Efficient coral DNA extraction method

Abstract

The invention belongs to the technical field of DNA extraction, and particularly relates to an efficient coral DNA extraction method. The invention provides a cell lysis solution, the cell lysis solution comprises PVP, sorbitol, sodium pyrosulfite and other substances, and the substances are specifically combined, so that mucoprotein and polysaccharide in a coral sample can be fully separated, and the DNA extraction quality is improved. On the basis, the invention also provides an efficient coral DNA extraction method, which not only improves the yield and quality of DNA extraction, but also shortens the experimental time. Compared with the prior art, the technical scheme provided by the invention has the advantages of low cost, short process and large-scale application, provides a standardized solution for coral genetics and genomics research, and has important scientific research value for coral biological research and protection.

Description

Technical Field

[0001] The invention belongs to the technical field of DNA extraction, and particularly relates to an efficient method for extracting coral DNA. Background Art

[0002] Coral reefs, often called the "tropical rainforests of the ocean," cover only 0.2% of the ocean surface, yet support 30% of marine life, making them a vital core of biodiversity in marine ecosystems. Reef-building corals, primarily composed of stony corals (Hexacoralia), perform irreplaceable ecological functions, providing habitat and food sources for other marine life, maintaining complex food chains and ecological balance. Coral reefs mitigate wave energy through their structure, protecting coastlines from erosion. During their growth, corals absorb and fix large amounts of carbon dioxide, playing a vital role in the carbon cycle and helping to regulate global climate. Furthermore, coral reef ecosystems sustain fisheries in surrounding waters, and the tourism they generate holds significant economic and cultural value.

[0003] However, the survival of stony corals currently faces severe challenges. With global warming, rising sea temperatures cause corals to lose their symbiotic algae, resulting in "bleaching." Long-term, sustained high temperatures can make the bleaching process irreversible, leading to coral mortality. Ocean acidification is another threat. As the ocean absorbs atmospheric carbon dioxide, the accumulation of calcium carbonate in stony coral skeletons is hindered, reducing skeletal density and thus impacting coral reef growth and resilience. Human activities such as overfishing, the use of explosives and cyanide fishing, coastal development, and pollutant emissions have also caused varying degrees of damage to coral reef ecosystems. It is reported that approximately 70% of coral reefs worldwide are threatened to varying degrees. Therefore, there is an urgent need to strengthen research and conservation of stony corals. DNA extraction is a cornerstone of molecular biology research. Obtaining whole-genome sequencing of coral DNA can help elucidate coral species classification, phylogenetic history, and population genetic structure. Analyzing the interactions between corals and their symbiotic algae and studying their responses to environmental changes can provide a scientific basis for developing effective conservation and restoration strategies.

[0004] However, due to the unique characteristics of these samples, DNA extraction from stony corals remains a recognized technical challenge for researchers. First, stony corals have a rigid exoskeleton, requiring grinding and pulverization to access their internal soft tissues for DNA extraction, which presents additional sample pretreatment challenges. Second, even after cleaning, coral tissues still contain a large amount of mucus, which comprises amino acids, glycoproteins, various polysaccharides, and various bioactive substances. Substances such as mucin can make the extraction system too viscous, making it difficult for impurities such as proteins and cell debris to be separated by centrifugation and effectively separated from the DNA in the supernatant. Currently, a variety of commercial kits have been used for coral DNA extraction, including marine organism DNA extraction kits, rapid DNA extraction kits, and actinomycete DNA extraction kits. However, these kits are not optimized for coral samples. Mucus can clog nucleic acid purification columns, and the DNA extraction yields of these kits using adsorption columns do not reach the same level as non-adsorption column methods. The phenol-chloroform method is a commonly used DNA extraction method, but it also fails to effectively separate interfering substances in corals, resulting in poor protein extraction and affecting the quality of the resulting DNA. Therefore, there is an urgent need to find a method that can effectively remove mucus impurities and efficiently extract stony coral DNA to meet the needs of high-throughput sequencing and precise ecological monitoring. Summary of the Invention

[0005] The present invention aims to provide an efficient coral DNA extraction method that can effectively remove mucus impurities and obtain high DNA yield and concentration.

[0006] The present invention provides a cell lysate, which comprises the following components: 80-100 mM Tris-HCl, 45-50 mM EDTA, 1%-2% v / v SDS solution, 1%-2% v / v PVP, 0.2-0.4 M sorbitol, 1 wt%-1.5 wt% sodium metabisulfite and 0.5-0.8 M sodium chloride.

[0007] The present invention provides the use of the cell lysis solution described in the above technical solution in improving the efficiency of sample DNA extraction.

[0008] Preferably, the sample includes: coral.

[0009] The present invention provides an efficient method for extracting coral DNA, comprising the following steps: (1) Pre-freezing and freezing-grinding the coral sample in sequence to obtain a ground sample; (2) shaking the ground sample and the cell lysis solution according to claim 1 to obtain a mixed solution; (3) adding EDTA and proteinase K to the mixed solution in sequence, treating the mixture in a water bath and performing a first centrifugation to obtain a first supernatant; (4) mixing the first supernatant with ammonium acetate, performing a first incubation and a second centrifugation to obtain a second supernatant; (5) mixing the second supernatant with anhydrous ethanol and performing a third centrifugation, and mixing the first precipitate with 70% ethanol by volume and performing a fourth centrifugation to obtain a second precipitate; (6) The air-dried second precipitate is resuspended and incubated for the second time to obtain coral DNA.

[0010] Preferably, the mass volume ratio of the coral sample, cell lysate, EDTA, proteinase K and ammonium acetate is 1 g: 90-100 μL: 15-20 μL: 2-3 μL: 25-30 μL; The concentration of the EDTA is 0.5-0.8M; The concentration of the ammonium acetate is 7.5-8M.

[0011] Preferably, the conditions of the first centrifugation and the second centrifugation include: a rotation speed of 12000-14000g, and a time of 10-12min; The conditions of the third centrifugation include: a rotation speed of 8000-10000g and a time of 5-8min; The conditions of the fourth centrifugation include: a rotation speed of 8000-10000 g and a time of 1-2 min.

[0012] Preferably, the volume ratio of the second supernatant to anhydrous ethanol is 1:1; The anhydrous ethanol includes: anhydrous ethanol that has been pre-cooled at -20°C for 10-20 minutes.

[0013] Preferably, the conditions of the water bath treatment include: temperature of 60-65°C, time of 2-3h; The conditions of the first incubation include: a temperature of 4-6°C and a time of 8-10 minutes; The second incubation conditions include: a temperature of 35-37° C. and a time of 25-30 min.

[0014] Preferably, the freeze grinding comprises: rotary grinding and static standing; The speed of the rotary grinding is 2000-2500 r / min; the time of the rotary grinding is 20-25 s; The standing time is 20-25s; The freezing grinding is performed 2-3 times.

[0015] Preferably, the resuspension is performed with nuclease-free water.

[0016] Beneficial effects: The present invention provides a cell lysate, which comprises the following components: the cell lysate comprises the following components: 80-100 mM Tris-HCl, 45-50 mM EDTA, 1%-2% v / v SDS solution, 1%-2% v / v PVP, 0.2-0.4 M sorbitol, 1 wt%-1.5 wt% sodium metabisulfite and 0.5-0.8 M sodium chloride.

[0017] The present invention adds additional reagents for coral mucus to the cell lysate. PVP (polyvinyl pyrrolidone) can not only effectively remove polyphenol contamination in the system, but also effectively separate polysaccharides; sorbitol helps reduce the interference of polysaccharides in the mucus; sodium metabisulfite, as an antioxidant, can prevent polyphenols in the mucus from oxidizing and binding to nucleic acids, further improving the quality of DNA extraction.

[0018] In addition, the present invention also provides an efficient method for extracting coral DNA. Compared with the existing technology, the technical solution provided by the present invention has the following advantages: (1) The system is precisely designed for stony coral samples. The hard coral skeleton is completely crushed by tissue grinding, so that the internal soft tissue and cell lysate are fully in contact, which reduces the DNA degradation caused by sample heating during other processing processes and the sample waste caused by incomplete crushing of the coral skeleton.

[0019] (2) The present invention additionally uses ammonium acetate as a salting-out agent, which is not only conducive to promoting protein precipitation, but also can form a complex with polysaccharides, and has an excellent separation effect on mucin and various polysaccharides in coral mucus; in addition, the use of the cell lysate described in the above technical solution effectively solves the problem of difficult extraction and purification of stony coral DNA; at the same time, the above solution is low-cost and has a short process, and can be applied to large-scale sample extraction, which has important scientific research value for the biological research and protection of corals.

[0020] (3) The present invention not only improves the efficiency of the DNA extraction process but also optimizes the operation steps, thereby reducing DNA degradation during the process. By pre-cooling the reagents, quickly air-drying the ethanol, and adjusting the RNase treatment to the end, the experimental time is shortened to obtain high-quality DNA for subsequent analysis.

[0021] (4) The present invention is applicable to multiple groups of stony coral species and can be promoted as a universal solution. It has the advantages of low cost, short process, and large-scale application, providing a standardized solution for coral genetics and genomics research. BRIEF DESCRIPTION OF THE DRAWINGS

[0022] In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the following briefly introduces the drawings required for use in the embodiments. Obviously, the drawings described below are only some embodiments of the present invention. For ordinary technicians in this field, other drawings can be obtained based on these drawings without paying any creative work.

[0023] Figure 1 The agarose gel electrophoresis diagram of the DNA of the stony coral provided by the present invention. DETAILED DESCRIPTION

[0024] In order to further illustrate the present invention, the solutions provided by the present invention are described in detail below with reference to the accompanying drawings and embodiments, but they should not be construed as limiting the scope of protection of the present invention.

[0025] In the examples and comparative examples, the stony coral samples included Acropora hyacinthus (Acropora Acropora hyacinthus ) and Porites (Pteris lutea) Porites lutea ) two methods, that is, different stony coral samples were treated with the same method.

[0026] Example 1 (Ammonium acetate salting-out-anhydrous ethanol precipitation method) An efficient method for extracting coral DNA, the steps are: (1) Take 5 g of liquid nitrogen-frozen stony coral sample and place it in a ball mill. Pre-freeze with liquid nitrogen for 3 min until the ball mill is completely cooled. Then install the ball mill in a medium-throughput tissue grinder. (2) Grinding at a speed of 2000 r / min, grinding for 20 s and stopping for 20 s, repeating three times to obtain a powdered tissue; (3) Transfer the ground tissue into a 2 mL centrifuge tube, add 500 μL of cell lysis buffer (cell lysis buffer consists of 100 mM Tris-HCl, 50 mM EDTA, 1% v / v SDS solution, 2% v / v PVP, 0.4 M sorbitol, 1 wt% sodium metabisulfite and 0.5 M sodium chloride), and shake to mix; (4) Add 100 μl of 0.5 M EDTA and 10 μl of proteinase K to the centrifuge tube from step (3), mix thoroughly by inverting, and incubate in a water bath at 65°C for 2-3 hours. Manually invert the tube every hour to improve the incubation effect. After incubation, centrifuge at 12,000 g for 10 minutes to remove coral skeletal fragments. Aspirate the supernatant and add it to a new 1.5 mL centrifuge tube. (5) Place the centrifuge tube in an ice box, add 150 μL of 7.5 M ammonium acetate, vortex to mix, incubate at 4°C for 10 min, and centrifuge at 12,000 g for 10 min to separate impurities such as proteins and cell debris; (6) Carefully aspirate the supernatant to avoid aspirating the precipitated impurities, add the supernatant to a new 1.5 mL centrifuge tube, and repeat the separation of impurities once more; (7) Add anhydrous ethanol that has been pre-cooled at -20°C for 10 min at a ratio of 1:1 to the supernatant, gently invert and mix 50 times, and then centrifuge at 8000 g for 5 min to precipitate the precipitated DNA; (8) Pour off the supernatant and add 1 mL of -20°C pre-cooled 70% ethanol to wash the DNA pellet. Gently invert to mix thoroughly and centrifuge at 8000 g for 1 min. (9) Pour off the supernatant and centrifuge briefly to allow the remaining supernatant to concentrate at the bottom of the centrifuge tube. Use a small-scale pipette to remove as much of the remaining supernatant as possible. (10) Place the centrifuge tube in an ice box, turn on the maximum wind speed of the clean bench, lower the baffle to increase the wind speed, and place the ice box at an angle at the air outlet to dry the residual ethanol in the precipitate. At the same time, avoid over-drying the DNA so that it is difficult to dissolve again; (11) After air-drying the ethanol, add 50 μL of nuclease-free water, invert and mix to redissolve the DNA, and centrifuge briefly to concentrate the liquid at the bottom of the centrifuge tube; (12) Add RNase after boiling in a water bath for 15 minutes and incubate in a metal bath at 37°C for 30 minutes to obtain the final target DNA sample. If the subsequent experiment does not have strict requirements for RNA, the extracted DNA sample can also be placed at 4°C overnight to promote the degradation of RNA in the sample.

[0027] Comparative Example 1 (High-pressure flushing method) A method for extracting coral DNA, comprising the following steps: The only difference between Comparative Example 1 and Example 1 is that steps (1) (2); The process of steps (1) and (2) is as follows: (1) Place 5 g of stony coral sample in a 50 mL centrifuge tube and rinse the sample with a tooth irrigator to separate the soft tissue from the skeleton to obtain the rinsing solution; (2) Centrifuge the obtained flushing fluid at 8000g for 10 minutes, discard the supernatant, and obtain the precipitated coral soft tissue.

[0028] The precipitated coral soft tissue was subsequently processed according to steps (3) to (12) in Example 1.

[0029] Comparative Example 2 (Unmodified Lysis Solution Method) A method for extracting coral DNA, comprising the following steps: The only difference between Comparative Example 2 and Example 1 is that the components of the cell lysate are different; The cell lysate in Comparative Example 2 consisted of 100 mM Tris-HCl, 50 mM EDTA and 1% v / v SDS solution.

[0030] Comparative Example 3 (Adsorption Column Method) A method for extracting coral DNA, comprising the following steps: The operation process of steps (1) to (4) is the same as that of steps (1) to (4) in Example 1.

[0031] (5) Pipette 600 μL of supernatant and add it to the adsorption column in the collection tube. Centrifuge at 12,000 rpm for 1 min and discard the liquid in the collection tube. (6) Add 500 μL of deproteinized rinse solution to the adsorption column, centrifuge at 12,000 rpm for 1 min, and discard the liquid in the collection tube; (7) Add 500 μL of washing solution to the adsorption column, centrifuge at 12,000 rpm for 1 min, discard the liquid in the collection tube, and repeat the washing process once; (8) Transfer the adsorption column to a 1.5 mL centrifuge tube, add 50 μL of elution buffer to the adsorption column, incubate at room temperature for 2 minutes, and centrifuge at 12,000 rpm for 2 minutes to obtain the final target DNA sample.

[0032] Comparative Example 4 (phenol-chloroform method) A method for extracting coral DNA, comprising the following steps: The operation process of steps (1) to (4) is the same as that of steps (1) to (4) in Example 1.

[0033] (5) Add 300 μL of phenol to a 1.5 mL centrifuge tube, aspirate 600 μL of supernatant, and then add 300 μL of chloroform-isoamyl alcohol (24:1). Shake gently for 20 minutes to avoid violent shaking that may break the DNA chain. Centrifuge at room temperature at 12,000 rpm for 10 minutes. (6) Repeat (5) twice, carefully aspirating the supernatant to avoid aspirating the middle protein layer; (7) Aspirate 600 μL of the supernatant, add 600 μL of chloroform-isoamyl alcohol, shake gently for 20 min, and centrifuge at 8,000 rpm for 10 min at room temperature; (8) Add anhydrous ethanol that has been pre-cooled at -20°C for 10 min at a ratio of 1:1 to the supernatant, gently invert and mix 50 times, and then centrifuge at 8000 g for 5 min to precipitate the precipitated DNA; (9) Pour off the supernatant and add 1 mL of -20°C pre-cooled 70% ethanol to wash the DNA pellet. Gently invert to mix, and centrifuge at 8000 g for 1 min. (10) Pour off the supernatant and centrifuge briefly to allow the remaining supernatant to concentrate at the bottom of the centrifuge tube. Use a small-scale pipette to remove as much of the remaining supernatant as possible. (11) Place the centrifuge tube in an ice box, turn on the maximum wind speed of the clean bench, lower the baffle to increase the wind speed, and place the ice box at an angle at the air outlet to dry the residual ethanol in the precipitate. At the same time, avoid over-drying the DNA so that it is difficult to dissolve again; (12) After air-drying the ethanol, add 50 μL of nuclease-free water, invert and mix to redissolve the DNA, and briefly centrifuge to concentrate the liquid at the bottom of the centrifuge tube; (13) Add RNase after boiling in a water bath for 15 minutes and incubate in a metal bath at 37°C for 30 minutes to obtain the final target DNA sample. If the subsequent experiment does not have strict requirements for RNA, the extracted DNA sample can also be placed at 4°C overnight to promote the degradation of RNA in the sample.

[0034] The DNA concentrations of the stony coral DNA extracted from Example 1 and Comparative Examples 1-4 were compared by Nanoodorp (diluted tenfold). See Tables 1 and Figure 1 (exist Figure 1 In Example 1 and Comparative Examples 1-4, the first bands respectively represent the electrophoresis results of Acropora; the second bands respectively represent the electrophoresis results of Porites).

[0035] Table 1 Effects of different treatment methods on DNA concentration

[0036] From Table 1 and Figure 1 It can be seen that the effect of magnetic bead crushing in a centrifuge tube is inferior to that of crushing in a ball mill, and it is unable to crush the harder coral skeleton into powder. When the same amount of sample is used for extraction, the latter has a higher DNA yield; When DNA was extracted using unmodified lysis buffer, the final sample was severely contaminated due to the inability to exclude interference from coral secretions such as mucin and polysaccharides. Figure 1 It can be seen that the DNA extracted by the unmodified lysis buffer method contains a large amount of protein contamination; When DNA is extracted using the adsorption column method, the DNA yield obtained is lower than that of other methods, and is still interfered by coral secretions such as mucin and polysaccharides, resulting in a certain amount of contamination in the final sample. Figure 1 It can be seen that the concentration of DNA extracted by the adsorption column method is low.

[0037] When DNA is extracted using the phenol-chloroform method, the DNA concentration is lower due to interference from coral secretions such as mucin and polysaccharides. In addition, according to the absorbance ratio, the DNA samples obtained by the phenol-chloroform method contain more contamination. Figure 1It can be seen that the quality of DNA extracted by the phenol-chloroform method is poor, and protein contamination can be observed in electrophoresis.

[0038] Although the above embodiment provides a detailed description of the present invention, it is only a part of the embodiments of the present invention, not all of the embodiments. People can also obtain other embodiments based on this embodiment without creativity, and these embodiments all fall within the scope of protection of the present invention.

Claims

1. A cell lysis solution, characterized in that The cell lysate comprises the following components: 80-100 mM Tris-HCl, 45-50 mM EDTA, 1%-2% v / v SDS solution, 1%-2% v / v PVP, 0.2-0.4 M sorbitol, 1 wt%-1.5 wt% sodium metabisulfite and 0.5-0.8 M sodium chloride.

2. Use of the cell lysate according to claim 1 in improving the efficiency of DNA extraction from samples.

3. The use according to claim 2, characterized in that The samples include: coral.

4. An efficient method for extracting coral DNA, characterized in that: The steps include: (1) Pre-freezing and freezing-grinding the coral sample in sequence to obtain a ground sample; (2) shaking the ground sample and the cell lysis solution according to claim 1 to obtain a mixed solution; (3) adding EDTA and proteinase K to the mixed solution in sequence, treating the mixture in a water bath and performing a first centrifugation to obtain a first supernatant; (4) mixing the first supernatant with ammonium acetate, performing a first incubation and a second centrifugation to obtain a second supernatant; (5) mixing the second supernatant with anhydrous ethanol and performing a third centrifugation, and mixing the first precipitate with 70% ethanol by volume and performing a fourth centrifugation to obtain a second precipitate; (6) The air-dried second precipitate is resuspended and incubated for the second time to obtain coral DNA.

5. The method for extracting coral DNA according to claim 4, wherein The mass volume ratio of the coral sample, cell lysate, EDTA, proteinase K and ammonium acetate is 1 g: 90-100 μL: 15-20 μL: 2-3 μL: 25-30 μL; The concentration of the EDTA is 0.5-0.8M; The concentration of the ammonium acetate is 7.5-8M.

6. The method for extracting coral DNA according to claim 4, wherein The conditions of the first centrifugation and the second centrifugation respectively include: a speed of 12000-14000g, and a time of 10-12min; The conditions of the third centrifugation include: a rotation speed of 8000-10000g and a time of 5-8min; The conditions of the fourth centrifugation include: a rotation speed of 8000-10000 g and a time of 1-2 min.

7. The method for extracting coral DNA according to claim 4, wherein The volume ratio of the second supernatant to anhydrous ethanol is 1:1; The anhydrous ethanol includes: anhydrous ethanol that has been pre-cooled at -20°C for 10-20 minutes.

8. The method for extracting coral DNA according to claim 4, wherein The conditions of the water bath treatment include: temperature of 60-65°C and time of 2-3h; The conditions of the first incubation include: a temperature of 4-6°C and a time of 8-10 minutes; The second incubation conditions include: a temperature of 35-37° C. and a time of 25-30 min.

9. The method for extracting coral DNA according to claim 4, wherein The freezing grinding comprises: rotating grinding and standing; The speed of the rotary grinding is 2000-2500 r / min; the time of the rotary grinding is 20-25 s; The standing time is 20-25s; The freezing grinding is performed 2-3 times.

10. The method for extracting coral DNA according to claim 4, wherein The resuspension was performed with nuclease-free water.

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