Efficient nucleic acid modification innovative technology and method

Abstract

The invention provides an efficient nucleic acid modification innovation technology and method, and belongs to the technical field of biology, the efficient nucleic acid modification innovation technology and method comprise the steps of nucleic acid sample preparation, modification reagent selection, modification reaction, modification post-treatment, quality detection and the like, the purity of a nucleic acid sample is not lower than 95%, and the integrity is good; the modification reagent comprises a chemical modification agent, an enzyme preparation and the like, and has specific requirements on purity and activity; the modification reaction temperature, time, pH value and reaction system composition are strictly stipulated; the modification post-treatment adopts chromatography, electrophoresis, precipitation and the like, so that the nucleic acid purity can be effectively improved; and the quality detection adopts the methods of spectral analysis, gel electrophoresis, sequencing and the like to ensure that the modification efficiency is not lower than 85% and the specificity reaches 90% or above.

Description

Technical Field

[0001] The present invention belongs to the field of biotechnology, and particularly relates to innovative technologies and methods for efficient nucleic acid modification. Background Art

[0002] In the current field of biotechnology, nucleic acid modification technology is crucial. However, there are many significant problems with existing nucleic acid modification technologies. Firstly, it is difficult to ensure the purity and integrity of nucleic acid samples, which may affect the effect and accuracy of subsequent modification reactions. Secondly, the selection of modification reagents often lacks pertinence, resulting in low modification efficiency and poor specificity. Moreover, the conditions of modification reactions are not precisely controlled, making the modification effect unstable and difficult to meet the actual application requirements. In addition, the post-modification treatment methods cannot effectively remove unreacted modification reagents and other impurities, resulting in low nucleic acid purity. Finally, the quality detection methods are not accurate and reliable enough to comprehensively evaluate the quality of modified nucleic acids, thus limiting the application of nucleic acid modification technology in many fields.

[0003] To solve the above problems, the present invention provides an innovative technology and method for efficient nucleic acid modification. By strictly controlling the preparation process of nucleic acid samples, ensuring that the sample purity is not less than 95% and the integrity is good, reasonably selecting modification reagents according to different modification purposes and nucleic acid types to improve modification efficiency and specificity, precisely controlling the conditions of modification reactions, including temperature, time, pH value and reaction system composition, to ensure the stability of modification effects, adopting effective post-modification treatment methods such as chromatography, electrophoresis and precipitation methods, etc., which can effectively remove unreacted modification reagents and impurities, and improve the nucleic acid purity to more than 99%, using accurate and reliable quality detection methods such as spectral analysis, gel electrophoresis and sequencing to comprehensively evaluate the quality of modified nucleic acids, so that the modification efficiency is not less than 85% and the specificity reaches more than 90%. Summary of the Invention

[0004] The purpose of the present invention is to provide an innovative technology and method for efficient nucleic acid modification, aiming to solve the problems in the prior art that it is difficult to ensure the purity and integrity of nucleic acid samples, which may affect the effect and accuracy of subsequent modification reactions. In addition, the selection of modification reagents often lacks pertinence, resulting in low modification efficiency and poor specificity.

[0005] To achieve the above purpose, the present invention provides the following technical solutions:

[0006] An innovative technology and method for efficient nucleic acid modification, characterized by comprising the following steps:

[0007] S1. Preparation of nucleic acid samples, where the selected nucleic acid samples have a purity of not less than 95%, and the integrity is detected by gel electrophoresis without obvious degradation bands and the main band is clear.

[0008] S2. Selection of modification reagents. The modification reagents include chemical modifiers, enzyme preparations, etc. The purity of the chemical modifier is not less than 98%, and the activity of the enzyme preparation is not less than 100 U / mg.

[0009] S3. Conducting the modification reaction. The reaction temperature is in the range of 30°C - 37°C, the reaction time is 2 hours, the pH value is in the range of 7.0 - 7.5, and the ratio of the nucleic acid sample to the modification reagent in the reaction system composition is 1:5.

[0010] S4. Post-treatment after modification. Chromatography, electrophoresis, precipitation and other methods are used for post-treatment, and the purity of the nucleic acid after treatment is not less than 99%.

[0011] S5. Quality detection. Spectral analysis, gel electrophoresis, sequencing and other methods are used for detection. The modification efficiency is not less than 85%, and the specificity is verified to be more than 90% through specific experimental methods.

[0012] As a preferred embodiment of the present invention, the nucleic acid sample can be derived from biological tissues, cell cultures or artificial synthesis.

[0013] As a preferred embodiment of the present invention, the modification reagents should be reasonably selected according to different modification purposes and nucleic acid types.

[0014] As a preferred embodiment of the present invention, the conditions of the modification reaction should be strictly controlled within the specified parameter range to ensure the modification effect.

[0015] As a preferred embodiment of the present invention, the post-treatment method after modification should be able to effectively remove unreacted modification reagents and other impurities and improve the nucleic acid purity.

[0016] As a preferred embodiment of the present invention, the quality detection method should be accurate and reliable and be able to comprehensively evaluate the quality of the modified nucleic acid.

[0017] Compared with the prior art, the beneficial effects of the present invention are:

[0018] 1. In this solution, in terms of the nucleic acid sample: By various purification methods, it is ensured that the purity of the nucleic acid sample is not less than 95% and the integrity is good. For example, for DNA samples, phenol-chloroform extraction combined with ethanol precipitation is used, and for RNA samples, after extraction with TRIzol method, they are treated with RNase-free DNase, and then detected by detection methods such as agarose gel electrophoresis, nucleic acid quantifier, ultraviolet spectrophotometer and agarose denaturing gel electrophoresis, which ensures the effect and accuracy of the subsequent modification reaction;

[0019] In terms of modification reagents: The modification reagents are reasonably selected according to different modification purposes and nucleic acid types, which improves the modification efficiency and specificity. For example, specific methylation reagents are selected for methylation modification, and for fluorescence labeling, appropriate dNTP analogs with fluorescent groups or fluorescently labeled RNA probes are selected according to the nucleic acid type. At the same time, ensure that the purity of the chemical modifier is not less than 98%, and the activity of the enzyme preparation is not less than 100 U / mg;

[0020] In terms of modification reaction conditions: The conditions of the modification reaction are precisely controlled, including temperature, time, pH value, and reaction system composition, ensuring the stability of the modification effect. Use precise temperature control equipment such as a constant temperature water bath or a PCR instrument to ensure that the reaction temperature is stable within the range of 30°C - 37°C, use a timing device to accurately control the reaction time to 2 hours, use an accurate pH meter to measure and adjust the pH value within the range of 7.0 - 7.5 through a buffer solution, and use a precision pipette to ensure that the ratio of the nucleic acid sample to the modification reagent is 1:5;

[0021] In terms of post-modification treatment: Effective post-modification treatment methods such as chromatography, electrophoresis, and precipitation are used, which can effectively remove unreacted modification reagents and impurities, and improve the nucleic acid purity to more than 99%. For example, in chromatography, a suitable chromatographic column and mobile phase are selected according to the properties of the nucleic acid and the modification reagent; in electrophoresis, a suitable gel concentration and electrophoresis conditions are selected; in precipitation, when using ethanol or isopropanol to precipitate nucleic acids, the precipitate is washed multiple times, etc.;

[0022] In terms of quality inspection: Accurate and reliable quality inspection methods such as spectral analysis, gel electrophoresis, and sequencing are used to comprehensively evaluate the quality of the modified nucleic acid, so that the modification efficiency is not less than 85%, and the specificity reaches more than 90%. For example, in spectral analysis, a high-quality ultraviolet spectrophotometer is used to measure the absorbance of the nucleic acid at 260 nm and 280 nm multiple times and calculate the A260 / A280 ratio; in gel electrophoresis, a high-resolution agarose gel or polyacrylamide gel is used and appropriate molecular weight standards and control samples are set; in sequencing analysis, a high-throughput sequencing platform is selected and deep sequencing and bioinformatics analysis are carried out, and multiple repeated experiments are also carried out. Specific implementation mode

[0023] Next, in combination with the embodiments of the present invention, the technical solutions in the embodiments of the present invention will be clearly and completely described. Obviously, the described embodiments are only a part of the embodiments of the present invention, rather than all embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those of ordinary skill in the art without creative efforts shall fall within the protection scope of the present invention.

[0024] Example 1

[0025] The present invention provides the following technical solutions:

[0026] An innovative technology and method for efficient nucleic acid modification, characterized by the following steps:

[0027] S1. Preparation of nucleic acid samples: The selected nucleic acid samples have a purity of not less than 95%, and the integrity is detected by gel electrophoresis without obvious degradation bands and with clear main bands.

[0028] S2. Selection of modification reagents: The modification reagents include chemical modifiers, enzyme preparations, etc. The purity of the chemical modifiers is not less than 98%, and the activity of the enzyme preparations is not less than 100 U / mg.

[0029] S3. Conducting the modification reaction: The reaction temperature is in the range of 30°C - 37°C, the reaction time is 2 hours, the pH value is in the range of 7.0 - 7.5, and the ratio of nucleic acid samples to modification reagents in the reaction system composition is 1:5.

[0030] S4. Post - modification treatment: Chromatography, electrophoresis, precipitation and other methods are used for post - treatment, and the purity of the nucleic acid after treatment is not less than 99%.

[0031] S5. Quality detection: Spectral analysis, gel electrophoresis, sequencing and other methods are used for detection. The modification efficiency is not less than 85%, and the specificity is verified to be over 90% through specific experimental methods.

[0032] In a specific embodiment of the present invention, 1. Preparation of nucleic acid samples (S1):

[0033] DNA sample processing: Tissue fragmentation and cell lysis (applicable to DNA from tissue sources);

[0034] Cut the tissue from sources such as animal liver tissue into small pieces, put them into a suitable lysis solution, and enzymes such as proteinase K can be added. Incubate at an appropriate temperature (such as about 55°C) for several hours to fully lyse the tissue and release the nucleic acid.

[0035] Protein removal and precipitation:

[0036] Use the phenol - chloroform extraction method: Mix the lysed sample with an equal volume of phenol - chloroform, shake vigorously and then centrifuge (such as 12000 rpm, 10 minutes). At this time, the protein will enter the organic phase and the nucleic acid will be in the supernatant.

[0037] Take the supernatant, add 2 volumes of absolute ethanol and 1 / 10 volume of 3M sodium acetate (pH 5.2), gently invert and mix well, and it can be placed at - 20°C for several hours or overnight to precipitate the DNA.

[0038] Purity detection:

[0039] Centrifuge to collect the precipitated DNA, wash it with 70% ethanol, dry it, and dissolve it in an appropriate amount of TE buffer.

[0040] Observe the integrity of DNA bands by agarose gel electrophoresis, and at the same time use a nucleic acid quantifier to measure the concentration and purity of DNA to ensure that the purity is not less than 95%.

[0041] RNA sample processing:

[0042] Extraction and removal of DNA contamination: Use the TRIzol method to extract RNA: Add samples from sources such as cell cultures to TRIzol reagent, operate according to the reagent instructions, after fully lysing the cells, add chloroform, centrifuge (such as 12,000 rpm, 15 minutes), and aspirate the upper aqueous phase (containing RNA).

[0043] Add RNase-free DNase for treatment to remove possible DNA contamination, and incubate at an appropriate temperature (such as 37 °C) for a certain period of time (such as 30 minutes).

[0044] Purity and integrity detection

[0045] Measure the absorbance ratio (A260 / A280) of RNA at 260 nm and 280 nm by ultraviolet spectrophotometer to judge the purity, which should generally be between 1.8 - 2.0.

[0046] Use agarose denaturing gel electrophoresis to observe the integrity of RNA bands to ensure that there are no obvious degraded bands and the main band is clear.

[0047] II. Selection of modification reagents (S2):

[0048] Selection of chemical modifiers: Screen according to the required modification type. For example, for methylation modification:

[0049] Consult relevant literature and materials to understand the characteristics and scope of application of different methylation reagents.

[0050] Select a specific methylation reagent, such as S-adenosylmethionine (SAM) and its analogs, etc., to ensure that its purity is not less than 98%. At the same time, consider its stability, and it can be stored under appropriate conditions (such as -20 °C or lower temperature, protected from light, etc.).

[0051] Selection of enzyme preparations: Consider the specificity, stability and reaction conditions of the enzyme. For example, for phosphorylation modification of nucleic acids, a suitable kinase may need to be selected.

[0052] Ensure that the activity of the enzyme preparation is not less than 100 U / mg. Consult the product instructions of the enzyme to understand its storage conditions (such as a specific temperature range, buffer requirements, etc.), and store the enzyme preparation according to the requirements to ensure its activity.

[0053] III. Conducting the modification reaction (S3):

[0054] Temperature control: Use precise temperature control equipment, such as a constant temperature water bath or a PCR instrument.

[0055] Place the reaction system into the equipment, set the temperature within the range of 30°C - 37°C, and continuously monitor the temperature during the reaction to ensure temperature stability.

[0056] Time control: Use a timing device, such as a timer or an instrument with a timing function.

[0057] Set the reaction time to 2 hours. After the reaction starts, the timing device begins to count, ensuring the accuracy of the reaction time.

[0058] pH value adjustment: Use an accurate pH meter to measure the pH value of the reaction system.

[0059] According to the measurement results, adjust the pH value within the range of 7.0 - 7.5 by adding an appropriate amount of buffer (such as Tris-HCl buffer, etc.). During the reaction, the pH value can be measured regularly to ensure its stability.

[0060] Reagent ratio control: Use a precise pipette to accurately measure nucleic acids and modification reagents.

[0061] Measure according to the ratio of nucleic acid sample to modification reagent of 1:5. In actual operation, it can be appropriately adjusted according to the concentration of nucleic acid and the activity of the modification reagent. For example, if the nucleic acid concentration is high, the amount of modification reagent can be appropriately reduced, but the ratio should be kept within a reasonable range.

[0062] IV. Post-modification treatment (S4):

[0063] Chromatography treatment: Select a suitable chromatographic column and mobile phase according to the properties of nucleic acids and modification reagents.

[0064] For small molecule modification reagents, such as small molecule metabolites of some chemical modifiers, a reverse-phase chromatographic column (such as a C18 column) can be used for separation and purification.

[0065] Prepare the chromatographic column, equilibrate the chromatographic column with the mobile phase, and inject the reaction sample into the chromatographic column.

[0066] Set a suitable flow rate (such as 1 ml / min) and detection wavelength (determined according to the characteristics of the modification reagent and nucleic acid), and collect the eluate containing purified nucleic acid.

[0067] For macromolecule modification reagents, such as some enzyme-modified complexes, a gel filtration chromatographic column can be used.

[0068] Select a gel filtration chromatographic column with a suitable pore size and equilibrate the column with a buffer.

[0069] Inject the sample into the column, elute at an appropriate flow rate (such as 0.5 ml / min), and collect the eluate containing the purified nucleic acid.

[0070] Electrophoresis treatment: Select appropriate gel concentration and electrophoresis conditions.

[0071] For agarose gel electrophoresis, if the modified nucleic acid has a large molecular weight, a lower concentration of agarose gel (such as 0.8%) can be selected; if the molecular weight is small, a higher concentration (such as 2%) can be chosen.

[0072] Prepare the gel, add the sample into the gel wells, and set an appropriate voltage (such as 100 V) for electrophoresis.

[0073] After electrophoresis, observe the gel electrophoresis pattern under ultraviolet light, separate the modified nucleic acid band from the unreacted substances, and the target nucleic acid band can be cut out for recovery (if further research is needed).

[0074] For polyacrylamide gel electrophoresis, select appropriate gel concentration and cross - linking degree according to the nucleic acid molecular weight, and perform electrophoresis and band separation according to the standard operating procedure.

[0075] Precipitation treatment: Use ethanol or isopropanol to precipitate nucleic acids.

[0076] If ethanol precipitation is used, add 2 volumes of absolute ethanol and 1 / 10 volume of 3M sodium acetate (pH 5.2) to the reacted sample, gently invert and mix well, and place at - 20 °C for several hours or overnight to precipitate the nucleic acid.

[0077] If isopropanol precipitation is used, add an equal volume of isopropanol and an appropriate amount of salt (such as 0.1M sodium chloride), gently invert and mix well, and place at low temperature for a period of time to precipitate the nucleic acid.

[0078] The precipitated nucleic acid is collected by centrifugation (such as 12000 rpm, 10 minutes), and dissolved in an appropriate amount of buffer to obtain highly purified modified nucleic acid.

[0079] V. Quality detection (S5):

[0080] Spectral analysis: Use a high - quality ultraviolet spectrophotometer.

[0081] Add the purified nucleic acid sample into a cuvette and measure the absorbance of the nucleic acid at 260 nm and 280 nm.

[0082] Calculate the A260 / A280 ratio, take the average value of multiple measurements to ensure the accuracy and stability of the results, and judge whether the purity of the nucleic acid meets the requirements.

[0083] Gel electrophoresis: Use high - resolution agarose gel or polyacrylamide gel.

[0084] Prepare a gel, add molecular weight standards and control samples, and add the purified nucleic acid sample into the gel wells.

[0085] Set an appropriate voltage for electrophoresis. After electrophoresis, observe the gel electrophoresis pattern under ultraviolet light to visually observe the integrity and modification effect of the nucleic acid. By comparing with the molecular weight standards, determine whether the size of the nucleic acid meets the expectations.

[0086] Sequencing analysis: Select a high-throughput sequencing platform, such as the Illumina sequencing platform, etc.

[0087] Process the purified nucleic acid sample according to the requirements of the platform, such as constructing a sequencing library, etc.

[0088] Perform deep sequencing on the modified nucleic acid. Through bioinformatics analysis, accurately evaluate the efficiency and specificity of the modification. At the same time, conduct multiple repeated experiments to ensure the reliability of the results.

[0089] Specificity verification: Detect using specific antibodies or probes.

[0090] If it is a modification of a certain protein binding site on the nucleic acid, the corresponding specific antibody can be used to incubate with the modified nucleic acid, and the specificity can be verified by detecting the antibody binding situation.

[0091] Or conduct functional experiments, such as performing a transcriptional activity test on the modified nucleic acid, etc., to verify whether the modified nucleic acid has the expected biological function and ensure that the specificity reaches more than 90%.

[0092] Specifically in this protocol, the nucleic acid sample can be derived from biological tissues, cell cultures, or artificial synthesis.

[0093] In this example: Processing of nucleic acid samples derived from biological tissues: Tissue disruption and cell lysis: Cut animal liver tissues, etc. into small pieces, put them into a suitable lysis solution, and enzymes such as proteinase K can be added. Incubate at an appropriate temperature (such as about 55°C) for several hours to fully lyse the tissue and release nucleic acids.

[0094] Protein removal and precipitation:

[0095] Phenol-chloroform extraction method: Mix the lysed sample with an equal volume of phenol-chloroform, shake vigorously and then centrifuge (such as 12000 rpm, 10 minutes). The protein enters the organic phase and the nucleic acid is in the supernatant.

[0096] Ethanol precipitation: Take the supernatant, add 2 volumes of absolute ethanol and 1 / 10 volume of 3M sodium acetate (pH 5.2), gently invert and mix well, and place at -20°C for several hours or overnight to precipitate the DNA.

[0097] Purity detection:

[0098] Centrifuge to collect the precipitated DNA, wash it with 70% ethanol, air dry it, and dissolve it in an appropriate amount of TE buffer.

[0099] Observe the integrity of the DNA bands by agarose gel electrophoresis, and at the same time use a nucleic acid quantifier to measure the DNA concentration and purity to ensure that the purity is not less than 95%.

[0100] Treatment of nucleic acid samples derived from cell cultures:

[0101] Cell lysis: Use cell lysis buffer to lyse cells from sources such as cultured cancer cells.

[0102] Nucleic acid purification: Perform subsequent nucleic acid purification steps.

[0103] Treatment of artificially synthesized nucleic acid samples: Strictly control the conditions during the synthesis process to ensure that the synthesized nucleic acid has the required sequence and purity.

[0104] Integrity detection: Detect the integrity of the nucleic acid by methods such as gel electrophoresis to ensure that there are no obvious degradation bands and the main band is clear.

[0105] Specifically in this protocol, the modification reagents should be reasonably selected according to different modification purposes and nucleic acid types.

[0106] In this example: If the modification purpose is to fluorescently label the nucleic acid, for DNA-type nucleic acids, dNTP analogs with fluorescent groups can be selected as modification reagents and incorporated into the newly synthesized DNA strand under the action of DNA polymerase to achieve fluorescent labeling. For RNA-type nucleic acids, fluorescently labeled dNTPs can be used as markers, and then fluorescent labeling can be carried out through reverse transcription reactions or RT-PCR. If the modification purpose is to methylate the nucleic acid, for DNA, a methyltransferase can be selected as the modification reagent to transfer a methyl group to a specific base; for RNA, specific methylases can be used to methylate specific nucleotides.

[0107] Specifically in this protocol, the conditions of the modification reaction should be strictly controlled within the specified parameter range to ensure the modification effect.

[0108] In this example: Temperature control: Use a high-precision constant temperature incubator or heating module.

[0109] Place the reaction system into the device, set the temperature in the range of 30°C - 37°C, and continuously monitor the temperature during the reaction to ensure temperature stability.

[0110] Time control: Use a timing device, such as a timer or an instrument with a timing function.

[0111] Set the reaction time to 2 hours. After the reaction starts, the timing device begins to time to ensure the accuracy of the reaction time.

[0112] pH value adjustment: Measure the pH value of the reaction system using an accurate pH meter.

[0113] According to the measurement results, adjust the pH value within the range of 7.0 - 7.5 by adding an appropriate amount of buffer solution (such as Tris-HCl buffer solution, etc.). During the reaction process, the pH value can be measured regularly to ensure its stability.

[0114] Reagent ratio control: Use a precise pipette to accurately measure nucleic acids and modification reagents.

[0115] Measure according to the ratio of nucleic acid sample to modification reagent of 1:5. In actual operation, it can be appropriately adjusted according to the concentration of nucleic acid and the activity of the modification reagent, but the ratio should be within a reasonable range.

[0116] In this specific protocol, the post-modification treatment method should be able to effectively remove unreacted modification reagents and other impurities, and improve the purity of nucleic acids.

[0117] In this example: Chromatography treatment: Select a suitable chromatographic column according to the properties of nucleic acids and modification reagents. For small molecule modification reagents, a high-performance liquid chromatography column can be selected.

[0118] Equilibrate the chromatographic column with the mobile phase, and inject the reacted sample into the chromatographic column.

[0119] Set appropriate flow rate (such as 1 ml / min) and detection wavelength (determined according to the characteristics of the modification reagent and nucleic acid), collect the eluate containing purified nucleic acids, and separate the unreacted modification reagent from the nucleic acid by optimizing the composition and flow rate of the mobile phase.

[0120] Electrophoresis treatment: Select appropriate gel concentration and electrophoresis conditions according to the molecular weight of nucleic acids.

[0121] For agarose gel electrophoresis, if the molecular weight of the modified nucleic acid is large, select a lower concentration agarose gel (such as 0.8%), and if the molecular weight is small, select a higher concentration (such as 2%).

[0122] For polyacrylamide gel electrophoresis, select appropriate gel concentration and cross-linking degree according to the molecular weight of nucleic acids, and perform electrophoresis and band separation according to the standard operating procedure.

[0123] Prepare the gel, add the sample into the gel wells, and set an appropriate voltage (such as 100 V) for electrophoresis.

[0124] After electrophoresis, observe the gel electrophoresis pattern under ultraviolet light, separate the modified nucleic acid band from the unreacted substances, and the target nucleic acid band can be cut out for recovery.

[0125] Precipitation method: Use ethanol or isopropanol to precipitate nucleic acids.

[0126] If ethanol precipitation is used, add 2 volumes of absolute ethanol and 1 / 10 volume of 3M sodium acetate (pH 5.2) to the reacted sample, gently invert and mix well, and place at -20 °C for several hours or overnight to precipitate nucleic acids.

[0127] If isopropanol precipitation is used, add an equal volume of isopropanol and an appropriate amount of salt (such as 0.1M sodium chloride), gently invert and mix well, and place at low temperature for a period of time to precipitate nucleic acids.

[0128] The precipitated nucleic acids are collected by centrifugation (such as 12000 rpm, 10 minutes), dissolved in an appropriate amount of buffer, and the residual unreacted modification reagents and impurities are removed by washing the precipitate multiple times to obtain highly purified modified nucleic acids.

[0129] Specifically, in this protocol, the quality detection method should be accurate and reliable, and be able to comprehensively evaluate the quality of the modified nucleic acids.

[0130] In this example: Spectral analysis: Use a high-quality ultraviolet spectrophotometer.

[0131] Add the purified nucleic acid sample to a cuvette and measure the absorbance of the nucleic acid at 260 nm and 280 nm.

[0132] Calculate the A260 / A280 ratio, take the average of multiple measurements to ensure the accuracy and stability of the results, and judge whether the purity of the nucleic acid meets the requirements based on this.

[0133] Gel electrophoresis: Use a high-resolution agarose gel or polyacrylamide gel.

[0134] Prepare the gel, add molecular weight standards and control samples, and add the purified nucleic acid sample to the gel wells.

[0135] Set an appropriate voltage for electrophoresis. After electrophoresis, observe the gel electrophoresis pattern under ultraviolet light, clearly observe the integrity and modification effect of the nucleic acid, and determine whether the size of the nucleic acid meets the expectation by comparing with the molecular weight standards.

[0136] Sequencing analysis: Select a high-throughput sequencing platform, such as the Illumina sequencing platform, etc.

[0137] Process the purified nucleic acid sample according to the platform requirements, such as constructing a sequencing library, etc.

[0138] Perform deep sequencing on the modified nucleic acids, accurately evaluate the efficiency and specificity of the modification through bioinformatics analysis, and conduct multiple repeated experiments at the same time to ensure the reliability of the results.

[0139] Finally, it should be noted that the above are only preferred embodiments of the present invention and are not used to limit the present invention. Although the present invention has been described in detail with reference to the foregoing embodiments, those skilled in the art can still modify the technical solutions recorded in the foregoing embodiments or perform equivalent replacements for some of the technical features. Any modification, equivalent replacement, improvement, etc. made within the spirit and principle of the present invention shall be included within the protection scope of the present invention.

Claims

1. An innovative technology and method for efficient nucleic acid modification, characterized in that: The following steps are involved: S1. Preparation of nucleic acid samples. The purity of the selected nucleic acid samples shall not be less than 95%. The integrity shall be detected by gel electrophoresis with no obvious degradation bands and clear main bands. S2. Selection of modification reagents. Modification reagents include chemical modifiers and enzyme preparations. The purity of chemical modifiers should not be less than 98%, and the activity of enzyme preparations should not be less than 100U / mg. S3. The modification reaction is carried out at a temperature in the range of 30°C-37°C, a reaction time of 2 hours, a pH value in the range of 7.0-7.5, and a ratio of nucleic acid sample to modification reagent in the reaction system composition of 1:

5. S4. Post-modification treatment: post-treatment by chromatography, electrophoresis, precipitation and other methods, the purity of the treated nucleic acid is not less than 99%. S5. Quality detection is carried out by spectral analysis, gel electrophoresis, sequencing and other methods. The modification efficiency is not less than 85%, and the specificity is verified to be more than 90% through specific experimental methods.

2. The innovative technology and method for efficient nucleic acid modification according to claim 1, characterized in that: Nucleic acid samples can come from biological tissues, cell cultures or be artificially synthesized.

3. The innovative technology and method for efficient nucleic acid modification according to claim 2, characterized in that: Modification reagents should be selected reasonably according to different modification purposes and nucleic acid types.

4. The innovative technology and method for efficient nucleic acid modification according to claim 3, characterized in that: The conditions of the modification reaction should be strictly controlled within the specified parameter range to ensure the modification effect.

5. The innovative technology and method for efficient nucleic acid modification according to claim 4, characterized in that: The post-modification treatment method should be able to effectively remove unreacted modification reagents and other impurities to improve the purity of nucleic acids.

6. The innovative technology and method for efficient nucleic acid modification according to claim 5, characterized in that: The quality detection method should be accurate and reliable, and be able to comprehensively assess the quality of the modified nucleic acid.