wall of biological samples and release the molecules in the cells into the solution. It is widely used in biomedicine, molecular biology, pharmacy, microbiology and biochemical analysis. Lysis solution can be divided according to different classification methods: according to the purpose: lysis solution for different types of biological samples (such as cells, bacteria, animal tissues); according to the chemical composition: extract solution, enzyme lysis solution, biological lysis solution, etc.; according to the preparation method: mechanical lysis solution, ultrasonic lysis solution, etc.

[0003] Traditional lysis buffers (such as SDS-based or CTAB-based) usually rely on a single detergent and have low lysis efficiency, especially for plant cell walls and protein-rich animal tissues. Incomplete lysis leads to low DNA yield. Traditional lysis buffers have limited ability to remove polyphenols, polysaccharides and proteins. In particular, plant samples are seriously contaminated with polyphenols and polysaccharides, which affects DNA purity. Impurities may inhibit downstream experiments (such as PCR, enzyme digestion, etc.). At the same time, the nuclease activity in traditional lysis buffers is not completely inhibited, resulting in DNA degradation during the extraction process. When the lysis time is long, the DNA integrity is further reduced. Summary of the invention

[0004] 1. Technical issues to be resolved

[0005] In view of the shortcomings of the prior art, the present invention provides a lysis solution for rapid extraction of animal and plant genomes and an application method thereof, which solves the problems of low lysis efficiency, limited removal capacity of polyphenols, polysaccharides and proteins, incomplete inhibition of nuclease activity and slow extraction of traditional lysis solutions.

[0006] (II) Technical solution

[0007] To achieve the above objectives, the present invention is implemented through the following technical scheme: a lysis solution for rapid extraction of animal and plant genomes, composed of the following parts by mass: 20-25 parts of Tris-Hcl, 10-15 parts of LICI, 5-10 parts of EDTA, 1-3 parts of 3-sulfopropyl hexadecyl dimethyl betaine, 1-3 parts of (NH4)2SO4, and 30-40 parts of ddH2O.

[0008] Preferably, it includes the following preparation steps; S1. Weigh an appropriate amount of Tris-HCl, LiCl, EDTA, 3-sulfopropyl hexadecyl dimethyl betaine, and (NH4)2SO4, dissolve them in ddH2O to obtain a first mixed solution, and adjust the pH and concentration of each component; S2. Use ddH2O to make the solution reach the required volume, stir well until all components are dissolved to obtain a lysis solution; S3. Filter the lysis solution with a 0.22 μm filter membrane to remove impurities, and store it at room temperature after aliquoting.

[0009] An application method of a lysis solution for rapidly extracting genomes of animals and plants includes the following application steps:

[0010] S1. Weigh 50 mg - 80 mg of tissue samples, add 500 - 700 μL of lysis solution, grind well, centrifuge for 1 min, and keep the lysis temperature at 65 °C;

[0011] S2. Take the supernatant, add 1 mL of ethanol, and centrifuge for 1 min;

[0012] S3. Add 75% alcohol to wash twice and air dry;

[0013] S4. Add 80 - 100 μL to obtain genomic DNA.

[0014] (III) Beneficial effects

[0015] The present invention provides a lysis solution for rapidly extracting genomes of animals and plants and an application method thereof.

[0016] It has the following beneficial effects:

[0017] 1. 3-sulfopropyl hexadecyl dimethyl betaine and (NH4)2SO4 in the lysis solution formula of the present invention can effectively destroy cell membranes and nuclear membranes, release DNA. Moreover, the components of the lysis solution prepared by the whole invention are clear, the preparation is simple, and DNA precipitation or column purification can be directly carried out after lysis. At the same time, this lysis solution is applicable to various sample types, including animal tissues and plant tissues, without the need to adjust the formula for different sample types. Compared with traditional lysis solutions, this optimized formula has the advantages of high lysis efficiency, wide application range, high DNA purity, fast extraction speed, and good DNA integrity. Specific embodiments

[0018] The technical solutions in the embodiments of the present invention will be clearly and completely described below. Obviously, the described embodiments are only a part of the embodiments of the present invention, rather than all the embodiments. All other embodiments obtained by those of ordinary skill in the art based on the embodiments of the present invention without creative efforts shall fall within the protection scope of the present invention.

[0019] Example 1:

[0020] An embodiment of the present invention provides a lysis solution for rapidly extracting animal and plant genomes, which is composed of the following parts by mass: 20-25 parts of Tris-HCl, 10-15 parts of LiCl, 5-10 parts of EDTA, 1-3 parts of CHAPS (3-sulfopropylhexadecyldimethylbetaine), 1-3 parts of (NH4)2SO4, and 30-40 parts of ddH2O. The concentrations of the respective materials in its specific application in this preparation process are as follows:

[0021] Tris-HCl: 50 mM (pH 8.0);

[0022] LiCl: 0.8 M;

[0023] EDTA: 0.4 M;

[0024] 3-sulfopropylhexadecyldimethylbetaine: 1%;

[0025] (NH4)2SO4: 0.1 M.

[0026] The lysis solution capable of rapidly extracting animal and plant genomes includes the following preparation steps;

[0027] S1. Weigh an appropriate amount of Tris-HCl, LiCl, EDTA, 3-sulfopropylhexadecyldimethylbetaine, and (NH4)2SO4 and dissolve them in ddH2O to obtain a first mixed solution, and adjust the pH and concentration of each component;

[0028] S2. Make up the volume of the solution to the required volume with ddH2O, and stir well until all components are dissolved to obtain the lysis solution;

[0029] S3. Filter the lysis solution with a 0.22 μm filter membrane to remove impurities, and after aliquoting, store it at room temperature.

[0030] EDTA chelates divalent metal ions (such as Mg2+, Ca 2 +), inhibits the activity of DNase, and prevents DNA degradation. PVP (polyvinylpyrrolidone) can bind polyphenolic substances in plant samples and prevent DNA degradation caused by polyphenol oxidation. LiCl can selectively precipitate DNA and reduce RNA contamination. If further removal of RNA is required, RNase A can be added. After adding proteinase K, the lysis time can be shortened to 10-15 minutes (or even shorter), and the synergistic effect of mercaptoethanol and CHAPS can further accelerate the lysis process;

[0031] An application method of a lysis solution for rapidly extracting animal and plant genomes includes the following application steps:

[0032] S1. Weigh 50 mg - 80 mg of tissue sample, add 500 - 700 μL of lysis buffer, grind thoroughly, centrifuge for 1 min, and maintain the lysis temperature at 65 °C;

[0033] S2. Take the supernatant, add 1 mL of ethanol, and centrifuge for 1 min;

[0034] S3. Wash twice with 75% alcohol and air dry;

[0035] S4. Add 80 - 100 μL to obtain genomic DNA.

[0036] Example 2:

[0037] Use the SDS lysis buffer in the prior art;

[0038] Comparative Example 1:

[0039] Select animal samples: mouse liver and muscle; plant samples: Arabidopsis thaliana leaves and rice seeds; Take two 50 mg of animal tissues, grind them into powder in liquid nitrogen, then take two 100 mg of leaves or seeds, grind them into powder in liquid nitrogen, and successively add 500 μL of lysis buffer (Example 1 and Example 2) to the two tissue powders, vortex to mix evenly, incubate at 55 - 65 °C for 10 - 15 minutes, add an equal volume of chloroform - isoamyl alcohol (24:1), vortex to mix evenly, let stand at room temperature for 5 minutes, then centrifuge at 12000 rpm for 10 minutes, take the supernatant, add 0.6 times the volume of isopropanol or 2 times the volume of ice - cold ethanol, gently mix, let stand at room temperature for 5 minutes, or place at - 20 °C for 5 minutes, centrifuge at 12000 rpm for 10 minutes, discard the supernatant, wash the DNA precipitate with 70% ethanol, centrifuge and discard the supernatant, air dry at room temperature, and finally dissolve the DNA precipitate in 50 - 100 μL of TE buffer or ddH2O;

[0040] Comparison of DNA yield:

[0041] Detect the DNA yield by quantitative PCR or fluorescence quantitative method, and the results are shown in Table 1:

[0042] Table 1

[0043]

[0044] Comparison of DNA purity:

[0045] Use an ultraviolet spectrophotometer to detect the ratios of A260 / A280 and A260 / A230, and the results are shown in Table 2:

[0046] Table 2

[0047]

[0048] Comparison of DNA integrity:

[0049] The integrity of DNA was detected by agarose gel electrophoresis, and the results are shown in Table 3 as follows:

[0050] Table 3

[0051]

[0052] In summary, through comparative experiments and chart demonstrations, the lysis solution prepared by the present invention is superior to the traditional lysis solution in terms of DNA yield, purity, and integrity. It is particularly suitable for the extraction of animal and plant genomes. The optimized lysis solution has higher efficiency and better compatibility with downstream experiments, meeting the experimental requirements for high-throughput and complex samples.

[0053] Although the embodiments of the present invention have been shown and described, those of ordinary skill in the art can understand that various changes, modifications, substitutions, and variations can be made to these embodiments without departing from the principle and spirit of the present invention. The scope of the present invention is defined by the appended claims and their equivalents.

Claims

1. A lysis solution for rapid extraction of animal and plant genomes, characterized in that: The invention is composed of the following parts by mass: 20-25 parts of Tris-HCl, 10-15 parts of LICI, 5-10 parts of EDTA, 1-3 parts of 3-sulfopropyl hexadecyl dimethyl betaine, 1-3 parts of (NH4)2SO4 and 30-40 parts of ddH2O.

2. The lysate for rapid extraction of animal and plant genomes according to claim 1, characterized in that: The method comprises the following preparation steps: S1. Weigh appropriate amounts of Tris-HCl, LICI, EDTA, 3-sulfopropyl hexadecyl dimethyl betaine, and (NH4)2SO4 and dissolve them in ddH2O to obtain a mixed solution 1, and adjust the pH and concentration of each component; S2. Add ddH2O to the solution to the required volume and stir thoroughly until all components are dissolved to obtain a lysate; S3. Filter the lysate with a 0.22 μm filter membrane to remove impurities, divide the solution into smaller pieces and store at room temperature.

3. An application method for rapid extraction of animal and plant genome lysate, characterized in that: The application steps include: S1. Weigh 50mg-80mg tissue sample, add 500-700μL lysis buffer and grind thoroughly, centrifuge for 1min, and keep the lysis temperature at 65℃; S2, take the supernatant, add 1 mL of ethanol, and centrifuge for 1 min; S3, rinse twice with 75% alcohol and let dry; S4. Add 80-100 μL to obtain genomic DNA.

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