Method for efficiently extracting genome DNA (Deoxyribose Nucleic Acid) of Snaketail

Abstract

The invention discloses a method for efficiently extracting genome DNA (Deoxyribose Nucleic Acid) of ophiurocephalus serratus. The method comprises the following steps: mixing a sample with a lysis solution and metal balls, crushing the mixture at-20 to-10 DEG C for 250-350s at 40-60Hz, and placing the crushed sample at-20 to 4 DEG C; cracking the sample at the temperature of 50-60 DEG C for 4-6 minutes; centrifuging at 2-4 DEG C, taking a supernatant, adding an extraction agent, and uniformly mixing; centrifuging at 2-4 DEG C, taking an upper-layer solution, adding an extracting agent, and uniformly mixing; centrifuging at 2-4 DEG C, taking an upper-layer solution, adding isopropanol, uniformly mixing, and standing at-20--15 DEG C for 20-30 minutes; centrifuging at 2-4 DEG C, discarding the supernatant, washing the precipitate with an ethanol solution, and dissolving with an eluent; the lysis solution contains CTAB (Cetyltrimethyl Ammonium Bromide), protease K and beta-mercaptoethanol. According to the invention, the problem of high-mineralization echinodermis sample treatment is solved, and a high-quality nucleic acid template and a new technology are provided for research on ocean invertebrate genetics.

Description

TECHNICAL FIELD

[0001] The application belongs to the technical field of nucleic acid extraction, and particularly relates to a method for efficiently extracting Ophiuroidea genomic DNA. BACKGROUND

[0002] As the most species-diverse group in the phylum Echinodermata, Ophiuroidea has a global distribution ranging from intertidal to deep-sea habitats, exhibiting complex environmental adaptation mechanisms and diverse reproductive strategies, and thus has important model significance in animal evolutionary biology research. This group not only forms multiple environmental adaptation phenotypes through the co-evolution of body surface pigmentation and pattern structure, but also becomes a classic model for tissue regeneration research due to the significant regeneration ability of its arm and foot organs. Notably, although Ophiuroidea only has a simple neural network system composed of basal disc nerve ring and radial nerve cords, it can achieve coordinated movement and feeding behavior of multiple arms through distributed neural regulation, which has provided an innovative bionics paradigm for the research and development of bionic soft robot motion control systems. In the molecular analysis of Ophiuroidea-specific biological mechanisms, the preparation of high-quality genomic DNA is a prerequisite for the study of transcriptome sequencing, functional gene cloning, and epigenetic research.

[0003] The current echinoderm genomics research technology system has significant limitations: although mature DNA extraction protocols have been established for Holothuroidea and Echinoidea, there is still a gap in DNA isolation techniques for Ophiuroidea-specific tissue characteristics. The high-density calcareous ossicles of Ophiuroidea epidermal tissue and the mucopolysaccharides-rich body wall tissue pose a double challenge to traditional columnar purification methods: the lysis system cannot effectively penetrate the calcified structure, limiting the lysis efficiency; meanwhile, the polysaccharide-protein complex competitively inhibits DNA capture, accompanied by residual endogenous nucleases. This technical defect results in two significant characteristics of the final DNA product: insufficient yield (<50 ng / mg tissue) and abnormal fragment distribution (main peak <10 kb), which cannot meet the quality requirements of long-read sequencing technologies such as PacBio SMRT or Oxford Nanopore for high-molecular-weight DNA (>50 kb). In addition, the conventional protocol lacks effective low-temperature protection measures during mechanical disruption, causing DNA degradation during the extraction process, which seriously affects the reliability of subsequent genome assembly. SUMMARY

[0004] Based on the common problems of the existing marine invertebrate genome DNA extraction technology system, such as limited cracking efficiency, insufficient purity of nucleic acid product, and DNA degradation caused by nuclease residue or oxidative damage, the present application aims to establish a high-efficiency extraction method for Ophiuroidea genome DNA, improve the purity and concentration of DNA product, and effectively inhibit the activity of nucleic acid hydrolase and free radical-mediated DNA chain breakage during the extraction process, thereby providing high-quality nucleic acid template for Ophiuroidea functional genomics research and long-read sequencing technology application.

[0005] The first object of the present application is to provide a method for efficiently extracting Ophiuroidea genome DNA, which comprises the following steps: mixing the sample with a lysis solution and metal balls, then crushing at 40-60Hz for 250-350s at-20 to-10℃, and placing the crushed sample at-20 to 4℃; lysing the sample at 50-60℃ for 4-6min; centrifuging at 2-4℃ to obtain the supernatant, mixing with an extraction agent; centrifuging at 2-4℃ to obtain the upper solution, mixing with an extraction agent; centrifuging at 2-4℃ to obtain the upper solution, mixing with isopropanol, and standing at-20 to-15℃ for 20-30min; centrifuging at 2-4℃ to discard the supernatant, washing the precipitate with an ethanol solution, and dissolving with an eluent; the lysis solution contains CTAB solution, proteinase K and beta-mercaptoethanol.

[0006] Preferably, the lysis solution is CTAB solution: 20mg / mL proteinase K: beta-mercaptoethanol in a volume ratio of 800:30:20, and the CTAB solution is 2-3% CTAB solution in a mass-volume ratio.

[0007] Preferably, the first extraction agent is phenol: chloroform: isopropyl alcohol in a volume ratio of 25:24:1, and the second extraction agent is chloroform: isopropyl alcohol in a volume ratio of 24:1.

[0008] Preferably, the sample is Echiurus brevispinus.

[0009] Preferably, the placement at-20 to 4℃ is on ice.

[0010] Preferably, the metal balls are 2-3mm steel balls.

[0011] Preferably, the centrifugation at 2-4℃ is 12000rpm centrifugation for 5-10min.

[0012] Preferably, the following steps are included: mixing the Acaudina molpadiica with a lysis solution, 3mm steel beads, and then crushing at 50Hz for 300s at -20 to -10°C, and then placing the crushed sample on ice; placing the sample in a 56°C water bath for lysis for 5min; centrifuging at 12000rpm at 4°C for 5min to take the supernatant, adding an extraction agent with a volume ratio of phenol:chloroform:isopropyl alcohol=25:24:1, and mixing; centrifuging at 12000rpm at 4°C for 10min to take the upper solution, adding an extraction agent with a volume ratio of chloroform:isopropyl alcohol=24:1, and mixing; centrifuging at 12000rpm at 4°C for 10min to take the upper solution, adding isopropyl alcohol, and mixing, and then placing at -20°C for 20min; centrifuging at 12000rpm at 4°C for 10min to discard the supernatant, and then washing the precipitate with a 75% ethanol solution and dissolving with an eluent; the lysis solution is a volume ratio of CTAB solution:20mg / mL protease K:β-mercaptoethanol=800:30:20, and the CTAB solution is a mass-volume ratio of 2% CTAB solution.

[0013] Preferably, the specific steps are as follows:

[0014] A, setting the water bath to 56°C in advance, and precooling the centrifuge at 4°C in advance, and placing the sample carrier in the crusher in advance to precool at -20°C;

[0015] B, adding 3 3mm steel beads to a 2mL centrifuge tube, and then sequentially adding 800μL CTAB solution, 30μL protease K, 20μL β-mercaptoethanol, and 0.1g Acaudina molpadiica, and crushing at 50Hz for 300s using the crusher, and then temporarily storing the crushed sample on ice, and the protease K has a concentration of 20mg / mL, and the CTAB solution is a mass-volume ratio of 2% CTAB solution;

[0016] C, placing the sample in a 56°C water bath for lysis for 5min, and inverting and mixing every two minutes during lysis;

[0017] D, centrifuging at 12000rpm at 4°C for 5min to discard the precipitate, and taking the supernatant, and placing in a new 2mL centrifuge tube, and adding 800μL of an extraction agent with a volume ratio of phenol:chloroform:isopropyl alcohol=25:24:1, and slowly inverting and mixing, and centrifuging at 12000rpm at 4°C for 10min, and then standing until obvious layering occurs, and a layer of film is generated at the junction of the upper and lower layers;

[0018] E, taking the upper solution and placing in a new 2mL centrifuge tube, and adding 800μL of an extraction agent with a volume ratio of chloroform:isopropyl alcohol=24:1, and slowly inverting and mixing, and centrifuging at 12000rpm at 4°C for 10min;

[0019] F, the supernatant was discarded, and the precipitate was retained; 1 mL of 75% ethanol was slowly added along the wall of the tube, and then the 75% ethanol solution was slowly sucked out; 1 mL of 75% ethanol was again slowly added along the wall of the tube; centrifugation was performed at 12000 rpm and 4 DEG C for 10 min; the supernatant was discarded, and the precipitate was retained;

[0020] G, the supernatant was discarded, and the precipitate was retained; 1 mL of 75% ethanol was slowly added along the wall of the tube, and then the 75% ethanol solution was slowly sucked out; 1 mL of 75% ethanol was again slowly added along the wall of the tube; centrifugation was performed at 12000 rpm and 4 DEG C for 10 min; the supernatant was discarded, and the precipitate was retained;

[0021] H, the EP tube was opened and placed at room temperature to dry;

[0022] I, 50 μL of Buffer TE was added to dissolve the DNA.

[0023] The second object of the present application is to provide the use of any of the above methods in the extraction of genomic DNA of Ophiuroidea.

[0024] The genomic DNA extraction scheme for Ophiuroidea established in the present application significantly improves the quality and integrity of nucleic acid extracted from Ophiuroidea arm samples by improving the composition and operation parameters of the lysis buffer, and combining mechanical crushing and low-temperature treatment methods.

[0025] Advantages of the present application:

[0026] In this study, we successfully constructed a genomic DNA extraction technique system for dermis layer of high calcification tissue of Ophiuroidea by optimizing the components of lysis buffer system and operation parameters. In view of the dense barrier characteristics of calcareous ossicles, we used low temperature coupled with high intensity mechanical disruption (frequency 50 Hz, time 300 s) to make the bone tissue into powder, combined with modified lysis buffer, and increased the amount of proteinase K to realize the dissociation of bone plate microstructure and improve the DNA release efficiency by 6.2 times compared with the conventional scheme. In order to maintain the integrity of DNA molecules, the low temperature control strategy was implemented throughout the process: the sample block in the disrupter was pre-cooled at-20℃, and low temperature centrifugation was used. After agarose gel electrophoresis and Nanodrop 2000 joint detection, the genomic DNA extracted by this scheme has stable purity (Nanodrop determination A260 / A280=1.92, A260 / A230=2.04), and the concentration and integrity of the obtained DNA are significantly better than those of the conventional method, which fully meets the requirements of PacBio SMRT sequencing and whole genome methylation analysis and other precise molecular experiments. This scheme solves the problem of high mineralization of echinoderm samples, provides high-quality nucleic acid templates for molecular evolution and comparative genomics research, and promotes the technological innovation of marine invertebrate genetics. The establishment of this technical system provides a solution for the molecular evolution research and comparative genomics of high mineralization species of Echinodermata, thus opening up a new way for marine animal DNA extraction technology. BRIEF DESCRIPTION OF DRAWINGS

[0027] Figure 1 The amount of DNA extracted by the optimized CTAB method was significantly higher than that by the kit method. ** indicates that there is a significant difference between groups, p<0.01.

[0028] Figure 2 is the agarose gel electrophoresis map of DNA extracted by different methods from Echis carinatus Ophiura; Lane M: Marker, Lane 1-3: genomic DNA extraction kit for marine animal tissue, Lane 4-6: optimized CTAB method. DETAILED DESCRIPTION

[0029] The following examples are further illustrations of the present application and are not intended to limit the present application.

[0030] Example 1: Optimized CTAB method

[0031] (I) Test material preparation

[0032] (1) Reagents

[0033] CTAB extraction solution (Biosharp BL1192A), proteinase K (20 mg / mL), β-mercaptoethanol, phenol:chloroform:isoamyl alcohol (25:24:1, volume ratio), chloroform:isoamyl alcohol (24:1, volume ratio), isoamyl alcohol, 75% ethanol, Buffer TE. The commercial item numbers of the reagents used are shown in Table 1.

[0034] Table 1 Optimized CTAB method - experimental reagent information table

[0035]

[0036] (2) Consumables

[0037] 1.5 mL centrifuge tube, 2 mL centrifuge tube, steel beads (3 mm), incubator box.

[0038] (3) Instruments

[0039] Cryomill, water bath, benchtop high-speed large-capacity centrifuge (Eppendorf 5810R), clean bench, pipette (100, 1000 μL range)

[0040] (II) Test steps for sea cucumber DNA extraction (1) Set the water bath to 56°C in advance, and pre-cool the centrifuge to 4°C in advance, and place the sample carrier in the cryomill in -20°C pre-cooling in advance;

[0041] (2) Add 3 steel beads to a 2 mL centrifuge tube, then add 800 μL of CTAB solution, 30 μL of proteinase K, 20 μL of β-mercaptoethanol, and 0.1 g of Echinocactum echinatus, and use the cryomill to crush the sample at 50 Hz for 300 s, and store the crushed sample on ice;

[0042] (3) Place the sample in a 56°C water bath and lyse for 5 min (invert and mix every two minutes during the lysis period);

[0043] (4) Centrifuge at 12000 rpm and 4°C for 5 min, discard the precipitate, take the supernatant, and place it in a new 2 mL centrifuge tube, add 800 μL of phenol:chloroform:isoamyl alcohol (25:24:1), slowly invert and mix, centrifuge at 12000 rpm and 4°C for 10 min, do not shake after centrifugation, slowly place the centrifuge tube on the rack, and a clear layering can be observed, and a layer of film will be produced at the junction between the upper and lower layers;

[0044] (5) Slowly aspirate the upper layer solution (do not aspirate the middle film), place it in a new 2 mL centrifuge tube, add 800 μL of chloroform:isoamyl alcohol (24:1), slowly invert and mix, and centrifuge at 12000 rpm and 4°C for 10 min;

[0045] (6) Pipette the upper layer solution into a new 1.5 mL EP tube, add 600 μL of isopropanol, gently invert the tube up and down to mix thoroughly, then place it at -20 °C and let it stand for 20 min. Centrifuge at 12,000 rpm for 10 min at 4 °C;

[0046] (7) Discard the supernatant and retain the precipitate; slowly add 1 mL of 75% ethanol along the inner wall of the tube, then slowly aspirate the 75% ethanol solution. Again, slowly add 1 mL of 75% ethanol along the inner wall of the tube. Centrifuge at 12,000 rpm for 10 min at 4 °C, discard the supernatant and retain the precipitate;

[0047] (8) Open the EP tube and let it dry at room temperature;

[0048] (9) Add 50 μL of Buffer TE to dissolve the DNA.

[0049] Example 2: Method using a genomic DNA extraction kit for marine animal tissues (abbreviated as "kit method")

[0050] Use a genomic DNA extraction kit for marine animal tissues (TIANGEN DP324-02) to extract the DNA of the arms of Ophiocoma scolopendrina. The supplier and product number of the reagents used are shown in Table 2. The specific steps are as follows:

[0051] (1) Take 0.1 g of the arms of Ophiocoma scolopendrina and put it into a centrifuge tube containing 200 μL of GA buffer, and vortex for 15 sec;

[0052] (2) Add 20 μL of Proteinase K (20 mg / mL) solution, vortex to mix thoroughly, and briefly centrifuge to remove the water droplets on the inner wall of the tube cap. Incubate at 56 °C for 0.5 h until the tissue is completely dissolved. Briefly centrifuge to remove the water droplets on the inner wall of the tube cap, and then proceed to the next step;

[0053] (3) Add 200 μL of buffer GB, invert the tube thoroughly to mix, incubate at 70 °C for 10 min. After the solution becomes clear, briefly centrifuge to remove the water droplets on the inner wall of the tube cap;

[0054] (4) Add 200 μL of absolute ethanol, invert the tube thoroughly to mix, and briefly centrifuge to remove the water droplets on the inner wall of the tube cap;

[0055] (5) Transfer the solution and flocculent precipitate obtained in the previous step into an adsorption column CB3 (the adsorption column is placed in the collection tube), centrifuge at 12,000 rpm for 30 sec, discard the waste liquid, and put the adsorption column CB3 back into the collection tube;

[0056] (6) Add 500 μL of buffer GD to the adsorption column CB3, centrifuge at 12,000 rpm for 30 sec, discard the waste liquid, and place the adsorption column CB3 into the collection tube;

[0057] (7) Add 600 μL of rinse solution PW to the adsorption column CB3, centrifuge at 12000 rpm for 30 sec, discard the waste, and place the adsorption column CB3 into the collection tube;

[0058] (8) Repeat operation step 7;

[0059] (9) Place the adsorption column CB3 back into the collection tube, centrifuge at 12000 rpm for 2 min, discard the waste. Place the adsorption column CB3 at room temperature for several minutes to dry the residual rinse solution in the adsorption material completely.

[0060] (10) Transfer the adsorption column CB3 into a clean centrifuge tube, add 50 μL of elution buffer TE to the middle of the adsorption membrane, place at room temperature for 2-5 min, centrifuge at 12000 rpm for 2 min, and collect the solution into the centrifuge tube.

[0061] Table 2 Kit method - experimental reagent information table

[0062]

[0063] Experimental example: DNA quality detection and comparative analysis:

[0064] Determine the DNA concentration, OD260 / 280 ratio and OD260 / 230 ratio of Example 1 (optimized CTAB method) and Example 2 (kit method) by using a micro-nucleic acid spectrophotometer (Nanodrop), and determine the integrity of the DNA by using a 1.2% agarose gel electrophoresis.

[0065] Results and explanations

[0066] 1. Determine the concentration and spectral ratio of the DNA extracted by two different methods by using a micro-nucleic acid spectrophotometer (Nanodrop), and the results are shown in Table 3. It can be seen that the amount of DNA extracted by the optimized CTAB method is significantly higher than that by the kit method. Figure 1 The OD260 / 230 value of the kit method is 1.9, while the OD260 / 230 value of the optimized CTAB method is 2.04, which indicates that the kit method may be contaminated by organic solvents and salt ions, while the OD260 / 230 value of the optimized CTAB method is within the qualified range.

[0067] Table 3 DNA quality of the snake tail extracted by different methods

[0068]

[0069] 2. The integrity of the DNA extracted by the above two methods was detected by 1.2% agarose gel electrophoresis. As can be seen from Figure 2 , the DNA extracted using the Marine Animal Tissue Genomic DNA Extraction Kit method (TIANGEN / Tiangen Biochemical DP324-02) showed serious degradation, while the DNA extracted by the optimized CTAB method showed better integrity.

[0070] In summary, the brittle star DNA extraction technology of the present invention can effectively obtain high-quality DNA. Aiming at the dense barrier characteristics of calcareous ossicles, this technology uses low temperature combined with high-intensity mechanical crushing (frequency 50 Hz, time 300 sec) to make the bone tissue into powder, combines with a modified lysis buffer, and increases the dosage of proteinase K, so as to achieve the dissociation of the bone plate microstructure and increase the DNA release efficiency by 6.2 times compared with the traditional method. In order to maintain the integrity of DNA molecules, a low-temperature control strategy was implemented throughout the process: the sample carrier block in the crusher was pre-cooled at -20 °C in advance, and low-temperature centrifugation was used. Through the combined detection of agarose gel electrophoresis and ultra-micro spectrophotometer (Nanodrop 2000), the purity of the genomic DNA extracted by this protocol was stable (Nanodrop measurement A260 / A280 = 1.92, A260 / A230 = 2.04), and the concentration and integrity of the obtained DNA were significantly better than those of the traditional method, fully meeting the requirements of precision molecular experiments such as PacBio SMRT sequencing and whole-genome methylation analysis. The establishment of this technical system provides an effective solution for the molecular evolution research and comparative genomics of highly mineralized species in the phylum Echinodermata, thus opening up a new way for the DNA extraction technology of marine animals.

Claims

1. A method for efficiently extracting genomic DNA of Echinodermata, characterized by, The method comprises the following steps: mixing the sample with a lysis solution and metal balls, crushing the mixture at 40-60 Hz for 250-350 s at-20 to-10 ℃, and then placing the crushed sample at-20 to 4 ℃; lysing the sample at 50-60 ℃ for 4-6 min; centrifuging at 2-4 ℃ to obtain supernatant, mixing with an extraction agent, centrifuging at 2-4 ℃ to obtain upper solution, mixing with an extraction agent, centrifuging at 2-4 ℃ to obtain upper solution, mixing with isopropyl alcohol, and then standing at-20 to-15 ℃ for 20-30 min; centrifuging at 2-4 ℃ to discard supernatant, washing the precipitate with an ethanol solution, and then dissolving the precipitate in an eluent; and the lysis solution comprises CTAB, proteinase K and β-mercaptoethanol.

2. The method of claim 1, wherein, The lysis solution is a solution with a volume ratio of CTAB solution: 20 mg / mL proteinase K: β-mercaptoethanol = 800:30:20, and the CTAB solution is a 2-3% CTAB solution with a mass-volume ratio.

3. The method of claim 1, wherein, The first extraction agent is a solution with a volume ratio of phenol: chloroform: isopentanol = 25:24:1, and the second extraction agent is a solution with a volume ratio of chloroform: isopentanol = 24:

1.

4. The method of claim 1, wherein, The sample is a centipede snake tail.

5. The method of claim 1, wherein, The step of placing the sample at-20 to 4 ℃ is placing the sample on ice.

6. The method of claim 1, wherein, The metal balls are 2-3 mm steel balls.

7. The method of claim 1, wherein, The centrifuging at 2-4 ℃ is centrifuging at 12000 rpm for 5-10 min.

8. The method of claim 1, wherein, The method comprises the following steps: mixing the sample with a lysis solution and metal balls, crushing the mixture at 40-60 Hz for 250-350 s at-20 to-10 ℃, and then placing the crushed sample at-20 to 4 ℃; lysing the sample at 50-60 ℃ for 4-6 min; centrifuging at 2-4 ℃ to obtain supernatant, mixing with an extraction agent, centrifuging at 2-4 ℃ to obtain upper solution, mixing with an extraction agent, centrifuging at 2-4 ℃ to obtain upper solution, mixing with isopropyl alcohol, and then standing at-20 to-15 ℃ for 20-30 min; centrifuging at 2-4 ℃ to discard supernatant, washing the precipitate with an ethanol solution, and then dissolving the precipitate in an eluent; and the lysis solution comprises CTAB, proteinase K and β-mercaptoethanol.

9. The method of claim 1, wherein, The method comprises the following steps: A. Set the water bath to 56 ℃ in advance, precool the centrifuge to 4 ℃, and precool the sample carrier in the crusher to-20 ℃; B. Add 3 3 mm steel balls to a 2 mL centrifuge tube, and then add 800 μL of CTAB solution, 30 μL of proteinase K, 20 μL of β-mercaptoethanol, and 0.1 g of centipede snake tail in sequence, crush the mixture using the crusher at 50 Hz for 300 s, and then temporarily store the crushed sample on ice, wherein the concentration of the proteinase K is 20 mg / mL, and the CTAB solution is a 2% CTAB solution with a mass-volume ratio; C. Place the sample in a 56 ℃ water bath, and invert the sample every two minutes during the lysis for 5 min. D, centrifugation at 12000 rpm, 4℃ for 5 min, discard the precipitate, take the supernatant, put it in a new 2 mL centrifuge tube, add 800 μL of phenol: chloroform: isoamyl alcohol = 25:24:1 in volume ratio, mix slowly up and down, centrifugation at 12000 rpm, 4℃ for 10 min, after centrifugation, stand until obvious stratification appears, and a layer of film will appear at the junction of the upper and lower layers; E, take the upper layer solution and put it in a new 2 mL centrifuge tube, add 800 μL of chloroform: isoamyl alcohol = 24:1 in volume ratio, mix slowly up and down, centrifugation at 12000 rpm, 4℃ for 10 min; F, take the upper layer solution and put it in a new 1.5 mL EP tube, add 600 μL of isoamyl alcohol, mix slowly up and down, after mixing, put it in -20℃, stand for 20 min, centrifugation at 12000 rpm, 4℃ for 10 min; G, discard the supernatant, keep the precipitate; slowly add 1 mL of 75% ethanol along the tube wall, then slowly suck out the 75% ethanol solution, again slowly add 1 mL of 75% ethanol along the tube wall, centrifugation at 12000 rpm, 4℃ for 10 min, discard the supernatant, keep the precipitate; H, open the EP tube and put it in room temperature to dry; I, add 50 μL of Buffer TE to dissolve the DNA.

10. The use of the method of any one of claims 1-9 in the extraction of genomic DNA of the class Ophiurida.

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