Transformation kit for DNA methylation detection pretreatment and application thereof

Abstract

The invention relates to a transformation kit for DNA methylation detection pretreatment and application thereof, and belongs to the technical field of gene detection. The conversion kit provided by the invention comprises a conversion solution, a desulfonation solution, a washing solution, an eluent and magnetic beads, the conversion solution is composed of a conversion reagent, a protective agent and a catalyst, the conversion reagent comprises sodium hydrogen sulfite, ammonium sulfite and ammonium hydrogen sulfite, the protective agent comprises dithiothreitol, quinodimethacrylate, hydroquinone, trehalose and spermine, and the catalyst comprises sodium hydroxide and sodium hydroxide. The catalyst comprises tetraethylenepentamine pentahydrochloride and tetraethylammonium chloride, and the desulfonation liquid comprises sodium hydroxide, sodium chloride, isopropanol, and any one of triton X-100, Tween and glycerol. When the transformation kit method provided by the invention is applied to sample pretreatment of DNA methylation detection, the transformation time can be shortened to be within 10 minutes, meanwhile, high transformation rate and high recovery rate are ensured, and the transformation rate is up to 99.9%.

Description

Technical Field

[0001] The present invention relates to the technical field of gene detection, and in particular to a conversion kit for DNA methylation detection pretreatment and its application. Background Art

[0002] DNA methylation is a form of DNA chemical modification that can change genetic expression without changing the DNA sequence. DNA methylation refers to the covalent binding of a methyl group to the 5th carbon position of cytosine in genomic CpG dinucleotides under the action of DNA methyltransferase. A large number of studies have shown that DNA methylation can cause changes in chromatin structure, DNA conformation, DNA stability, and the interaction mode between DNA and proteins, thereby controlling gene expression.

[0003] Currently, the basic route for detecting methylated cytosine is to convert unmethylated cytosine into uracil, while methylated cytosine remains unchanged, and then determine the methylation modification state of cytosine in DNA molecules by means of PCR, sequencing, or gene chips. The mainstream technologies are the enzymatic conversion of New England Biolabs (abbreviated as NEB), and the representative product is NEBNext Enzymatic Methl-seq Conversion Module, as well as the chemical conversion of Zymo Reaserch (abbreviated as Zymo), and the representative products are EZ DNA Methylation Gold Kit and EZ DNA Methylation-LightningKit. The enzymatic conversion of NEB depends on two important enzymatic reactions. The first step is to provide protection by oxidizing 5-methylcytosine and 5-hydroxymethylcytosine by TET2, and the second step is to complete the conversion by deaminating cytosine to uracil by the deaminase APOBEC. However, the expression purification and storage stability of enzyme proteins will affect the performance of the enzyme and thus the performance of the conversion. Moreover, the enzymatic reaction requires certain conditions and time, and subsequent recovery and purification steps will be introduced, making the whole conversion reaction unstable and cumbersome; while the chemical conversion of Zymo mainly treats DNA with bisulfite, and then converts unmethylated cytosine into uracil through a desulfonation reaction, while methylated cytosine remains unchanged during the conversion process.

[0004] Although the principle of bisulfite conversion is very simple and the bisulfite chemical reagent itself is relatively stable, there are still some serious drawbacks: (1) DNA will be severely degraded under the conditions of high temperature, low pH, and long reaction time in bisulfite solution. Especially for trace DNA samples, traditional bisulfite conversion will greatly reduce the sensitivity of subsequent detection; (2) And traditional bisulfite conversion reactions require overnight (>16h). The reaction duration not only causes severe degradation of DNA samples but also has many impacts on actual detection work. Even the representative product of Zymo, the EZ DNA Methylation Gold Kit, requires 2 hours and 40 minutes of DNA treatment, and the optimized EZ DNA Methylation-Lightning Kit of this company also requires 1 hour and 10 minutes of DNA treatment. The long-term and intense reaction will deepen the damage to the sample DNA and the complexity of actual detection work; (3) The recovery rate of bisulfite conversion products is low. On the one hand, it is because of the degradation effect of sulfurous acid treatment on DNA, and on the other hand, bisulfite will damage the magnetic bead recovery system, resulting in poor magnetic bead recovery effect, and a large amount of DNA will be lost, sometimes even reaching 50-90%.

[0005] In summary, the enzymatic conversion of NEB has unstable enzyme proteins, long enzymatic reaction time, cumbersome steps, and harsh conditions. The chemical conversion of Zymo also has a long reaction time, which poses a great challenge to the detection of samples, especially trace DNA. Both methods have their own inevitable defects. Summary of the Invention

[0006] To solve the above technical problems, the present invention provides a conversion kit for DNA methylation detection pretreatment, including a conversion solution, a desulfonation solution, a washing solution, an elution solution, and magnetic beads. The conversion solution includes a conversion reagent, a protective agent, and a catalyst. The conversion reagent includes sodium bisulfite, ammonium bisulfite, and ammonium metabisulfite. The protective agent includes dithiothreitol, quinol dimethacrylate, hydroquinone, trehalose, and spermine. The catalyst includes tetraethylenepentamine pentahydrochloride and tetraethylammonium chloride. The desulfonation solution includes sodium hydroxide, sodium chloride, and isopropanol, and any one of Triton X-100, Tween, and glycerol.

[0007] The first object of the present invention is to provide a conversion kit, including a conversion solution, a desulfonation solution, a washing solution, an elution solution, and magnetic beads, wherein:

[0008] The conversion solution includes a conversion reagent, a protective agent, and a catalyst;

[0009] The conversion reagent includes sodium bisulfite, ammonium bisulfite, and ammonium metabisulfite;

[0010] The protective agent includes dithiothreitol, quinol dimethacrylate, hydroquinone, trehalose and spermine. The molar concentration of quinol dimethacrylate is 1 - 10 mM, the molar concentration of trehalose is 1 - 100 mM, and the molar concentration of spermine is 1 - 10 mM;

[0011] The catalyst includes tetraethylenepentamine pentahydrochloride and tetraethylammonium chloride;

[0012] The desulfonation solution includes sodium hydroxide, sodium chloride and isopropanol, and any one of Triton X - 100, Tween, and glycerol;

[0013] The washing solution includes ethanol, Tris - HCl buffer and EDTA;

[0014] The elution solution includes Tris - HCl buffer and EDTA.

[0015] Furthermore, the molar concentration of sodium bisulfite in the conversion reagent is 1 - 2 M, the molar concentration of ammonium bisulfite is 0.1 - 1 M, and the molar concentration of ammonium bisulfite is 6 - 10 M.

[0016] Furthermore, adding a protective agent to the conversion solution can increase the stability of the conversion reagent and the stability of the conversion.

[0017] Furthermore, the concentration of dithiothreitol in the protective agent is 1 - 100 mM, and the molar concentration of hydroquinone is 0.5 - 100 mM.

[0018] Furthermore, the protective agent acts as an antioxidant to protect the reagent from being oxidized and inactivated during formulation and storage for long - term preservation. At the same time, it acts as a free - radical scavenger to protect DNA fragments from breaking and degrading during the conversion process.

[0019] Furthermore, the catalyst is composed of polyamine and quaternary ammonium salt, both of which are catalysts for the bisulfite conversion reaction and can accelerate the reaction process.

[0020] Furthermore, the molar concentration of tetraethylenepentamine pentahydrochloride in the catalyst is 1 - 10 mM, and the molar concentration of tetraethylammonium chloride is 1 - 10 mM.

[0021] Furthermore, the molar concentration of sodium hydroxide in the desulfonation solution is 0.1 - 1 M, the molar concentration of sodium chloride is 100 - 500 mM, and the concentration of isopropanol is 30 - 70%.

[0022] Furthermore, the magnetic beads are carboxyl magnetic beads, amino magnetic beads, silicon - hydroxyl magnetic beads or silica - membrane magnetic beads, and the mass concentration is 5 - 15%.

[0023] Furthermore, the particle size of the magnetic beads is 100 - 1000 nanometers.

[0024] The second object of the present invention is to provide the application of the above conversion kit in the pretreatment of DNA methylation detection.

[0025] Furthermore, it includes the following steps:

[0026] Step S1: Mix the DNA sample to be detected with the conversion solution, centrifuge to the bottom of the tube, and perform PCR thermal cycling reaction at 98 °C for 5 - 10 minutes to obtain a reaction product;

[0027] Step S2: Add magnetic beads to the reaction product, mix well by oscillation or pipetting, incubate for 1 - 10 minutes, then magnetically attract until the solution is clear, and remove the supernatant;

[0028] Step S3: Add washing solution to the precipitate, mix well by oscillation or pipetting, magnetically attract until the solution is clear, and remove the supernatant;

[0029] Step S4: Add desulfonation solution to the precipitate, mix well by oscillation or pipetting, incubate at room temperature or 37 °C for 5 - 20 minutes, then magnetically attract until the solution is clear, and remove the supernatant;

[0030] Step S5: Repeat Step S3 twice;

[0031] Step S6: Dry the precipitate for 1 - 10 minutes, add elution solution, mix well by oscillation or pipetting, centrifuge, and heat and elute at room temperature or 37 °C for 1 - 5 minutes, then magnetically attract until the solution is clear to obtain a conversion product.

[0032] Furthermore, in Step S2, the mass concentration of the added magnetic beads is 5 - 15%.

[0033] Advantages of the present invention:

[0034] (1) The conversion reagent provided by the present invention does not require a traditional binding solution, which not only simplifies the purification and recovery steps but also ensures the recovery rate of the sample;

[0035] (2) The combination of protective agents in the conversion reagent provided by the present invention not only effectively protects the stability during the conversion process but also ensures the stability of the conversion reagent;

[0036] (3) Applying the conversion kit provided by the present invention to the sample pretreatment of DNA methylation detection compresses the conversion time within 10 minutes and improves the sensitivity of subsequent detection of the conversion product;

[0037] (4) Applying the conversion kit provided by the present invention to the sample pretreatment of DNA methylation detection, the recovery rate reaches over 90%, and the product conversion rate reaches 99.9%. Description of the Drawings

[0038] To make the content of the present invention easier to be clearly understood, the following further details the present invention according to specific embodiments of the present invention in conjunction with the accompanying drawings, where:

[0039] Figure 1 is the process schematic diagram of the present invention;

[0040] Figure 2 is the comparison chart of ACTB-1 amplification results in Example 8 of the present invention;

[0041] Figure 3 is the comparison chart of ACTB-2 amplification results in Example 8 of the present invention;

[0042] Figure 4 is the comparison chart of results of different transformation reagents in Example 9 of the present invention. Specific Embodiments

[0043] The following further describes the present invention in conjunction with the accompanying drawings and specific embodiments, so that those skilled in the art can better understand the present invention and be able to implement it, but the embodiments cited are not intended to limit the present invention.

[0044] The preparation of the main solutions in the present invention is as follows:

[0045] Transformation reagent: one or more components of sodium bisulfite, ammonium sulfite, and ammonium bisulfite, where sodium bisulfite is 1-2M, ammonium sulfite is 0.1-1M, and ammonium bisulfite is 6-10M;

[0046] Protective agent: dithiothreitol 1-100 mM, quinol dimethacrylate 1-10 mM, hydroquinone 0.5-100 mM, trehalose 1-100 mM, spermine 1-10 mM;

[0047] Catalyst: tetraethylenepentamine pentahydrochloride 1-10 mM, tetraethylammonium chloride 1-10 mM;

[0048] Magnetic beads: carboxyl magnetic beads, amino magnetic beads, silanol magnetic beads, silica gel membrane magnetic beads, with a mass concentration of 5-15% and a particle size of 100-1000 nm;

[0049] Desulfonation solution: sodium hydroxide 0.1-1M, sodium chloride 100-500 mM, Triton X-100, Tween, or glycerol 5%, isopropanol 30-70%;

[0050] Washing solution: Tris-HCl (pH = 6-8) 10 mM, EDTA 1 mM, ethanol 70-80%;

[0051] Elution solution: Tris-HCl (pH = 7.5-9) 10 mM, EDTA 1 mM.

[0052] Example 1

[0053] Take 100 ng of DNA extracted from pancreatic cancer tissue, transform it using the following method, purify and recover the transformed DNA with magnetic beads, and quantitatively determine the concentration of the recovered DNA using the Yeasen ssDNA Qubit reagent and calculate the recovery rate (recovery rate = recovered concentration × elution volume / amount of DNA input before transformation × 100%, the same below). The test results are shown in Table 1.

[0054] The transformation and recovery steps of the method of the present invention are as follows:

[0055] (1) Components of relevant solutions

[0056] Transformation solution: sodium bisulfite 1 M, ammonium sulfite 0.1 M, ammonium bisulfite 6 M, dithiothreitol 1 mM, quinol dimethacrylate 1 mM, hydroquinone 0.5 mM, trehalose 1 mM, spermine 1 mM, tetraethylenepentamine pentahydrochloride 1 mM, tetraethylammonium chloride 1 mM.

[0057] Magnetic beads: The selected magnetic beads are silicon hydroxyl magnetic beads, with a mass concentration of 5 - 15% and a particle size of 200 nm;

[0058] Desulfonation solution: sodium hydroxide 1 M, sodium chloride 500 mM, Tween 5%, isopropanol 30%;

[0059] Washing solution: Tris - HCl (pH = 6 - 8) 10 mM, EDTA 1 mM, ethanol 80%;

[0060] Elution solution: Tris - HCl (pH = 7.5 - 9) 10 mM, EDTA 1 mM;

[0061] (2) Add a certain volume of sample DNA to be processed into a 200 μL centrifuge tube, add 180 μL of the above - prepared transformation solution, make up the volume to 200 μL with nuclease - free water, mix the liquid thoroughly by oscillation, and centrifuge instantaneously to the bottom of the tube;

[0062] (3) Divide the liquid into two equal parts and transfer them to another 200 μL centrifuge tube, cover the tube cap, and centrifuge instantaneously to the bottom of the tube;

[0063] (4) Perform a thermal cycling reaction on a PCR thermal cycler with the conditions of 98 °C for 5 - 10 min;

[0064] (5) Transfer the reaction product to a 1.5 mL centrifuge tube, add 10 μL of magnetic beads, mix well by oscillation or pipetting, and incubate at room temperature for 1 - 10 min;

[0065] (6) After instantaneous centrifugation, place the centrifuge tube on a magnetic rack, magnetically attract for 1 - 10 min. After the solution in the centrifuge tube becomes clear, carefully remove the supernatant;

[0066] (7) Add 100 - 400 μL of freshly prepared washing solution into the centrifuge tube, mix well by shaking or pipetting, centrifuge briefly and then place the centrifuge tube on the magnetic stand. Magnetically attract for 1 - 10 min. After the solution in the centrifuge tube becomes clear, carefully remove the supernatant.

[0067] (8) Add 50 - 200 μL of desulfonation solution into the centrifuge tube, mix well by shaking or pipetting, and incubate at 37 °C for 5 - 20 min.

[0068] (9) Centrifuge briefly and then place the centrifuge tube on the magnetic stand. Magnetically attract for 1 - 10 min. After the solution in the centrifuge tube becomes clear, carefully remove the supernatant.

[0069] (10) Add 100 - 400 μL of freshly prepared washing solution into the centrifuge tube, mix well by shaking or pipetting, centrifuge briefly and then place the centrifuge tube on the magnetic stand. Magnetically attract for 1 - 10 min. After the solution in the centrifuge tube becomes clear, carefully remove the supernatant.

[0070] (11) Repeat step (7) twice.

[0071] (12) Keep the centrifuge tube on the magnetic stand and dry at room temperature for 1 - 10 min.

[0072] (13) Add 20 μL of elution solution into the centrifuge tube, mix well by shaking or pipetting, centrifuge briefly and then elute at room temperature or 37 °C for 1 - 5 min.

[0073] (14) Place the centrifuge tube on the magnetic stand and magnetically attract for 1 - 5 min. After the solution in the centrifuge tube becomes clear, carefully aspirate 20 μL of the supernatant into a new 1.5 mL centrifuge tube.

[0074] Comparative Example 1

[0075] This comparative example is the same as Example 1 in terms of DNA sample, contents of each solution component, transformation and recovery methods, except that: after the transformation is completed, the binding solution is added and mixed well with the transformation mixture and magnetic beads. The binding solution includes 8 M guanidine hydrochloride, 1 M sodium chloride, 10 mM Tris - HCl, and 1 mM EDTA. The DNA recovered after transformation in this comparative example was quantitatively analyzed for concentration using the Yeasen ssDNA Qubit reagent and the recovery rate was calculated. The test results are shown in Table 1.

[0076] Comparative Example 2

[0077] This comparative example is the same as Example 1 in terms of the DNA sample, the content of each solution component, the transformation and recovery methods, with the only difference being that after the transformation is completed, a binding solution is added and mixed well with the transformation mixture and magnetic beads. The binding solution includes 6M guanidine isothiocyanate, 1M sodium chloride, 10mM Tris-HCl, and 1mM EDTA. The DNA recovered after the transformation of this comparative example was quantitatively analyzed for concentration using the Yeasen ssDNA Qubit reagent, and the recovery rate was calculated. The test results are shown in Table 1.

[0078] Table 1 Comparison of recovery rates between Example 1 and Comparative Examples 1-2

[0079] Recovery Concentration Recovery Rate Example 1 4.32 ng / μL 86.4％ Comparative Example 1 4.05 ng / μL 81.0％ Comparative Example 2 4.17 ng / μL 83.4％

[0080] From the results in Table 1, the transformation reagent and transformation method provided by the present invention ensure a certain recovery rate without the need to add a binding solution, simplify the experimental steps, reduce the transformation cost, and improve the efficiency of the transformation process.

[0081] Example 2

[0082] 1 μg of DNA extracted from pancreatic cancer tissue was mixed evenly with 1 ng of unmethylated Lambda DNA, and then fragmented using a Covaris M220 ultrasonic disruptor. The fragmented DNA (100 ng) was transformed using the following different transformation solutions respectively. The DNA recovered after the transformation was quantitatively analyzed for concentration using the Yeasen ssDNA Qubit reagent, and the recovery rate was calculated. 10 ng of the transformed DNA was taken respectively and used with the DNAMethylation Library Kit for Illumina V3 kit from Novoprotein to construct libraries. The libraries were quantitatively analyzed for concentration using the Yeasen dsDNA Qubit reagent, and the library yield was calculated. Then, the libraries were sent to the Illumina platform for sequencing. The conversion rate of the unmethylated C bases of Lambda DNA was analyzed by bioinformatics. The test results are shown in Table 2. The conversion rate is defined as (1 - the ratio of the number of times the base on Lambda DNA is measured as C to the total number of sequencing times at the C site) × 100%.

[0083] The transformation solutions in this example are as follows:

[0084] Transformation solution A: 1.056M sodium bisulfite, 0.144M ammonium bisulfite, 5.86M ammonium bisulfite, 1mM dithiothreitol, 1mM quinol dimethacrylate, 0.5mM hydroquinone, 1mM trehalose, 1mM spermine, 1mM tetraethylenepentamine pentahydrochloride, 1mM tetraethylammonium chloride;

[0085] Conversion solution B: Sodium bisulfite 1.527 M, ammonium sulfite 0.473 M, ammonium bisulfite 6.93 M, dithiothreitol 1 mM, quinol dimethacrylate 1 mM, hydroquinone 0.5 mM, trehalose 1 mM, spermine 1 mM, tetraethylenepentamine pentahydrochloride 1 mM, tetraethylammonium chloride 1 mM;

[0086] Conversion solution C: Sodium bisulfite 1.983 M, ammonium sulfite 0.917 M, ammonium bisulfite 9.87 M, dithiothreitol 1 mM, quinol dimethacrylate 1 mM, hydroquinone 0.5 mM, trehalose 1 mM, spermine 1 mM, tetraethylenepentamine pentahydrochloride 1 mM, tetraethylammonium chloride 1 mM;

[0087] The content of each other solution component, the conversion and recovery steps are the same as those in Example 1.

[0088] The steps for single-strand methylation library construction are as follows:

[0089] (1) Add 10 ng of the converted DNA, make up the volume to 20 μL with Dilution Buffer, incubate at 95 °C for 2 min on a PCR instrument, and quickly place on ice for 2 min;

[0090] (2) Sequentially add 3'-end ligation buffer, ligation enzyme mixture, and adapter, pipette and mix well and centrifuge briefly, perform the reaction on a PCR instrument at 37 °C for 15 min, 95 °C for 2 min, and hold at 4 °C;

[0091] (3) Sequentially add single-strand extension primer, single-strand extension enzyme mixture, perform the reaction on a PCR instrument at 98 °C for 1 min, 62 °C for 2 min, 65 °C for 5 min, and hold at 4 °C;

[0092] (4) After the reaction is completed, purify the reaction product with 1.2× VAHTS DNA Clean Beads;

[0093] (5) Sequentially add 5'-end ligation mixture, adapter to the purified product, perform the reaction on a PCR instrument at 25 °C for 15 min, and hold at 4 °C;

[0094] (6) After the reaction is completed, purify the reaction product with 1× VAHTS DNA Clean Beads;

[0095] (7) Sequentially add Index Primers, VAHTS HiFi Amplification Mix V3 to the purified product, perform the reaction on a PCR instrument at 95 °C for 3 min, (98 °C for 30 s, 60 °C for 15 s, 72 °C for 30 s) for a total of 9 cycles, 72 °C for 5 min, and hold at 4 °C;

[0096] After the reaction is completed, the reaction product is purified with 0.85×VAHTS DNA Clean Beads, and the purified product is the single-stranded methylation library. The test results are shown in the following table:

[0097] Table 2 Results of recovery rate and conversion rate in Example 2

[0098] Reagent Recovery Concentration Recovery Rate Library Yield Conversion Rate Conversion Solution A 4.22 ng / μL 84.4％ 328 ng 98.4％ Conversion Solution B 4.38 ng / μL 87.6％ 403 ng 99.2％ Conversion Solution C 4.59 ng / μL 91.8％ 453 ng 99.5％

[0099] Judging from the results in Table 2, in Example 2, Transformation Liquids A, B, and C respectively selected the low, medium, and high concentration values of each solution component content of the transformation reagent disclosed in the present invention. Combining the transformation results of Transformation Liquids A, B, and C, it can prove the effectiveness of the content of each solution component of the reagent disclosed in the present invention, and the highest conversion rate of the transformation reagent of the present invention can reach 99.5%.

[0100] Example 3

[0101] Take 100 ng of DNA extracted from pancreatic cancer tissue and perform transformation using the following method. The DNA recovered after transformation is quantitatively determined for concentration with the Yeasen ssDNA Qubit reagent and the recovery rate is calculated. The test results are shown in Table 3.

[0102] The transformation liquid in this example is as follows:

[0103] Transformation Liquid: Sodium bisulfite 1.983 M, ammonium sulfite 0.917 M, ammonium bisulfite 9.87 M, dithiothreitol 1 mM, quinol dimethacrylate 1 mM, hydroquinone 0.5 mM, trehalose 1 mM, spermine 1 mM, tetraethylenepentamine pentahydrochloride 1 mM, tetraethylammonium chloride 1 mM;

[0104] The content of each other solution component, the transformation and recovery steps are the same as those in Example 1, except that: the selected magnetic beads are silicon hydroxyl magnetic beads, with a mass concentration of 5-15% and a particle size of 500 nm.

[0105] Example 4

[0106] This example is the same as Example 3 in terms of DNA sample, content of each solution component, transformation and recovery method, except that: the selected magnetic beads are silicon hydroxyl magnetic beads with a particle size of 100 nm; the DNA sample after transformation and recovery in this example is quantitatively determined for concentration with the Yeasen ssDNA Qubit reagent and the recovery rate is calculated. The test results are shown in Table 3.

[0107] Example 5

[0108] This example is the same as Example 3 in terms of the DNA sample, the content of each solution component, the transformation and recovery methods, except that: the selected magnetic beads are silicon hydroxyl magnetic beads with a particle size of 200 nm; the DNA sample after transformation and recovery in this example is quantitatively analyzed for concentration using the Yeasen ssDNA Qubit reagent, and the recovery rate is calculated. The test results are shown in Table 3.

[0109] Example 6

[0110] This example is the same as Example 3 in terms of the DNA sample, the content of each solution component, the transformation and recovery methods, except that: the selected magnetic beads are silicon hydroxyl magnetic beads with a mass concentration of 5 - 15% and a particle size of 1000 nm; the DNA sample after transformation and recovery in this example is quantitatively analyzed for concentration using the Yeasen ssDNA Qubit reagent, and the recovery rate is calculated. The test results are shown in Table 3.

[0111] Table 3 Comparison of recovery rate results of Examples 3 - 6

[0112] Recovery Concentration Recovery Rate Example 3 4.65 ng / μL 93.0％ Example 4 4.57 ng / μL 91.4％ Example 5 4.58 ng / μL 91.6％ Example 6 4.58 ng / μL 91.6％

[0113] From the results in Table 3, Examples 3 - 6 selected magnetic beads with different particle sizes used in the transformation reagent disclosed in the present invention, ranging from 100, 200, 500 to 1000 nm from low to high. Combining with the experimental results, it can prove the effectiveness of the selected magnetic bead particle size of the transformation reagent disclosed in the present invention, maintaining the recovery rate at a relatively high level.

[0114] Example 7

[0115] Take 100 ng of DNA extracted from pancreatic cancer tissue, use the following transformation solution for transformation, purify and recover the transformed DNA with magnetic beads, quantitatively analyze the concentration of the recovered DNA using the Yeasen ssDNA Qubit reagent, and calculate the recovery rate. The test results are shown in Table 4.

[0116] The transformation solution in this example is as follows:

[0117] The selected transformation solution is sodium bisulfite 1.983 M, ammonium sulfite 0.917 M, ammonium bisulfite 9.87 M, dithiothreitol 100 mM, quinol dimethacrylate 10 mM, hydroquinone 100 mM, trehalose 100 mM, spermine 10 mM, tetraethylenepentamine pentahydrochloride 1 mM, tetraethylammonium chloride 1 mM;

[0118] The content of the remaining solution components, the transformation and recovery steps are the same as those in Example 3.

[0119] Comparative Example 3

[0120] This comparative example is the same as Example 7 in terms of the DNA sample, the content of each solution component, and the transformation and recovery methods, with the only difference being that the transformation solution only contains 1.983 M sodium bisulfite, 0.917 M ammonium sulfite, 10 M ammonium bisulfite, 1 mM tetraethylenepentamine pentahydrochloride, and 1 mM tetraethylammonium chloride, and does not contain the protective agent component; the DNA sample after transformation and recovery in this comparative example was quantitatively analyzed for concentration using the Yeasen ssDNA Qubit reagent and the recovery rate was calculated. The test results are shown in Table 4.

[0121] Comparative Example 4

[0122] This comparative example is the same as Comparative Example 3 in terms of the DNA sample, the content of each solution component, and the transformation and recovery methods, with the only difference being that only 100 mM dithiothreitol is added; the DNA sample after transformation and recovery in this comparative example was quantitatively analyzed for concentration using the Yeasen ssDNA Qubit reagent and the recovery rate was calculated. The test results are shown in Table 4.

[0123] Comparative Example 5

[0124] This comparative example is the same as Comparative Example 3 in terms of the DNA sample, the content of each solution component, and the transformation and recovery methods, with the only difference being that only 100 mM dithiothreitol and 10 mM quinol dimethacrylate are added; the DNA sample after transformation and recovery in this comparative example was quantitatively analyzed for concentration using the Yeasen ssDNA Qubit reagent and the recovery rate was calculated. The test results are shown in Table 4.

[0125] Comparative Example 6

[0126] This comparative example is the same as Comparative Example 3 in terms of the DNA sample, the content of each solution component, and the transformation and recovery methods, with the only difference being that only 10 mM quinol dimethacrylate and 100 mM hydroquinone are added; the DNA sample after transformation and recovery in this comparative example was quantitatively analyzed for concentration using the Yeasen ssDNA Qubit reagent and the recovery rate was calculated. The test results are shown in Table 4.

[0127] Comparative Example 7

[0128] This comparative example is the same as Comparative Example 3 in terms of the DNA sample, the content of each solution component, and the transformation and recovery methods, with the only difference being that only 100 mM hydroquinone and 100 mM trehalose are added; the DNA sample after transformation and recovery in this comparative example was quantitatively analyzed for concentration using the Yeasen ssDNA Qubit reagent and the recovery rate was calculated. The test results are shown in Table 4.

[0129] Comparative Example 8

[0130] This comparative example is the same as Comparative Example 3 in terms of DNA samples, the content of each solution component, and the transformation and recovery methods, with the only difference being that only 100 mM trehalose and 10 mM spermine are added; the DNA sample after transformation and recovery in this comparative example is quantitatively analyzed for concentration using the Yeasen ssDNA Qubit reagent, and the recovery rate is calculated. The test results are shown in Table 4.

[0131] Table 4 Comparison of Recovery Rates between Example 7 and Comparative Examples 3 - 8

[0132]

[0133]

[0134] Referring to Table 4, Example 7 and Comparative Examples 3 - 8 selected different protectants, their different concentrations, and different combinations used in the transformation reagent disclosed in the present invention. Considering the experimental results, it can prove the effectiveness of the protectant combination selected for the transformation reagent disclosed in the present invention.

[0135] Example 8

[0136] Take 100 ng of DNA extracted from pancreatic cancer tissue and use the following transformation solution for transformation. The transformed DNA is purified and recovered using magnetic beads, and the recovered DNA is quantitatively analyzed for concentration using the Yeasen ssDNA Qubit reagent, and the recovery rate is calculated. The experimental results are shown in Table 8.

[0137] The transformation solution in this example is as follows:

[0138] The selected transformation solution is 1.983 M sodium bisulfite, 0.917 M ammonium bisulfite, 9.87 M ammonium hydrogen sulfite, 100 mM dithiothreitol, 10 mM quinol dimethacrylate, 100 mM hydroquinone, 100 mM trehalose, 10 mM spermine, 10 mM tetraethylenepentamine pentahydrochloride, 10 mM tetraethylammonium chloride;

[0139] The content of the remaining solution components, as well as the transformation and recovery steps, are the same as those in Example 3.

[0140] In addition, according to the sequence of the ACTB gene on the human genome, two different probe primers are designed according to the rule of converting unmethylated C to U (amplified to T), named ACTB - 1 and ACTB - 2, and methylation - specific qPCR detection is performed on the transformed DNA. The test results are shown in Table 7.

[0141] The transformation procedure of the EZ DNA Methylation - Lightning Kit from Zymo Research for comparison is 98℃ for 8 min, 54℃ for 60 min, 4℃ Hold. After transformation, purification is carried out according to its instruction manual. The specific purification steps are as follows:

[0142] (1) Add 600 μL of M-Binding Buffer to the adsorption column, transfer the transformation product to the adsorption column, invert it up and down 10 times, and centrifuge at 12,000 rpm for 30 s;

[0143] (2) Add 100 μL of M-Wash Buffer to the adsorption column (make sure to add anhydrous ethanol), and centrifuge at 12,000 rpm for 30 s;

[0144] (3) Add 200 μL of L-Desulphonation Buffer to the adsorption column, incubate at room temperature for 15 - 20 min, and centrifuge at 12,000 rpm for 30 s;

[0145] (4) Add 200 μL of M-Wash Buffer to the adsorption column, and centrifuge at 12,000 rpm for 30 s;

[0146] (5) Repeat step (4) once;

[0147] (6) Centrifuge the empty column for 2 min, transfer the adsorption column to a new 1.5 mL centrifuge tube and let it dry for about 5 min;

[0148] (7) Add 20 μL of M-Elution Buffer to the adsorption column, let it stand at room temperature for 3 min, centrifuge at 12,000 rpm for 2 min, and collect the filtrate, which is the transformed DNA.

[0149] III. qPCR Detection

[0150] (1) Configure the reaction system according to the following table:

[0151] Table 5 qPCR Reaction System

[0152] Reagent Volume / μL 2×PCR Mix 10 Primer Probe Mix (Final Concentration 0.4 μM) 1.8 Converted DNA 5 Nuclease-Free Water 3.2

[0153] (2) Perform qPCR reaction according to the following table:

[0154] Table 6 qPCR Reaction Conditions

[0155]

[0156] (3) The CT values of the qPCR detection of the DNA samples are shown in the following table. Among them, the amplification results of ACTB-1 are as Figure 2 shown, and the amplification results of ACTB-2 are as Figure 3 shown:

[0157] Table 7 Results of Recovery Rate and Conversion Rate in Example 9

[0158] Reagent Recovery Concentration Recovery Rate ACTB-1 ACTB-2 Zymo Research 2.56 ng / μL 51.2％ 26.81 27.22 Example 8 4.73 ng / μL 94.6％ 26.31 27.03

[0159] From the results, when using the transformation reagent disclosed in the present invention and the Zymo Research EZ DNAMethylation-Lightning Kit to transform DNA samples simultaneously for parallel comparison, on the basis of shortening the transformation time, the DNA recovery rate after transformation is increased, and the CT value detected by qPCR also indicates an improvement in its detection sensitivity, which can prove the effectiveness of the content of each solution component of the transformation reagent disclosed in the present invention.

[0160] Comparative Example 9

[0161] This comparative example is the same as the DNA sample, the content of each solution component, the transformation and recovery methods in Example 8, except that: tetraethylenepentamine pentahydrochloride and tetraethylammonium chloride are not added; the DNA sample after transformation and recovery in this comparative example is quantitatively analyzed for concentration with the Yeasen ssDNA Qubit reagent and the recovery rate is calculated. The test results are shown in Table 8.

[0162] Comparative Example 10

[0163] This comparative example is the same as the DNA sample, the content of each solution component, the transformation and recovery methods in Example 8, except that: tetraethylenepentamine pentahydrochloride is not added; the DNA sample after transformation and recovery in this comparative example is quantitatively analyzed for concentration with the Yeasen ssDNA Qubit reagent and the recovery rate is calculated. The test results are shown in Table 8.

[0164] Comparative Example 11

[0165] This comparative example is the same as the DNA sample, the content of each solution component, the transformation and recovery methods in Example 8, except that: tetraethylammonium chloride is not added; the DNA sample after transformation and recovery in this comparative example is quantitatively analyzed for concentration with the Yeasen ssDNA Qubit reagent and the recovery rate is calculated. The test results are shown in Table 8.

[0166] Table 8 Comparison of recovery rates between Example 8 and Comparative Examples 9 - 11

[0167] Recovery Concentration Recovery Rate Example 8 4.73 ng / μL 94.6％ Comparative Example 9 2.27 ng / μL 45.4％ Comparative Example 10 2.91 ng / μL 58.2％ Comparative Example 11 3.03 ng / μL 60.6％

[0168] Referring to Table 5, Example 8 and Comparative Examples 9 - 11 selected different catalysts and their different concentrations and different combinations used in the transformation reagent disclosed in the present invention. From the test results of Example 8 and Comparative Examples 9 - 11, it can be proved that the selected catalyst combination of the transformation reagent disclosed in the present invention is effective, and can greatly increase the DNA yield after transformation, and increase the recovery rate to more than 90%.

[0169] Example 9

[0170] Mix 1 μg of DNA extracted from pancreatic cancer tissue thoroughly with 1 ng of unmethylated Lambda DNA. Use a Covaris M220 ultrasonic disruptor to fragment the DNA. Then, transform 100 ng of the fragmented DNA using the following methods respectively. The DNA recovered after transformation is quantitatively analyzed for concentration using the Yeasen ssDNA Qubit reagent. Take 10 ng of the transformed DNA each and use the DNAMethylation Library Kit for Illumina V3 kit from Novoprotein to construct single-stranded methylation libraries. The library construction steps refer to those described in Example 2. After the library construction is completed, the concentration of the library is quantitatively analyzed using the Yeasen dsDNA Qubit reagent, and then it is sent to the Illumina platform for sequencing. The sequencing results are bioinformatics-analyzed for the conversion rate of unmethylated C bases in Lambda DNA. The test results are shown in Table 9.

[0171] I. The transformation solution in this example is as follows:

[0172] The selected transformation solution is sodium bisulfite 1.983 M, ammonium bisulfite 0.917 M, ammonium hydrogen sulfite 9.87 M, dithiothreitol 100 mM, quinuclidinyl methacrylate 10 mM, hydroquinone 100 mM, trehalose 100 mM, spermine 10 mM, tetraethylenepentamine pentahydrochloride 10 mM, tetraethylammonium chloride 10 mM.

[0173] The content of each other solution component, the transformation and recovery steps are the same as those in Example 3.

[0174] II. The transformation procedure of the EZ DNA Methylation-Lightning Kit from Zymo Research for comparison is 98 °C for 8 min, 54 °C for 60 min, and hold at 4 °C. After the transformation is completed, purification is carried out according to its instruction manual. The specific purification steps refer to those described in Example 8.

[0175] Table 9 Results of recovery rate and conversion rate in Example 9

[0176] Reagent Recovery Concentration Recovery Rate Library Yield Conversion Rate Zymo Research 2.23 ng / μL 44.6％ 438 ng 99.3％ Example 9 4.71 ng / μL 94.2％ 652 ng 99.9％

[0177] Combined with the results in Table 9 and Figure 4 Looking at the results, when using the transformation reagent disclosed in the present invention and the EZDNA Methylation-Lightning Kit from Zymo Research to simultaneously transform DNA samples for parallel comparison, not only the DNA recovery rate is greatly improved, but the conversion rate also has a significant increase, which can prove the effectiveness of the content of each solution component of the transformation reagent disclosed in the present invention.

[0178] Obviously, the above embodiments are merely examples for clear illustration and not limitations on the implementation manners. For those of ordinary skill in the art, other different forms of changes or modifications can be made based on the above description. It is not necessary and impossible to enumerate all implementation manners here. And the obvious changes or modifications derived therefrom still fall within the protection scope of the present invention.

Claims

1. A transformation kit, characterized in that: It includes conversion solution, desulfurization solution, washing solution, elution solution and magnetic beads, wherein: The conversion solution includes a conversion reagent, a protective agent and a catalyst; The conversion reagents include sodium bisulfite, ammonium sulfite and ammonium bisulfite; The protective agent includes dithiothreitol, quinol dimethacrylate, hydroquinone, trehalose and spermine, wherein the molar concentration of the quinol dimethacrylate is 1-10 mM, the molar concentration of the trehalose is 1-100 mM, and the molar concentration of the spermine is 1-10 mM; The catalyst includes tetraethylenepentamine pentahydrochloride and tetraethylammonium chloride; The desulfurization solution includes sodium hydroxide, sodium chloride and isopropanol, and any one of Triton X-100, Tween and glycerol; The washing solution includes ethanol, Tris-HCl buffer and EDTA; The elution solution includes Tris-HCl buffer and EDTA.

2. The conversion kit according to claim 1, characterized in that: The molar concentration of sodium bisulfite in the conversion reagent is 1-2M, the molar concentration of ammonium sulfite is 0.1-1M, and the molar concentration of ammonium bisulfite is 6-10M.

3. The conversion kit according to claim 1, characterized in that: The concentration of dithiothreitol in the protective agent is 1-100 mM, and the molar concentration of hydroquinone is 0.5-100 mM.

4. The transformation kit according to claim 1, characterized in that: The molar concentration of tetraethylenepentamine pentahydrochloride in the catalyst is 1-10mM, and the molar concentration of tetraethylammonium chloride is 1-10mM.

5. The conversion kit according to claim 1, characterized in that: The molar concentration of sodium hydroxide in the desulfurization solution is 0.1-1M, the molar concentration of sodium chloride is 100-500mM, and the concentration of isopropanol is 30-70%.

6. The transformation kit according to claim 1, characterized in that: The magnetic beads are any one of carboxyl magnetic beads, amino magnetic beads, silanol magnetic beads or silica membrane magnetic beads.

7. The transformation kit according to claim 1, characterized in that: The particle size of the magnetic beads is 100-1000 nanometers.

8. Use of the conversion kit according to any one of claims 1 to 7 in pretreatment for DNA methylation detection.

9. The use according to claim 8, characterized in that: The following steps are involved: Step S1, mixing the DNA sample to be tested with the conversion solution, centrifuging to the bottom of the tube, and performing PCR thermal cycling reaction at 98°C for 5-10 minutes to obtain a reaction product; Step S2, adding magnetic beads to the reaction product, shaking or pipetting to mix, incubating for 1-10 minutes, and then magnetically aspirating until the solution is clear, and removing the supernatant; Step S3, adding washing solution to the precipitate, shaking or blowing to mix, magnetically aspirating until the solution is clear, and removing the supernatant; Step S4, adding desulfurization solution to the precipitate, shaking or pipetting to mix, incubating at room temperature or 37°C for 5-20 minutes, and then magnetically aspirating until the solution is clear, and removing the supernatant; Step S5, repeat step S3 twice; Step S6, the precipitate is dried for 1-10 minutes, an eluent is added, and the mixture is shaken or blown to mix. After centrifugation, the mixture is heated and eluted at room temperature or at 37° C. for 1-5 minutes, and magnetically adsorbed until the solution is clear to obtain the conversion product.

10. The use according to claim 9, characterized in that: In step S2, the mass concentration of added magnetic beads is 5-15%.

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