Method for extracting DNA and its use

Abstract

The application provides a DNA extraction method and application thereof. The DNA extraction method comprises the following steps: crushing cells of a sample to obtain a first mixture; mixing the first mixture with a hexammine cobalt (III) chloride solution and an adsorption carrier, performing first incubation, and obtaining a first incubation product; washing and dissolving the first incubation product to obtain DNA of the sample. The method can solve the problems of the prior art, such as complexity of the DNA extraction method and low quality of the obtained DNA, and is suitable for the field of DNA extraction.

Description

Technical Field

[0001] This invention relates to the field of DNA extraction, and more specifically, to a method for DNA extraction and its application. Background Technology

[0002] Illumina's high-throughput sequencing platform has become a mainstream technology platform in fields such as genomics research, clinical molecular diagnostics, crop breeding, and biodiversity analysis due to its advantages such as high sequencing throughput, excellent accuracy, and low unit data cost. However, the Illumina library construction process has extremely strict requirements on the quality of the input DNA, which must meet the following core indicators: (1) high purity, i.e., no residual impurities such as proteins, polysaccharides, phenols, RNA, and excess salt ions; (2) high molecular weight, i.e., low degree of DNA fragmentation, with N50 usually ≥50 kb, to ensure the uniformity and coverage depth of the library construction; (3) no enzyme inhibition, i.e., the extracted products do not contain inhibitors that can inhibit tagging, PCR amplification, or ligation reactions. Failure to meet any of these indicators may lead to library construction failure, decreased sequencing data quality, reduced effective data rate, or increased adapter contamination rate.

[0003] Currently, the mainstream methods for extracting plant genomic DNA and constructing Illumina libraries mainly fall into the following three categories:

[0004] (1) Conventional CTAB-phenol / chloroform extraction method: This method utilizes CTAB to form a complex with nucleic acids under high salt conditions, and then removes proteins and lipids through phenol-chloroform-isoamyl alcohol extraction. Although it has a certain ability to lyse complex plant tissues (such as old leaves or xylem rich in polysaccharides and phenols), the operation process is cumbersome, requiring multiple centrifugations and repeated extraction with organic solvents. This not only poses a significant risk of DNA mechanical shearing, but also easily leads to a sharp drop in recovery rate, especially in low input samples. Moreover, toxic reagents such as phenol and chloroform pose a potential threat to the health of experimental personnel and environmental safety. In addition, residual chloroform and salt ions often inhibit subsequent tagmentation enzyme reactions and PCR amplification, affecting the efficiency of library construction.

[0005] (2) Silica gel membrane column extraction: This method is based on the specific binding of nucleic acids to silica gel membranes under high salt and low pH conditions, which enables rapid purification. It is simple to operate, time-saving, and suitable for standardized procedures. However, it is highly dependent on the sample lysis efficiency. For plant samples with thick cell walls, high polysaccharide content, or hard tissues (such as poplar wood and old tea leaves), the DNA adsorption efficiency is significantly reduced, and the recovery rate is generally less than 50%. At the same time, the cost of column reagents is high, and the cost of a single extraction is about 5-10 times that of traditional methods, making it difficult to apply to large-scale sample screening.

[0006] (3) Modified solid-phase extraction: Some commercial kits use magnetic beads or novel adsorption materials to replace silica membranes, which improves the recovery rate to some extent, but still depends on the adequacy of the pre-lysis step, and has limited ability to remove plant-derived polysaccharides and secondary metabolites, often resulting in a low A260 / A230 ratio (<1.8), affecting the stability of library construction. In addition, the above methods generally lack an active protection mechanism for the integrity of DNA molecules (especially the N50 value), and high molecular weight DNA is still easily broken in the grinding, centrifugation, washing and other steps.

[0007] In summary, existing plant genomic DNA extraction technologies generally suffer from problems such as complex processes, high reagent toxicity, low recovery rates for low-input samples, severe DNA fragmentation, and residual impurities inhibiting enzyme reactions. In particular, when processing difficult-to-lyse tissues or trace samples, it is difficult to simultaneously meet the three major requirements of high purity, high integrity, and low cost. Summary of the Invention

[0008] The main objective of this invention is to provide a method for DNA extraction and its application, in order to solve the problems of complex DNA extraction methods and low quality of DNA extraction obtained in the prior art.

[0009] To achieve the above objectives, according to a first aspect of the present invention, a method for DNA extraction is provided, the method comprising: disrupting the cells of a sample to obtain a first mixture; mixing the first mixture with a solution of hexaamminecobalt(III) trichloride and an adsorbent carrier, performing a first incubation to obtain a first incubation product; washing the first incubation product and dissolving it to obtain the DNA of the sample.

[0010] Furthermore, the concentration of the above-mentioned hexaamminecobalt(III) trichloride solution is 15-25 mM.

[0011] Further, the adsorbent carrier includes one or more of glass beads, ceramic beads, or silica beads; preferably, the glass beads are selected from one or more of borosilicate glass beads, soda-lime glass beads, quartz glass beads, or high borosilicate glass beads; more preferably, the adsorbent carrier is the borosilicate glass beads; even more preferably, the diameter of the borosilicate glass beads is 2-3 mm; preferably, the surface of the adsorbent carrier is an unmodified original surface or a surface modified; preferably, the surface modification includes one or more of silanization treatment, hydroxylation treatment, or hydrophilization treatment.

[0012] Further, the first mixture is mixed with a solution of hexaamminecobalt(III) trichloride and borosilicate glass beads and then shaken to obtain the first incubation product; preferably, the shaking frequency is 8-15 rpm; preferably, the first incubation time is 5-20 min.

[0013] Further, the cell disruption method includes: mixing the sample with a lysis buffer and performing a second incubation to obtain the first mixture; preferably, the lysis buffer includes one or more of CTAB, β-mercaptoethanol, EDTA or Tris-HCl.

[0014] Furthermore, the temperature for the second incubation is 45~75℃, and the incubation time is 15~60min.

[0015] Further, the washing process includes: sequentially performing a first wash and a second wash on the first incubation product; the first wash includes mixing the first incubation product with a first washing solution, allowing it to stand, and then centrifuging to obtain a first washed product; the second wash includes mixing the first washed product with a second washing solution, allowing it to stand, and then centrifuging to obtain a second washed product; preferably, both the first and second washing solutions include alcohol solvents and nonionic surfactants; preferably, the alcohol solvent is selected from one or more of ethanol, isopropanol, or methanol; preferably, the nonionic surfactant is selected from one or more of Tween-20, Triton X-100, or NP-40; preferably, in the first wash, the standing time is 20-60 s, the centrifugation frequency is 6000-10000×g, and the centrifugation time is 1.5-3 min; preferably, in the second wash, the standing time is 20-60 s, the centrifugation frequency is 10000-13000×g, and the centrifugation time is 0.5-1.5 min.

[0016] Further, the above dissolution includes mixing the second washing product with the dissolving solution and performing a third incubation to obtain the DNA of the above sample; preferably, the above dissolving solution includes Tris-HCl and / or EDTA; preferably, the temperature of the above third incubation is 30~40°C and the time is 5~20 min.

[0017] Furthermore, the samples mentioned above include plant tissues.

[0018] To achieve the above objectives, according to a second aspect of the present invention, the application of DNA extracted using the above-described DNA extraction method in the construction of a gene library is provided.

[0019] The present invention utilizes a method where sample cells are lysed and then mixed with a solution of hexaamminecobalt(III) trichloride and borosilicate glass beads for a first incubation. The strong electrostatic neutralization of DNA phosphate groups by CoHex(III) promotes the formation of helical aggregates of genomic DNA, which are then efficiently adsorbed onto the surface of the glass beads. After gentle washing and low-shear dissolution, high-integrity DNA enrichment without phenol-chloroform extraction is achieved. Compared with existing technologies, this method reduces DNA fragmentation, improves DNA recovery rate and purity, and the obtained DNA can be directly used for Illumina library construction. Compared to existing DNA extraction methods, this method is simpler to operate, has lower extraction costs, and produces higher-quality DNA, making it more suitable for widespread application. Attached Figure Description

[0020] The accompanying drawings, which form part of this application, are used to provide a further understanding of the invention. The illustrative embodiments of the invention and their descriptions are used to explain the invention and do not constitute an undue limitation of the invention. In the drawings:

[0021] Figure 1 The diagram shows the DNA extraction results of Example 1 of this application specification.

[0022] Figure 2 The diagram shows the DNA extraction results of Example 2 of this application specification.

[0023] Figure 3 The diagram shows the DNA extraction results of Example 3 of this application specification.

[0024] Figure 4 The diagram shows the DNA extraction results of Example 4 of this application specification.

[0025] Figure 5 The diagram shows the DNA extraction results of Example 5 of this application specification. Detailed Implementation

[0026] It should be noted that, unless otherwise specified, the embodiments and features described in this application can be combined with each other. The present invention will now be described in detail with reference to the embodiments.

[0027] As mentioned in the background section, existing DNA extraction methods are complex and cumbersome, and the quality of the extracted DNA is unstable, making it difficult to construct Illumina libraries. Therefore, in this application, the inventors have attempted to develop a new DNA extraction method, and thus proposed a series of protection schemes.

[0028] In a first typical embodiment of this application, a method for DNA extraction is provided, the method comprising: disrupting the cells of a sample to obtain a first mixture; mixing the first mixture with a solution of hexaamminecobalt(III) trichloride (CoHex(III)) and an adsorbent carrier, performing a first incubation to obtain a first incubation product; and washing the first incubation product to obtain the DNA of the sample.

[0029] This application utilizes CoHex(III) to rapidly neutralize the negative charge of DNA in the cell lysate (the first mixture mentioned above) through strong electrostatic attraction, promoting the aggregation of DNA segments and the formation of a precipitate. This precipitate is efficiently adsorbed onto the added borosilicate glass beads. Since the RNA in the first mixture has different secondary structures and phosphate group densities, it remains soluble under low-concentration CoHex(III) conditions. Therefore, the DNA extraction method of this application eliminates the need for RNase to remove RNA, thus separating RNA-free DNA and avoiding the introduction of RNase reagents. This method also eliminates the need for phenol-chloroform extraction, achieving high recovery rates and low fragmentation of DNA from low-input samples, and is directly compatible with Illumina standard library construction workflows.

[0030] This application introduces a hexaamminecobalt(III) chloride solution and borosilicate glass beads into the lysate (the first mixture mentioned above) after cell disruption for a first incubation. This allows the hexaamminecobalt(III) chloride metal ions to efficiently neutralize the negative charge of DNA molecules, inducing their selective accumulation on the glass bead surface and the formation of stable aggregates. This achieves the physical separation of DNA from proteins, polysaccharides, and phenolic impurities. This process completely replaces the traditional phenol-chloroform extraction step, avoiding the shearing damage to the DNA backbone caused by strong organic solvents, and significantly improving the integrity and yield of genomic DNA. Subsequently, the glass beads are washed to remove residual salts and impurities, and a conventional resolution step yields high-purity, high-molecular-weight genomic DNA. The DNA extraction method of this application is simple to operate, requires no centrifuge columns or complex equipment, reduces operational risks and environmental pollution, and has potential for large-scale application. In a preferred embodiment, the concentration of the hexaamminecobalt(III) chloride solution is 15-25 mM. Preferably, it is 20-23 mM, more preferably 20 mM.

[0031] In this application, the concentration of hexaamminecobalt(III) trichloride solution is controlled within the range of 15-25 mM to promote good charge density and stability of hexaamminecobalt(III) ions in the solution. Through strong electrostatic attraction, the negative charge on nucleic acids is rapidly neutralized, promoting DNA aggregation and precipitation. This ensures stable adsorption of DNA onto the surface of borosilicate glass beads and avoids non-specific protein or polysaccharide co-precipitation due to excessive concentration, thereby further improving the purity and integrity of DNA recovery. At the same time, within this concentration range, the synergistic effect with the first incubation conditions ensures uniform and controllable DNA aggregation, avoiding DNA breakage or uneven adsorption caused by local ionic strength imbalance. This further improves the high yield and high molecular weight characteristics of DNA in the subsequent resolution step, and improves the fragmentation and low purity problems caused by the traditional phenol-chloroform method.

[0032] In a preferred embodiment, the adsorbent carrier comprises one or more of glass beads, ceramic beads, or silica beads; preferably, the glass beads are selected from one or more of borosilicate glass beads, soda-lime glass beads, quartz glass beads, or high borosilicate glass beads; more preferably, the adsorbent carrier is the borosilicate glass beads; even more preferably, the diameter of the borosilicate glass beads is 2-3 mm; preferably, the surface of the adsorbent carrier is an unmodified original surface or a surface modified; preferably, the surface modification includes one or more of silanization treatment, hydroxylation treatment, or hydrophilization treatment.

[0033] The above-mentioned adsorption carriers can be used to enrich and adsorb DNA. Optionally, adsorption carriers with surface modifications can be used to enhance the adsorption stability of DNA aggregates on the carrier surface and improve the DNA recovery rate.

[0034] Optionally, the above-mentioned adsorption carrier includes borosilicate glass beads. In this application, the diameter of the borosilicate glass beads is controlled at 2-3 mm, which allows the sample, such as plant tissue, to undergo sufficient and gentle mechanical disruption under the first incubation conditions, avoiding excessive shearing and DNA fragmentation due to excessively small particle size. Furthermore, at this diameter, its surface can provide a large specific surface area for efficient adsorption of [Co(NH3)6]³ + The DNA helical aggregates formed after neutralization of charge; at the same time, this diameter size is conducive to the separation of glass beads from impurities by centrifugation in the subsequent washing step, avoiding the impact of washing uniformity due to excessively large particles settling too quickly, or DNA loss due to difficulty in recovery due to excessively small particles. Thus, in the overall process, it works synergistically with the hexaamminecobalt(III) trichloride solution and the first incubation system to improve the DNA capture efficiency, recovery rate and integrity, and achieve stable extraction of high-purity and high-yield plant genomic DNA under phenol-chloroform-free conditions.

[0035] In a preferred embodiment, the first mixture is mixed with a solution of hexaamminecobalt(III) trichloride and borosilicate glass beads and then shaken to obtain a first incubation product; preferably, the shaking frequency is 8-15 rpm; preferably, the first incubation time is 5-20 min.

[0036] This application preferably utilizes a synergistic effect of hexaamminecobalt(III) chloride solution and borosilicate glass beads to achieve efficient aggregation and selective adsorption of plant genomic DNA. In the aforementioned first incubation, the oscillation frequency is controlled at 8-15 rpm and the incubation time is controlled at 5-20 min, allowing the ions in the hexaamminecobalt(III) chloride solution to fully form a stable complex with DNA molecules under mild conditions, while avoiding mechanical shearing of DNA caused by violent oscillation, thus improving DNA integrity and recovery rate. The first incubation system of this application, without introducing toxic solvents such as phenol and chloroform, inhibits nuclease activity and microbial contamination, reduces the residual rate of impurities, and makes it easier for subsequent DNA to be released from the surface of the glass beads while maintaining high purity, thereby further improving the reproducibility of extraction results, biosafety, and stability of downstream library construction and sequencing.

[0037] In a preferred embodiment, the cell disruption method includes: mixing the sample with a lysis buffer and performing a second incubation to obtain a first mixture. Preferably, the sample is subjected to tissue disruption before the second incubation; preferably, the tissue disruption method includes any disruption method known to those skilled in the art, such as grinding or mechanical disruption, and this application does not impose any limitation.

[0038] In a preferred embodiment, the lysis buffer includes one or more of CTAB, β-mercaptoethanol, EDTA, or Tris-HCl. Preferably, the lysis buffer of this application contains 2% CTAB, 1.4M NaCl, 25mM EDTA (pH 8.0), and 10 mM Tris-HCl (pH 8.0). Those skilled in the art can choose any lysis buffer according to actual needs, and all are applicable to this application.

[0039] In a preferred embodiment, the temperature for the second incubation is 45~75°C, and the incubation time is 15~60 min.

[0040] In this application, by incubating the sample with the above-mentioned lysis buffer at 45~75°C (i.e., the second incubation), the cell wall and membrane structure are effectively broken down and genomic DNA is released. In this embodiment, the temperature of the second incubation is set to 45~75°C and the time is controlled to 15~60 min. The setting of this temperature and time window is based on the optimization of the balance between cell lysis efficiency and DNA integrity: if the temperature is too low, the lysis may be incomplete and the DNA may not be released sufficiently; if the temperature is too high or the incubation time is too long, it may aggravate heat-induced DNA breakage or promote residual nuclease activity, thereby reducing the quality of DNA.

[0041] In a preferred embodiment, washing includes: sequentially performing a first wash and a second wash on the first incubation product; the first wash includes centrifuging the first incubation product with a first washing solution after standing to obtain a first washed product; the second wash includes centrifuging the first washed product with a second washing solution after standing to obtain a second washed product; preferably, both the first and second washing solutions include alcohol solvents and nonionic surfactants; preferably, the alcohol solvent is selected from one or more of ethanol, isopropanol, or methanol; preferably, the nonionic surfactant is selected from one or more of Tween-20, Triton X-100, or NP-40; preferably, in the first wash, the standing time is 20-60 s, the centrifugation frequency is 6000-10000×g, and the centrifugation time is 1.5-3 min; preferably, in the second wash, the standing time is 20-60 s, the centrifugation frequency is 10000-13000×g, and the centrifugation time is 0.5-1.5 min. Preferably, both the first and second washing solutions are 70% ethanol in 0.1% Tween-20.

[0042] In this embodiment, by performing the first and second washing steps separately and using specific combinations of washing solutions and centrifugation parameters, efficient and gentle decontamination of DNA aggregates adsorbed on the surface of borosilicate glass beads is achieved: the first washing removes most of the protein and polysaccharide impurities with low centrifugation force at 6000~10000×g for 1.5~3min, avoiding DNA breakage due to excessive shearing force; the second washing increases the centrifugation frequency to 10000~13000×g and shortens the time to 0.5~1min, so that the washing solution removes residual salts and lipids while reducing mechanical disturbance of DNA on the glass bead surface, maintaining the structural integrity of DNA helical aggregates, improving the purity of DNA recovery and the stability of the N50 value, and providing a reliable template for subsequent high-throughput sequencing or long fragment amplification.

[0043] In a preferred embodiment, dissolution includes mixing the second washing product with a dissolving solution and performing a third incubation to obtain the DNA of the sample; preferably, the dissolving solution includes Tris-HCl and / or EDTA; preferably, the temperature of the third incubation is 30~40°C and the time is 5~20 min.

[0044] In a preferred embodiment, the DNA dissolution step includes mixing the glass bead precipitate (i.e., the second washing product) after the second washing with a dissolving solution, and then performing a third incubation at a specific temperature to achieve desorption and complete dissolution of DNA from the glass bead surface, obtaining a genomic DNA product with high integrity and high purity. The dissolving solution preferably contains Tris-HCl and / or EDTA, preferably 10 mM Tris-HCl (pH 8.0), and optionally further contains 0.5 mM EDTA (pH 8.0). In this dissolving solution system, Tris-HCl provides a stable neutral buffer environment, maintaining the integrity of the DNA structure and avoiding alkaline hydrolysis caused by pH fluctuations; EDTA, as a metal ion chelating agent, further inhibits residual nuclease activity, preventing DNA degradation during resolution, thereby improving DNA stability. Those skilled in the art can select the appropriate solvent system according to the sample type or downstream application requirements.

[0045] Using the DNA extraction method described in this application, the A260 / A280 ratio is between 1.8 and 2.0, and the A260 / A230 ratio is between 2.0 and 2.2. Agarose gel electrophoresis shows no obvious diffusion of the DNA bands, and the main band is greater than 10K. The samples to be tested include nucleic acids extracted from various plant samples, and their illumin library pass rate is ≥90%.

[0046] In a preferred embodiment, the sample includes, but is not limited to, plant tissue.

[0047] The samples applicable to this application include, but are not limited to, tissue materials of various plant sources, covering, but not limited to, leaves, stems, roots, anthers, plumules, seeds, callus, xylem, phloem, fruits, seed coats, and spores, or trace tissues (such as anthers, endosperm, and single-cell calluses) from model plants such as rice, wheat, and Arabidopsis thaliana. The method of this application overcomes the technical bottlenecks of traditional methods in processing the above-mentioned samples, such as low recovery rate, severe fragmentation, and high impurity residue, through a CoHex(III)-mediated electrostatic coagulation mechanism and a mild glass bead adsorption strategy, demonstrating excellent universality and stability. Those skilled in the art can flexibly select applicable samples according to the plant species, tissue type, and sample input volume, all of which are within the scope of protection of this invention.

[0048] The DNA extraction method described in this application is based on the CTAB lysis system to release and preliminarily purify DNA from plant samples. It is suitable for Illumina library construction with low input sample volumes (cells, tissues, blood, <100mg), and eliminates the need for phenol-chloroform extraction. The method is simple to operate, low in cost, environmentally friendly and safe, and yields high-purity extracted DNA (A260 / A280 1.8-2.0), which meets the sequencing requirements of the Illumina platform. It is suitable for rapid detection of clinical samples and low-quality samples and has good prospects for commercialization.

[0049] It should be noted that the DNA extraction method described in this application constructs a tandem integrated "aggregation-adsorption-purification" system. CoHex(III) selectively neutralizes the negative charge of DNA in a CTAB / NaCl / Tris composite lysis buffer environment, inducing it to form helical aggregates. These aggregates are simultaneously captured in situ by the glass bead surface during their formation and are then purified efficiently through subsequent differentiated washing steps.

[0050] In a second typical embodiment of this application, the application of DNA extracted by the above-described DNA extraction method in the construction of a gene library is provided.

[0051] In the DNA extraction method of this application, the charge neutralization effect of CoHex(III) in the extraction system works synergistically with the decontamination ability of CTAB, combined with the gentle adsorption of DNA precipitate by glass beads, to construct a "lysis-coagulation-adsorption-purification" process that can seamlessly connect to library construction. The extracted DNA does not require additional purification steps and can be directly connected to Illumina DNA Prep or PCR-Free Prep kits, simplifying the process and shortening the time consumption.

[0052] The DNA extraction method of this application has the following advantages:

[0053] (1) Ease of operation: DNA is attracted by strong electrostatic attraction of CoHex (III) to form helical aggregates, which are gently adsorbed onto glass beads, eliminating the need for cumbersome multiple organic solvent extractions.

[0054] (2) Low fragmentation: DNA N50≥50 kb, which is more than 50% higher than the CTAB extraction method, meeting the requirements of Illumina library construction for high molecular weight DNA;

[0055] (3) High recovery rate: The amount of DNA in low input samples (<100mg) is increased by more than 30% compared with the conventional CTAB extraction method;

[0056] (4) High purity: A260 / A280=1.8-2.0, A260 / A230=2.0-2.2.

[0057] The beneficial effects of this application will be explained in more detail below with reference to specific embodiments.

[0058] The sample and reagent information for this embodiment is as follows:

[0059] 1. Experimental Samples

[0060] Three types of typical plant samples were selected to verify the universality of the method:

[0061] Easily lysable samples: Arabidopsis thaliana young leaves (50 mg / sample, fresh weight) and rice seedling leaves (80 mg / sample, fresh weight).

[0062] Difficult-to-lyse samples: Poplar wood tissue (fresh weight 100 mg / part), old tea leaves (fresh weight 100 mg / part).

[0063] Low-input samples: Arabidopsis callus (fresh weight 20 mg / part) and rice anthers (fresh weight 10 mg / part).

[0064] 2. Experimental reagents

[0065] CoHex(III): Hexaamminecobalt(III) trichloride, purity ≥98% (Sigma-Aldrich); CoHex(III) is hexaamminecobalt(III) trichloride. Add 0.268g of CoHex(III) powder to 8mL of Nuclease-Free Water, mix well to dissolve, and then add to a final volume of 10mL. Filter through a 0.22µm PVDF membrane for sterilization to prepare a 100mM stock solution. Store in the dark at low temperature. CTAB lysis buffer: Contains 2% CTAB, 1.4M NaCl, 20mM EDTA (pH 8.0), 2% β-mercaptoethanol (2-ME), and 100mM Tris-HCl (pH 8.0). Prepare fresh before use.

[0066] 100mM CoHex(III) stock solution: Weigh 0.268g of CoHex(III) powder, add 8mL of Nuclease-Free Water and mix well to dissolve. After complete dissolution, add to a final volume of 10mL. Filter through a 0.22μm PVDF membrane for sterilization and store at 4℃ protected from light.

[0067] Washing solution: 70% ethanol containing 0.1% Tween-20, prepared fresh for each use.

[0068] Elution buffer: 10 mM Tris-HCl (pH 8.0).

[0069] 48Kb Gene Ruler High Range DNA Ladder (Thermo Fisher).

[0070] Standard CTAB extraction reagents: phenol-chloroform-isoamyl alcohol (25:24:1), anhydrous ethanol, 3M sodium acetate (pH 5.2).

[0071] Commercially available column extraction kits;

[0072] VAHTS Universal Plus DNA Library Prep Kit V4 (Vazyme).

[0073] Example 1

[0074] This embodiment uses young leaves of Arabidopsis thaliana as a sample, and the specific steps are as follows:

[0075] Sample pretreatment: Take 50 mg of tender Arabidopsis thaliana leaves, place them in a pre-cooled mortar, add liquid nitrogen and grind them quickly into powder, then immediately transfer them to a 1.5 mL centrifuge tube;

[0076] Lysis: Add 600 μL CTAB lysis buffer to a centrifuge tube, vortex for 1 min to mix, and incubate in a 65°C water bath for 30 min, vortexing once every 10 min (10 s each time) to promote complete cell lysis.

[0077] Preliminary separation: Centrifuge at 13000×g at room temperature for 2 min, and carefully aspirate the supernatant (about 500 μL) into a new 1.5 mL centrifuge tube, avoiding aspiration of precipitate;

[0078] DNA aggregation and adsorption: Add 100 μL of 100 mM CoHex (III) stock solution (final concentration 20 mM) to the supernatant, then add 2 2 mm borosilicate glass beads, place the centrifuge tube on a three-dimensional mixer, and incubate at room temperature for 10 min at 9 rpm to promote the formation of helical aggregates of DNA and adsorption onto the surface of the glass beads.

[0079] First wash: Add 600 μL of washing solution to the centrifuge tube, let stand for 30 seconds, centrifuge at 8000×g at room temperature for 2 minutes, discard the supernatant, and retain the glass bead precipitate;

[0080] Second wash: Add 600 μL of washing solution again, let stand for 30 seconds, centrifuge at 12000×g at room temperature for 1 minute, remove the residual liquid with a pipette, and dry at room temperature for 30 seconds;

[0081] DNA dissolution: Add 50 μL of elution buffer to the centrifuge tube and incubate at 37°C for 10 min, gently tapping the centrifuge tube 3 times during this period to promote complete DNA dissolution; Collection and detection: Centrifuge at 12000×g at room temperature for 1 min, and aspirate the supernatant (containing genomic DNA) into a new centrifuge tube. Detect DNA concentration and purity (A260 / A280, A260 / A230) using a nucleic acid detection instrument. Detect DNA integrity by agarose gel electrophoresis. The results are shown in Table 1 and... Figure 1 As shown.

[0082] Table 1. Data on DNA extraction purity from young Arabidopsis leaves

[0083]

[0084] Example 2: Effect of different final concentrations of CoHex(III) on DNA extraction efficiency

[0085] This embodiment uses rice seedling leaves as samples, only changing the final concentration of CoHex(III), with the remaining steps the same as in Example 1, to explore the optimal CoHex(III) concentration. The specific groupings are as follows:

[0086] Group 1: CoHex(III) final concentration 8 mM;

[0087] Group 2: CoHex(III) final concentration 15 mM;

[0088] Group 3: CoHex(III) final concentration 20 mM;

[0089] Group 4: CoHex(III) final concentration 25 mM;

[0090] After extraction, the concentration, total amount, purity, and integrity of each DNA group were measured, and the results are shown in Table 2 and... Figure 2 As shown.

[0091] Table 2 Comparison of DNA extraction effects at different final concentrations of CoHex(III)

[0092]

[0093] Results analysis: As the final concentration of CoHex(III) increased, the DNA concentration, total amount, and integrity all showed a trend of first increasing and then stabilizing. The extraction effect was optimal at a final concentration of 20 mM, with a total DNA amount of 6.45 μg, A260 / A280 = 1.93, A260 / A230 = 2.03, all meeting high purity standards. The DNA N50 was 52.3 kb, indicating the lowest degree of fragmentation. No significant improvement in extraction effect was observed beyond 20 mM; therefore, 20 mM was determined to be the optimal final concentration for CoHex(III).

[0094] Example 3: The effect of different borosilicate glass bead diameters on DNA integrity

[0095] This embodiment uses poplar wood tissue as a sample, changing only the diameter of the borosilicate glass beads, while the remaining steps are the same as in Example 1 (final CoHex concentration 20mM), to explore the optimal glass bead diameter. The specific groupings are as follows:

[0096] Group 1: Glass beads with a diameter of 1mm;

[0097] Group 2: Glass beads with a diameter of 2mm;

[0098] Group 3: Glass beads with a diameter of 3mm;

[0099] Group 4: Glass beads with a diameter of 4mm.

[0100] After extraction, DNA integrity was detected by agarose gel electrophoresis, and concentration and purity were analyzed using a nucleic acid detector. The results are shown in Table 3. Figure 3 As shown.

[0101] Table 3 Comparison of DNA extraction effects with different glass bead diameters

[0102]

[0103] Results analysis: A 2mm glass bead diameter resulted in the optimal DNA extraction efficiency, achieving the highest concentration and total amount (9.93μg), with a DNA N50 of 53.7kb. The clear, non-diffuse bands on agarose gel electrophoresis indicated optimal DNA integrity. 1mm glass beads, due to their small size, resulted in overly tight DNA entanglement, increased spatial tension, and poor DNA integrity. 3mm and 4mm glass beads, being too large, reduced DNA adsorption efficiency. Therefore, 2mm was determined to be the optimal glass bead diameter.

[0104] Figure 3 A comparison of DNA integrity extracted from different borosilicate glass bead diameters; lane M is the DNA ladder, and lanes 1-4 correspond to glass bead diameters of 1mm, 2mm, 3mm, and 4mm, respectively.

[0105] Example 4

[0106] 1. Experimental Design

[0107] Three types of samples (Arabidopsis callus, rice anthers, and old tea leaves) were selected, and DNA was extracted using the following three methods:

[0108] Method A: The method of Example 1;

[0109] Method B: Conventional CTAB extraction method (including phenol-chloroform extraction step);

[0110] Method C: Commercially available column extraction kit method (follow the kit instructions).

[0111] Three replicates were set up for each sample. After extraction, the purity and integrity of DNA were tested. Then, the library was constructed using the VAHTSUniversal Plus DNA Library Prep Kit V4 (Vazyme) and the library pass rate and sequencing data quality were tested.

[0112] 2. Test Results

[0113] (1) Comparison of DNA extraction effects

[0114] The results are shown in Table 4 and Figure 4 As shown.

[0115] Table 4 Comparison of DNA extraction effects of the three methods

[0116]

[0117] (2) Comparison of library construction and sequencing results

[0118] The results are shown in Table 5.

[0119] Table 5. Comparison of sequencing data quality for library construction using three methods.

[0120]

[0121] 3. Results Analysis

[0122] (1) DNA extraction effect: The method of Example 1 of this application (method A) performed best in all types of samples. The low input amount samples (Arabidopsis callus, rice anthers) improved by more than 50% compared with the conventional CTAB method (method B) and by more than 20% compared with the column extraction method (method C). From the agarose gel spot application, the DNA integrity of different treatments was observed. The DNA N50 of this method was ≥50kb, and the fragmentation degree was significantly reduced compared with methods B and C. The A260 / A280 and A260 / A230 detection data were better than those of methods B and C.

[0123] (2) Library construction and sequencing results: Although the nucleic acids extracted from different samples by methods A, B and C were used to construct WGS small fragment libraries and all passed the library test, in terms of sequencing Q30, effective data volume and adapter contamination rate, method A was better than methods B and C. This indicates that the extracted DNA had no obvious impurities and could be directly adapted to the Illumina library construction process.

[0124] (3) Convenience and operational advantages: Method A does not require phenol-chloroform extraction, and the operation steps are reduced by 4 steps compared with Method B and by 2 steps compared with Method C, reducing the time consumption by more than 30%. At the same time, the cost is only one-fifth of that of Method C, which has significant practical value and commercial transformation prospects.

[0125] Example 5

[0126] 1. Experimental Design

[0127] Two types of samples (Arabidopsis thaliana leaves and rice leaves) were selected, and DNA was extracted using the following two methods:

[0128] Method A: The method of Example 1 is used, except that no adsorption carrier is used;

[0129] Method B: The method of Example 1 is adopted.

[0130] After extraction, the purity and integrity of the DNA were tested.

[0131] 2. Test Results

[0132] (1) Comparison of DNA extraction effects

[0133] The results are shown in Table 6 and Figure 5 As shown.

[0134] Table 6 Comparison of DNA extraction effects between the two methods

[0135]

[0136] As can be seen from the above description, the embodiments of the present invention achieve the following technical effects: By introducing a DNA aggregation and adsorption mechanism through the synergistic effect of CoHex(III) and borosilicate glass beads, this application eliminates the highly toxic and cumbersome phenol-chloroform extraction step in the traditional CTAB method, thereby improving the safety and environmental friendliness of the DNA extraction method; under mild incubation and low-frequency oscillation conditions, mechanical shearing of DNA during the extraction process is avoided, and the N50 value of the extracted product is stably maintained at 50. The DNA extraction method yields over 30% more DNA than conventional CTAB extraction, making it suitable for obtaining high-integrity DNA from low-input, difficult-to-lyse plant samples. The obtained DNA has high purity (A260 / A280 1.8-2.0, A260 / A230 2.0-2.2) and no residual enzyme inhibition, allowing direct entry into Illumina standard library construction with a library construction success rate ≥90%. Sequencing data Q30 values, effective output, and adapter contamination control are all superior to traditional methods. Furthermore, the proposed DNA extraction method is simple, time-efficient, and uses reagents at only one-fifth the cost of commercially available column extraction methods. It requires no special equipment and is easily scalable for use in research, agricultural breeding, clinical testing, and other scenarios. This represents a unified breakthrough in DNA extraction safety, integrity, purity, efficiency, and economy, providing a stable, reliable, and scalable upstream solution for high-throughput sequencing.

[0137] The above description is merely a preferred embodiment of the present invention and is not intended to limit the invention. Various modifications and variations can be made to the present invention by those skilled in the art. Any modifications, equivalent substitutions, improvements, etc., made within the spirit and principles of the present invention should be included within the scope of protection of the present invention.

Claims

1. A method for DNA extraction, characterized in that, The method includes: The sample was subjected to cell disruption to obtain a first mixture; The first mixture was mixed with a solution of hexaamminecobalt(III) chloride and an adsorbent carrier, and then incubated for the first time to obtain the first incubation product. The first incubation product was washed and dissolved to obtain the DNA of the sample.

2. The method according to claim 1, characterized in that, The concentration of the hexaamminecobalt(III) trichloride solution is 15-25 mM.

3. The method according to claim 1, characterized in that, The adsorption carrier includes one or more of glass beads, ceramic beads, or silica beads; Preferably, the glass beads are selected from one or more of borosilicate glass beads, soda-lime glass beads, quartz glass beads, or high borosilicate glass beads; More preferably, the adsorption carrier is the borosilicate glass bead; More preferably, the diameter of the borosilicate glass beads is 2-3 mm; Preferably, the surface of the adsorbent carrier is an unmodified original surface or a surface that has undergone surface modification treatment; Preferably, the surface modification includes one or more of silanization, hydroxylation, or hydrophilization treatments.

4. The method according to claim 1, characterized in that, The first mixture was mixed with a solution of hexaamminecobalt(III) trichloride and borosilicate glass beads and then shaken to obtain the first incubation product; Preferably, the frequency of the oscillation is 8-15 rpm; Preferably, the first incubation time is 5-20 minutes.

5. The method according to claim 1, characterized in that, The cell disruption method includes: mixing the sample with a lysis buffer and performing a second incubation to obtain the first mixture; Preferably, the lysis buffer includes one or more of CTAB, β-mercaptoethanol, EDTA, or Tris-HCl.

6. The method according to claim 1, characterized in that, The second incubation temperature is 45~75℃, and the second incubation time is 15~60min.

7. The method according to claim 1, characterized in that, The washing process includes: sequentially performing a first washing and a second washing on the first incubation product; The first washing process includes mixing the first incubation product with a first washing solution, allowing it to stand, and then centrifuging to obtain the first washing product. The second washing process involves mixing the first washing product with a second washing solution, allowing it to stand, and then centrifuging to obtain the second washing product. Preferably, both the first washing solution and the second washing solution include alcohol solvents and nonionic surfactants; Preferably, the alcohol solvent is selected from one or more of ethanol, isopropanol, or methanol; Preferably, the nonionic surfactant is selected from one or more of Tween-20, Triton X-100, or NP-40; Preferably, in the first washing, the settling time is 20-60 seconds, the centrifugation frequency is 6000-10000×g, and the centrifugation time is 1.5-3 minutes. Preferably, in the second washing process, the settling time is 20-60 seconds, the centrifugation frequency is 10000-13000×g, and the centrifugation time is 0.5-1.5 minutes.

8. The method according to claim 1, characterized in that, The dissolution process includes mixing the second washing product with the dissolving solution, followed by a third incubation to obtain the DNA of the sample. Preferably, the solution comprises Tris-HCl and / or EDTA; Preferably, the third incubation temperature is 30~40℃ and the time is 5~20min.

9. The method according to any one of claims 1-8, characterized in that, The samples included plant tissues.

10. The application of DNA extracted by the method of any one of claims 1 to 9 in the construction of a gene library.

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