A method for recovering single stranded nucleic acids from PAG

Abstract

The application discloses a method for recovering single-stranded nucleic acid from PAG, comprising the following steps: preparing a swelling solution by using deionized water and anhydrous ethanol as a mixed solvent and sodium hydroxide as a solute; mixing polyacrylamide gel fragments containing target fragments with the swelling solution, then incubating, and then collecting the polyacrylamide gel fragments after incubation; soaking the polyacrylamide gel fragments after incubation in pure water for swelling to obtain a gel swelling solution; and concentrating and purifying the gel swelling solution to obtain a collection, i.e. a target nucleic acid fragment. The method provided by the application increases the negative charge between the side chain groups of acrylamide, uses classical repulsion to make high-resolution and structure-dense PAG highly swell, so that the PAG no longer tightly 'wraps' single-stranded nucleic acid, thereby increasing the probability of elution or escape of single-stranded nucleic acid, and finally achieving the purpose of improving the recovery rate of single-stranded nucleic acid.

Description

Technical Field

[0001] This invention relates to the field of biochemistry, and more specifically to a method for recovering single-stranded nucleic acids from PAG. Background Technology

[0002] Single-stranded nucleic acids (single-stranded DNA, single-stranded RNA, single-stranded LNA, etc.) possess unique functionalities. The bases on a single-stranded nucleic acid are "naked," forming the structural basis for base pairing and the specific recognition of nucleic acids by other molecules. Single-stranded DNA (or RNA) can act as probes, aptamers, and other core components, completing biochemical reactions such as biosensing, nucleic acid amplification and detection, and expression regulation. Therefore, single-stranded nucleic acids play a crucial role in scientific research, drug development, and disease treatment. For example, microRNA (miRNA) is a non-coding single-stranded RNA approximately 22 nt in length. Different miRNA molecules are similar in size but differ in sequence composition. Some types of miRNA participate in the regulation of basic metabolic processes such as cell cycle and cell differentiation, while others play important roles in key life activities such as individual development and pathological evolution. Therefore, miRNAs are of great significance in research and practice in life sciences, medicine, and other fields. Among them, RNA, especially that which exists in a covalently closed form, occupies an important position in current life science research. In addition, commonly used single-stranded nucleic acids include mRNA, which constitutes vaccines; DNAzymes, used for heavy metal detection or RNA; and molecular beacons (MBs), which generate detection signals. Unlike double-stranded nucleic acid molecules with a stable double helix structure, the sequence composition (base arrangement and number of bases) almost completely determines the secondary structure of single-stranded nucleic acids, and further affects the interactions between single-stranded nucleic acid molecules and between single-stranded nucleic acid molecules and other substances. A difference of a single base can cause significant functional differences in single-stranded nucleic acid molecules. Therefore, high-purity single-stranded nucleic acids are of great significance for scientific research and production activities.

[0003] However, single-stranded nucleic acids synthesized chemically or enzymatically often require further identification and purification via gel electrophoresis. Polyacrylamide gel (PAG) is the primary method used. PAG is a highly hydrophilic three-dimensional network gel polymerized from acrylamide monomers and cross-linking agents (such as methylene acrylamide). Polyacrylamide gel electrophoresis (PAGE) using PAG as a carrier is the most widely used electrophoresis technique. Compared with another gel electrophoresis technique, agarose gel electrophoresis, this technology has two main advantages: 1. Polyacrylamide gel electrophoresis has higher resolution, identifying different nucleic acid fragments at the level of individual base molecular weight differences. It can also distinguish nucleic acid elements with the same molecular weight and sequence but different conformations (such as single-stranded linear nucleic acids and single-stranded circular nucleic acids, single-stranded DNA loops with different numbers of intertwines, etc.) based on the differences in nucleic acid spatial structure. In contrast, the highest resolution of agarose gel electrophoresis is only 100-bp, which cannot achieve high-resolution nucleic acid separation. 2. Since single-stranded nucleic acids tend to fold to form certain secondary structures, if they are not denatured (breaking the hydrogen bonds between or within molecules), they are prone to forming polymers, which appear as diffuse bands in the lanes, affecting the identification and analysis of single-stranded nucleic acids. Therefore, PAGE with added nucleic acid denaturants such as urea and formamide is used for the denaturation of nucleic acid fragments and the analysis and preparation of denatured nucleic acid fragments. However, agarose gel is prone to rubberization reaction with denaturants such as urea and formamide, and cannot be used to analyze single-stranded DNA and single-stranded RNA under deformable conditions.

[0004] Thanks to these characteristics, denaturing PAG (or denaturing PAGE electrophoresis) has become a primary method for separating single-stranded nucleic acids. For example, PAGE purification, commonly used in the chemical synthesis of linear single-stranded DNA (l-ssDNA) such as primers, involves recovering DNA fragments using polyacrylamide gels after synthesis. Single-stranded circular DNA (c-ssDNA) and single-stranded circular RNA (c-ssRNA), which cannot be chemically synthesized, require high-resolution denaturing PAG for identification and preparation to ensure high purity. PAG preparation uses acrylamide and methylene acrylic acid (bisacrylamide) as raw materials. Free radicals generated by a catalytic initiator break the unsaturated bonds of the monomers, and repeated addition reactions complete the polymerization. Therefore, unlike agarose gels, PAG has a carbon-carbon single bond (CC) backbone with amide groups as side chains, resulting in greater chemical stability.

[0005] However, efficiently and easily recovering nucleic acid fragments from PAG is challenging, as the complexity of the procedures is far greater than that of recovering DNA from agarose gels. This is because PAG tightly encapsulates nucleic acids, making it difficult for nucleic acid molecules to be eluted from the gel. In contrast, nucleic acid fragments are easier to recover from agarose gels. This is because agarose enzymes are commonly used to treat agarose gels. Agarose enzymes hydrolyze the glycosidic bonds on the backbone of large agarose molecules, degrading agarose into soluble monosaccharides and thoroughly disrupting the gel (transforming it from a gel to a solution). During this process, the gel network system encapsulating nucleic acids is degraded, releasing the nucleic acid fragments to be recovered into the solution, making them "free" and thus easier to recover in large quantities. The backbone of the PAG molecular network is composed of carbon-carbon single bonds, which have extremely high chemical stability. It is difficult to destroy the PAG backbone and its network structure through physical, chemical, or biological methods. Even after treating PAG using some methods, nucleic acid molecules remain tightly entangled in the gel network, resulting in low recovery efficiency. The current method for recovering nucleic acids from PAGs is mainly the gel crushing-pure water immersion method. The core steps of this method include: 1. Crushing the gel fragments (containing the target nucleic acid); 2. Swelling the fragments in pure water for an extended period; 3. Recovering the nucleic acid fragments from the pure water. The principle of this method is to utilize the random motion of nucleic acid molecules, increasing the contact area between the gel and pure water, thereby increasing the probability of nucleic acid fragments escaping from the gel. This method undoubtedly suffers from drawbacks such as low nucleic acid recovery rate and long operation time. Furthermore, the recovered nucleic acid is prone to residual unreacted small molecules such as acrylamide monomers, urea, and formamide, which severely affect subsequent reactions. More importantly, the escape rate of nucleic acid fragments from the PAG largely depends on the size of the pores in the PAG: the higher the resolution of the PAG, the smaller the pores, and the slower the nucleic acid fragments escape. For example, it is difficult to recover large amounts of nucleic acid fragments from PAGs with an acrylamide mass dispersion of 15% or higher using the crushing-immersion method. Another method for recovering nucleic acid fragments from PAGs includes adding the excised PAG containing the target fragment to the wells of an agarose gel for a second electrophoresis, and then recovering the target DNA fragment from the agarose gel. This method mainly utilizes the effect of electric current to accelerate the escape rate of nucleic acids from the PAG, thereby improving the recovery rate. However, because PAG fragments are very small and their density is close to that of aqueous solutions, they are easily dispersed in the wells, causing the target product bands to diffuse in the agarose gel, ultimately affecting the recovery rate. Furthermore, performing a second electrophoresis on the excised PAG is difficult, more likely to cause RNA degradation, and also easily leads to secondary contamination of the sample. Summary of the Invention

[0006] The main objective of this invention is to provide a method for recovering single-stranded nucleic acids from PAG, aiming to offer a simple and efficient method for recovering single-stranded nucleic acids from PAG while ensuring the recovery rate and purity of the single-stranded nucleic acids.

[0007] To achieve the above objectives, this invention proposes a method for recovering single-stranded nucleic acids from PAG, comprising the following steps:

[0008] A swelling solution was prepared using deionized water and anhydrous ethanol as a mixed solvent and sodium hydroxide as a solute.

[0009] The polyacrylamide gel fragments containing the target fragments were mixed with the swelling solution and incubated, and then the incubated polyacrylamide gel fragments were collected.

[0010] The incubated polyacrylamide gel fragments were soaked in pure water to swell, resulting in a gel swelling solution.

[0011] The gel swelling solution is concentrated and purified to obtain a collection, which yields the target nucleic acid fragment.

[0012] Optionally, in the step of preparing the swelling solution using deionized water and anhydrous ethanol as a mixed solvent and sodium hydroxide as a solute:

[0013] In the mixed solvent, the volume ratio of deionized water to anhydrous ethanol is 1:1 to 4, and the pH of the mixed solvent is 10 to 12.

[0014] Optionally, in the step of mixing polyacrylamide gel fragments containing the target fragment with the swelling solution and then incubating them, followed by collecting the incubated polyacrylamide gel fragments:

[0015] Each 1 mg of the polyacrylamide gel fragment is mixed with 5 to 100 mL of the swelling solution.

[0016] Optionally, in the step of mixing polyacrylamide gel fragments containing the target fragment with the swelling solution and then incubating them, followed by collecting the incubated polyacrylamide gel fragments:

[0017] The incubation temperature is 65–85°C and the incubation time is 30–120 min.

[0018] Optionally, in the step of soaking the incubated polyacrylamide gel fragments in pure water to swell and obtain a gel swelling solution:

[0019] Each 1 mg of the polyacrylamide gel fragment is soaked in 5 to 25 mL of the purified water.

[0020] Optionally, in the step of soaking the incubated polyacrylamide gel fragments in pure water to swell and obtain a gel swelling solution:

[0021] The swelling temperature is 25–37°C and the swelling time is 2–8 hours.

[0022] Optionally, in the step of concentrating and purifying the gel swelling solution to obtain a collection, i.e., obtaining the target nucleic acid fragment:

[0023] The concentration temperature is not higher than 50°C, and the concentration is to concentrate to 1 / 8 to 1 / 10 of the original volume of the supernatant.

[0024] Optionally, in the step of concentrating and purifying the gel swelling solution to obtain a collection, i.e., obtaining the target nucleic acid fragment, the purification includes:

[0025] The concentrated gel swelling solution was placed in an ultrafiltration tube for a first centrifugation, and the first filtrate was discarded. Then the ultrafiltration tube was inverted for a second centrifugation, and the second filtrate was collected.

[0026] After adding frozen anhydrous ethanol to the second filtrate, centrifuge, collect the precipitate, and remove any residual anhydrous ethanol.

[0027] Optionally, in the step of placing the concentrated gel swelling solution in an ultrafiltration tube for a first centrifugation, discarding the first filtrate, and then inverting the ultrafiltration tube for a second centrifugation to collect the second filtrate:

[0028] The centrifugal force for the first centrifugation is 12000–16000 g, and the centrifugation time is 8–12 min; and / or,

[0029] The centrifugal force for the second centrifugation is 800–1200 g, and the centrifugation time is 8–12 min.

[0030] Optionally, in the step of adding frozen anhydrous ethanol to the second filtrate, centrifuging, collecting the precipitate, and removing any residual anhydrous ethanol:

[0031] The volume of the frozen anhydrous ethanol is 4 to 6 times the volume of the second filtrate; and / or,

[0032] The centrifugation speed is 10,000 to 14,000 rpm and the centrifugation time is 8 to 12 minutes.

[0033] This invention innovatively proposes a method for flexibly controlling the volume of polyacrylamide (PAG) from the perspective of rapid hydrolysis of its side chains. This method increases the negative charge between the acrylamide side chain groups, utilizing classical repulsion to cause the high-resolution, densely structured PAG to swell significantly, preventing it from tightly "wrapping" single-stranded nucleic acids. This increases the probability of single-stranded nucleic acids being eluted or escaping from the PAG, ultimately improving the recovery rate of single-stranded nucleic acids. In addition to its high recovery rate, this invention, compared to other existing methods for recovering single-stranded nucleic acids from PAG, can effectively remove residual small molecules such as urea, formamide, and salt ions from deformed PAG, avoiding the influence of these impurities on subsequent reactions. Furthermore, this method is simple to operate, has a streamlined process, requires no complex equipment, and can separately meet the needs for recovering single-stranded DNA and single-stranded RNA from high-resolution PAG. Attached Figure Description

[0034] To more clearly illustrate the technical solutions in the embodiments of the present invention or the prior art, the drawings used in the description of the embodiments or the prior art will be briefly introduced below. Obviously, the drawings described below are only some embodiments of the present invention. For those skilled in the art, other related drawings can be obtained based on these drawings without creative effort.

[0035] Figure 1A A schematic flowchart of an embodiment of the method for recovering single-stranded nucleic acids from PAG provided by the present invention;

[0036] Figure 1B for Figure 1A A basic schematic diagram of the method for recovering single-stranded nucleic acids from PAG is provided.

[0037] Figure 2A Comparison of actual effects of PAG swelling using different swelling methods;

[0038] Figure 2B A comparison chart of the swelling rates of PAG swelled using different swelling methods;

[0039] Figure 3 This is a comparison chart of the recovery rates of DNA recovered in Example 1 and Example 2.

[0040] Figure 4 This is a comparison chart of the purity of the recovered DNA in Example 2 and Comparative Example 2;

[0041] Figure 5A The graph shows the purity test results of the recovered RNA in Comparative Example 3;

[0042] Figure 5BThis is a graph showing the purity test results of the recovered RNA in Example 3;

[0043] Figure 6 This is a comparison of electrophoretic images of the recovered RNA in Example 3 and Comparative Example 3;

[0044] Figure 7 This is a diagram of the secondary structure of the DNA or RNA used in the examples.

[0045] The realization of the objective, functional features and advantages of the present invention will be further explained in conjunction with the embodiments and with reference to the accompanying drawings. Detailed Implementation

[0046] To make the objectives, technical solutions, and advantages of the embodiments of the present invention clearer, the technical solutions in the embodiments of the present invention will be clearly and completely described below. Where specific conditions are not specified in the embodiments, conventional conditions or conditions recommended by the manufacturer shall apply. Where the manufacturers of reagents or instruments are not specified, they are all conventional products that can be purchased commercially. Furthermore, the meaning of "and / or" throughout the text includes three parallel solutions; for example, "A and / or B" includes solution A, or solution B, or a solution where both A and B are satisfied simultaneously. In addition, the technical solutions of the various embodiments can be combined with each other, but this must be based on the ability of those skilled in the art to implement them. When the combination of technical solutions is contradictory or cannot be implemented, it should be considered that such a combination of technical solutions does not exist and is not within the scope of protection claimed by the present invention. Based on the embodiments of the present invention, all other embodiments obtained by those skilled in the art without creative effort are within the scope of protection of the present invention.

[0047] Our research team discovered that accelerating the escape rate of nucleic acid fragments from the gel is key to improving DNA recovery from PAG. Based on this, we propose a method for recovering single-stranded nucleic acids from PAG. Figure 1A The image shows an embodiment of the method for recovering single-stranded nucleic acids from PAG provided by the present invention. Figure 1B The basic principle of the recycling method provided by this invention. See also... Figure 1A As shown, in this embodiment, the method for recovering single-stranded nucleic acids from PAG includes the following steps:

[0048] Step S10: Prepare a swelling solution using deionized water and anhydrous ethanol as a mixed solvent and sodium hydroxide as a solute;

[0049] Step S20: Mix the polyacrylamide gel fragments containing the target fragment with the swelling solution and incubate them, then collect the incubated polyacrylamide gel fragments.

[0050] Step S30: Soak the incubated polyacrylamide gel fragments in pure water to swell, and obtain a gel swelling solution;

[0051] Step S40: Concentrate and purify the gel swelling solution to obtain the collected material, which is the target nucleic acid fragment.

[0052] This invention innovatively proposes a method for flexibly controlling the volume of polyacrylamide (PAG) from the perspective of rapid hydrolysis of its side chains. This method increases the negative charge between the acrylamide side chain groups, utilizing classical repulsion to cause the high-resolution, dense PAG to swell significantly, preventing it from tightly "wrapping" single-stranded nucleic acids. This increases the probability of single-stranded nucleic acids being eluted or escaping from the PAG, ultimately improving the recovery rate of single-stranded nucleic acids. In addition to its high recovery rate, this method, compared to other existing methods for recovering single-stranded nucleic acids from PAG, effectively removes residual urea, formamide, and salt ions from the modified PAG, avoiding the influence of these impurities on subsequent reactions. Furthermore, this method is simple to operate, requires fewer steps, and eliminates the need for complex equipment. It can also meet the needs for recovering single-stranded DNA and single-stranded RNA from high-resolution PAGs.

[0053] First, deionized water and wastewater ethanol are mixed in a certain proportion as a solvent, and sodium hydroxide is used as a solute to prepare a swelling solution, which is used as a swelling agent for PAG. In some embodiments of the present invention, the volume ratio of deionized water to anhydrous ethanol in the mixed solvent is 1:1 to 4, and the pH of the mixed solvent is 10 to 12.

[0054] Then, the PAG fragments containing the target fragment (cut from the intact gel under UV light) were mixed with the prepared swelling solution and incubated at a certain temperature. After incubation, the PAG fragments were collected, and the changes during the incubation process were as follows. Figure 1BAs shown. In some embodiments of the present invention, the ratio of the PAG fragments to the swelling solution is: 1 mg of the PAG fragments to 5-100 mL of the swelling solution. Additionally, in some embodiments of the present invention, the incubation temperature is 65-85°C, the incubation time is 30-120 min, and the heating method is preferably water bath heating. Furthermore, it should be noted that in some embodiments of the present invention, if the PAG fragments in step S20 are relatively intact (e.g., PAG that has not been crushed or chopped), they can be collected directly after incubation; that is, the mixed solution after incubation of the PAG fragments and the swelling solution is used as the product for further processing. If the PAG fragments in step S20 are relatively small and dispersed (e.g., PAG that has been crushed or chopped), the PAG fragments can be collected by centrifugation, and the swelling solution after incubation can be discarded. In some embodiments of the present invention, the centrifugation conditions are preferably 20-30°C, 10000-15000 rpm, and 5-15 min.

[0055] Next, the incubated PAG fragments are rapidly washed 2-3 times with ultrapure water. Specifically, the amount of ultrapure water used for washing the PAG fragments is 10 mL per wash. For crushed or shredded PAG, the added ultrapure water can be removed by centrifugation for rapid washing. In some embodiments of the present invention, the preferred centrifugation conditions are 20-30°C, 10000-15000 rpm, and 20-40 s. Subsequently, the washed PAG fragments are mixed with pure water in a certain proportion and allowed to stand at room temperature for a period of time to obtain a gel swelling solution. In some embodiments of the present invention, the mixing ratio of the PAG fragments with pure water is: 1 mg of the polyacrylamide gel fragments is soaked in 5-25 mL of pure water. In addition, in some embodiments of the present invention, the swelling temperature is 25-37°C and the swelling time is 2-8 h.

[0056] Referring to the TE swelling method in "Method for Recovering DNA from Polyacrylamide Gel After Electrophoresis" (CN100410372C) as a comparison, the swelling rate of the gel after treatment with the method provided in this invention is compared. The steps of the TE swelling method are as follows:

[0057] (1) Prepare PAG with an acrylamide mass fraction of 15% and containing 8M urea and 25% formamide (V:V);

[0058] (2) PAG was mixed with TE solution (10mM Tris-HCl, 1mM EDTA, pH=8.0) at a ratio of 1g:25mL and incubated at 25℃ and 65℃ respectively. The weight of the gel was recorded and the swelling rate was calculated.

[0059] Figure 2A , Figure 2B The figure shows a comparison between the TE swelling method and the swelling method provided by this invention (the volume ratio of anhydrous ethanol to deionized water in the swelling solution is 1:1, the ratio of gel to swelling solution is 1g:25mL, the incubation temperature is 85℃, the incubation time is 30min, the ratio of gel to pure water is 1g:25mL, the soaking temperature is 25℃, and the soaking time is 8h). Figure 2A Images and swelling rate measurements of PAG swelled at room temperature (25℃) and high temperature (65℃) using the TE swelling method are shown. Figure 2B This image shows a physical sample of PAG swelled using the swelling method of this invention, along with the results of swelling rate determination. Figure 2A and Figure 2B It is known that both methods can achieve PAG swelling to some extent, but the effects of different swelling treatments on the PAG swelling rate vary significantly. Specifically, the TE swelling method cannot achieve efficient PAG swelling at room temperature or high temperature, with a maximum swelling rate of only 25%. In contrast, the swelling method provided by this invention can achieve a maximum PAG swelling rate of 150%. Since the migration rate of nucleic acids in the gel is affected by the pore size of the gel network—larger pores result in faster nucleic acid migration, and smaller pores result in slower migration—and the pore size of the swollen gel inevitably increases, with a higher swelling rate corresponding to a greater increase in pore size, the swelling method provided by this invention is more conducive to the migration of nucleic acid molecules.

[0060] After obtaining the gel swelling solution, the gel swelling solution is then concentrated. In some embodiments of the present invention, the concentration temperature is not higher than 50°C, the concentration ratio is 1 / 8 to 1 / 10 of the original volume of the supernatant, and the concentration method can be centrifugation or rotary evaporation. Subsequently, the obtained concentrate is purified to obtain the target fragment nucleic acid. Specifically, in some embodiments of the present invention, the purification in step S40 includes the following steps:

[0061] Step S401: Place the concentrated gel swelling solution in an ultrafiltration tube for a first centrifugation, discard the first filtrate, then invert the ultrafiltration tube for a second centrifugation, and collect the second filtrate.

[0062] Step S402: Add frozen anhydrous ethanol to the second filtrate, centrifuge, collect the precipitate and remove the residual anhydrous ethanol.

[0063] The concentrated gel swelling solution is placed in an ultrafiltration tube for a first centrifugation, and the first filtrate is discarded. The ultrafiltration tube is then inverted for a second centrifugation, and the second filtrate is collected. This second filtrate is then concentrated by low-temperature centrifugation to increase the concentration of single-stranded nucleic acids or to produce a dry powder for long-term storage. In some embodiments of the present invention, the centrifugal force for the first centrifugation is 12000–16000 g, and the centrifugation time is 8–12 min. In other embodiments, the centrifugal force for the second centrifugation is 800–1200 g, and the centrifugation time is 8–12 min. Furthermore, the molecular weight cutoff of the ultrafiltration tube depends on the length (molecular weight) of the single-stranded nucleic acid to be separated. For example, to recover single-stranded nucleic acids with a length of 100 nt, it is recommended to use an ultrafiltration tube with a molecular weight cutoff of no more than 10000 Da for purification.

[0064] In some embodiments of the present invention, the low-temperature centrifugation involves adding frozen anhydrous ethanol to the second filtrate and then centrifuging. After centrifugation, the precipitate is collected, and after the ethanol in the precipitate evaporates, a dry powder of the single-stranded nucleic acid recovered to the target fragment is obtained. The volume of the frozen anhydrous ethanol is 4 to 6 times the volume of the second filtrate. Furthermore, the centrifugation speed is 10,000 to 14,000 rpm, and the centrifugation time is 8 to 12 minutes. This maintains the temperature of the low-temperature centrifugation process at approximately 4°C. Finally, the obtained dry powder of single-stranded nucleic acid is dissolved and diluted in TE solution (10 mM Tris-HCl; 1 mM EDTA; pH = 8.0) or other buffer solutions before use in subsequent experiments.

[0065] It should be noted that in some embodiments of the present invention, when the object to be recovered is single-stranded RNA, all solutions (including ultrapure water), containers, pipette tips, and other reagents or consumables that come into direct or indirect contact with RNA in all operational steps must be strictly treated with diethyl pyrocarbonate (DEPC) to inactivate RNase; and the operating environment should be clean and uncontaminated to avoid degradation of single-stranded RNA. Since ultrafiltration tubes cannot be treated with DEPC, the purification in step S40 uses 75% ethanol to wash the precipitate to remove excess salt ions and other soluble impurities. The specific steps are as follows: First, the concentrated gel swelling solution is centrifuged at low temperature. The low-temperature centrifugation method can be the same as described above, using the method of adding frozen anhydrous ethanol before centrifugation. After low-temperature centrifugation, the precipitate is collected, and the precipitate is washed with pre-cooled 75% ethanol. After evaporating the ethanol, RNA containing the target band is recovered.

[0066] The technical solution of the present invention will be further described in detail below with reference to specific embodiments and accompanying drawings. It should be understood that the following embodiments are only used to explain the present invention and are not intended to limit the present invention.

[0067] In the following examples, the preparation steps of PAG for denatured DNA are as follows: A certain amount of acrylamide and methylene acrylamide are mixed at a mass ratio of 29:1. A certain amount of urea and TBE buffer (10*) are added, and the volume is adjusted to 400 mL with ultrapure water, so that the total mass fraction of acrylamide is 20%, the urea concentration is 8M, the formamide concentration is 0.09M, and the EDTA concentration is 0.002M. Undissolved solid powder is carefully filtered to remove the solid powder, and the liquid (polyacrylamide gel) is collected for later use. Before use, a suitable concentration of 20% acrylamide gel solution is taken, and formamide solution is added to account for 25% of the total volume. The volume is adjusted to 40 mL with urea solution (8M, containing 0.09M Tris-boric acid and 0.002M EDTA, the same below). 40 μL of TEMED and 400 μL of 10% (m:V) ammonium persulfate aqueous solution are added, and the mixture is stirred evenly and poured into a gel casting frame. After the reaction is complete, the PAG is taken out for subsequent experiments. It should be noted that the preparation method and reagent dosage for ordinary PAG not used for denatured DNA are the same as the above method, but urea and formamide are not added, and pure aqueous solution is used for preparation.

[0068] In the following examples, the weighing method was used to observe PAG at different swelling stages. The steps are as follows:

[0069] The swelling rate of PAG was tested using gravimetric analysis. The weighed PAG was immersed in pure water or an alkaline alcohol solution and allowed to swell for a certain period. After removal, the solution on the surface of the PAG was absorbed with filter paper, and then it was weighed again. The swelling rate (SR) of PAG was calculated as follows:

[0070] SR% = (W S -W0) / W0%

[0071] Where W0 is the weight of PAG before swelling, W S This represents the weight of PAG after swelling.

[0072] In the following examples, the steps of polyacrylamide gel electrophoresis (denaturing or non-denaturing) and staining imaging are as follows:

[0073] A certain concentration of PAG was prepared for electrophoresis (vertical electrophoresis apparatus, Beijing Liuyi). The electrophoresis voltage was 300V, and the electrophoresis time was determined by the fragment size. After electrophoresis, SYBR Green II staining was performed for 15 minutes, and the electrophoresis results were analyzed in a gel imaging system (Bio-rad, USA).

[0074] In the following examples, the recovery rate of nucleic acids recovered from PAG was determined using an absorbance measurement method at 260 nm. The steps are as follows:

[0075] Keeping the sample volume constant before and after, use the following formula:

[0076] C S / C0%=Abs -s / Abs -0 (C: Sample concentration; Abs: Absorbance of the sample at a wavelength of 260 nm; C0 and Abs) -0 The sample concentration and corresponding absorbance before gel recovery; C S With Abs -s The recovery rate was calculated by taking the concentration of the recovered sample and the corresponding absorbance value. Note: Absorbance value determination method: 2 μL of sample was pipetted into a Nano Drop (Thermo Fisher, USA) each time, and the Abs of the sample was obtained by detection. Pure water was used as the empty space. The concentration of the sample could then be calculated.

[0077] Table 1 Sequence Information

[0078]

[0079] Note: L-ssDNA sequences and c-ssDNA sequences with similar names are identical in base arrangement and composition. For example, the 42-nt L-ssRNA and 42-nt c-ssRNA sequences both have the following composition:

[0080] 5'-rGrGrUrGrCrArCrGrArArArCrGrUrGrCrArCrCrUrGrUrCrCrGrGrUrArArArArUrArCrCrGrGrArCrA-3'.

[0081] However, c-ssDNA or c-ssRNA does not have a free 5' or 3' end.

[0082] Example 1

[0083] (1) Prepare PAG with 15% acrylamide by mass, containing 8M urea and 25% formamide (V:V).

[0084] (2) Prepare 1-ssDNA (42-nt, 90-nt and 200-nt, respectively, with sequences shown in Table 1) purchased or prepared in the early stage, and perform electrophoresis with PAG prepared in step (1). After electrophoresis, stain the gel, place it under a UV lamp to observe the bands, and cut off the gel containing the target band.

[0085] (3) Take the gel fragments cut off in step (2) and the swelling solution (pH=12.0, the volume ratio of anhydrous ethanol to deionized water is 1:1) and mix them at a ratio of 1g:25mL. Incubate at 85℃ for 30min, then wash quickly with ultrapure water 2-3 times. The amount of ultrapure water used for each wash is 10mL / wash. Centrifuge at 25℃, 12000rpm, and 30s.

[0086] (4) Place the washed gel fragments in pure water at a ratio of 1g:25mL and swell at 25℃ for 8h. Collect the pure water solution to obtain the gel swelling solution.

[0087] (5) The gel swelling solution (total 45 mL) obtained by vacuum low-temperature rotary concentration method was concentrated at 50℃ for 4 h to obtain a concentrated solution of 5 mL. Then, the concentrated solution was placed in an ultrafiltration tube with a molecular weight cutoff of 3K for the first centrifugation at 14000 g for 10 min. The first filtrate was discarded. The ultrafiltration tube was then inverted for the second centrifugation at 1000 g for 10 min. The second filtrate was collected. Five times the volume of frozen anhydrous ethanol was added to the second filtrate for low-temperature centrifugation at 4℃, 12000 rpm for 10 min. The precipitate was collected, and the ethanol was evaporated to obtain the single-stranded nucleic acid (in dry powder form) from which the target fragment was recovered.

[0088] Comparative Example 1

[0089] (1) Prepare PAG with 15% acrylamide by mass, containing 8M urea and 25% formamide (V:V).

[0090] (2) Prepare 1-ssDNA (42-nt, 90-nt and 200-nt, respectively, with sequences shown in Table 1) purchased or prepared in the early stage, and perform electrophoresis with PAG prepared in step (1). After electrophoresis, stain the gel, place it under a UV lamp to observe the bands, and cut off the gel containing the target band.

[0091] (3) Take the gel cut off in step (2) and crush it gently. Mix it with TE solution (10mM Tris-HCl, 1mM EDTA, pH=8.0) at a ratio of 1g:25mL. Incubate at 65℃ and collect the TE solution.

[0092] (4) Wash the TE solution obtained in step (3) with ultrapure water 2 to 3 times. The amount of ultrapure water used for each wash is 10 mL / wash. The centrifugation conditions are 25℃, 12000 rpm, and 30 s.

[0093] (5) The washed TE solution (45 mL in total) was concentrated using a vacuum low-temperature rotary concentration method at 50 °C for 4 h to obtain a concentrated solution of 5 mL. Then, the concentrated solution was placed in an ultrafiltration tube with a molecular weight cutoff of 3 K for the first centrifugation at 4000 g for 10 min, and the first filtrate was discarded. The ultrafiltration tube was then inverted for the second centrifugation at 1000 g for 10 min, and the second filtrate was collected. Five times the volume of frozen anhydrous ethanol was added to the second filtrate for low-temperature centrifugation at 4 °C, 12000 rpm for 10 min, and the precipitate was collected. After evaporating the ethanol, the recovered single-stranded nucleic acid (in dry powder form) was obtained.

[0094] TE solution was added to the nucleic acid dry powder recovered in Example 1 and Comparative Example 1, and the volume was adjusted to the original sample volume. The absorbance of the recovered sample at 260 nm was analyzed by nanodrop, converted into concentration, and the recovery rate was calculated.

[0095] Figure 3 The figure shows the DNA recovery rates of Example 1 and Comparative Example 1, respectively. Figure 3 It can be seen that both Example 1 and Comparative Example 1 can recover DNA to a certain extent, but the effects of different swelling treatment methods on DNA recovery rates vary significantly. Specifically, when PAG was swollen with TE solution, the recovery rates of 42-nt, 90-nt, and 200-nt DNA were 24.24%, 24.21%, and 25.15%, respectively. However, after using the swelling method of this invention, the recovery rates of 42-nt, 90-nt, and 200-nt DNA were 60.91%, 65.18%, and 64.18%, respectively, which are much higher than the DNA recovery rates after using the TE swelling method. This is because after treatment with the swelling method provided by this invention, the pores of the PAG gel increase, and the restriction on the free movement of DNA molecules in the solution is reduced. This facilitates the "escape" of DNA fragments from the gel to the pure aqueous solution via the concentration gradient. Compared with traditional methods of recovering DNA from swollen gels, this method can achieve more efficient recovery of single-stranded DNA from high-resolution PAG.

[0096] Example 2

[0097] (1) Prepare denatured PAG with 15% acrylamide by mass, containing 8M urea and 25% formamide (V:V); prepare non-denatured PAG with 15% acrylamide by mass, without 8M urea or 25% formamide (V:V).

[0098] (2) Two hairpin DNA segments (molecular weights of 50-nt and 42-nt, respectively, sequences are shown in Table 1, and secondary structures are shown in Table 1) were ligated using DNA ligase. Figure 7As shown), dumbbell-shaped c-ssDNA (molecular weight 92-nt) was prepared; the 92-nt dumbbell-shaped c-ssDNA was electrophoresed and identified using denatured PAG and non-denatured PAG prepared in step (1). After electrophoresis, the gel was stained, and the bands were observed under a UV lamp. The gel containing the target band was cut off.

[0099] (3) Take the gel fragments cut off in step (2) and the swelling solution (pH=12.0, the volume ratio of anhydrous ethanol to deionized water is 1:1) and mix them at a ratio of 1g:25mL. Incubate at 85℃ for 120min, then wash quickly with ultrapure water 2-3 times. The amount of ultrapure water used for each wash is 10mL / wash. The centrifugation conditions are 25℃, 12000rpm, and 30s.

[0100] (4) Place the washed gel fragments in pure water at a ratio of 1g:25mL and swell at 25℃ for 8h. Collect the pure water solution to obtain the gel swelling solution.

[0101] (5) The gel swelling solution (total 45 mL) obtained by vacuum low-temperature rotary concentration method was concentrated at 50℃ for 4 h to obtain a concentrated solution of 5 mL. Then, the concentrated solution was placed in an ultrafiltration tube with a molecular weight cutoff of 3K for the first centrifugation at 14000 g for 10 min. The first filtrate was discarded. The ultrafiltration tube was then inverted for the second centrifugation at 1000 g for 10 min. The second filtrate was collected. Five times the volume of frozen anhydrous ethanol was added to the second filtrate for low-temperature centrifugation at 4℃, 12000 rpm for 10 min. The precipitate was collected, and the ethanol was evaporated to obtain the single-stranded nucleic acid (in dry powder form) from which the target fragment was recovered.

[0102] Comparative Example 2

[0103] (1) Prepare denatured PAG with 15% acrylamide by mass, containing 8M urea and 25% formamide (V:V); prepare non-denatured PAG with 15% acrylamide by mass, without 8M urea or 25% formamide (V:V).

[0104] (2) Two hairpin DNA segments (molecular weights of 50-nt and 42-nt, respectively, sequences are shown in Table 1, and secondary structures are shown in Table 1) were ligated using DNA ligase. Figure 7 As shown), dumbbell-shaped c-ssDNA (molecular weight 92-nt) was prepared; the 92-nt dumbbell-shaped c-ssDNA was electrophoresed and identified using denatured PAG and non-denatured PAG prepared in step (1). After electrophoresis, the gel was stained, and the bands were observed under a UV lamp. The gel containing the target band was cut off.

[0105] (3) Take the gel cut off in step (2) and crush it gently. Mix it with TE solution (10mM Tris-HCl, 1mM EDTA, pH=8.0) at a ratio of 1g:25mL. Incubate at 65℃ and collect the TE solution.

[0106] (4) Wash the TE solution obtained in step (3) with ultrapure water 2 to 3 times. The amount of ultrapure water used for each wash is 10 mL / wash. The centrifugation conditions are 25℃, 12000 rpm, and 30 s.

[0107] (5) The washed TE solution (45 mL in total) was concentrated using a vacuum low-temperature rotary concentration method at 50 °C for 4 h to obtain a concentrated solution of 5 mL. Then, the concentrated solution was placed in an ultrafiltration tube with a molecular weight cutoff of 3 K for the first centrifugation at 14000 g for 10 min, and the first filtrate was discarded. The ultrafiltration tube was then inverted for the second centrifugation at 1000 g for 10 min, and the second filtrate was collected. Five times the volume of frozen anhydrous ethanol was added to the second filtrate for low-temperature centrifugation at 4 °C, 12000 rpm for 10 min, and the precipitate was collected. After evaporating the ethanol, the single-stranded nucleic acid recovered from the target (in dry powder form) was obtained.

[0108] TE solution was added to the nucleic acid dry powder recovered in Example 2 and Comparative Example 2, and the volume was adjusted to the original sample volume. The purity of the recovered samples was analyzed by 15% denaturing gel electrophoresis.

[0109] Figure 4 The figure shows the purity of the recovered DNA in Example 2 and Comparative Example 2, determined by... Figure 4 It can be seen that both Example 1 and Comparative Example 1 can recover DNA to a certain extent, but the effects of different swelling treatment methods on the purity of the recovered DNA vary significantly. Specifically, when using non-denaturing PAG for electrophoresis, 90-nt l-ss DNA and c-ss DNA with similar molecular weights cannot be distinguished. Figure 4 Therefore, denaturing PAG electrophoresis must be used to determine the purity of c-ssDNA (Lane 1 and Lane 2). Figure 4 As shown in Lanes 3 and 4, in denatured and high-resolution PAGs, the band positions of both 90-nt l-ssDNA and c-ssDNA differed significantly. And as... Figure 4 As shown in Lanes 5-9, the 90-nt samples recovered using the TE swelling method (Comparative Example 2) contained both c-ssDNA and l-ssDNA. Figure 4In the case of Lane 6 and Lane 9, the swelling method provided by this invention did not yield 1-ssDNA in the recovered 90-nt samples. This is because conventional swelling methods, in order to increase the swelling rate, utilize heating or other treatments to increase the random movement speed of DNA molecules, thereby promoting the free leakage of DNA from the gel network. Therefore, this requires prolonged incubation of PAG fragments containing the target fragment at high temperatures, which carries a high risk of damaging the integrity of the single-stranded DNA structure. In contrast, the swelling method provided by this invention is an alkaline-alcohol swelling method, which completes gel swelling at room temperature after a brief alkaline-alcohol treatment. The main reaction conditions are milder, less likely to damage the DNA structure, and therefore can obtain target single-stranded DNA fragments with higher purity. Furthermore, Figure 4 The test results also demonstrate to some extent that the heating incubation conditions (85°C, 120 min) and the alkaline alcohol (pH = 12, 50% ethanol) solution provided in the embodiments of the present invention did not cause significant damage to the DNA structure.

[0110] Example 3

[0111] (1) Prepared modified PAG with 15% acrylamide by mass, containing 8M urea and 25% formamide (V:V).

[0112] (2) Linear RNA substrates (molecular weight 46-nt, sequence shown in Table 1, secondary structure shown in Table 1) were ligated using RNA ligase. Figure 7 As shown), the cyclization was completed by filling in the nick, resulting in single-stranded circular RNA (c-ssRNA, molecular weight 46-nt). The 46-nt c-ssRNA was electrophoresed and identified using the denatured PAG prepared in step (1). After electrophoresis, the gel was stained, and the bands were observed under a UV lamp. The gel containing the target l-ssRNA band and the c-ssRNA band was cut off.

[0113] (3) Take the gel fragments cut off in step (2) and the swelling solution (pH=12.0, the volume ratio of anhydrous ethanol to deionized water is 1:1) and mix them at a ratio of 1g:25mL. Incubate at 85℃ for 120min, then wash quickly with ultrapure water 2-3 times. The amount of ultrapure water used for each wash is 10mL / wash. The centrifugation conditions are 25℃, 12000rpm, and 30s.

[0114] (4) Place the washed gel fragments in pure water at a ratio of 1g:25mL and swell at 25℃ for 8h. Collect the pure water solution to obtain the gel swelling solution.

[0115] (5) The gel swelling solution (total 45 mL) was concentrated using a vacuum low-temperature rotary concentration method at a concentration temperature of 50 °C for 4 h until it was reduced to 5 mL to obtain a concentrated solution. Then, 5 times the volume of frozen anhydrous ethanol was added to the concentrated solution for low-temperature centrifugation at 4 °C, 12000 rpm for 10 min. The precipitate was collected and washed with pre-cooled 75% ethanol. After evaporating the ethanol, the l-ssRNA or c-ssRNA of the target fragment was obtained (in dry powder form).

[0116] Comparative Example 3

[0117] (1) Prepared modified PAG with 15% acrylamide by mass, containing 8M urea and 25% formamide (V:V).

[0118] (2) Linear RNA substrates (molecular weight 46-nt, sequence shown in Table 1, secondary structure shown in Table 1) were ligated using RNA ligase. Figure 7 As shown), the cyclization was completed by filling in the nick, resulting in single-stranded circular RNA (c-ssRNA, molecular weight 46-nt). The 46-nt c-ssRNA was electrophoresed and identified using the denatured PAG prepared in step (1). After electrophoresis, the gel was stained, and the bands were observed under a UV lamp. The gel containing the target l-ssRNA band and the c-ssRNA band was cut off.

[0119] (3) Take the gel cut off in step (2) and crush it gently. Mix it with TE solution (10mM Tris-HCl, 1mM EDTA, pH=8.0) at a ratio of 1g:25mL. Incubate at 65℃ for 8h and collect the TE solution.

[0120] (4) Wash the TE solution obtained in step (3) with ultrapure water 2 to 3 times. The amount of ultrapure water used for each wash is 10 mL / wash. The centrifugation conditions are 25℃, 12000 rpm, and 30 s.

[0121] (5) The washed TE solution (total 45 mL) was concentrated using a vacuum low-temperature rotary concentration method at 50 °C for 4 h until it was reduced to 5 mL to obtain a concentrated solution. Then, 5 times the volume of frozen anhydrous ethanol was added to the concentrated solution for low-temperature centrifugation at 4 °C, 12000 rpm for 10 min. The precipitate was collected and washed with pre-cooled 75% ethanol. After evaporating the ethanol, the l-ssRNA or c-ssRNA of the target fragment was obtained (in dry powder form).

[0122] TE solution was added to the RNA powder recovered in Example 3 and Comparative Example 3, and the volume was adjusted to the original sample volume. The absorbance of the recovered sample in the range of 200-300 nm was scanned by nanodrop to analyze whether there was contamination by other biomacromolecules (proteins, sugars, etc.). The purity of the recovered RNA (whether there were other nucleic acid fragments of different molecular weights) was analyzed by 15% denaturing gel electrophoresis.

[0123] Figure 5A , Figure 5B and Figure 6 The image shows a comparison between Example 3 and Comparative Example 3. The absorbance of the recovered RNA in the 200–300 nm range was measured as follows. Figure 5A (Comparative Example 3) and Figure 5B As shown in Example 3, by Figure 5A and Figure 5B It can be seen that the RNA sample recovered in Comparative Example 3 has a certain absorption peak in the 200-240 nm range, which is much higher than the absorption peak at 260 nm, indicating that the recovered RNA contains a relatively large amount of impurities. In contrast, the RNA sample recovered in Example 3 only has a characteristic absorption peak at 260 nm, and no characteristic absorption peaks are observed at the wavelengths corresponding to impurities such as polysaccharides and proteins (230 nm, 280 nm), indicating that the recovered RNA has higher purity. Based on this, the RNA concentration was estimated from the absorbance value at 260 nm and compared with the amount of RNA before recovery to calculate the recovery rate. The results show that the RNA recovery rate in Example 3 is as high as 61%, which is much higher than the RNA recovery rate in Comparative Example 3.

[0124] Figure 6 The electrophoresis results of the RNA samples recovered in Example 3 and Comparative Example 3 are shown below. Lanes 1-6 represent l-ssRNA before recovery, l-ssRNA in Comparative Example 3, l-ssRNA in Example 3, c-ssRNA before recovery, c-ssRNA in Comparative Example 3, and c-ssRNA in Example 3, respectively. Figure 6It can be seen that the proportion of non-target bands in the recovered samples decreased significantly. For example, in Lane 1, the proportion of 46-nt l-ssRNA in all lane bands was 80%, and in Lane 3, the proportion of 46-nt l-ssRNA in all lane bands was 90%. That is, the content of impurity bands in Lane 3 was lower, indicating that the RNA recovery method provided by this invention can effectively improve the purity of RNA. Analysis of the cyclization products under the three conditions showed that the purity of c-ssRNA before recovery was approximately 70%, and after recovery using the method of this invention, its purity exceeded 90%. Furthermore, compared to the purity of each RNA sample in Comparative Example 3, the purity of both l-ssRNA and c-ssRNA in Example 3 was higher. Figure 5A The absorbance measurement results shown in the figure are consistent with Figure 5B The purity analysis of RNA bands shows that the method provided by this invention can improve the recovery rate of single-stranded RNA while also improving the purity of the recovered RNA.

[0125] Example 4

[0126] (1) Prepare PAG with 15% acrylamide by mass, containing 8M urea and 25% formamide (V:V).

[0127] (2) Prepare 1-ssDNA (42-nt, 90-nt and 200-nt, respectively, with sequences shown in Table 1) purchased or prepared in the early stage, and perform electrophoresis with PAG prepared in step (1). After electrophoresis, stain the gel, place it under a UV lamp to observe the bands, and cut off the gel containing the target band.

[0128] (3) Take the gel fragments cut off in step (2) and the swelling solution (pH=12.0, the volume ratio of anhydrous ethanol to deionized water is 1:1) and mix them at a ratio of 1g:5mL. Incubate at 85℃ for 60min, then wash quickly with ultrapure water 2-3 times. The amount of ultrapure water used for each wash is 10mL / wash. Centrifuge at 25℃, 12000rpm, and 30s.

[0129] (4) Place the washed gel fragments in pure water at a ratio of 1g:5mL, swell at 30℃ for 6h, collect the pure water solution, and obtain the gel swelling solution.

[0130] (5) The gel swelling solution (total 40 mL) obtained by vacuum low-temperature rotary concentration method was concentrated at 50℃ for 4 h to obtain a concentrated solution of 5 mL. Then, the concentrated solution was placed in an ultrafiltration tube with a molecular weight cutoff of 3K for the first centrifugation at 12000 g for 12 min. The first filtrate was discarded. The ultrafiltration tube was then inverted for the second centrifugation at 800 g for 12 min. The second filtrate was collected. Four times the volume of frozen anhydrous ethanol was added to the second filtrate for low-temperature centrifugation at 4℃, 10000 rpm for 12 min. The precipitate was collected, and the ethanol was evaporated to obtain the single-stranded nucleic acid (in dry powder form) from which the target fragment was recovered.

[0131] Example 5

[0132] (1) Prepare denatured PAG with 15% acrylamide by mass, containing 8M urea and 25% formamide (V:V); prepare non-denatured PAG with 15% acrylamide by mass, without 8M urea or 25% formamide (V:V).

[0133] (2) Two hairpin DNA segments (molecular weights of 50-nt and 42-nt, respectively, sequences are shown in Table 1, and secondary structures are shown in Table 1) were ligated using DNA ligase. Figure 7 As shown), dumbbell-shaped c-ssDNA (molecular weight 92-nt) was prepared; the 92-nt dumbbell-shaped c-ssDNA was electrophoresed and identified using denatured PAG and non-denatured PAG prepared in step (1). After electrophoresis, the gel was stained, and the bands were observed under a UV lamp. The gel containing the target band was cut off.

[0134] (3) Take the gel fragments cut off in step (2) and the swelling solution (pH=12.0, the volume ratio of anhydrous ethanol to deionized water is 1:1) and mix them at a ratio of 1g:50mL. Incubate at 70℃ for 80min, then wash quickly with ultrapure water 2-3 times. The amount of ultrapure water used for each wash is 10mL / wash. The centrifugation conditions are 25℃, 12000rpm, and 30s.

[0135] (4) Place the washed gel fragments in pure water at a ratio of 1g:10mL, swell at 35℃ for 4h, collect the pure water solution, and obtain the gel swelling solution.

[0136] (5) The gel swelling solution (total 40 mL) obtained by vacuum low-temperature rotary concentration method was concentrated at 50℃ for 4 h to obtain a concentrated solution. Then, the concentrated solution was placed in an ultrafiltration tube with a molecular weight cutoff of 3K for the first centrifugation at 16000 g for 8 min, and the first filtrate was discarded. The ultrafiltration tube was then inverted for the second centrifugation at 1200 g for 8 min, and the second filtrate was collected. Six times the volume of frozen anhydrous ethanol was added to the second filtrate for low-temperature centrifugation at 4℃, 14000 rpm for 8 min, and the precipitate was collected. After evaporating the ethanol, the single-stranded nucleic acid of the target fragment was obtained (in dry powder form).

[0137] Example 6

[0138] (1) Prepared modified PAG with 15% acrylamide by mass, containing 8M urea and 25% formamide (V:V).

[0139] (2) Linear RNA substrates (molecular weight 46-nt, sequence shown in Table 1, secondary structure shown in Table 1) were ligated using RNA ligase. Figure 7 As shown), the cyclization was completed by filling in the nick, resulting in single-stranded circular RNA (c-ssRNA, molecular weight 46-nt). The 46-nt c-ssRNA was electrophoresed and identified using the denatured PAG prepared in step (1). After electrophoresis, the gel was stained, and the bands were observed under a UV lamp. The gel containing the target l-ssRNA band and the c-ssRNA band was cut off.

[0140] (3) Take the gel fragments cut off in step (2) and the swelling solution (pH=12.0, the volume ratio of anhydrous ethanol to deionized water is 1:1) and mix them at a ratio of 1g:100mL. Incubate at 80℃ for 30min, then wash quickly with ultrapure water 2-3 times. The amount of ultrapure water used for each wash is 10mL / wash. Centrifuge at 25℃, 12000rpm, and 30s.

[0141] (4) Place the washed gel fragments in pure water at a ratio of 1g:20mL, swell at 37℃ for 2h, collect the pure water solution, and obtain the gel swelling solution.

[0142] (5) The gel swelling solution (total 45 mL) was concentrated using a vacuum low-temperature rotary concentration method at a concentration temperature of 50 °C for 4 h until it was reduced to 5 mL to obtain a concentrated solution. Then, 5 times the volume of frozen anhydrous ethanol was added to the concentrated solution for low-temperature centrifugation at 4 °C, 12000 rpm for 10 min. The precipitate was collected and washed with pre-cooled 75% ethanol. After evaporating the ethanol, the l-ssRNA or c-ssRNA of the target fragment was obtained (in dry powder form).

[0143] The above are merely preferred embodiments of the present invention and do not limit the patent scope of the present 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 patent protection scope of the present invention.

Claims

1. A method for recovering single-stranded nucleic acids from PAG, characterized in that, Includes the following steps: A swelling solution was prepared using deionized water and anhydrous ethanol as a mixed solvent and sodium hydroxide as a solute. The polyacrylamide gel fragments containing the target fragments were mixed with the swelling solution and incubated, and then the incubated polyacrylamide gel fragments were collected. The incubated polyacrylamide gel fragments were soaked in pure water to swell, resulting in a gel swelling solution. The gel swelling solution is concentrated and purified to obtain a collection, which yields the target nucleic acid fragment. In the step of preparing the swelling solution using deionized water and anhydrous ethanol as a mixed solvent and sodium hydroxide as a solute: In the mixed solvent, the volume ratio of deionized water to anhydrous ethanol is 1:1 to 4, and the pH of the mixed solvent is 10 to 12.

2. The method for recovering single-stranded nucleic acids from PAG as described in claim 1, characterized in that, In the step of mixing polyacrylamide gel fragments containing the target fragment with the swelling solution and then incubating them, followed by collecting the incubated polyacrylamide gel fragments: Each 1 mg of the polyacrylamide gel fragment is mixed with 5 to 100 mL of the swelling solution.

3. The method for recovering single-stranded nucleic acids from PAG as described in claim 1, characterized in that, In the step of mixing polyacrylamide gel fragments containing the target fragment with the swelling solution and then incubating them, followed by collecting the incubated polyacrylamide gel fragments: The incubation temperature is 65~85℃ and the incubation time is 30~120min.

4. The method for recovering single-stranded nucleic acids from PAG as described in claim 1, characterized in that, In the step of soaking the incubated polyacrylamide gel fragments in pure water to swell and obtain a gel swelling solution: Each 1 mg of the polyacrylamide gel fragment is soaked in 5 to 25 mL of the purified water.

5. The method for recovering single-stranded nucleic acids from PAG as described in claim 1, characterized in that, In the step of soaking the incubated polyacrylamide gel fragments in pure water to swell and obtain a gel swelling solution: The swelling temperature is 25~37℃ and the swelling time is 2~8h.

6. The method for recovering single-stranded nucleic acids from PAG as described in claim 1, characterized in that, In the step of concentrating and purifying the gel swelling solution to obtain the collected material, i.e., obtaining the target nucleic acid fragment: The concentration temperature is no higher than 50°C, and the concentration is to concentrate to 1 / 8 to 1 / 10 of the original volume of the supernatant.

7. The method for recovering single-stranded nucleic acids from PAG as described in claim 1, characterized in that, In the step of concentrating and purifying the gel swelling solution to obtain a collected sample, i.e., obtaining the target nucleic acid fragment, the purification includes: The concentrated gel swelling solution was placed in an ultrafiltration tube for a first centrifugation, and the first filtrate was discarded. Then the ultrafiltration tube was inverted for a second centrifugation, and the second filtrate was collected. After adding frozen anhydrous ethanol to the second filtrate, centrifuge, collect the precipitate, and remove any residual anhydrous ethanol.

8. The method for recovering single-stranded nucleic acids from PAG as described in claim 7, characterized in that, In the step of placing the concentrated gel swelling solution in an ultrafiltration tube for a first centrifugation, discarding the first filtrate, and then inverting the ultrafiltration tube for a second centrifugation to collect the second filtrate: The centrifugal force for the first centrifugation is 12000~16000g, and the centrifugation time is 8~12min; and / or, The centrifugal force for the second centrifugation is 800~1200g, and the centrifugation time is 8~12min.

9. The method for recovering single-stranded nucleic acids from PAG as described in claim 7, characterized in that, In the step of adding frozen anhydrous ethanol to the second filtrate, centrifuging, collecting the precipitate, and removing any residual anhydrous ethanol: The volume of the frozen anhydrous ethanol is 4 to 6 times the volume of the second filtrate; and / or, The centrifugation speed is 10000~14000 rpm and the centrifugation time is 8~12 min.

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