Extraction method and application of high molecular weight turbellarian worm genome DNA

Abstract

The invention discloses an extraction method and application of high-molecular-weight turbellarian worm genome DNA, and belongs to the field of turbellarian worm genome extraction.The turbellarian worm genome DNA extraction is achieved by improving a lysate formula and removing ethylenediaminetetraacetic acid and increasing Mg < 2 + > on the basis of a conventional reagent in a classic Tris-HCl-phenol-chloroform method to inhibit activity of deoxyribonuclease II in a turbellarian worm body to inhibit degradation of genome DNA, and the high-molecular-weight turbellarian worm genome DNA is obtained. Meanwhile, ribonuclease A is added, RNA in cells can be degraded in the tissue cell digestion process, so that the step of two times of purification is omitted, the loss of DNA yield is reduced, experimental steps are simplified, especially the step of DNA purification is reduced, the problem of degradation in the extraction process of the turbellarian turbellarian worm genome DNA is solved, and the genome DNA yield (gt; 10 micrograms per turbellarian worm), and the turbellarian worm genome ultra-long sequencing or PacBio high-fidelity sequencing requirements are met.

Description

Technical Field

[0001] The present invention relates to a method for extracting high-molecular-weight planarian genomic DNA and its application, belonging to the field of gene extraction. Background Art

[0002] The individual of Dugesia japonica is relatively small (0.8 - 1.5 cm), but there are significant differences in genomic sequences among populations and individuals, with a high content of repetitive sequences. Sequencing a mixture of multiple planarians poses a great challenge to the assembly of the genome of this species. High-molecular-weight genomic DNA is a prerequisite for planarian genome sequencing and assembly. However, currently, commercial kits and methods such as silica and cellulose membrane adsorption cannot obtain high-molecular-weight genomic DNA from planarians. So far, the successful acquisition of planarian genomic DNA has been reported using Dugesia mediterranea as the material, and its extraction methods include:

[0003] Classical Tris-HCl-phenol-chloroform method: Add a buffer solution of Tris-HCl containing ethylenediaminetetraacetic acid, proteinase K, and sodium dodecyl sulfate to 50 Dugesia mediterranea (~0.8 cm) and lyse the planarian tissue in a water bath at 55°C; add phenol-chloroform to denature proteins, and separate genomic DNA from proteins according to the characteristics that DNA dissolves in water and proteins dissolve in the organic phase; subsequently, precipitate DNA using ammonium acetate and isopropanol, wash the DNA with 70% ethanol, and dissolve the genomic DNA in TE buffer. To meet the requirements of genome sequencing, the genomic DNA needs to be further digested with ribonuclease and precipitated with isopropanol to obtain purified genomic DNA. However, this method is not applicable to Dugesia japonica with rich intracellular deoxyribonuclease II. Using this method, the genomic DNA of Dugesia japonica is prone to large-scale degradation, resulting in short DNA fragments. Moreover, the planarian genomic DNA extracted by this method undergoes two precipitations, with a low yield, and cannot meet the requirements of ultra-long sequencing or PacBio high-fidelity sequencing of a single planarian genome (>10 μg / worm, 3 cells);

[0004] Guanidinium thiocyanate method: 20 planarians (∼1 cm) were added to guanidinium thiocyanate lysis buffer (GTC buffer) and lysed on ice; phenol-chloroform solution was added to denature proteins and release DNA into the aqueous phase; chloroform was added to remove a small amount of phenol in the DNA solution, and frozen isopropanol and sodium chloride were added to precipitate DNA, and the DNA was washed with 70% ethanol. Since guanidinium thiocyanate is a strong denaturing agent, this method not only inhibits the activity of deoxyribonuclease, but also greatly protects the RNA in cells, resulting in a large amount of RNA in the extracted DNA. Subsequently, ribonuclease is needed to digest the RNA to obtain purified genomic DNA. Although high-molecular-weight genomic DNA can be obtained by this method, the yield is extremely low (<3.0 μg / worm), which cannot meet the requirements of ultra-long sequencing of a single planarian genome or PacBio high-fidelity sequencing (>10 μg / worm).

[0005] Different from the stable diploid karyotype (2n = 8) of Dugesia mediterranea, there are quite large differences in the chromosome numbers among different individuals and populations of Dugesia japonica. Even in the same population, different individuals also show karyotype polymorphism, with diploid (2n = 16), triploid (3n = 24) and mixed-ploid (2n&3n) individuals coexisting; the extremely high AT content and extensive repetitive sequences in the planarian genome have significantly accelerated chromosome evolution. Therefore, sequencing a single planarian is crucial for the accurate assembly of the Dugesia japonica genome. Moreover, Dugesia japonica contains abundant deoxyribonuclease II in its body, which has strong activity and a fast DNA cleavage rate, and can cleave DNA at 0-55 °C, resulting in a large amount of degradation of genomic DNA during the process of the classical planarian Tris-HCl-phenol-chloroform method for genomic DNA extraction. In addition, ultra-long sequencing of the planarian genome or PacBio high-fidelity sequencing requires a DNA yield of >10 μg per single planarian. At present, both of the above two methods need to be purified twice, and their yields cannot meet the sequencing requirements, and it is impossible to obtain sufficient high-molecular-weight DNA for ultra-long sequencing or PacBio high-fidelity sequencing from a single Dugesia japonica, which greatly limits the assembly, annotation and downstream applications of the Dugesia japonica genome. Summary of the Invention

[0006] In view of this, the present invention provides a method for extracting high-molecular-weight genomic DNA from a single planarian. By removing ethylenediaminetetraacetic acid from the lysis buffer and increasing Mg 2+ Inhibit the activity of deoxyribonuclease II in planarians to inhibit the degradation of genomic DNA, and at the same time simplify the experimental steps, especially reduce the DNA purification steps, so as to reduce DNA loss and increase the yield of genomic DNA from a single planarian, meeting the requirements of ultra-long sequencing of a single planarian genome or PacBio high-fidelity sequencing (>10 μg / worm).

[0007] To achieve the above object, the present invention adopts the following technical solutions:

[0008] A method for extracting high molecular weight planarian genomic DNA, comprising the following steps:

[0009] (1) Take a single planarian, wash it with sterile deionized water, and then add N-acetylcysteine solution to remove the mucus on the surface of the planarian;

[0010] (2) After freezing the planarian in step (1) with liquid nitrogen, add the frozen planarian to the lysis solution for digestion, and add sodium dodecyl sulfate after restoring to room temperature;

[0011] (3) Add a mixture of phenol, chloroform and isoamyl alcohol with the same volume as the system solution to denature the protein, and release the genomic DNA according to the characteristics that DNA is soluble in the aqueous phase and protein is soluble in the organic phase, and centrifuge to take the aqueous phase;

[0012] (4) Add a mixture of chloroform and isoamyl alcohol with the same volume as the aqueous phase to the aqueous phase to remove a small amount of phenol in the aqueous phase, and centrifuge again to take the supernatant;

[0013] (5) Add ammonium acetate and isopropanol to the supernatant to precipitate DNA, and centrifuge to take the precipitate;

[0014] (6) Wash the precipitate with 70% ethanol, then centrifuge instantaneously and dry it, and add TE buffer solution to dissolve the DNA, where the TE buffer solution is 10 mM Tris-HCl, 1 mM ethylenediaminetetraacetic acid, pH 8.0.

[0015] On the basis of the above technical solutions, the present invention further limits the solution as follows:

[0016] Further, the final concentration of N-acetylcysteine aqueous solution in step (1) is 0.5%.

[0017] Further, the lysis solution in step (2) is a lysis solution containing 10 - 40 mM Mg 2+ and ribonuclease A. Preferably, the composition of the lysis solution is: 20 mM Tris pH 8.0, 100 mM sodium chloride, 0.2 mg / ml proteinase K, 0.2% β-mercaptoethanol, 20 mM Mg 2+ , 4 μg / μl ribonuclease A;

[0018] The addition amount of the lysis solution is 1 ml of the lysis solution for every 20 mg of planarian tissue;

[0019] The concentration of sodium dodecyl sulfate is 10%, and the addition amount is such that the content of sodium dodecyl sulfate added to the system is 1%.

[0020] Further, in the mixed solution of phenol, chloroform and isopentyl alcohol in step (3), the volume ratio of phenol:chloroform:isopentyl alcohol is 25:24:1.

[0021] Further, in the mixed solution of chloroform and isopentyl alcohol in step (4), the volume ratio of chloroform:isopentyl alcohol is 24:1.

[0022] Further, step (2) also includes the operation of adding to the lysis solution, pipetting until there are no obvious tissue clumps, and then adding sodium dodecyl sulfate.

[0023] Further, the number of centrifugations in step (4) is 2 times to ensure that there is no phenol contamination in the aqueous phase.

[0024] Further, in step (5), the addition amount of ammonium acetate is 0.3 times the volume of the supernatant, and the addition amount of isopropanol is 0.7 - 1.0 times the volume of the supernatant.

[0025] The present invention also provides the application of the above method for extracting high - molecular - weight planarian genomic DNA in ultra - long sequencing and / or PacBio high - fidelity sequencing.

[0026] The beneficial effects of the present invention are as follows:

[0027] For the conventional method for extracting genomic DNA from planarians (such as *Dugesia mediterranea*), such as the guanidine thiocyanate lysis method, although it can obtain high - molecular - weight genomic DNA, the yield is extremely low (<~3 μg / worm), and a large amount of RNA is mixed in the genomic DNA, and further digestion and purification of DNA are required; another classic Tris - HCl - phenol - chloroform method will cause a large amount of DNA degradation in *Dugesia japonica*. The main reason is that the content and activity of deoxyribonuclease II in *Dugesia japonica* cells are high, and the temperature range for its cleavage action is large (0 - 55 °C).

[0028] The method of the present invention for rapidly extracting >10 μg of genomic DNA from a single *Dugesia japonica* (~1 cm, <20 mg / worm) is to improve the lysis solution formula, remove ethylenediaminetetraacetic acid on the basis of conventional reagents in the classic Tris - HCl - phenol - chloroform method, and add an appropriate concentration of Mg 2+ , Mg 2+ As an inhibitor of deoxyribonuclease II activity, its addition can inhibit the activity of the main deoxyribonuclease II in planarian cells; however, the addition of Mg 2+ can also activate other deoxyribonucleases (such as deoxyribonuclease I, TatD deoxyribonuclease, etc.) at the same time. Therefore, selecting an appropriate concentration of Mg 2+ can inhibit the activity of the main deoxyribonuclease II in planarian cells without over - activating other deoxyribonucleases, thereby obtaining high - molecular - weight DNA. Through Mg2+ In the concentration gradient experiment, it was found that the lysis solution contained 10 - 40 mM Mg 2+ High molecular weight genomic DNA can be obtained, and 20 mM Mg 2+ is the optimal concentration for stable extraction of high molecular weight DNA. Concentrations less than 10 mM or greater than 50 mM will cause degradation of genomic DNA;

[0029] In addition, ribonuclease A was added to the lysis solution in the present invention, which can degrade RNA in cells during the digestion of tissue cells, thus eliminating two purification steps, reducing the loss of DNA yield, simplifying the experimental steps, and obtaining > 10 μg of high molecular weight DNA from a single planarian. Description of the Drawings

[0030] Figure 1 This is the electrophoretic detection comparison diagram of the DNA extracted in Example 1 and Comparative Examples 1 - 2 of the present invention;

[0031] Figure 2 This is the electrophoretic detection comparison diagram of the DNA extracted in Example 1 and Comparative Example 3 of the present invention;

[0032] Figure 3 This is the electrophoretic detection comparison diagram of the DNA extracted in Example 2 and Comparative Example 4 of the present invention. Detailed Description of the Invention

[0033] Next, the technical solutions in the embodiments of the present invention will be clearly and completely described in conjunction with the accompanying drawings in the embodiments of the present invention. Obviously, the described embodiments are only a part of the embodiments of the present invention, rather than all of the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those of ordinary skill in the art without creative efforts fall within the scope of protection of the present invention.

[0034] Example 1

[0035] A method for extracting high molecular weight planarian genomic DNA, using Dugesia japonica collected from Yiyuan County, Zibo City, Shandong Province as a sample, and the operation steps are as follows:

[0036] 1. Take 1 planarian about 1 cm long and wash it 2 times with sterilized deionized water, and weigh it (~10 mg);

[0037] 2. Add a solution of N - acetylcysteine with a final concentration of 0.5% to remove the mucus on the surface of the planarian;

[0038] 3. Quick - freeze in liquid nitrogen, add 1 ml of lysis solution containing different concentrations of Mg 2+ and ribonuclease A (20 mM Tris pH8.0, 100 mM sodium chloride, 0.2 mg / ml proteinase K, 0.2% β - mercaptoethanol, 0 - 50 mM Mg2+ , 4 μg / μl ribonuclease A), pipette repeatedly until there are no large tissue clumps in the solution. Then add 10% sodium dodecyl sulfate to a final concentration of 1%;

[0039] 4. Place the tissue suspension in a 55 °C water bath and digest for about 4 h (or overnight);

[0040] 5. Add an equal volume of phenol:chloroform:isoamyl alcohol (25:24:1) mixture, and rotate at the minimum speed (∼80 rpm) on a rotary shaker at room temperature for 10 min;

[0041] 6. Centrifuge at 10,000 × g for 5 min at 4 °C and take the supernatant;

[0042] 7. Add an equal volume of chloroform:isoamyl alcohol (24:1) mixture, and rotate at the minimum speed (∼80 rpm) on a rotary shaker at room temperature for 10 min;

[0043] 8. Centrifuge at 10,000 × g for 10 min at 4 °C and take the supernatant;

[0044] 9. Add 0.3 volume of sodium acetate and 0.7 - 1.0 volume of isopropanol to the supernatant, centrifuge at 5500 × g for 20 min, and discard the supernatant;

[0045] 10. Add 1 ml of 70% ethanol and wash 3 times;

[0046] 12. Air dry for 3 min;

[0047] 13. Add 200 μl of TE buffer and dissolve overnight at 4 °C;

[0048] 14. The next day, use a NanoDrop spectrophotometer and a QubitTM fluorometer to measure the DNA concentration and purity;

[0049] 15. Quantify 200 ng of genomic DNA for agarose gel electrophoresis and pulsed field gel electrophoresis.

[0050] After electrophoresis, use a gel imager to detect the quality of DNA extraction. The results are as Figure 1 shown, Figure 1 in which, A, genomic DNA detected by agarose gel electrophoresis; lane E, genomic DNA of Dugesia japonica extracted using the classical Tris-HCl-phenol-chloroform method (Comparative Example 1); Mg 2+ (0 - 50 mM), using different concentrations of Mg 2+Genomic DNA extracted with the improved lysis solution; GTC, genomic DNA extracted using the guanidine thiocyanate lysis method (Comparative Example 2); 1, 2, parallel samples of single planarians using the guanidine thiocyanate lysis method; M1, 1kb DNA Marker; M2, λ-HindⅢ digest DNA Marker; B, pulsed-field gel electrophoresis to detect the above genomic DNA; Lane E, genomic DNA extracted using the classical Tris-HCl-phenol-chloroform method (Comparative Example 1); Mg 2+ (0 - 50 mM), genomic DNA extracted with the improved lysis solution containing different concentrations of Mg 2+ ; GTC, genomic DNA extracted using the guanidine thiocyanate lysis method (Comparative Example 2); 1, 2, parallel samples of single planarians using the guanidine thiocyanate lysis method; M, LambdaPFG Ladder; Figure 1 As can be seen in 2+ , adding Mg 2+ to the lysis solution has a protective effect on the DNA fragments of *Dugesia japonica*. 20 mM Mg

[0051] is the optimal concentration for extracting the genome. The length of the extracted genomic DNA > 60 Kb, and there is no RNA contamination in the DNA. The genomic DNA amount of a single planarian > 10 μg. Figure 2 And as can be seen from 2+ , the method of the present invention can stably extract planarian genomic DNA multiple times. Once the Mg Figure 2 concentration in the lysis solution exceeds 50 mM, the genomic DNA will degrade ( 2+ left); 20 mM Mg Figure 2 is the optimal concentration for extracting the genome and has good repeatability ( Figure 2 , A, agarose gel electrophoresis to detect genomic DNA, 20, genomic DNA extracted with 20 mM Mg 2+ lysis solution; 50, genomic DNA extracted with 50 mM Mg 2+ lysis solution; 1 - 8, parallel samples of genomic DNA extracted from single planarians using 20 mM Mg 2+ lysis solution; B, pulsed-field gel electrophoresis to detect the above extracted genomic DNA.

[0052] Example 2

[0053] Method for extracting high-molecular-weight planarian genomic DNA, using *Dugesia japonica* collected from Boshan County, Zibo City, Shandong Province (belonging to different water systems from Yiyuan County) as samples, the operation steps are as follows:

[0054] 1. Take 1 planarian about 1 cm long and wash it 2 times with sterilized deionized water, and weigh it (<20 mg);

[0055] 2. Add 0.5% N-acetylcysteine solution to remove the mucus on the surface of planarians;

[0056] 3. Quickly freeze in liquid nitrogen, add 1 ml of lysis buffer containing different concentrations of Mg 2+ and ribonuclease A (20 mM Tris pH 8.0, 100 mM sodium chloride, 0.2 mg / ml proteinase K, 0.2% β-mercaptoethanol, 0 - 50 mM Mg 2+ , 4 μg / μl ribonuclease A), and pipette repeatedly until there are no large tissue clumps in the solution. Then add 10% sodium dodecyl sulfate to a final concentration of 1%;

[0057] 4. Place the tissue suspension in a 55°C water bath and digest for about 4 h (or overnight);

[0058] 5. Add an equal volume of phenol:chloroform:isoamyl alcohol (25:24:1) mixture, and rotate at the minimum speed (~80 rpm) on a rotor shaker at room temperature for 10 min;

[0059] 6. Centrifuge at 10,000×g for 5 min at 4°C, and take the supernatant;

[0060] 7. Add an equal volume of chloroform:isoamyl alcohol (24:1) mixture, and rotate at the minimum speed (~80 rpm) on a rotor shaker at room temperature for 10 min;

[0061] 8. Centrifuge at 10,000×g for 10 min at 4°C, and take the supernatant;

[0062] 9. Add 0.3 volume of ammonium acetate and 0.7 - 1.0 volume of isopropanol to the supernatant, centrifuge at 5500×g for 20 min, and discard the supernatant;

[0063] 10. Add 1 ml of 70% ethanol and wash 3 times;

[0064] 11. Centrifuge instantaneously for 5 s;

[0065] 12. Air dry naturally for 3 min;

[0066] 13. Add 200 μl of TE buffer and dissolve overnight at 4°C;

[0067] 14. The next day, use a NanoDrop spectrophotometer and a QubitTM fluorometer to measure the DNA concentration and purity;

[0068] 15. Quantify 200 ng of genomic DNA for agarose gel electrophoresis and pulsed-field gel electrophoresis.

[0069] After electrophoresis, use a gel imager to detect the quality of DNA extraction. The results are as Figure 3 shown.Figure 3 Among them, A, genomic DNA was detected by agarose gel electrophoresis; lane E, genomic DNA of *Dugesia japonica* (collected from Boshan) extracted by the classical Tris-HCl-phenol-chloroform method (Comparative Example 4); Mg 2+ (0 - 50 mM), genomic DNA extracted using a modified lysis solution containing different concentrations of Mg 2+ ; M1, 1Kb DNA Marker; M2, λ-HindⅢ digest DNA Marker; B, the above genomic DNA was detected by pulsed field gel electrophoresis. M, LambdaPFG Ladder; Figure 3 As can be seen in [Figure], adding Mg 2+ to the lysis solution has a protective effect on the DNA fragments of *Dugesia japonica* collected from another water system in Zibo. 20 mM Mg 2+ is the optimal concentration for extracting genomic DNA. The length of the extracted genomic DNA > 60 Kb, and there is no RNA contamination in the DNA. The genomic DNA amount of a single planarian can reach 12 μg.

[0070] Comparative Example 1

[0071] The genomic DNA of *Dugesia japonica* collected from Yiyuan County, Zibo City, Shandong Province was extracted by the classical Tris-HCl-phenol-chloroform method. The operation steps are as follows:

[0072] 1. Take 1 planarian about 1 cm long and wash it twice with sterilized deionized water, and weigh it (< 20 mg);

[0073] 2. Add 0.5% N-acetylcysteine solution to remove the mucus on the surface of the planarian;

[0074] 3. Quick-freeze in liquid nitrogen, add 900 μl of frozen lysis solution containing ethylenediaminetetraacetic acid and ribonuclease A (20 mM Tris pH 8.0, 100 mM sodium chloride, 0.2 mg / ml proteinase K, 0.2% β-mercaptoethanol, 50 nM ethylenediaminetetraacetic acid, 4 μg / μl ribonuclease A), and pipette repeatedly until there are no large tissue blocks in the solution. Then add 100 μl of 10% sodium dodecyl sulfate to a final concentration of 1%;

[0075] 4. Place the tissue suspension in a 55 °C water bath and digest for about 4 h (or overnight);

[0076] 5. Add an equal volume of phenol:chloroform:isoamyl alcohol (25:24:1) mixture, and rotate at the minimum speed (~80 rpm) on a rotary shaker at room temperature for 10 min;

[0077] 6. Centrifuge at 4 °C, 10000×g for 5 min, and take the supernatant;

[0078] 7. Add an equal volume of chloroform:isoamyl alcohol (24:1) mixture, and rotate it on a rotor shaker at the minimum speed (~80 rpm) for 10 min at room temperature;

[0079] 8. Centrifuge at 10,000×g for 10 min at 4℃, and take the supernatant;

[0080] 9. Add 0.3 times the volume of ammonium acetate and 0.7 - 1.0 times the volume of isopropanol to the supernatant, centrifuge at 5,500×g for 20 min, and discard the supernatant;

[0081] 10. Add 1 ml of 70% ethanol and wash 3 times;

[0082] 11. Centrifuge instantaneously for 5 s;

[0083] 12. Air dry naturally for 3 min

[0084] 13. Add 200 μl of TE buffer and dissolve overnight at 4℃;

[0085] 14. The next day, use a NanoDrop spectrophotometer and a QubitTM fluorometer to measure the DNA concentration and purity;

[0086] 15. Quantify 200 ng of genomic DNA for agarose gel electrophoresis and pulsed-field gel electrophoresis;

[0087] After electrophoresis, use a gel imager to detect the quality of DNA extraction. The results are as Figure 1 (left) lane E shows that the genomes extracted by this method are all highly degraded, and no high-molecular-weight genomic DNA is obtained.

[0088] Comparative Example 2

[0089] Extract genomic DNA of Dugesia japonica collected from Yiyuan County, Zibo City, Shandong Province by the guanidine thiocyanate lysis method. The operation steps are as follows:

[0090] 1. Take 1 Dugesia japonica about 1 cm long, wash it 2 times with sterilized deionized water, and weigh it (<20 mg);

[0091] 2. Add 0.5% N-acetylcysteine solution to remove the mucus on the surface of Dugesia japonica;

[0092] 3. Add 300 μl of frozen GTC buffer (4 M guanidine thiocyanate, 25 mM sodium chloride, 0.5% N-lauroylsarcosine, 7% β-mercaptoethanol), and lyse on ice for 30 min until there are no tissue blocks;

[0093] 4. Add 200 μl of TE buffer to make the total volume 500 μl;

[0094] 5. Add 500 μl of phenol:chloroform:isoamyl alcohol (25:24:1) mixture, centrifuge at 10,000×g for 10 min, and take the supernatant;

[0095] 7. Add an equal volume of chloroform:isoamyl alcohol (24:1) mixture, centrifuge at 4 °C and 10,000×g for 10 min, and take the supernatant;

[0096] 9. Add 500 μl of pre-cooled isopropanol and 500 μl of 5 M sodium chloride, and place on ice for 10 - 30 min;

[0097] 10. Centrifuge at the maximum speed at room temperature for 15 min, and take the supernatant;

[0098] 11. Add 1 ml of 70% ethanol and wash once;

[0099] 12. Centrifuge instantaneously for 5 s;

[0100] 13. Air-dry naturally for 3 min;

[0101] 14. Add 200 μl of TE buffer and dissolve overnight at 4 °C;

[0102] 15. The next day, use a NanoDrop spectrophotometer and a QubitTM fluorometer to measure the DNA concentration and purity;

[0103] 16. Quantify 200 ng of genomic DNA for agarose gel electrophoresis and pulsed-field gel electrophoresis.

[0104] After electrophoresis, use a gel imager to detect the quality of DNA extraction. The results are as Figure 1 (right) shows that although this extraction method can obtain high-molecular-weight DNA, its yield is low, only ~3 μg, and a large amount of RNA contamination is contained in the genomic DNA.

[0105] Comparative Example 3

[0106] 50 mM Mg 2+ Extract the genomic DNA of Dugesia japonica collected from Yiyuan County, Zibo City, Shandong Province using the lysis solution. The operation steps are as follows:

[0107] 1. Take 1 planarian about 1 cm long, wash it twice with sterilized deionized water, and weigh it (<20 mg);

[0108] 2. Add 0.5% N-acetylcysteine solution to remove the mucus on the surface of the planarian;

[0109] 3. Quick-freeze in liquid nitrogen, add 900 μl of frozen lysis buffer containing ethylenediaminetetraacetic acid and ribonuclease A (20 mM Tris pH 8.0, 100 mM NaCl, 0.2 mg / ml proteinase K, 0.2% β-mercaptoethanol, 50 mM Mg 2+ , 4 μg / μl ribonuclease A), and pipette repeatedly until there are no large tissue chunks in the solution. Then add 100 μl of 10% sodium dodecyl sulfate to a final concentration of 1%;

[0110] 4. Place the tissue suspension in a 55 °C water bath and digest for about 4 h (or overnight);

[0111] 5. Add an equal volume of phenol:chloroform:isoamyl alcohol (25:24:1) mixture, and rotate at the minimum speed (~80 rpm) on a rotary shaker at room temperature for 10 min;

[0112] 6. Centrifuge at 10,000 × g for 5 min at 4 °C and take the supernatant;

[0113] 7. Add an equal volume of chloroform:isoamyl alcohol (24:1) mixture, and rotate at the minimum speed (~80 rpm) on a rotary shaker at room temperature for 10 min;

[0114] 8. Centrifuge at 10,000 × g for 10 min at 4 °C and take the supernatant;

[0115] 9. Add 0.3 times the volume of ammonium acetate and 0.7 - 1.0 times the volume of isopropanol to the supernatant, centrifuge at 5500 × g for 20 min, and discard the supernatant;

[0116] 10. Add 1 ml of 70% ethanol and wash 3 times;

[0117] 11. Centrifuge instantaneously for 5 s;

[0118] 12. Air-dry naturally for 3 min;

[0119] 13. Add 200 μl of TE buffer and dissolve overnight at 4 °C;

[0120] 14. The next day, measure the DNA concentration and purity using a NanoDrop spectrophotometer and a QubitTM fluorometer;

[0121] 15. Quantify 200 ng of genomic DNA for agarose gel electrophoresis and pulsed-field gel electrophoresis.

[0122] After electrophoresis, use a gel imager to detect the quality of DNA extraction. The results are as Figure 2 (left) shown. The genomic DNA extracted by this method shows partial degradation, and its DNA is compared with 20 mM Mg 2+The DNA extracted by the lysis solution has a slightly smaller molecular weight, from which it can be known that the lysis solution contains Mg 2+ When the concentration exceeds 50 mM, it will cause degradation of genomic DNA.

[0123] Comparative Example 4

[0124] Extract the genomic DNA of Dugesia japonica collected from Boshan County, Zibo City, Shandong Province (belonging to different water systems from Yiyuan County) by the classic Tris-HCl-phenol-chloroform method. The operation steps are as follows:

[0125] 1. Take 1 planarian about 1 cm long and wash it 2 times with sterilized deionized water, and weigh it (<20 mg);

[0126] 2. Add 0.5% N-acetylcysteine solution to remove the mucus on the surface of the planarian;

[0127] 3. Quick-freeze in liquid nitrogen, add 900 μl of frozen lysis solution containing ethylenediaminetetraacetic acid and ribonuclease A (20 mM Tris pH 8.0, 100 mM sodium chloride, 0.2 mg / ml proteinase K, 0.2% β-mercaptoethanol, 50 nM ethylenediaminetetraacetic acid, 4 μg / μl ribonuclease A), and pipette repeatedly until there are no large tissue chunks in the solution. Then add 100 μl of 10% sodium dodecyl sulfate to a final concentration of 1%;

[0128] 4. Place the tissue suspension in a 55°C water bath and digest for about 4 h (or overnight);

[0129] 5. Add an equal volume of phenol:chloroform:isoamyl alcohol (25:24:1) mixture, and rotate it on a rotary shaker at the minimum speed (~80 rpm) at room temperature for 10 min;

[0130] 6. Centrifuge at 4°C, 10000×g for 5 min, and take the supernatant;

[0131] 7. Add an equal volume of chloroform:isoamyl alcohol (24:1) mixture, and rotate it on a rotary shaker at the minimum speed (~80 rpm) at room temperature for 10 min;

[0132] 8. Centrifuge at 4°C, 10000×g for 10 min, and take the supernatant;

[0133] 9. Add 0.3 times the volume of ammonium acetate, 0.7 - 1.0 times the volume of isopropanol to the supernatant, centrifuge at 5500×g for 20 min, and discard the supernatant;

[0134] 10. Add 1 ml of 70% ethanol and wash 3 times;

[0135] 11. Centrifuge instantaneously for 5 s;

[0136] 12. Air dry naturally for 3 min

[0137] 13. Add 200 μl of TE buffer and dissolve overnight at 4 °C;

[0138] 14. The next day, measure the DNA concentration and purity using a NanoDrop spectrophotometer and a QubitTM fluorometer;

[0139] 15. Quantify 200 ng of genomic DNA for agarose gel electrophoresis and pulsed-field gel electrophoresis;

[0140] After electrophoresis, use a gel imager to detect the quality of DNA extraction. The results are as Figure 3 shown in lane E. The genomes extracted by this method were all highly degraded, and no high-molecular-weight genomic DNA was obtained.

Claims

1. A method for extracting high-molecular-weight planarian genomic DNA, characterized in that, It includes the following steps: (1) Take a single planarian, wash it with sterilized deionized water, and then add it to an N-acetylcysteine solution to remove the mucus on the surface of the planarian. (2) Freeze the planarian in step (1) with liquid nitrogen, then add the frozen planarian to a lysis solution for digestion. After restoring to room temperature, immediately add sodium dodecyl sulfate. (3) Add a mixed solution of phenol, chloroform, and isoamyl alcohol with an equal volume to denature the protein and release genomic DNA, and centrifuge to take the aqueous phase. (4) Add a mixed solution of chloroform and isoamyl alcohol with an equal volume to the aqueous phase to remove phenol, and centrifuge again to take the supernatant. (5) Add ammonium acetate and isopropanol to the supernatant to precipitate DNA, and centrifuge to take the precipitate. (6) Wash the precipitate with 70% ethanol, dry it, and add TE buffer to dissolve the DNA.

2. The extraction method according to claim 1, wherein In step (1), the final concentration of the N-acetylcysteine aqueous solution is 0.5%.

3. The extraction method according to claim 1, characterized in that, In step (2), the lysis solution is a lysis solution containing 10-40 mM Mg 2+ and ribonuclease A; The addition amount of the lysis solution is 1 ml of the lysis solution for every 20 mg of planarian tissue. The concentration of sodium dodecyl sulfate is 10%, and the addition amount is such that the content of sodium dodecyl sulfate in the system is 1%.

4. The extraction method according to claim 3, characterized in that, In step (2), the lysis solution consists of: 20 mM Tris pH 8.0, 100 mM sodium chloride, 0.2 mg / ml proteinase K, 0.2% β-mercaptoethanol, 20 mM Mg 2+ , and 4 μg / μl ribonuclease A.

5. The extraction method according to claim 1, characterized in that, In the mixed solution of phenol, chloroform, and isoamyl alcohol in step (3), the volume ratio of phenol:chloroform:isoamyl alcohol is 25:24:

1.

6. The extraction method according to claim 1, wherein In the mixed solution of chloroform and isoamyl alcohol in step (4), the volume ratio of chloroform:isoamyl alcohol is 24:

1.

7. The extraction method according to claim 1, wherein Step (2) also includes the operation of blowing and beating until there are no obvious tissue lumps after adding it to the lysis solution and then adding sodium dodecyl sulfate.

8. The extraction method according to claim 1, wherein The number of centrifugations in step (4) is 2 times.

9. The extraction method according to claim 1, wherein In step (5), the addition amount of ammonium acetate is 0.3 times the volume of the supernatant, and the addition amount of isopropanol is 0.7 - 1.0 times the volume of the supernatant.

10. Use of the method for extracting high-molecular-weight planarian genomic DNA according to any one of claims 1 to 9 in ultra-long sequencing and / or PacBio high-fidelity sequencing.

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