Two methods for simultaneously and efficiently extracting DNA (Deoxyribonucleic Acid) and RNA (Ribonucleic Acid) based on TRIZOL lysis buffer

Abstract

The invention belongs to the technical field of nucleic acid extraction, and particularly relates to two methods for simultaneously and efficiently extracting DNA and RNA based on TRIZOL lysate. A transformation buffer is developed on the basis of a TRIZOL lysis solution, the lysis solution is changed from acidity to nearly neutral, DNA can be dissociated from chromatin to a water phase, two methods (a chloroform phase separation method and a transformation buffer method) which are suitable for the lysis solution and can extract DNA and RNA at the same time are developed at the same time, efficient and high-purity separation of DNA and RNA is achieved, and the method is suitable for large-scale industrial production. And the purity can be compatible with the highest standard of nucleic acid mass spectrum and high-throughput sequencing. According to the method, a principle and a method which are different from those of a traditional TRIZOL method for extracting RNA and DNA are adopted, so that the problem that irreversible low-quality low-purity DNA is generated when DNA is prepared through the traditional TRIZOL method is solved, and the method meets the processing requirement of a sample high-throughput automatic platform.

Description

Technical Field

[0001] The invention belongs to the technical field of nucleic acid extraction, and specifically relates to two methods for simultaneously and efficiently extracting DNA and RNA based on TRIZOL lysis solution. Background Art

[0002] Life science research usually requires the simultaneous extraction of DNA and RNA from the same tissue in order to obtain the corresponding genomic and transcriptomic information to analyze its functional mechanism. At present, the technology for efficient extraction and separation of individual components is very mature, so it can usually be achieved by manually dividing the sample and extracting the DNA and RNA components separately. However, for limited highly heterogeneous tissues (such as early embryonic tissue and tumor tissue), especially needle biopsies in actual medical diagnosis, it is impossible to divide the sample equally to obtain the corresponding DNA and RNA information at the same time. Therefore, it is crucial to find and develop methods and technologies that can extract DNA and RNA at the same time.

[0003] The TRIZOL method for RNA extraction has long been considered the "gold standard" for RNA purification. At the same time, the TRIZOL method established a prototype for the simultaneous purification of RNA / DNA / protein in the same tissue, and it is still in use today. However, the experimental process of DNA extraction by the TRIZOL method is very cumbersome and time-consuming, and only low-quality and low-purity DNA can be harvested, which cannot meet downstream applications. Therefore, the application of the TRIZOL method in DNA extraction is limited, and it has also become the focus of improvement for current researchers. TRIZOL is a single-phase reagent based on acidic guanidine phenol. The industry believes that the principle of distinguishing DNA and RNA and achieving separate extraction is that under acidic guanidine conditions (pH is about 2), DNA and chromatin cannot be dissociated and exist in the organic phase or precipitate phase, while RNA is polar and exists in the aqueous phase. Therefore, the separation of different phases can achieve the separate extraction and purification of DNA and RNA. However, the acidic conditions make DNA highly coupled to chromatin proteins, making it difficult to subsequently dissociate DNA from this dense protein stack. It is speculated that this may be the reason why the TRIZOL method produces low-quality, low-purity DNA, which is also inevitable for this principle to distinguish between DNA and RNA and extract them separately.

[0004] TRIZOL has excellent ability to lyse a variety of complex samples, has a wide range of applications, and can be easily expanded to accommodate very large input volumes. This is an unparalleled advantage of TRIZOL compared to other lysing solutions. Therefore, if a method based on TRIZOL can be developed to simultaneously distinguish and extract DNA and RNA, it will have important application value. Although our previous patent CN118240814A established a principle and process for completely distinguishing DNA and RNA in a liquid phase in vitro, that is, if DNA and RNA can exist in a liquid phase at the same time, then there is a way to completely distinguish DNA and RNA. However, in the process of nucleic acid extraction from actual samples such as cells, an important problem usually faced is that although TRIZOL lysing solution has a strong lysing ability, only RNA exists in the aqueous phase, while DNA is almost irreversibly present in the precipitate, so in principle, TRIZOL cell lysate is not compatible with the principle established by patent CN118240814A. Therefore, the current difficulty and focus lies in how to extract RNA and DNA in the liquid phase simultaneously in TRIZOL cell lysis buffer. If this problem can be solved, on the one hand, the advantages of TRIZOL lysis can be utilized, and on the other hand, the principle of downstream silica gel extraction to distinguish DNA and RNA can be combined to avoid the disadvantage of irreversible damage caused by traditional TRIZOL method of DNA extraction, so as to develop a method for simultaneously and efficiently extracting and purifying DNA and RNA in TRIZOL cell lysis buffer.

[0005] In summary, although the professional extraction kits and methods for separating DNA and RNA separately on the market are very mature, they are still powerless for limited highly heterogeneous tissues because they cannot obtain DNA and RNA at the same time by equally dividing the sample. For example, the AllPrep kit represented by Qiagen can extract DNA and RNA at the same time, but the yield of the extracted nucleic acid is unstable and usually low, and the expensive price also limits its application. At the same time, although the prototype and principle of the experimental scheme for simultaneous extraction of RNA / DNA / protein based on TRIZOL reagent was established in 1993 and has been used to this day, the traditional TRIZOL method is only the gold standard for RNA extraction. The quality, yield, and purity of the DNA it produces are low, and it is only a crude product containing DNA. Multiple rounds of DNA purification processes are still required to be compatible with downstream experimental applications, but the DNA loss during the purification process is serious, so the purification recovery rate is low. Therefore, there is also a view that the damage to DNA extraction caused by the traditional TRIZOL method is irreversible. On the one hand, this is because DNA is unstable under acidic conditions and is easily depurinated and degraded, so the quality and yield of the prepared DNA are unstable; on the other hand, this is because the differentiation principle of the traditional TRIZOL method makes DNA densely coupled with chromatin proteins, and it is difficult to separate it efficiently from the proteins later. This is also the reason for the low purity of the extraction and is an unavoidable problem. In addition, although our previous invention patent CN118240814A established a principle and silica gel purification process for simultaneously and efficiently distinguishing DNA and RNA in the liquid phase, this principle is contrary to the traditional TRIZOL extraction principle, and the two cannot be directly compatible. Therefore, it is necessary to develop a method based on TRIZOL that can simultaneously distinguish and extract DNA and RNA. Summary of the invention

[0006] In order to overcome the deficiencies of the above-mentioned prior art, it is difficult to optimize the principles and schemes of extracting DNA and RNA based on the traditional TRIZOL method so as to efficiently and conveniently achieve the simultaneous separation and purification of high-purity and high-yield DNA and RNA. However, considering the strong lysis ability of the TRIZOL reagent and its universal use (it is easy to obtain in any laboratory and can be applied to the simultaneous extraction of DNA and RNA through improvement), the present invention has developed a conversion buffer based on TRIZOL, and adopted the newly discovered principle of distinguishing DNA and RNA based on silica gel, and designed and developed two methods that are compatible with the buffer and principle and can simultaneously and efficiently separate DNA and RNA, providing assistance and supplement to the field of simultaneous and efficient extraction and purification of DNA and RNA.

[0007] In order to achieve the above objectives, the present invention first developed a conversion buffer compatible with TRIZOL, proposed the concept of neutral TRIZOL buffer for the first time, and explored whether the buffer can indeed dissociate DNA and chromatin and extract them into the liquid phase while ensuring the quality and yield of RNA. Secondly, the present invention developed two purification methods and process schemes that are suitable for the lysate and can simultaneously extract DNA and RNA, and the extracted DNA and RNA are of high purity, with an OD of 1. 260 / 280 , O.D. 260 / 230 The parameters are good and compatible with the highest standards of nucleic acid mass spectrometry and high-throughput sequencing. At the same time, the extraction process is simple and the extraction time is short. The chloroform phase separation method extracts DNA for 14 minutes and RNA for 16 minutes, while the conversion method extracts DNA for 4 minutes and RNA for 6 minutes (the traditional TRIZOL RNA extraction takes more than 1 hour or even overnight, and the DNA extraction takes more than 2 hours, and requires subsequent multiple rounds of purification, which takes more than 1 day). It can be seen that the experimental processes of these two methods are simple and efficient, and can adapt to the processing requirements of the sample high-throughput automation platform. Finally, the two extraction methods were compared with the traditional TRIZOL method for extracting DNA and RNA. It was unexpectedly found that in addition to obtaining high-quality, high-purity, and high-yield DNA (the yield is 6-8 times that of traditional DNA), the quality and yield of the extracted RNA surpassed the RNA extracted by the traditional TRIZOL method, which can be increased by up to 52%. Therefore, the present invention will further promote the development of industry and technology in the field of nucleic acid extraction.

[0008] Specifically, the technical solution adopted by the present invention is: The first aspect of the present invention provides a kit for simultaneously and efficiently extracting DNA and RNA based on TRIZOL lysis solution, the kit comprising a neutral TRIZOL lysis solution, a DNA rinse solution, anhydrous ethanol, an RNA rinse solution 1 solution and an RNA rinse solution 2 solution; or comprising a neutral TRIZOL lysis solution, a DNA rinse solution, anhydrous ethanol, an RNA rinse solution 1 solution, an RNA rinse solution 2 solution and chloroform; The neutral TRIZOL lysis solution consists of a conversion buffer and a TRIZOL reagent, wherein the conversion buffer consists of 4M guanidine isothiocyanate and 1M Tris-HCl, and has a pH of 7.4; the DNA rinse solution consists of ethanol and NaCl, the RNA rinse solution 1 solution consists of ethanol and guanidine hydrochloride, and the RNA rinse solution 2 solution is an ethanol solution.

[0009] Preferably, the neutral TRIZOL lysis solution consists of conversion buffer and TRIZOL reagent in a volume ratio of 1:2.

[0010] Preferably, the DNA washing solution consists of 70% ethanol and 150 mM NaCl.

[0011] Preferably, the RNA rinse solution 1 consists of 80% ethanol and 1 M guanidine hydrochloride.

[0012] Preferably, the RNA rinse solution 2 is 80% ethanol.

[0013] The second aspect of the present invention provides a method for simultaneously and efficiently extracting DNA and RNA based on TRIZOL lysis solution. The method uses the kit described in the first aspect to extract DNA and RNA. The extraction process includes the following steps: S1. Add neutral TRIZOL lysis buffer to the cells and mix well to make neutral TRIZOL homogenate; S2, add chloroform to the neutral TRIZOL homogenate of S1, collect the supernatant by centrifugation, and incubate the supernatant with a silica column to allow the DNA to bind to the silica column; or add conversion buffer to the neutral TRIZOL homogenate of S1, mix well, and incubate the TRIZOL homogenate with a silica column to allow the DNA to bind to the silica column; S3, adding DNA rinsing solution to the silica gel column in S2 for rinsing, and then spinning and eluting with pure water to obtain a DNA sample; S4, collecting the flow-through of S3, adding anhydrous ethanol and incubating it with a new silica gel column to allow RNA to bind to the silica gel column; S5, adding RNA washing solution 1 solution to the silica gel column in S4 for washing; S6. Add RNA rinsing solution 2 to the silica gel column in S5 for rinsing, and then spin dry and elute with pure water to obtain an RNA sample.

[0014] Preferably, in S2, the volume ratio of chloroform to neutral TRIZOL homogenate is 1:5; the volume ratio of transformation buffer to neutral TRIZOL homogenate is 0.5-3:1.

[0015] Preferably, in S1, 1×10 5 – 1×10 7 Cells were lysed in 1 mL of TRIZOL standard by adding neutral TRIZOL lysis buffer to the cells.

[0016] Preferably, in S4, the volume ratio of the flow-through liquid to anhydrous ethanol is 1:1-2.

[0017] Preferably, the silica gel column is a silica gel column made of polypropylene.

[0018] Preferably, in S2, after the supernatant or TRIZOL homogenate is incubated with the silica gel column, it is centrifuged at 10000-20000 g for 30 seconds to 1 minute to allow the DNA to bind to the silica gel column.

[0019] Preferably, in S3, after adding the DNA rinsing solution, rinse at 10000-20000 g for 30 s-1 min, spin for 1-2 min, and elute with 10000-20000 g pure water for 1-3 min.

[0020] Preferably, in S6, after adding RNA rinsing solution 2, rinse at 10000-20000 g for 30 s-1 min, spin for 1-2 min, and elute with 10000-20000 g pure water for 1-3 min.

[0021] Preferably, in S4, after incubation with a new silica gel column, centrifugation is performed at 10000-20000 g for 30 seconds to 1 minute to allow the RNA to bind to the silica gel column.

[0022] Preferably, in S5, after adding RNA rinsing solution 1, rinse at 10000-20000g for 30s-1min.

[0023] Compared with the prior art, the present invention has the following beneficial effects: The present invention develops a conversion buffer based on TRIZOL lysis solution, and develops two methods (chloroform phase separation method and conversion buffer method) that are suitable for the lysis solution and can simultaneously extract DNA and RNA. The present invention adopts a principle and method different from the traditional TRIZOL method for extracting RNA and DNA, thereby solving the problem of irreversible low-quality and low-purity DNA produced by the traditional TRIZOL method for preparing DNA. At the same time, the conversion buffer developed by the present invention using the strong lysis ability of TRIZOL changes the lysis solution from acidic (approximately PH2) to nearly neutral, which is more conducive to the integrity and stability of DNA and RNA, and also allows DNA to dissociate from chromatin into the aqueous phase, which is also the key to preparing high-quality and high-purity DNA. Finally, the chloroform phase separation method and conversion buffer method developed by the present invention utilize the different binding abilities of DNA and RNA to silica gel in different PH, salt concentrations and ethanol to achieve efficient and high-purity separation of DNA and RNA, and its OD 260 / 280 , O.D. 260 / 230 The parameters are good, the purity is high, and it is compatible with the highest standards of nucleic acid mass spectrometry and high-throughput sequencing. In addition, the chloroform phase separation method and conversion buffer method developed by the present invention have simple operation procedures, adapt to the processing requirements of the sample high-throughput automated platform, and can provide new reference and supplement for the field of nucleic acid extraction. BRIEF DESCRIPTION OF THE DRAWINGS

[0024] Figure 1 The spectrum of DNA and RNA extracted from TRIZOL lysis buffer simultaneously (lane 1 is the eluent, M is the marker): Figure 2The graphs are of the simultaneous extraction of DNA and RNA from TRIZOL lysate based on different silica gel separation and purification processes (lane 1 is the eluate obtained by extraction using Example 1, lane 2 is the DNA eluate obtained by extraction using Example 2, lane 3 is the RNA eluate obtained by extraction using Example 2, and M is a marker); Figure 3 The figure is a graph of the simultaneous extraction of DNA and RNA from TRIZOL lysate using the chloroform phase separation and phenol removal method (lane 1 is the eluate obtained by extraction in Example 2, lane 2 is the RNA eluate obtained by extraction in Example 2, lane 3 is the DNA eluate obtained by extraction in Example 3, lane 4 is the RNA eluate obtained by extraction in Example 3, and M is a marker); Figure 4 The graph is the simultaneous extraction of DNA and RNA from TRIZOL lysate by adding conversion buffer (lanes 1-12 represent different experimental treatments, see Table 1 for details, M is Marker); Figure 5 The graphs show the simultaneous extraction of DNA and RNA from TRIZOL lysate using the traditional TRIZOL method, the conversion buffer method, and the chloroform phase separation and phenol removal method (lanes 1-6 represent different experimental treatments, see Table 2 for details, M is a marker).

[0025] Figures 1 to 5 In the figure, M is Marker, from large to small, they are 5000bp, 3000bp, 2000bp, 1500bp, 1000bp, 750bp, 500bp, 250bp and 100bp. DETAILED DESCRIPTION

[0026] The specific embodiments of the present invention are further described below. It should be noted that the description of these embodiments is used to help understand the present invention, but does not constitute a limitation of the present invention. In addition, the technical features involved in each embodiment of the present invention described below can be combined with each other as long as they do not conflict with each other.

[0027] The experimental methods in the following examples are conventional methods unless otherwise specified, and the experimental materials used in the following examples are commercially available unless otherwise specified.

[0028] In the following examples, the purification column used is from Zymo research, model C1004-50, and any silica gel column made of polypropylene material (which can resist the corrosion of phenol) on the market meets the requirements.

[0029] According to the principle of extracting DNA or RNA by the traditional TRIZOL method, it is impossible to use the traditional TRIZOL method, i.e., using solution phase separation (RNA exists in the aqueous phase, DNA exists in the precipitate phase or organic phase), to efficiently prepare RNA and DNA at the same time, because once DNA is coupled to chromatin proteins in the precipitate phase, the damage to the purity and quality of DNA is almost irreversible. At the same time, DNA is inherently unstable in the traditional TRIZOL lysis solution, and acidic conditions easily cause DNA to undergo depurination and degradation. However, considering that the TRIZOL method has excellent lysis ability, it has significant advantages to develop a method for separating DNA and RNA simultaneously based on TRIZOL. At the same time, our previous patent CN118240814A found that when using silica gel to extract nucleic acids, by controlling the ratio of pH, salt and ethanol, and the order of extraction during the silica gel binding process, it is possible to achieve complete and efficient separation of DNA and RNA in the liquid phase in vitro, and produce high-quality and high-purity DNA and RNA. Therefore, we have such an idea that in the process of nucleic acid extraction of actual samples such as cells, if we can find a way to make DNA and RNA exist in the water phase at the same time when TRIZOL is lysed, then the DNA in the water phase has been dissociated from the chromatin, which is the key prerequisite for preparing high-quality and high-purity DNA. Then, through the process of distinguishing DNA and RNA by silica gel, high-quality and high-purity DNA and RNA can be obtained efficiently and conveniently, thereby solving the problems that plague the field.

[0030] According to the traditional theory, the reason why DNA exists in the precipitate phase is probably because H + Neutralizing the charge in the backbone causes it to lose polarity and be unable to dissociate from chromatin, resulting in its presence in the precipitate phase. + content, then the charge of the DNA backbone cannot be neutralized and has polarity, then it can naturally dissociate from the chromatin protein and exist in the aqueous phase. In order to verify this hypothesis, the present invention has developed a conversion buffer (4M guanidine isothiocyanate, 1MTris-HCl, PH=7.4), first mixed it with the traditional TRIZOL reagent and then lysed the cells. As can be seen from Example 1, our hypothesis has been verified. This conversion buffer can indeed make DNA and RNA exist in the aqueous phase at the same time and can be separated. Then we began to try how to completely separate these two components using a simple separation and purification process. As can be seen from Example 2, if this neutral TRIZOL homogenate is directly subjected to the silica gel separation and purification process (i.e., the process established by patent CN118240814A), it is impossible to separate the DNA and RNA components. Considering that it may be due to the influence of phenol, the present invention further developed a chloroform phase separation method. As can be seen from Example 3, the chloroform phase separation method of the present invention achieves complete separation of DNA and RNA components, and OD260 / 280 , O.D. 260 / 230 The parameters are good, indicating that the extracted DNA and RNA have high purity, and can be compatible with the highest standards of nucleic acid mass spectrometry and high-throughput sequencing. The time is 14min for DNA and 16min for RNA. However, considering that chloroform needs centrifugal stratification, the operation requires certain skills, and then further considers developing a method for directly distinguishing by silica gel without phase separation, so that the operation is also simpler and the time is also shorter. Therefore, after adding 1 / 2-3 times the volume of conversion buffer, the present invention directly walks the experimental flow of silica gel distinguishing DNA and RNA, and surprisingly finds that the bottleneck that genomic DNA cannot be directly separated and purified by silica gel can be overcome by adding conversion buffer, and its extraction rate can be restored to between 57.2%-87.4%. When adding 2 times and 3 times the volume of conversion buffer, the recovery rate of more than 80% can be reached, and this conversion method is shorter in time, and DNA is 4min and RNA is 6min. Therefore, the present invention develops two methods for efficiently separating and purifying DNA and RNA, wherein the chloroform phase separation method can obtain DNA with higher yield, and the conversion method can obtain RNA with higher yield. Compared with the traditional TRIZOL method for RNA extraction, it was unexpectedly found that the yield and purity of DNA extracted by the method of the present invention far exceeded the traditional DNA. In terms of yield improvement, the conversion method was 6.61 times, and the chloroform phase separation method was 7.52 times. Even more unexpectedly, the RNA extracted by the method of the present invention was 40-60% higher than the RNA extracted by the traditional TRIZOL method (the gold standard for RNA extraction in the field). Therefore, the present invention will greatly promote the technical development in the field of nucleic acid extraction and the development of related industries.

[0031] In order to further clearly present the method of the present invention for simultaneously and efficiently extracting DNA and RNA based on TRIZOL lysis solution, a detailed description is given below in conjunction with Examples 1-4.

[0032] Example 1: Design of transformation buffer and verification of simultaneous extraction of DNA and RNA from TRIZOL lysate 1. The experimental operation is as follows: (1) Mix the transformation buffer (4 M guanidine thiocyanate, 1 M Tris-HCl, pH = 7.4) and TRIZOL reagent in a ratio of 1:2 (e.g. 1 mL transformation buffer plus 2 mL TRIZOL) to make a neutral TRIZOL lysis solution; (2) According to 1×10 5 – 1×10 7 The cells were dissolved in 1 mL of TRIZOL. 400 μL of neutral TRIZOL lysis buffer was added to human 293T cells and mixed by pipetting to obtain a neutral TRIZOL homogenate. (3) Add an equal volume of anhydrous ethanol (400 μL) and mix well; (4) Incubate the TRIZOL homogenate with the silica gel column for 1 min and centrifuge at 12,000 g for 30 s; (5) Add 400 μL of RNA washing solution 1 (80% ethanol, 1 M guanidine hydrochloride) and centrifuge at 12,000 g for 30 seconds for washing; (6) Add 700 μL of RNA washing solution 2 (80% ethanol) and rinse once by centrifugation at 12,000 g for 30 s; spin at room temperature for 1 min, elute with pure water at 12,000 g for 1 min, and retain the sample for quality inspection.

[0033] 2. Experimental results: from Figure 1 The experimental results show that adding neutral TRIZOL lysis buffer to the conversion buffer can extract genomic DNA from the sample while still maintaining the quality and stability of RNA. The star position in the figure is the genomic DNA band.

[0034] Example 2: Neutral TRIZOL lysis buffer cannot be used directly to achieve complete separation of DNA and RNA using the silica gel separation process 1. The experimental operation is as follows: The experimental operation of lane 1 was the same as that of Example 1, and the samples of lanes 2 and 3 were subjected to the following experimental operation: (1) Mix the transformation buffer (4 M guanidine thiocyanate, 1 M Tris-HCl, pH = 7.4) and TRIZOL reagent in a ratio of 1:2 (e.g. 1 mL transformation buffer plus 2 mL TRIZOL) to make a neutral TRIZOL lysis solution; (2) According to 1×10 5 – 1×10 7 The cells were dissolved in 1 mL of TRIZOL. 400 μL of neutral TRIZOL lysis buffer was added to human 293T cells and mixed by pipetting to obtain a neutral TRIZOL homogenate. (3) Directly incubate the TRIZOL homogenate with the silica column for 1 min and centrifuge at 12,000 g for 30 s; (4) Add 700 μL of DNA washing solution (70% ethanol + 150 mM NaCl), rinse once at 12,000 g for 30 s; spin for 1 min, elute with pure water at 12,000 g for 1 min. This is the separated DNA sample, and keep the sample for quality inspection; (5) Collect the flow-through from step (3), add 1 volume of anhydrous ethanol, incubate with a new silica gel column for 1 min, and centrifuge at 12000g for 30 s; (6) Add 400 μL of RNA washing solution 1 (80% ethanol, 1 M guanidine hydrochloride) and centrifuge at 12,000 g for 30 seconds for washing; (7) Add 700 μL of RNA rinse solution 2 (80% ethanol) and centrifuge at 12,000 g for 30 s to rinse once; spin at room temperature for 1 min, and elute with pure water at 12,000 g for 1 min. This is the RNA sample and should be retained for quality inspection.

[0035] 2. Experimental results: from Figure 2 It can be seen that lane 1 and Figure 1 The results are consistent, and the neutral TRIZOL lysis buffer can extract DNA and RNA at the same time. Lanes 2 and 3 are purification processes that use conventional silica gel to distinguish and separate DNA and RNA. However, as can be seen from the figure, it is impossible to completely separate DNA and RNA by directly using this purification process. There are no visible genomic DNA bands in the DNA component, and some RNA fragments also remain in this component; at the same time, the RNA extracted from the RNA component is also incomplete. The above experimental results show that it is impossible to completely separate DNA and RNA directly using the conventional silica gel purification process to distinguish nucleic acids.

[0036] Example 3: Efficient separation of DNA and RNA using chloroform phase separation and phenol removal 1. The experimental operation is as follows: The experimental operation of lanes 1 and 2 was the same as that of Example 2, and the experimental operation of lanes 3 and 4 was performed as follows: (1) Mix the transformation buffer (4 M guanidine thiocyanate, 1 M Tris-HCl, pH = 7.4) and TRIZOL reagent in a ratio of 1:2 (e.g. 1 mL transformation buffer plus 2 mL TRIZOL) to make a neutral TRIZOL lysis solution; (2) According to 1×10 5 – 1×10 7 The cells were dissolved in 1 mL of TRIZOL. 400 μL of neutral TRIZOL lysis buffer was added to human 293T cells and mixed by pipetting to obtain a neutral TRIZOL homogenate. (3) Add 1 / 5 volume of chloroform, i.e. 80 μL, and centrifuge at 12,000 g and 4 degrees for 10 min; (4) Incubate the supernatant with the silica gel column for 1 min and centrifuge at 12000g for 30 s; (5) Add 700 μL of DNA washing solution (70% ethanol + 150 mM NaCl), centrifuge at 12,000 g for 30 s to wash once; spin for 1 min, and elute with pure water at 12,000 g for 1 min. This is the separated DNA sample, and keep the sample for quality inspection; (6) Collect the flow-through from step (4), add 1 volume of anhydrous ethanol, incubate with a new silica gel column for 1 min, and centrifuge at 12000g for 30 s; (7) Add 400 μL of RNA washing solution 1 (80% ethanol, 1 M guanidine hydrochloride) and centrifuge at 12,000 g for 30 seconds for washing; (8) Add 700 μL of RNA rinse solution 2 (80% ethanol) and centrifuge at 12,000 g for 30 s to rinse once; spin at room temperature for 1 min, and elute with pure water at 12,000 g for 1 min. This is the RNA sample and should be retained for quality inspection.

[0037] 2. Experimental results: Figure 3 Based on the results of lanes 1 and 2, we speculate that the phenol component in the neutral TRIZOL homogenate may affect the complete separation of DNA and RNA in the downstream silica gel purification. Therefore, after removing the phenol by adding chloroform to obtain the supernatant, the experimental process of separating DNA and RNA by silica gel was directly carried out. The results showed that DNA and RNA could be separated efficiently ( Figure 3 Lanes 3 and 4), thus establishing a chloroform phase separation method for the efficient separation of DNA and RNA using neutral TRIZOL lysis buffer combined with silica gel.

[0038] Example 4: DNA and RNA can be separated directly using silica gel separation process by adding conversion buffer 1. The experimental operation is as follows: (1) Mix the transformation buffer (4 M guanidine thiocyanate, 1 M Tris-HCl, pH = 7.4) and TRIZOL reagent in a ratio of 1:2 (e.g. 1 mL transformation buffer plus 2 mL TRIZOL) to make a neutral TRIZOL lysis solution; (2) According to 1×10 5 – 1×10 7 The cells were dissolved in 1 mL of TRIZOL. 600 μL of neutral TRIZOL lysis buffer was added to human 293T cells. After pipetting and mixing, a neutral TRIZOL homogenate was obtained and divided into 6 equal parts (100 μL each). (3) According to Table 1, the corresponding conversion buffer (4 M guanidine isothiocyanate, 1 M Tris-HCl, pH = 7.4) was added for experimental treatment, wherein the components in lanes 11 and 12 were subjected to the experimental operation of Example 3, and the other lanes were subjected to the subsequent operations of step (4); (4) Incubate the TRIZOL homogenate with the silica gel column for 1 min and centrifuge at 12000 g for 30 s; (5) Add 700 μL of DNA washing solution (70% ethanol + 150 mM NaCl), centrifuge at 12,000 g for 30 s to wash once; spin for 1 min, and elute with pure water at 12,000 g for 1 min. This is the separated DNA sample, and keep the sample for quality inspection; (6) Collect the flow-through from step (4), add 1 volume of anhydrous ethanol, incubate with a new silica gel column for 1 min, and centrifuge at 12000g for 30 s; (7) Add 400 μL of RNA washing solution 1 (80% ethanol, 1 M guanidine hydrochloride) and centrifuge at 12,000 g for 30 seconds for washing; (8) Add 700 μL of RNA washing solution 2 (80% ethanol) and centrifuge at 12,000 g for 30 s to rinse once; spin at room temperature for 1 min, and elute with pure water at 12,000 g for 1 min. This is the RNA sample and should be retained for quality inspection.

[0039] 2. Experimental results: Depend on Figure 4 From the experimental results in Table 1, it can be seen that the ability of silica gel to distinguish between DNA and RNA can be gradually reversed by adding different volumes of conversion buffer. When the neutral TRIZOL homogenate is directly combined with the silica gel column, the DNA and RNA components cannot be distinguished well (lanes 1 and 2), and even no trace of genomic DNA can be seen in lane 1. However, when 1 / 2-3 times the volume of conversion buffer is added, the genomic DNA yield can be restored to between 57.2%-87.4% (the total amount of DNA extracted by chloroform phase separation is 1), especially when 2 and 3 times the volume of conversion buffer are added, the recovery rate can reach more than 80%. At the same time, it was unexpectedly discovered in the experiment that the RNA yield obtained by adding this conversion buffer was 1%-9% higher than that of the chloroform phase separation method, and the OD values ​​of DNA and RNA extracted by these two methods were similar. 260 / 280 , O.D. 260 / 230 The parameters were good and all met the purity requirements for high-throughput sequencing.

[0040] Table 1 Experimental treatment and results of simultaneous extraction of DNA and RNA from TRIZOL lysate by conversion buffer method and chloroform phase separation and phenol removal method Example 5: Comparison of the conventional TRIZOL method and the two methods of the present invention (conversion buffer method and chloroform phase separation phenol removal method) for simultaneous extraction of DNA and RNA 1. The experimental operation is as follows: 293T cells were collected and divided into three equal parts, and the experimental treatments were performed according to Table 2. The traditional TRIZOL method was performed according to the official instructions of TRIZOL reagent (15596026CN) of Thermo Fisher. The conversion buffer method and the chloroform phase separation method were performed according to the experimental operations of Example 4 and Example 3, respectively.

[0041] 2. Experimental results: The present invention transforms the traditional TRIZOL reagent into a neutral TRIZOL lysis solution (conversion buffer), and develops the corresponding conversion buffer method and chloroform phase separation method to simultaneously prepare DNA and RNA. It is unexpectedly found that the amount of RNA prepared by these two methods exceeds that of the traditional TRIZOL method (the gold standard for RNA extraction), with the conversion method increasing by 52% and the chloroform phase separation method increasing by 41%, and the OD 260 / 280 Greater than 2.0, OD 260 / 230 Greater than 2.0 ( Figure 5 ), which is an extremely high purity standard, all meet the purity requirements of downstream high-throughput sequencing. At the same time, the extracted DNA content, purity and yield all surpass the traditional TRIZOL method, and the DNA extracted by the traditional TRIZOL method, its gel running conditions and abnormal OD values ​​are consistent with the industry's belief that the traditional TRIZOL method of extracting DNA will produce irreversible low-quality and low-purity DNA crude products. For the quantification of the crude product, a DNA-specific fluorescent dye method was used for quantification to reduce the interference of impurities on DNA quantification. It was found that the DNA extracted by the method of the present invention far surpassed the traditional method in terms of gel running conditions and OD values. The yield of the conversion method is 6.61 times that of the traditional method, and the chloroform phase separation method is 7.52 times that of the traditional method. In addition, from the perspective of experimental operation and cycle, the traditional method extracts DNA for more than 2h, and requires multiple rounds of purification to adapt to downstream experimental applications. The process is cumbersome and the loss is serious. The two methods of the present invention are simple to operate, short in time, high in yield and quality, and directly compatible with the highest standards of high-throughput sequencing in the downstream.

[0042] The chloroform phase separation method takes 14 minutes to extract DNA and 16 minutes to extract RNA, while the conversion method takes 4 minutes to extract DNA and 6 minutes to extract RNA. It can be seen that the method of the present invention has incomparable advantages.

[0043] Table 2 Experimental treatment and results of the traditional TRIZOL method, the conversion buffer method, and the chloroform phase separation and phenol removal method for simultaneous extraction of DNA and RNA from TRIZOL lysate In summary, the present invention has developed a method for simultaneously and efficiently separating DNA and RNA using a silica gel column using TRIZOL as a lysis solution, and has developed a conversion buffer to convert traditional acidic TRIZOL into neutral TRIZOL. It is also the first time that the concept of a neutral TRIZOL lysis solution has been proposed. This lysis solution is more conducive to the integrity and stability of DNA and RNA; it can also avoid the irreversible damage to DNA caused by the traditional acidic TRIZOL extraction method. In principle, DNA is first released from chromatin into the aqueous phase, while RNA is also present in the aqueous phase. Subsequently, DNA and RNA are efficiently separated and purified by controlling the ratio of pH, salt and ethanol, and the order of extraction after combining with silica gel.

[0044] The embodiments of the present invention are described in detail above, but the present invention is not limited to the described embodiments. For those skilled in the art, various changes, modifications, substitutions and variations of these embodiments are made without departing from the principles and spirit of the present invention, and still fall within the protection scope of the present invention.

Claims

1. A kit for simultaneously and efficiently extracting DNA and RNA based on TRIZOL lysis buffer, characterized in that: The kit comprises a neutral TRIZOL lysis solution, a DNA rinse solution, anhydrous ethanol, an RNA rinse solution 1 solution and an RNA rinse solution 2 solution; or comprises a neutral TRIZOL lysis solution, a DNA rinse solution, anhydrous ethanol, an RNA rinse solution 1 solution, an RNA rinse solution 2 solution and chloroform; The neutral TRIZOL lysis solution is composed of a conversion buffer and a TRIZOL reagent, wherein the conversion buffer is composed of 4M guanidine thiocyanate and 1M Tris-HCl, and the pH value is 7.4; The DNA rinse solution is composed of ethanol and NaCl, the RNA rinse solution 1 is composed of ethanol and guanidine hydrochloride, and the RNA rinse solution 2 is an ethanol solution.

2. A kit for simultaneously and efficiently extracting DNA and RNA based on TRIZOL lysis solution according to claim 1, characterized in that: The neutral TRIZOL lysis solution is composed of conversion buffer and TRIZOL reagent in a volume ratio of 1:

2.

3. A kit for simultaneously and efficiently extracting DNA and RNA based on TRIZOL lysate according to claim 1, characterized in that: The DNA washing solution consists of 70% ethanol and 150 mM NaCl.

4. A kit for simultaneously and efficiently extracting DNA and RNA based on TRIZOL lysate according to claim 1, characterized in that: The RNA washing solution 1 consists of 80% ethanol and 1M guanidine hydrochloride.

5. A kit for simultaneously and efficiently extracting DNA and RNA based on TRIZOL lysis solution according to claim 1, characterized in that: The RNA washing solution 2 is 80% ethanol.

6. A method for simultaneously and efficiently extracting DNA and RNA based on TRIZOL lysis buffer, characterized in that: The kit according to any one of claims 1 to 5 is used to extract DNA and RNA, and the extraction process comprises the following steps: S1. Add neutral TRIZOL lysis buffer to the cells and mix well to make neutral TRIZOL homogenate; S2, add chloroform to the neutral TRIZOL homogenate of S1, collect the supernatant by centrifugation, and incubate the supernatant with a silica column to allow the DNA to bind to the silica column; or add conversion buffer to the neutral TRIZOL homogenate of S1, mix well, and incubate the TRIZOL homogenate with a silica column to allow the DNA to bind to the silica column; S3, adding DNA rinsing solution to the silica gel column in S2 for rinsing, and then spinning and eluting with pure water to obtain a DNA sample; S4, collecting the flow-through of S3, adding anhydrous ethanol and incubating it with a new silica gel column to allow RNA to bind to the silica gel column; S5, adding RNA washing solution 1 solution to the silica gel column in S4 for washing; S6. Add RNA rinsing solution 2 to the silica gel column in S5 for rinsing, and then spin dry and elute with pure water to obtain an RNA sample.

7. The method for simultaneously and efficiently extracting DNA and RNA based on TRIZOL lysis solution according to claim 6, characterized in that: In S2, the volume ratio of chloroform to neutral TRIZOL homogenate is 1:5; the volume ratio of transformation buffer to neutral TRIZOL homogenate is 0.5-3:

1.

8. The method for simultaneously and efficiently extracting DNA and RNA based on TRIZOL lysis solution according to claim 6, characterized in that: In S1, press 1×10 5 – 1×10 7 Cells were lysed in 1 mL of TRIZOL standard by adding neutral TRIZOL lysis buffer to the cells.

9. The method for simultaneously and efficiently extracting DNA and RNA based on TRIZOL lysis solution according to claim 6, characterized in that: In S4, the volume ratio of the flow-through liquid to anhydrous ethanol = 1:1-2.

10. The method for simultaneously and efficiently extracting DNA and RNA based on TRIZOL lysis solution according to claim 6, characterized in that: The silica gel column is a silica gel column made of polypropylene.

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