Super-long fragment DNA extraction kit and application

Abstract

The application discloses a Super-long fragment DNA extraction kit and application. The Super-long fragment DNA extraction kit is universal, can solve the DNA super-long fragment extraction of culture cells, cryopreserved cells, fresh animal tissues, cryopreserved animal tissues, common crops, polysaccharide and polyphenol plants and part of silica gel dry plant samples and the like, can guarantee the purity under the condition that the length reaches 500kb-1Mb, and is suitable for long read length sequencing with high requirements for the DNA fragment length and the DNA purity.

Description

Technical Field

[0001] This invention relates to the field of nucleic acid extraction technology, specifically to a Super-long fragment DNA extraction kit and its applications. Background Technology

[0002] Third-generation sequencing (NGS) technology developed based on first- and second-generation sequencing technologies. Sanger sequencing technology pioneered sequencing applications in 1977, and next-generation sequencing (NGS) technology was commercialized in 2005, significantly increasing throughput and reducing costs, thus driving many sequencing-based applications. However, read length limitations still existed, restricting some applications. To address these read length limitations, third-generation sequencing technologies, represented by Helicos' Heliscope single-molecule sequencer, Pacific Biosciences' SMRT technology, and Oxford Nanopore Technologies' nanopore single-molecule technology, gradually emerged. In recent years, third-generation sequencing technology has achieved significant breakthroughs. In April 2022, using long-read sequencing technology, the first complete gapless T2T human reference genome was completed, solving challenges such as highly repetitive sequences (8% of the genome), centromeres, and telomeres in the Human Genome Project. Long-read sequencing technology was subsequently named the 2022 Technology of the Year by *Nature Methods*. Currently, this technology has been widely applied in fields such as genetic disease research, transcriptomics research, and microbiome research. It can achieve genome-wide SV detection, accurately depict transcript diversity, and improve the accuracy of microbial species classification. In the future, the cost of third-generation sequencing technology will further decrease, and its accuracy will further improve, leading to its application in even more fields.

[0003] Super-long DNA fragments (typically referring to complete DNA molecules ≥50kb in length, and even reaching hundreds ofkb to Mb) are irreplaceable in long-read sequencing-based applications because they preserve large-fragment structural information of the genome and reduce sequence splicing errors caused by breaks. Advances in their extraction techniques have directly driven research breakthroughs in multiple fields, finding wide applications in complex genome assembly, structural variation detection, and three-dimensional epigenetic studies.

[0004] Complex genomes, generally referring to genomes containing numerous repetitive sequences, polyploidy, and high heterozygosity, have long been constrained by the "splicing blind spots" (such as telomeres, centromeres, and repetitive sequence clusters) of short DNA fragments. Super-long DNA fragments can traverse these regions, significantly improving assembly continuity and integrity.

[0005] Structural variations (such as large insertions / deletions, inversions, translocations, duplications, etc., typically ≥50bp in length) are a major cause of genetic diseases, cancer, and other illnesses. However, short DNA fragments are difficult to cross the variation region, easily leading to missed detections or misdiagnoses. Super-long DNA fragments can directly cover the complete variation region, and combined with long-read sequencing technology, they enable highly sensitive and accurate detection of structural variations (SVs).

[0006] DNA epigenetic modifications (such as methylation and hydroxymethylation) and higher-order chromatin structures (such as topological association domains (TADs) and chromatin loops) exhibit long-distance correlations. Super-long DNA fragments can retain this "spatial information," providing support for epigenetic regulation research at the single-molecule level.

[0007] Super-long fragment DNA extraction is the core step in connecting samples with long-read analysis technology. Its applications cover multiple fields, from basic scientific research (genomics, evolutionary biology) to clinical diagnostics and industrial biotechnology. It directly promotes the leap from "fragmented analysis" to "complete genome level" in life science research and is a key supporting technology for achieving "precise interpretation of the code of life".

[0008] Existing commercial nucleic acid extraction kits are mainly designed for traditional next-generation sequencing, and have certain limitations in terms of fragment length, extraction purity, and applicable sample types, making it difficult to meet the diverse needs of long-length sequencing.

[0009] Traditional cultured cells and cryopreserved cells, due to their relatively simple tissue structures, generally use relatively uniform extraction methods. Fresh animal tissues and cryopreserved animal tissues, containing more extracellular matrix, are more difficult to extract than cell-type samples, and their extraction methods are specific and cannot be universally applied to cell types. Furthermore, plant tissues, especially polysaccharide and polyphenol plant samples, contain polysaccharide, polyphenol, and secondary metabolites, which drastically increase the difficulty of extraction. Currently, there is no effective solution for extracting ultra-long DNA fragments from polysaccharide and polyphenol samples.

[0010] Currently, there are some commercially available kits and extraction methods developed for long-length DNA applications. These methods are primarily suited for common cell and animal tissue samples, mainly relying on silica purification and anion exchange resins to reduce shearing forces on the DNA during extraction, thus obtaining longer DNA fragments. However, due to the high technical difficulty, only a small number of these methods are currently commercially available, and they are compatible with a limited range of sample types. They also require a high level of expertise from technicians and are easily affected by various factors, making it difficult to achieve the expected extraction results. Summary of the Invention

[0011] The purpose of this invention is to provide a Super-long fragment DNA lysis buffer, a Super-long fragment DNA kit, and their applications. The lysis buffer enables lysis to achieve a length of 500kb-1Mb, and the kit's separation and purification system ensures extraction purity, thus meeting the requirements for long-length sequencing where both DNA fragment length and purity are critical.

[0012] In view of this, the solution of the present invention is as follows:

[0013] The first aspect of this invention is to provide a Super-long fragment DNA lysis buffer, comprising a lysis buffer, a proteinase K, and a separation buffer; the lysis buffer comprises, by weight percentage: 10% Buffer A, 2.5% Buffer B, 0.1% Buffer C, 0.1% Buffer D, 10-20% sucrose, 0.5-1% PVP, 0.5-2% β-mercaptoethanol, and the balance being water; the separation buffer comprises, by weight percentage: 86% Buffer E, 7.5% Buffer F, 0.5-2% β-mercaptoethanol, 0.1-0.5% RNase, and the balance being water; wherein:

[0014] The concentration of proteinase K is 10-20 mg / ml;

[0015] The Buffer A consists of 100-500 mM Tris HCl at pH 7.0, 100-500 mM EDTA, 1-2 M sodium chloride, 1-2 M potassium chloride, 1-2 M lithium chloride, and 5 vol% BSA.

[0016] The Buffer B is a surfactant with a volume concentration of 50-60%;

[0017] The Buffer C is spermine with a concentration of 1-3M;

[0018] The Buffer D is spermidine with a concentration of 1-3M;

[0019] The Buffer E consists of 100-200 mM Tris HCl at pH 7.0, 100-200 mM EDTA, 1-2 M sodium chloride, 0.6-1 M lithium chloride, and 0-2 vol% CTAB.

[0020] The Buffer F consists of 0-25% anionic surfactant, 25-62.5% nonionic surfactant, and the balance water by volume percentage.

[0021] Furthermore, in Buffer B, the surfactant is selected from at least one of Triton X-100, NP40, and SLS.

[0022] Furthermore, in Buffer F: the anionic surfactant is at least one of SLS and SDS; and / or, the nonionic surfactant is selected from at least one of Tween20, NP40 and Triton X-100.

[0023] A second aspect of the present invention is to provide a Super-long fragment DNA extraction kit, comprising the lysis buffer described in the first aspect and DNA separation and purification reagents.

[0024] Further, the DNA separation and purification reagent includes DNA separation solution A, DNA separation solution B, Buffer L, and EB buffer; wherein:

[0025] The DNA separation solution A includes DNA extraction phenol reagent, chloroform, and isoamyl alcohol; the DNA separation solution B includes chloroform and isoamyl alcohol.

[0026] The Buffer L consists of 100-200 mM Tris HCl at pH 7.0, 100-200 mM EDTA, 1-2 M potassium chloride, 1-2 M sodium chloride, 2-3 vol% PVP40000, and 0.5-2 vol% Tween20.

[0027] Further, the volume ratio of DNA extraction phenol reagent to chloroform and isoamyl alcohol in DNA separation solution A is 25:24:1; and / or, the volume ratio of chloroform to isoamyl alcohol in DNA separation solution B is 24:1.

[0028] A third aspect of the invention is to provide the use of the lysis buffer described in the first aspect or the kit described in the second aspect in the preparation of super-long fragment DNA from lysed tissues.

[0029] Furthermore, the tissue is selected from cultured cells, cryopreserved cells, fresh animal tissue, cryopreserved animal tissue, crop tissue, polysaccharide and polyphenol plant samples, or silica gel dried plant samples.

[0030] A fourth aspect of the present invention is to provide a method for preparing Super-long fragment DNA, the method using the Super-long fragment DNA lysis buffer described in the first aspect; the steps include:

[0031] S1. Grind the tissue material at low temperature to depolymerize it;

[0032] S2. Add lysis buffer to the tissue obtained in S1, mix, and incubate at low temperature on a mixer.

[0033] S3. Filter the product obtained from S2 incubation, centrifuge the filtrate and collect the precipitate;

[0034] S4. Take the precipitate obtained by centrifugation, add proteinase K, and mix until there are no obvious clumps;

[0035] S5. Add the product obtained in S4 to the separation buffer, incubate at 40-55℃ for 0.5-2h, cool and centrifuge to collect the supernatant;

[0036] S6. The product obtained in S5 is separated and purified to obtain the Super-long fragment DNA.

[0037] Furthermore, steps S2-S3 are repeated multiple times.

[0038] Furthermore, the preparation method uses the kit described in the second aspect, and the separation and purification process includes: sequentially using DNA separation solution A and DNA separation solution B to remove impurities from the product obtained in S5, then performing alcohol precipitation on the separated DNA, dissolving it in EB buffer, adding Buffer L for purification, centrifuging to collect the precipitate, and then performing alcohol precipitation again to obtain the purified Super-long fragment DNA.

[0039] Compared with the prior art, the beneficial effects of the present invention are as follows:

[0040] The lysis buffer described in this invention is based on a unique buffer system for the extraction and purification of high molecular weight DNA. Under the action of the lysis buffer, the cell wall of the tissue sample is removed and the cell membrane is destroyed, releasing high molecular weight DNA. Then, the released DNA is further lysed by the separation buffer to remove residual unlysed proteins and other impurities, effectively improving the yield of Super-long fragment DNA.

[0041] The kit described in this invention provides lysis systems of varying degrees, and the multi-round purification system solves the problems of obtaining DNA length and purity, ensuring that the extracted DNA meets the requirements for subsequent ultra-long sequencing. The lysis buffer and separation buffer utilize a combination of various ionic and nonionic surfactants, solving the problem of using a single kit for extracting multiple types of samples, avoiding the need to change kits for different samples. While maintaining high-quality results, this invention achieves a universal extraction process for various sample types, significantly improving efficiency; it also greatly reduces usage costs, lowers usage bottlenecks, enables more applications, and promotes the development of related scientific research.

[0042] The extraction method described in this invention can achieve the extraction of Super-long fragment DNA from various types of samples (cultured cells, frozen cells, fresh animal tissues, frozen animal tissues, common crops, polysaccharide and polyphenol plants, and some silica gel dried plant samples). Through multiple lysis processes using this universal kit and multiple removal of impurities using organic reagent extraction and other methods, a high-yield and high-purity DNA product is finally obtained. Attached Figure Description

[0043] Figure 1 The results of electrophoretic analysis of DNA fragment sizes obtained from tissue extraction in this embodiment of the invention are shown.

[0044] Figure 2 The results of electrophoretic analysis of DNA fragment sizes obtained from the extraction of another part of the tissue in the embodiments of the invention are shown. Detailed Implementation

[0045] The technical solutions of the present invention will be clearly and completely described below with reference to the embodiments of the present invention. Obviously, the described embodiments are only some embodiments of the present invention, and not all embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those of ordinary skill in the art without creative effort are within the scope of protection of the present invention.

[0046] In one embodiment, a method for extracting super-long fragment DNA is proposed, the specific steps of which are as follows:

[0047] 1. Low-temperature liquid nitrogen grinding of tissue materials to depolymerize the tissue;

[0048] 2. Use lysis buffer to lyse and remove the cell wall and disrupt the cell membrane, releasing high molecular weight DNA;

[0049] 3. Use separation buffer to further lyse the released DNA to remove residual unlysed proteins and other impurities;

[0050] 4. Use DNA separation solution A to remove impurities produced during lysis;

[0051] 5. Use DNA separation solution B to further remove residual impurities from the lysis process;

[0052] 6. Use Buffer L to purify high molecular weight DNA to obtain high-purity DNA.

[0053] Specifically, the components of each buffer or separation solution are:

[0054] The lysis buffer comprises, by mass percentage: 10% Buffer A, 2.5% Buffer B, 0.1% Buffer C, 0.1% Buffer D, 10-20% sucrose, 0.5-1% PVP, 0.5-2% β-mercaptoethanol, and the balance being water;

[0055] The separation buffer consists of the following components by mass percentage: 86% Buffer E, 7.5% Buffer F, 0.5-2% β-mercaptoethanol, 0.1-0.5% RNase, and the balance being water;

[0056] in:

[0057] The Buffer A consists of 100-500 mM Tris HCl at pH 7.0, 100-500 mM EDTA, 1-2 M sodium chloride, 1-2 M potassium chloride, 1-2 M lithium chloride, and 5 vol% BSA.

[0058] The Buffer B is a surfactant with a volume concentration of 50-60%;

[0059] The Buffer C is spermine with a concentration of 1-3M;

[0060] The Buffer D is spermidine with a concentration of 1-3M;

[0061] The Buffer E consists of 100-200 mM Tris HCl at pH 7.0, 100-200 mM EDTA, 1-2 M sodium chloride, 0.6-1 M lithium chloride, and 0-2 vol% CTAB.

[0062] The Buffer F consists of 0-25% anionic surfactant, 25-62.5% nonionic surfactant, and the balance water by volume percentage.

[0063] The above Buffers A to E are all aqueous solutions, that is, buffers with the above contents or concentrations are prepared using water as a solvent.

[0064] In the above embodiments, the extraction method can solve the problem of extracting Super-long fragment DNA from various types of samples, including cultured cells, frozen cells, fresh animal tissues, frozen animal tissues, common crops, polysaccharide and polyphenol plants, and some silica gel dried plant samples. It can obtain high-quality extraction results and meet the purity requirements when the length reaches 500kb-1Mb. It is suitable for long sequencing with high requirements for DNA fragment length and high DNA purity.

[0065] In the above embodiments, the extraction method can effectively improve efficiency while ensuring high-quality results. It can use a general extraction process to handle various types of samples, which greatly improves efficiency. Furthermore, it significantly reduces usage costs and lowers usage bottlenecks, enabling more applications to be realized.

[0066] In a preferred embodiment, the surfactant in Buffer B is selected from at least one of Triton X-100, NP40, and SLS.

[0067] In a preferred embodiment, in Buffer F: the anionic surfactant is at least one of SLS and SDS; the nonionic surfactant is at least one of Tween20, NP40 and Triton X-100.

[0068] In a preferred embodiment, the DNA separation solution A comprises DNA extraction phenol reagent, chloroform, and isoamyl alcohol, with a preferred volume ratio of 25:24:1; the DNA separation solution B comprises chloroform and isoamyl alcohol, with a preferred volume ratio of 24:1.

[0069] The Buffer L consists of 100-200 mM Tris HCl at pH 7.0, 100-200 mM EDTA, 1-2 M potassium chloride, 1-2 M sodium chloride, 2-3 vol% PVP40000, and 0.5-2 vol% Tween20.

[0070] Example 1

[0071] A method for extracting super-long fragment DNA, comprising the following steps:

[0072] 1) Take approximately 0.2g of tissue and grind it at low temperature using liquid nitrogen;

[0073] 2) After the liquid nitrogen has evaporated, transfer the ground tissue material to 30ml of the prepared Lysis buffer and mix thoroughly to avoid tissue clumps.

[0074] 3) Place the Lysis buffer reaction system horizontally in a prepared ice box, and then incubate it on a horizontal mixer for 20 minutes. Set the mixer speed to 10-20 rpm / min.

[0075] 4) Prepare two 50ml centrifuge tubes, place the 70um cell strainer into the 50ml centrifuge tubes, place them in an ice box to keep them at a low temperature for later use, and pre-cool the centrifuge to 4°C for later use;

[0076] 5) After the Lysis buffer reaction system has been incubated, filter it through a 70µm cell strainer and collect the filtrate;

[0077] 6) Centrifuge the collected filtrate at 3000g for 15 minutes at 4℃;

[0078] 7) After centrifugation, discard the supernatant and collect the precipitate at the bottom;

[0079] 8) Add 30 ml of Lysis buffer to the bottom precipitate and resuspend it repeatedly by pipetting and aspirating with a 5 ml pipette until there are no obvious clumps or particles.

[0080] 9) After resuspending, centrifuge at 4℃ and 60g for 2 min, then transfer the supernatant to a new 50ml tube, taking care to avoid aspirating the bottom precipitate.

[0081] 10) Centrifuge the transferred supernatant at 2500g for 15 min at 4°C;

[0082] 11) After centrifugation, discard the supernatant and collect the precipitate at the bottom;

[0083] 12) Add 200ul of Buffer G to the bottom sediment and mix quickly with a wide-mouth pipette until there are no obvious lumps;

[0084] 13) Transfer the resuspension to the prepared separation buffer using a wide-mouth pipette tip, and mix by blowing and aspiration;

[0085] 14) Transfer the separation buffer reaction system to a water bath and incubate at 50°C for 1 hour, gently inverting and mixing once every 20 minutes;

[0086] 15) After incubation, place on ice to cool rapidly for 5 minutes;

[0087] 16) After cooling is complete, centrifuge at 4℃ and 5000g for 5 minutes;

[0088] 17) Transfer the supernatant to a new 15ml centrifuge tube, add an equal volume of DNA separation solution A, and place on a horizontal shaker to react fully for 15 minutes;

[0089] 18) Centrifuge at 5000g for 10 min at room temperature, transfer the supernatant to a new 15ml tube, add an equal volume of DNA separation solution B, and place on a horizontal shaker to react fully for 15 min;

[0090] 19) Centrifuge at 5000g for 10 min at room temperature, transfer the supernatant to a new centrifuge tube, add 0.8 times the volume of isopropanol, gently invert to mix, and let stand for 5-10 min;

[0091] 20) Centrifuge at 5000g for 10 min at room temperature, discard the supernatant, add 1 ml of 80% ethanol to the bottom precipitate, and transfer the entire amount to a new 1.5 ml centrifuge tube;

[0092] 21) Perform instant centrifugation, collect the precipitate at the bottom of the centrifuge tube, discard the alcohol, and try to avoid aspirating the precipitate;

[0093] 22) Add 1 ml of 80% ethanol to the precipitate at the bottom and invert the container 3-5 times.

[0094] 23) Perform a brief centrifugation, collect the precipitate at the bottom of the centrifuge tube, discard the alcohol, and try to avoid aspirating the precipitate;

[0095] 24) Open the tube cap and allow the alcohol to evaporate for 2-3 minutes;

[0096] 25) Add 100 μL of Buffer EB and dissolve the precipitate at room temperature for 2-4 hours until completely dissolved;

[0097] 26) Prepare Buffer L reagent, invert to mix well, and then centrifuge;

[0098] 27) Add an equal volume of Buffer L reagent to the above DNA sample, mix thoroughly, and let stand for 10 min;

[0099] 28) Centrifuge at 10000 g for 30 min at room temperature, discard the supernatant, and collect the precipitate at the bottom;

[0100] 29) Add 200 μl of freshly prepared 80% ethanol, centrifuge at 10000g for 2 min at room temperature, and discard the supernatant;

[0101] 30) Add 200 μl of freshly prepared 80% ethanol again, centrifuge at 10000g for 2 min at room temperature, and discard the supernatant;

[0102] 31) Open the tube cap and allow the alcohol to evaporate for 2-3 minutes;

[0103] 32) Add 100 μL of EB to the precipitate and dissolve the precipitate at room temperature for 2-4 hours until completely dissolved;

[0104] 33) Use a wide-mouth pipette tip to aspirate and spit the DNA solution 20-30 times to thoroughly mix the DNA solution;

[0105] 34) Use Nanodrop and Qubit to detect the DNA solution and determine the DNA concentration and purity;

[0106] 35) Use a pulsed electrophoresis apparatus to perform electrophoresis and determine the DNA length distribution;

[0107] 36) Preserve the remaining high molecular weight DNA at 4°C for 1-2 weeks. For long-term storage, freeze at -20°C.

[0108] In this embodiment, the composition of each reagent is as follows:

[0109] The mass percentages of the components in the Lysis buffer are as follows: 10% Buffer A, 2.5% Buffer B, 0.1% Buffer C, 0.1% Buffer D, 17.1% sucrose, 1% PVP, 2% β-mercaptoethanol, and the remainder water.

[0110] Buffer A consists of: 500mM Tris HCl at pH 7.0, 500mM EDTA, 1M sodium chloride, 1M potassium chloride, 2M lithium chloride, and 5% BSA.

[0111] Buffer B consists of 60 wt% Triton X-100;

[0112] Buffer C consists of 3M spermine;

[0113] Buffer D consists of 3M spermidine;

[0114] The components of the separation buffer, by mass percentage, are: 86% Buffer E, 7.5% Buffer F, 2% β-mercaptoethanol, 0.4% RNase, and the balance being water;

[0115] Buffer E consists of: 200 mM Tris HCl at pH 7.0, 200 mM EDTA, 2 M sodium chloride, 1 M lithium chloride, and 2% CTAB;

[0116] Buffer F consists of: 20% SLS, 20% NP40, 20% SDS, and 5% Tween20;

[0117] Buffer G consists of 20 mg / ml of proteinase K;

[0118] Buffer L consists of: 200 mM Tris HCl at pH 7.0, 200 mM EDTA, 1.2 M sodium chloride, 2% PVP40000, and 2% Tween20.

[0119] Buffer EB consists of 200 mM Tris HCl at pH 7.0.

[0120] Example 2

[0121] A method for extracting super-long DNA fragments, the steps of which are the same as in Example 1, and the composition of each reagent is as follows:

[0122] The mass percentages of the components in the Lysis buffer are as follows: 10% Buffer A, 2.5% Buffer B, 0.1% Buffer C, 0.1% Buffer D, 17.1% sucrose, 1% PVP, 2% β-mercaptoethanol, and the remainder water.

[0123] Buffer A contains 100 mM Tris HCl at pH 7.0, 100 mM EDTA, 2 M sodium chloride, 1 M potassium chloride, 1 M lithium chloride, and 5% BSA by volume.

[0124] Buffer B contains 20 wt% Triton X-100 and 40 wt% NP40;

[0125] Buffer C contains 2M of spermine;

[0126] Buffer D contains 3M spermidine;

[0127] The components of the separation buffer, by mass percentage, are: 86% Buffer E, 7.5% Buffer F, 2% β-mercaptoethanol, 0.4% RNase, and the balance being water;

[0128] Buffer E consists of: 200 mM Tris HCl at pH 7.0, 200 mM EDTA, 2 M sodium chloride, and 0.6 M lithium chloride;

[0129] Buffer F consists of: 18.75 wt% NP40, 18.75 wt% SLS, 12.5 wt% Tween20, and 12.5 wt% Triton X-100;

[0130] Buffer G contains 20 mg / ml of proteinase K;

[0131] Buffer L contains 100 mM Tris HCl at pH 7.0, 100 mM EDTA, 1.2 M potassium chloride, 2 wt% PVP40000, and 2 wt% Tween20.

[0132] Buffer EB contains 200 mM Tris HCl at pH 7.0.

[0133] Example 3

[0134] A method for extracting super-long DNA fragments, the steps of which are the same as in Example 1, and the composition of each reagent is as follows:

[0135] A method for extracting super-long DNA fragments, the steps of which are the same as in Example 1, and the composition of each reagent is as follows:

[0136] The mass percentages of the components in the Lysis buffer are as follows: 10% Buffer A, 2.5% Buffer B, 0.1% Buffer C, 0.1% Buffer D, 15% sucrose, 1% PVP, 2% β-mercaptoethanol, and the remainder water.

[0137] Buffer A contains 300 mM Tris HCl at pH 7.0, 300 mM EDTA, 2 M sodium chloride, 1 M potassium chloride, 1 M lithium chloride, and 5% BSA by volume.

[0138] Buffer B contains 20 wt% Triton X-100, 20 wt% NP40, and 10 wt% SLS;

[0139] Buffer C contains 3M of spermine;

[0140] Buffer D contains 2M spermidine;

[0141] The components of the separation buffer, by mass percentage, are: 86% Buffer E, 7.5% Buffer F, 2% β-mercaptoethanol, 0.4% RNase, and the balance being water;

[0142] Buffer E consists of: 200 mM Tris HCl at pH 7.0, 200 mM EDTA, 2 M sodium chloride, and 0.6 M lithium chloride;

[0143] Buffer F consists of: 18.75 wt% NP40, 18.75 wt% SLS, 12.5 wt% Tween20, and 12.5 wt% Triton X-100;

[0144] Buffer G contains 15 mg / ml of proteinase K;

[0145] Buffer L contains 100 mM Tris HCl at pH 7.0, 100 mM EDTA, 1.2 M potassium chloride, 2 wt% PVP40000, and 1 wt% Tween20.

[0146] Buffer EB contains 200 mM Tris HCl at pH 7.0.

[0147] Comparative Example 1

[0148] A method for extracting super-long fragment DNA, the steps of which are the same as in Example 1, except that the composition of each reagent is different from that in Example 1, neither Lysis buffer component Buffer A nor Separation buffer component Buffer E contains lithium chloride.

[0149] Comparative Example 2

[0150] A method for extracting super-long fragment DNA, the steps of which are the same as in Example 1, except that the composition of each reagent is different from that in Example 1, the Lysis buffer component does not contain Buffer C and Buffer D.

[0151] Comparative Example 3

[0152] A method for extracting super-long fragment DNA, the steps of which are the same as in Example 1, except that the composition of each reagent differs from that in Example 1, the Lysis buffer component does not contain Buffer B.

[0153] Comparative Example 4

[0154] A method for extracting super-long fragment DNA, the steps of which are the same as in Example 1, except that the composition of each reagent differs from that in Example 1, the separation buffer component does not contain Buffer E.

[0155] Based on Example 1 above, tissue DNA was extracted from different samples, and the DNA concentration and purity are shown in Table 1. The DNA length distribution is shown in... Figure 1-2 As shown in the image (analysis method: pulsed electrophoresis), the extracted DNA fragments are clearly larger than 200kb in size.

[0156] Table 1:

[0157]

[0158] Examples 2 and 3 performed the same extraction steps on the above samples, and analyzed the DNA concentration and purity of the extracted DNA. The DNA concentration and purity results of the extracted DNA from the same samples were not significantly different from those in Example 1. This indicates that Examples 1-3 can effectively extract and purify Super-long fragment DNA for different types of samples.

[0159] Comparative Examples 1-4 performed the same extraction steps on the samples above, and analyzed the DNA concentration and purity of the extracted DNA. The DNA concentration or purity results of the same samples differed significantly from those of Example 1. Specific differences in some samples are shown in Table 2.

[0160] Table 2:

[0161]

[0162]

[0163] It is clear from the comparative examples 1-4 above that when the buffer system lacks lithium chloride, spermine / spermine, and surfactant, the total amount, purity, and fragment length of the extracted DNA solution are all much lower than those obtained by the formal method, which cannot meet the requirements of downstream experiments.

[0164] Although embodiments of the invention have been shown and described, it will be understood by those skilled in the art that various changes, modifications, substitutions and alterations can be made to these embodiments without departing from the principles and spirit of the invention, the scope of which is defined by the appended claims and their equivalents.

Claims

1. A method for preparing super-long fragment DNA, characterized in that the steps include... include: S1. Grind the tissue material at low temperature to depolymerize it; S2. Add lysis buffer to the tissue obtained in S1, mix, and incubate at low temperature on a mixer. S3. Filter the product obtained from S2 incubation, centrifuge the filtrate and collect the precipitate; S4. Take the precipitate obtained by centrifugation, add proteinase K, and mix until there are no obvious clumps; S5. Add the product obtained in S4 to the separation buffer, incubate at 40-55℃ for 0.5-2h, cool and centrifuge to collect the supernatant; S6. The product obtained in S5 is separated and purified to obtain Super-long fragment DNA; The lysis buffer comprises, by mass percentage: 10% Buffer A, 2.5% Buffer B, 0.1% Buffer C, 0.1% Buffer D, 10-20% sucrose, 0.5-1% PVP, 0.5-2% β-mercaptoethanol, and the balance being water; wherein: Buffer A consists of 100-500mM Tris HCl at pH 7.0, 100-500mM EDTA, 1-2M sodium chloride, 1-2M potassium chloride, 1-2M lithium chloride, and 5 vol% BSA; Buffer B is a surfactant with a volume concentration of 50-60%; Buffer C is spermine with a concentration of 1-3M; and Buffer D is spermidine with a concentration of 1-3M. The concentration of proteinase K is 10-20 mg / ml; The separation buffer consists of the following components by mass percentage: 86% Buffer E, 7.5% Buffer F, 0.5-2% β-mercaptoethanol, 0.1-0.5% RNase, and the balance being water; Buffer E consists of 100-200mM Tris HCl at pH 7.0, 100-200mM EDTA, 1-2M sodium chloride, 0.6-1M lithium chloride, and 0-2 vol% CTAB; Buffer F consists of 0-25% anionic surfactant, 25-62.5% nonionic surfactant, and the balance being water. The reagents used for separation and purification in step S6 include DNA separation solution A, DNA separation solution B, Buffer L, and EB buffer; wherein: DNA separation solution A includes DNA extraction phenol reagent, chloroform, and isoamyl alcohol; DNA separation solution B includes chloroform and isoamyl alcohol; Buffer L consists of 100-200mM Tris HCl at pH 7.0, 100-200mM EDTA, 1-2M potassium chloride, 1-2M sodium chloride, 2-3 vol% PVP40000, and 0.5-2 vol% Tween 20.

2. The preparation method according to claim 1, characterized in that, In Buffer B, the surfactant is selected from at least one of Triton X-100, NP40, and SLS.

3. The preparation method according to claim 1, characterized in that, In the Buffer F: The anionic surfactant is at least one of SLS and SDS; and / or, The nonionic surfactant is selected from at least one of Tween20, NP40 and Triton X-100.

4. The preparation method according to claim 1, characterized in that, The volume ratio of DNA extraction phenol reagent to chloroform and isoamyl alcohol in the DNA separation solution A is 25:24:

1. And / or, the volume ratio of chloroform to isoamyl alcohol in the DNA separation solution B is 24:

1.

5. The preparation method according to claim 1, characterized in that, The separation and purification process includes: sequentially using DNA separation solution A and DNA separation solution B to remove impurities from the product obtained in S5, then performing alcohol precipitation on the separated DNA, dissolving it in EB buffer, adding Buffer L for purification, centrifuging to collect the precipitate, and then performing alcohol precipitation again to obtain the purified Super-long fragment DNA.

6. The preparation method according to claim 1, characterized in that, The tissues are selected from cultured cells, cryopreserved cells, fresh animal tissues, cryopreserved animal tissues, crop tissues, polysaccharide and polyphenol plants, or silica gel dried plant samples.

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