CN115786461A - DNA methylation conversion method, kit and application - Google Patents
DNA methylation conversion method, kit and application Download PDFInfo
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- CN115786461A CN115786461A CN202211516489.9A CN202211516489A CN115786461A CN 115786461 A CN115786461 A CN 115786461A CN 202211516489 A CN202211516489 A CN 202211516489A CN 115786461 A CN115786461 A CN 115786461A
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Abstract
The invention belongs to the technical field of biology, and discloses a DNA methylation transformation method, a kit and application. DNA methylation conversion method: reacting the DNA with a methylation conversion reagent, and deaminating unmethylated cytosine in the DNA into uracil by the methylation conversion reagent to obtain methylation conversion DNA; the methylation conversion reagent comprises at least one of pyrosulfite and bisulfite, and sodium hydroxide. The kit comprises a methylation conversion reagent, a DNA protective agent and a desulfurizing agent. The methylation conversion reagent provided by the invention optimizes the conversion method, comprises sodium hydroxide, can open the double strands of DNA, is convenient for methylation conversion, shortens the conversion time, reduces the degradation of the DNA and improves the conversion efficiency. The conversion method or the kit can be used for preparing methylation markers of samples to be detected so as to detect related diseases.
Description
Technical Field
The invention belongs to the technical field of biology, and particularly relates to a DNA methylation transformation method, a kit and application.
Background
DNA methylation refers to a chemical modification process in which a methyl group provided by SAM (S-adenosylmethionine) is added to the 5 th carbon atom of the 5'-CpG-3' cytosine of DNA by DNA methyltransferase (DNMT). DNA methylation is an important regulatory factor for transcription, and regulates gene expression by changing chromatin structure, DNA conformation, DNA stability, and the way DNA interacts with proteins. In recent years, a great deal of research shows that the occurrence of DNA abnormal methylation is closely related to the occurrence and the progress of tumor diseases.
There are many methods currently used to study DNA methylation. For example: (1) Various CpG island methylation detection methods developed based on bisulfite-modified DNA, such as post-bisulfite-treatment sequencing (BSP sequencing), methylation-specific PCR (MSP), methylation fluorescence detection PCR (Methylight PCR), and chip hybridization techniques; and (2) methylation sensitive restriction endonuclease method. Wherein, the bisulfite is used for modifying DNA as the basis and has wide application.
The principle of bisulfite modification is simple. The basic steps of the bisulfite modification process are: sodium hydroxide denatures double-stranded DNA to form single-stranded DNA, the single-stranded DNA reacts with bisulfite to deaminate, the treated DNA is desalted and desulfonated, and the modified DNA is purified and recovered. In this process, complete conversion of unmethylated cytosine and avoidance of DNA degradation and fragmentation as much as possible are the technical emphasis of bisulfite modification. However, the prior art and the corresponding commercial kit have the problems of long bisulfite modification time, low conversion efficiency, complex operation, low purification efficiency, low extraction efficiency, high DNA loss and the like.
Disclosure of Invention
The present invention is directed to solving at least one of the problems of the prior art described above. Therefore, the invention provides a DNA methylation conversion method, which can solve the technical problems of low conversion efficiency, high DNA loss in the conversion process and overlong time in the conversion process in the prior art.
The invention also provides a DNA methylation transformation kit.
The invention also provides an application of the DNA methylation conversion method or the DNA methylation conversion kit in preparation of the methylation marker of the sample to be detected.
According to one aspect of the present invention, there is provided a method for DNA methylation transformation, comprising the steps of:
reacting DNA with a methylation conversion reagent, wherein the methylation conversion reagent deaminates unmethylated cytosine in the DNA into uracil to obtain methylation conversion DNA;
wherein the methylation conversion reagent comprises sodium hydroxide and at least one of pyrosulfite and bisulfite.
According to a preferred embodiment of the present invention, at least the following advantages are provided:
the invention uses methylation conversion reagent to deaminate cytosine (C) that is not methylated in DNA into uracil (U), while methylated cytosine (mC) remains unchanged. The methylation conversion reagent comprises sodium hydroxide, and the sodium hydroxide can break hydrogen bonds between double chains in DNA molecules, stably and quickly open the double chains to form single chains, so that the methylation conversion reaction of the DNA is facilitated. Shortens the time of methylation conversion, reduces the degradation of DNA, and can improve the efficiency of methylation conversion. The method for DNA methylation transformation can provide a good sample for molecular biological detection, particularly clinical diagnosis, and the obtained methylation transformation DNA can effectively identify methylation sites in PCR amplification, sequencing and hybridization-based capture technologies.
In some embodiments of the invention, the metabisulfite comprises at least one of sodium metabisulfite, potassium metabisulfite, or magnesium metabisulfite.
In some embodiments of the invention, the bisulfite salt comprises at least one of sodium bisulfite, magnesium bisulfite, or ammonium bisulfite.
In some embodiments of the invention, the concentration of the pyrosulfite is 1 to 1.2mol/L.
In some embodiments of the invention, the concentration of bisulfite is 0.5 to 1.2mol/L.
In some embodiments of the invention, the concentration of sodium hydroxide is 0.005 to 0.2mol/L.
In some embodiments of the invention, the solvent of the methylation conversion reagent is water.
In some embodiments of the present invention, a DNA protecting agent is further added when the DNA reacts with the methylation conversion reagent.
DNA degradation is the most prominent side reaction in the process of methylation conversion, which can generate SO 3 - And O 2 H - And the free radicals react under certain conditions to oxidize and crack DNA, and the addition of DNA protecting agent into the methylation converting reagent can play the role of resisting oxidation, so as to protect DNA from degradation.
In some embodiments of the invention, the DNA protectant comprises at least one of hydroquinone, trehalose solution, agarose solution, dimethyl sulfoxide, ethylene glycol dimethyl ether, or 6-hydroxy-2,5,7,8-tetramethylchromane-2-carboxylic acid.
In some embodiments of the invention, the solvent of the DNA protectant is water.
In some embodiments of the present invention, the first and second electrodes are, the concentration of the DNA protective agent is 0.03-0.3 mol/L.
In some embodiments of the invention, the DNA, the methylation conversion reagent and the DNA protective agent are mixed and then reacted at 90-99 ℃ for 6-10 min, and then at 75-85 ℃ for 0.8-1.2 h; and finally, standing for 5-15 min at the temperature of 0-4 ℃ to obtain the methylation conversion DNA.
The methylation conversion reagent comprises sodium hydroxide, and the methylation conversion reaction is carried out under the heating condition after the DNA, the methylation conversion reagent and the DNA protective agent are mixed. The alkaline environment and the heating environment provided by the sodium hydroxide can break hydrogen bonds between double chains in DNA molecules, stably and quickly open the double chains to form single chains, so that DNA methylation conversion reaction is facilitated, the methylation conversion time is shortened, the degradation of DNA is reduced, and the methylation conversion efficiency is improved.
In some embodiments of the invention, after the DNA and the methylation conversion reagent are reacted, adding a desulfurizing agent to obtain the methylated and converted DNA.
The purpose of adding the desulfurizing agent is to remove unreacted methylation conversion reagent.
In some embodiments of the invention, the desulfurizing agent comprises a caustic solution, a sodium chloride solution, or an alcohol solution.
In some embodiments of the invention, the base solution comprises one of sodium hydroxide or Tris base.
In some preferred embodiments of the present invention, the desulfurizing agent is selected from sodium hydroxide.
In some embodiments of the invention, the concentration of the desulfurizing agent is 3 to 10mol/L.
In some embodiments of the invention, the solvent of the desulfurizing agent is water.
In some embodiments of the invention, after obtaining the methylated DNA, the methylated DNA is further purified.
In some embodiments of the invention, the purification method comprises any one of magnetic bead method, silica gel chromatography column method, permeate purification, and precipitation purification.
In some preferred embodiments of the present invention, the method of purification is performed by magnetic bead method.
The methylated and transformed DNA obtained after purification by the magnetic bead method has high quality and can be scaled up.
In some embodiments of the invention, the method of purification comprises the steps of:
s1: mixing the methylated and converted DNA with the magnetic bead suspension, and incubating to form a complex liquid containing a magnetic bead-methylated and converted DNA complex;
s2: carrying out magnetic adsorption on the composite liquid, and collecting a first precipitate; cleaning the first precipitate, and collecting a second precipitate by magnetic adsorption;
s3: cleaning and magnetically adsorbing the second precipitate, repeating the cleaning and magnetically adsorbing for one time, and collecting a third precipitate;
s4: collecting the magnetic bead-methylation conversion DNA complex, and drying; adding eluent, incubating, magnetically adsorbing, and collecting eluent to obtain purified methylated converted DNA.
In some embodiments of the invention, the magnetic bead-methylation conversion DNA complexes are contained in each of the first, second, and third precipitates.
In some embodiments of the present invention, in step S1, a binding solution and isopropanol are further added when the methylated conversion DNA is mixed with the magnetic bead suspension.
In some embodiments of the invention, the binding solution comprises 5 to 10M guanidine hydrochloride, 0.5 to 2mM sodium iodide, 1 to 5M citric acid, 10 to 40% ethanol solution by volume, and 100 to 150mM Tris-HCl.
The binding solution is used for adsorbing the methylated and converted DNA.
In some embodiments of the present invention, the mass concentration of the magnetic beads in the magnetic bead suspension is 5% to 15%.
In some embodiments of the invention, the magnetic beads have a particle size of 40 to 200nm.
In some embodiments of the invention, the magnetic beads include any one of hydroxyl magnetic beads, amino magnetic beads, and silica gel membrane magnetic beads.
In some preferred embodiments of the present invention, the magnetic beads are selected from the group consisting of the hydroxyl magnetic beads.
In some embodiments of the invention, the incubation temperature in step S1 is 5 to 40 ℃.
In some embodiments of the invention, the cleaning agent used to clean the first precipitate in step S2 comprises: ethanol with the volume concentration of 35-45%, 1-5M guanidine hydrochloride and 10-50 mM Tris-HCl;
or comprises the following steps: 5 to 20 percent of polyethylene glycol with volume concentration, 5 to 10 percent of sodium chloride with volume concentration, 0.1 to 1M of sodium acetate, 2 to 10mM of Ethylene Diamine Tetraacetic Acid (EDTA), 2 to 10mM of tris (hydroxymethyl) aminomethane, 10 to 20mM of guanidinium isothiocyanate with volume concentration of 40 to 50 percent of absolute ethyl alcohol.
In some embodiments of the invention, the pH of the cleaning agent used to clean the first precipitate in step S2 is 7.5 to 8.5.
In some embodiments of the invention, the cleaning agent used to clean the second precipitate in step S3 comprises: 70-80% alcohol, 10-50 mM Tris-HCl.
In some embodiments of the invention, the pH of the cleaning agent used to clean the second precipitate in step S3 is 8.0.
In some embodiments of the invention, the temperature of the drying in step S4 is 50 to 55 ℃.
In some embodiments of the invention, the incubation temperature in step S4 is 50 to 70 ℃.
In some embodiments of the invention, the eluent comprises: tris-HCl 8-12 mM, EDTA 0.8-1.2 mM.
In some embodiments of the invention, the pH of the eluent is 7.5 to 8.5.
In some embodiments of the invention, the eluate is enriched in the methylated transforming DNA.
According to a second aspect of the present invention, there is provided a DNA methylation conversion kit, comprising a methylation conversion reagent, wherein the methylation conversion reagent deaminates unmethylated cytosine in DNA into uracil to obtain a methylation converted DNA; the methylation conversion reagent comprises at least one of pyrosulfite and bisulfite and sodium hydroxide.
In some embodiments of the invention, the metabisulfite comprises at least one of sodium metabisulfite, potassium metabisulfite, or magnesium metabisulfite.
In some embodiments of the invention, the bisulfite salt comprises at least one of sodium bisulfite, magnesium bisulfite, or ammonium bisulfite.
In some embodiments of the invention, the concentration of the pyrosulfite is 1 to 1.2mol/L.
In some embodiments of the invention, the concentration of bisulfite is 0.5 to 1.2mol/L.
In some embodiments of the invention, the concentration of sodium hydroxide is 0.005 to 0.2mol/L.
The sodium hydroxide can break hydrogen bonds between double chains in DNA molecules, stably and quickly open the double chains to make the double chains become single chains, thereby facilitating DNA methylation conversion reaction, shortening methylation conversion time, reducing degradation of DNA and improving methylation conversion efficiency.
In some embodiments of the invention, the solvent of the methylation conversion reagent is water.
In some embodiments of the invention, the DNA methylation conversion kit further comprises a DNA protecting agent.
In some embodiments of the invention, the DNA protectant comprises at least one of hydroquinone, trehalose solution, agarose solution, dimethyl sulfoxide, ethylene glycol dimethyl ether, or 6-hydroxy-2,5,7,8-tetramethylchromane-2-carboxylic acid.
In some embodiments of the invention, the solvent of the DNA protectant is water.
In some embodiments of the invention, the concentration of the DNA protective agent is 0.03 to 0.3mol/L.
In some embodiments of the invention, the DNA methylation conversion kit further comprises a desulfurizing agent.
In some embodiments of the invention, the desulfurizing agent comprises a lye, a sodium chloride solution or an alcohol solution.
In some embodiments of the invention, the base solution comprises one of sodium hydroxide or Tris base.
In some preferred embodiments of the present invention, the desulfurizing agent is selected from sodium hydroxide.
In some embodiments of the invention, the concentration of the desulfurizing agent is 3 to 10mol/L.
In some embodiments of the invention, the solvent of the desulfurizing agent is water.
In some embodiments of the invention, the DNA methylation conversion kit further comprises a suspension of magnetic beads, a binding solution, a wash and an eluent.
In some embodiments of the present invention, the mass concentration of the magnetic beads in the magnetic bead suspension is 5% to 15%.
In some embodiments of the invention, the magnetic beads have a particle size of 40 to 200nm.
In some embodiments of the invention, the magnetic beads include any one of hydroxyl magnetic beads, amino magnetic beads, and silica gel membrane magnetic beads.
In some preferred embodiments of the present invention, the magnetic beads are selected from the group consisting of the hydroxyl magnetic beads.
In some embodiments of the invention, the binding solution comprises 5 to 10M guanidine hydrochloride, 0.5 to 2mM sodium iodide, 1 to 5M citric acid, 10 to 40% ethanol solution by volume, and 100 to 150mM Tris-HCl.
In some embodiments of the invention, the cleaning agent comprises a first cleaning agent and a second cleaning agent.
In some embodiments of the invention, the first cleaning agent comprises 35-45% ethanol, 1-5M guanidine hydrochloride, 10-50 mM Tris-HCl;
or comprises 5-20% of polyethylene glycol by volume concentration, 5-10% of sodium chloride by volume concentration, 0.1-1M of sodium acetate, 2-10 mM of EDTA, 2-10 mM of tris (hydroxymethyl) aminomethane, 10-20 mM of guanidinium isothiocyanate by volume concentration and 10-50% of absolute ethyl alcohol by volume concentration.
In some embodiments of the invention, the first cleaning agent has a pH of 7.5 to 8.5.
In some embodiments of the invention, the second cleaning agent comprises 70-80% ethanol, 10-50 mM Tris-HCl.
In some embodiments of the invention, the pH of the second cleaning agent is 8.0.
In some embodiments of the invention, the eluent comprises Tris-HCl in an amount of 8-12 mM, EDTA in an amount of 0.8-1.2 mM.
In some embodiments of the invention, the pH of the eluent is 7.5 to 8.5.
According to the third aspect of the invention, the method for DNA methylation conversion or the application of the kit for DNA methylation conversion in the preparation of methylation markers of DNA samples to be detected is provided.
In some embodiments of the present invention, the DNA sample to be tested comprises any one of plasma, serum, buccal swab sample, nasopharyngeal swab sample, urine, sputum, feces, biopsy tissue, and blood cells.
Specifically, the methylation conversion DNA of the DNA sample to be detected is prepared according to the DNA methylation conversion method and is used as the methylation marker for detecting related diseases.
Specifically, the DNA methylation kit is used for preparing methylation conversion DNA of the DNA sample to be detected, and the methylation conversion DNA is used as the methylation marker for detecting related diseases.
Drawings
The invention is further described with reference to the following figures and examples, in which:
FIG. 1 is a comparative graph of gel electrophoresis of products obtained by gene amplification after transformation using the DNA methylation transformation kits of example 1 (sodium metabisulfite) and the comparative example in the experimental examples of the present invention; wherein, M: DNA Ladder;1: the result of using the methylated converted DNA obtained after the transformation with the kit of Z company in the comparative example as a template for amplifying the gene; 2: blank control; 3: results of using the methylated converted DNA obtained after transformation with the kit 1 of example 1 as a template for gene amplification;
FIG. 2 is a comparative graph showing gel electrophoresis of products obtained by gene amplification after conversion using the DNA methylation conversion kit of example 2 (sodium bisulfite) and the comparative example in the experimental example of the present invention; wherein, M: DNA Ladder;1: the result of using the methylated converted DNA obtained after the transformation with the kit of Z company in the comparative example as a template for amplifying the gene; 2: blank control; 3: results of using the methylated converted DNA obtained after transformation with the kit 2 of example 2 as a template for gene amplification;
FIG. 3 is a comparison graph of gel electrophoresis of products obtained by gene amplification after transformation using the DNA methylation transformation kits of example 3 (using other different components) and comparative example in experimental examples of the present invention; wherein, M: DNA Ladder;1: blank control; 2: the result of using the methylated converted DNA obtained after the transformation with the kit of Z company in the comparative example as a template for amplifying the gene; 3: results of using the methylated converted DNA obtained after transformation with the kit 3 of example 3 as a template for gene amplification;
FIG. 4 is a comparative graph showing gel electrophoresis of products obtained by gene amplification after transformation using example 4 and several other DNA methylation transformation kits according to the experimental examples of the present invention; wherein, lane M: DNA Ladder; lane 1: blank control; lane 2: negative control; lane 3: a positive control; lane 4: using the methylated converted DNA obtained after the reagent in A is converted as a template to amplify the gene; lane 5: using the methylated converted DNA obtained after the reagent in the B is converted as a template to amplify the gene; lane 6: using the methylated converted DNA obtained after the reagent in the step C is converted as a template to amplify the gene; lane 7: using the methylation transformation DNA obtained after the reagent in the step D is transformed as a template amplification gene result; lane 8: the result of using the methylated converted DNA obtained after the transformation with the reagent in E as a template for amplifying the gene.
Detailed Description
The following detailed description of the embodiments of the invention is intended to be illustrative, and is not to be construed as limiting the invention.
In the description of the present invention, if there are first and second described only for the purpose of distinguishing technical features, it is not understood that relative importance is indicated or implied or that the number of indicated technical features or the precedence of the indicated technical features is implicitly indicated or implied.
In the description of the present invention, unless otherwise specifically limited, terms such as reaction and conversion are to be understood in a broad sense, and those skilled in the art can reasonably determine the specific meanings of the above terms in the present invention in combination with the specific contents of the technical solutions.
Reference throughout this specification to "one embodiment," "some embodiments," or similar language means that a particular feature, material, or characteristic described in connection with the embodiment is included in at least one embodiment of the present invention. In this specification, the schematic representations of the terms used above do not necessarily refer to the same embodiment. Furthermore, the particular features, materials, or characteristics described may be combined in any suitable manner in any one or more embodiments.
The test methods used in the examples are all conventional methods unless otherwise specified; the materials, reagents and the like used are commercially available reagents and materials unless otherwise specified.
The methylation detection of circulating tumor DNA comprises the first step of extracting free DNA from human plasma, the second step of methylating the DNA, and the third step of identifying methylated sites. The following examples utilize methylation transformation to methylate DNA in patient tissues and plasma, followed by purification using magnetic bead method.
The principle of the magnetic bead method is to provide a nanometer magnetic bead with a functional group marked on the surface, wherein the functional group can be adsorbed with nucleic acid, impurities such as protein and the like can not be adsorbed with the magnetic bead, and the magnetic bead can be gathered and dispersed under a magnetic field, so that the purpose of separating nucleic acid from impurities is realized. The efficiency of purification is related to the nature of the magnetic beads.
Example 1 DNA methylation conversion was carried out using the DNA methylation conversion kit 1 of this example (including a methylation conversion reagent containing sodium metabisulfite as a main component).
The specific process is as follows:
the DNA methylation conversion kit 1 comprises: methylation conversion reagents (sodium metabisulfite and sodium hydroxide), DNA protective agents (hydroquinone and ethylene glycol dimethyl ether), a desulfurizing agent (8M sodium hydroxide), a magnetic bead suspension (including silica-based hydroxyl magnetic beads), a binding solution (8M guanidine hydrochloride, 3M citric acid and 130mM Tris-HCl (pH = 8.0)), a first cleaning agent (40% ethanol by volume, 5M guanidine hydrochloride, 10mM Tris-HCl (pH = 8.0)), a second cleaning agent (75% ethanol by volume, 30mM Tris-HCl (pH = 8.0)), and an eluent (10 mM Tris-HCl (pH = 8.0), 1mM EDTA, and a final pH of 7.5-8.5).
S1: methylation conversion:
(1) Adding 18 mu L of DNA sample of the tissue to be treated of the patient into a 1.5mL centrifuge tube;
(2) Dissolving 1g of sodium metabisulfite in 5mL of deionized water to prepare a sodium metabisulfite solution, dissolving 0.1g of sodium hydroxide in 50mL of deionized water to prepare a sodium hydroxide solution, and adding 500 mu L of the sodium hydroxide solution into the sodium metabisulfite solution to prepare a methylation conversion reagent;
(3) Adding 200 μ L of methylation transformation reagent and 15 μ L of DNA protectant with concentration of 0.1M into the centrifuge tube, supplementing the transformation volume to 246 μ L with deionized water, vortex oscillating for about 30s, mixing, and slightly centrifuging; placing the centrifugal tube in a constant temperature metal bath kettle, reacting at 99 deg.C for 6min, then at 80 deg.C for 1h, finally slightly centrifuging the centrifugal tube, placing on ice, and keeping at 4 deg.C for 10min.
S2: and (3) desulfurization: adding 80 mu L of desulfurizer into the centrifuge tube after ice bath, reversing the upside and the downside for 5 times, mixing the mixture evenly, placing the mixture after slight centrifugation in a constant temperature metal bath kettle at 55 ℃ for incubation for 5min, and then obtaining the methylated DNA.
S3: magnetic bead method purification:
(1) Nucleic acid adsorption: adding 350 mu L of binding solution (preheated by 50 ℃ in advance), 450 mu L of isopropanol with the volume concentration of 100% and 20 mu L of magnetic bead suspension which is fully and uniformly oscillated into the methylated and converted DNA obtained in the S2, uniformly mixing the mixture by vortex oscillation for about 10S, slightly centrifuging the mixture, and standing and incubating the mixture at room temperature for 10min to form composite solution comprising a magnetic bead-methylated and converted DNA complex, wherein the methylated and converted DNA in the tissue is adsorbed on the magnetic bead at the moment, so that the enrichment of the methylated and converted DNA on the magnetic bead is realized; placing the centrifugal tube on a magnetic frame, performing magnetic adsorption for 5min, discarding the supernatant, and collecting the first precipitate;
(2) Washing nucleic acid: taking the centrifugal tube off the magnetic frame, adding 500 μ L of the first cleaning agent, performing vortex oscillation for 10s, mixing, slightly centrifuging, placing the centrifugal tube on the magnetic frame, performing magnetic adsorption for 2min, discarding the supernatant, and collecting the second precipitate;
taking the centrifugal tube off the magnetic frame, adding 500 μ L of second cleaning agent, performing vortex oscillation for about 10s, mixing, slightly centrifuging, placing the centrifugal tube on the magnetic frame, performing magnetic adsorption for 2min, and removing the supernatant; repeating the steps once, and collecting a third precipitate;
the first cleaning agent and the second cleaning agent are used for cleaning the sediment, so that substances except DNA adsorbed on the magnetic beads can be removed, and because the binding force between the methylated DNA and the magnetic beads in the magnetic bead-methylated DNA complex is weak, in order to avoid damaging the structure of the magnetic bead-methylated DNA complex, a slight centrifugal operation is adopted, wherein the slight centrifugal operation is a centrifugal operation with the rotating speed of less than or equal to 2000 rpm;
(3) Nucleic acid elution: taking off the centrifugal tube from the magnetic frame, slightly centrifuging, placing the centrifugal tube on the magnetic frame, magnetically adsorbing for 1min, and removing all supernatant; taking off the centrifugal tube from the magnetic frame, and drying in a 55 deg.C constant temperature metal bath for 3min; adding 20 μ L of eluent into the centrifuge tube, performing vortex oscillation for about 30s, mixing, and incubating in a constant temperature metal bath at 55 deg.C for 5min; and (3) placing the centrifugal tube on a magnetic frame, carrying out magnetic adsorption for 3min, and transferring the eluent into a clean centrifugal tube, wherein the eluent is the purified methylation conversion DNA.
Example 2 DNA methylation conversion was performed using the DNA methylation conversion kit 2 of this example (including a methylation conversion reagent containing sodium bisulfite as a main component).
The specific process is as follows:
the DNA methylation conversion kit 2 comprises: methylation conversion reagents (sodium bisulfite and sodium hydroxide), DNA protecting agents (hydroquinone and 6-hydroxy-2,5,7,8-tetramethylchromane-2-carboxylic acid), desulfurizing agent (8M sodium hydroxide), magnetic bead suspension (including silica-based hydroxyl magnetic beads), binding solution (8M guanidine hydrochloride, 3M citric acid, and 130mM Tris-HCl (pH = 8.0)), first cleaning agent (40% ethanol by volume, 5M guanidine hydrochloride, 10mM Tris-HCl (pH = 8.0)), second cleaning agent (75% ethanol by volume, 30mM Tris-HCl (pH = 8.0)), eluent (10 mM Tris-HCl (pH = 8.0), 1mM EDTA, final pH 7.5-8.5).
S1: methylation conversion:
(1) Adding 18 mu L of DNA sample of the plasma to be treated of the patient into a 1.5mL centrifuge tube;
(2) Dissolving 0.5g of sodium bisulfite in 5mL of deionized water to prepare a sodium bisulfite solution, dissolving 0.1g of sodium hydroxide in 50mL of deionized water to prepare a sodium hydroxide solution, and adding 500 mu L of the sodium hydroxide solution into the sodium bisulfite solution to prepare a methylation conversion reagent;
(3) Adding 200 μ L of methylation transformation reagent and 15 μ L of DNA protectant with concentration of 0.1M into the centrifuge tube, supplementing the transformation volume to 246 μ L with deionized water, vortex oscillating for about 30s, mixing, and slightly centrifuging; placing the centrifugal tube in a constant temperature metal bath kettle, reacting at 99 deg.C for 6min, then at 80 deg.C for 1h, centrifuging the centrifugal tube slightly, placing on ice, and keeping at 4 deg.C for 10min.
S2: and (3) desulfurization: adding 80 mu L of desulfurizer into the centrifuge tube after ice bath, reversing the upside and the downside for 5 times, mixing the mixture evenly, placing the mixture after slight centrifugation in a constant temperature metal bath kettle at 55 ℃ for incubation for 5min, and then obtaining the methylated DNA.
S3: magnetic bead method purification:
(1) Nucleic acid adsorption: adding 350 mu L of binding solution (preheated at 50 ℃ in advance), 450 mu L of isopropanol with the volume concentration of 100% and 20 mu L of magnetic bead suspension which is fully and uniformly oscillated into the methylated and converted DNA obtained in the step S2, carrying out vortex oscillation for about 10S to uniformly mix, carrying out slight centrifugation, standing and incubating for 10min at room temperature to form composite solution comprising magnetic bead-methylated and converted DNA complexes, wherein the methylated and converted DNA in the blood plasma is adsorbed on the magnetic beads, so that the enrichment of the methylated and converted DNA on the magnetic beads is realized; placing the centrifugal tube on a magnetic frame, performing magnetic adsorption for 5min, discarding the supernatant, and collecting the first precipitate;
(2) Washing nucleic acid: taking the centrifugal tube off the magnetic frame, adding 500 μ L of the first cleaning agent, performing vortex oscillation for 10s, mixing, slightly centrifuging, placing the centrifugal tube on the magnetic frame, performing magnetic adsorption for 2min, discarding the supernatant, and collecting the second precipitate;
taking the centrifugal tube off the magnetic frame, adding 500 μ L of second cleaning agent, performing vortex oscillation for about 10s, mixing, slightly centrifuging, placing the centrifugal tube on the magnetic frame, performing magnetic adsorption for 2min, and removing the supernatant; repeating the steps once, and collecting a third precipitate;
the first cleaning agent and the second cleaning agent are used for cleaning the sediment, so that substances except DNA adsorbed on the magnetic beads can be removed, and because the binding force between the methylated DNA and the magnetic beads in the magnetic bead-methylated DNA complex is weak, in order to avoid damaging the structure of the magnetic bead-methylated DNA complex, a slight centrifugal operation is adopted, wherein the slight centrifugal operation is a centrifugal operation with the rotating speed of less than or equal to 2000 rpm;
(3) Nucleic acid elution: taking off the centrifugal tube from the magnetic frame, slightly centrifuging, placing the centrifugal tube on the magnetic frame, magnetically adsorbing for 1min, and removing all supernatant; taking off the centrifugal tube from the magnetic frame, and drying in a 55 deg.C constant temperature metal bath for 3min; adding 20 μ L of eluent into the centrifuge tube, performing vortex oscillation for about 30s, mixing, and incubating in a constant temperature metal bath at 55 deg.C for 5min; and (3) placing the centrifugal tube on a magnetic frame, carrying out magnetic adsorption for 3min, and transferring the eluent into a clean centrifugal tube, wherein the eluent is the purified methylation conversion DNA.
Example 3 DNA methylation conversion was performed using the DNA methylation conversion kit 3 of this example (including a methylation conversion reagent, a desulfurizing agent, a detergent, and magnetic beads).
The specific process is as follows:
the DNA methylation conversion kit 3 comprises: methylation conversion reagents (sodium bisulfite and sodium hydroxide), DNA protecting agents (hydroquinone and 6-hydroxy-2,5,7,8-tetramethylchromane-2-carboxylic acid), desulfurizing agent (8M sodium hydroxide), magnetic bead suspension (including silica-based hydroxyl magnetic beads), binding solution (7M guanidine hydrochloride, 3M citric acid, and 125mM Tris-HCl (pH = 8.0)), first cleaning agent (40% ethanol by volume, 5M guanidine hydrochloride, 10mM Tris-HCl (pH = 8.0)), second cleaning agent (75% ethanol by volume, 30mM Tris-HCl (pH = 8.0)), eluent (10 mM Tris-HCl (pH = 8.0), 1mM EDTA, final pH 7.5-8.5).
S1: methylation conversion:
(1) Adding 18 mu L of DNA sample of the plasma to be treated of the patient into a 1.5mL centrifuge tube;
(2) Dissolving 0.5g of sodium bisulfite in 5mL of deionized water to prepare a sodium bisulfite solution, dissolving 0.1g of sodium hydroxide in 50mL of deionized water to prepare a sodium hydroxide solution, and adding 500 mu L of the sodium hydroxide solution into the sodium bisulfite solution to prepare a methylation conversion reagent;
(3) Adding 200 μ L of methylation transformation reagent and 14 μ L of DNA protectant with concentration of 0.1M into the centrifuge tube, supplementing the transformation volume to 246 μ L with deionized water, mixing by vortex oscillation for about 30s, and slightly centrifuging; placing the centrifugal tube in a constant temperature metal bath kettle, reacting at 99 deg.C for 6min, then at 80 deg.C for 1h, centrifuging the centrifugal tube slightly, placing on ice, and keeping at 4 deg.C for 10min.
S2: and (3) desulfurization: adding 80 mu L of desulfurizer into the centrifuge tube after ice bath, reversing the upside and the downside for 5 times, mixing the mixture evenly, placing the mixture after slight centrifugation in a constant temperature metal bath kettle at 50 ℃ for incubation for 10min, and then obtaining the methylated DNA.
S3: magnetic bead method purification:
(1) Nucleic acid adsorption: adding 350 mu L of binding solution (preheated by 50 ℃ in advance), 450 mu L of isopropanol with the volume concentration of 100% and 20 mu L of magnetic bead suspension which is fully and uniformly oscillated into the methylated and converted DNA obtained in the S2, uniformly mixing the mixture by vortex oscillation for about 10S, slightly centrifuging the mixture, and standing and incubating the mixture at room temperature for 10min to form composite solution comprising a magnetic bead-methylated and converted DNA complex, wherein the methylated and converted DNA in the plasma is adsorbed on the magnetic beads, so that the enrichment of the methylated and converted DNA on the magnetic beads is realized; placing the centrifugal tube on a magnetic frame, performing magnetic adsorption for 5min, discarding the supernatant, and collecting the first precipitate;
(2) Washing nucleic acid: taking the centrifugal tube off the magnetic frame, adding 500 μ L of the first cleaning agent, performing vortex oscillation for 10s, mixing, slightly centrifuging, placing the centrifugal tube on the magnetic frame, performing magnetic adsorption for 2min, discarding the supernatant, and collecting the second precipitate;
taking the centrifugal tube off the magnetic frame, adding 500 μ L of second cleaning agent, performing vortex oscillation for about 10s, mixing, slightly centrifuging, placing the centrifugal tube on the magnetic frame, performing magnetic adsorption for 2min, and removing the supernatant; repeating the steps once, and collecting a third precipitate;
the above-mentioned slight centrifugal operation is a centrifugal operation at a rotational speed of 2000rpm or less;
(3) Nucleic acid elution: taking off the centrifugal tube from the magnetic frame, slightly centrifuging, placing the centrifugal tube on the magnetic frame, magnetically adsorbing for 1min, and removing all supernatant; taking off the centrifugal tube from the magnetic frame, and drying in a constant temperature metal bath at 50 deg.C for 3min; adding 20 μ L of eluent into the centrifuge tube, performing vortex oscillation for about 30s, mixing, and incubating in a 55 deg.C constant temperature metal bath for 5min; and (3) placing the centrifugal tube on a magnetic frame, carrying out magnetic adsorption for 2min, and transferring the eluent into a clean centrifugal tube, wherein the eluent is the purified methylation conversion DNA.
Example 4 DNA methylation conversion was performed using the DNA methylation conversion kit 4 of this example, which included methylation conversion reagents (sodium bisulfite and sodium hydroxide) and a DNA protectant (hydroquinone).
Other reagents and methods were the same as in example 3.
Comparative example this comparative example provides a DNA methylation transformation kit from Zymo (Z for short) and a corresponding DNA methylation transformation method. The kit of the comparative example is different from the kit of example 3 in the components including the methylation conversion reagent, the desulfurizing agent, the cleaning agent and the magnetic beads.
Experimental examples this experimental example compares the performance of the kits of 4 examples and comparative examples, respectively.
The results of testing the comparative examples and comparative examples are as follows:
1. the kit of comparative example 1 (including a methylation conversion reagent containing sodium metabisulfite as a main component) was tested for methylation conversion efficiency with the kit of comparative example.
After the DNA methylation transformation kit 1 in example 1 and the DNA methylation transformation kit of the comparative example were used to perform methylation transformation on DNA derived from a tissue to be treated of a patient, primers were designed using the methylation transformed DNA as a template, and a plurality of cycles of methylation specific PCR (MMSP) were performed, and the amplification results were detected by agarose gel electrophoresis, as shown in FIG. 1. In FIG. 1, lane M: DNA Ladder; lane 1: the result of using the methylated converted DNA obtained after the transformation with the kit of Z company in the comparative example as a template for amplifying the gene; lane 2: blank control; lane 3: the result of amplifying the gene using the methylated DNA obtained after transformation with the kit 1 of example 1 as a template. As can be seen, the band in lane 3 is slightly brighter than the band in lane 1, indicating that the conversion efficiency using the DNA methylation conversion kit 1 of example 1 is higher than that of Z corporation.
2. The kit of comparative example 2 (including a methylation conversion reagent containing sodium bisulfite as a main component) was tested for methylation conversion efficiency with the kit of comparative example.
After the DNA methylation transformation kit 2 in example 2 and the DNA methylation transformation kit of the comparative example were used to perform methylation transformation on the DNA derived from the plasma to be treated of the patient, the methylation transformed DNA was used as a template, corresponding primers were designed, and a plurality of cycles of methylation specific PCR (MMSP) were performed, and the amplification results were detected by agarose gel electrophoresis, as shown in FIG. 2. In FIG. 2, lane M: DNA Ladder; lane 1: results of using the methylated converted DNA obtained after the transformation using the kit of Z company in comparative example as a template amplification gene; lane 2: blank control; lane 3: the result of using the methylated DNA obtained after transformation with the kit 2 of example 2 as a template for gene amplification. As can be seen, the band in lane 3 is slightly brighter than that in lane 1, indicating that the conversion efficiency using the DNA methylation conversion kit 2 of example 2 is higher than that of Z corporation.
The results show that the methylation transformation DNA prepared by the DNA methylation transformation kit has high methylation transformation efficiency, the methylation transformation DNA obtained by purification has high quality, and the methylation transformation DNA can be used for preparing methylation markers in tissues or blood plasma to further detect related diseases and has high detection efficiency.
3. The reagent kits of example 3 and comparative example (which differ in the methylation conversion reagent, desulfurizing agent, cleaning agent and magnetic beads) were tested for methylation conversion efficiency
a. The DNA methylation transformation kit 3 of example 3 and the DNA methylation transformation kit of comparative example (including methylation transformation reagent, desulfurizing agent, cleaning agent and magnetic beads) were selected and combined, after methylation transformation was performed on 6 genes (ACTB, CALCA, GRIK2, HOXA9 and HISTAH3A, SHOX), 6 methylation transformation DNAs were amplified by a quantitative PCR probe method using the methylation transformation DNAs as a template, and CT values of the respective genes were compared, with the results shown in Table 1 (wherein water is a negative control, a positive control refers to a 100% methylation transformed DNA standard, namely Methylated bisulfate transformed DNA, Z represents a DNA methylation transformation kit from Zymo, and Box 3 represents DNA methylation transformation kit 3).
TABLE 1
Table 1 compares the CT values of the respective genes, and shows that the methylation conversion reagent (1 and 3, 2 and 4 compared) and the desulfurizing agent (1 and 2, 3 and 4 compared) of the reagent kit 3 in example 3 are superior to those of Z when purification is performed by the magnetic bead method of Z; when the magnetic bead method of example 3 was used for purification, the methylation conversion reagent (5 and 7, 6 and 8 in comparison) and the desulfurizing agent (5 and 6, 7 and 8 in comparison) of the reagent kit 3 of example 3 were superior to those of Z corporation. Overall, the methylation conversion efficiency of the kit 3 of example 3 is superior to that of the kit of comparative example 1. In addition, the methylation conversion using the kit of Z company requires 12 to 16 hours of overnight treatment, whereas the methylation conversion using the kit of example 3 requires about 2 hours, which greatly shortens the treatment time.
b. After methylation transformation of 4 genes (ACTB, GRIK2, SHOX2, HISTAH 3A) using the DNA methylation transformation kit 3 of example 3 and the DNA methylation transformation kit of comparative example, multiplex methylation-specific PCR was performed using the methylation-transformed DNA as a template, and the amplification results were examined by gel electrophoresis at an agarose concentration of 2.5%; meanwhile, a quantitative PCR probe method is used for detecting the CT value, and the conversion efficiency is detected.
The results of gel electrophoresis are shown in FIG. 3, lane M: DNA Ladder; lane 1: blank control; lane 2: the result of using the methylated converted DNA obtained after the transformation with the kit of Z company in the comparative example as a template for amplifying the gene; lane 3: the result of using the methylated DNA obtained after transformation with the kit 3 of example 3 as a template for gene amplification. As can be seen, the band in lane 3 is slightly brighter than the band in lane 2.
The results of quantitative PCR are shown in Table 2.
TABLE 2
Table 2 compares CT values obtained by quantitative PCR amplification of each gene using the methylated DNA obtained by transformation using the two kits and the corresponding methods as templates, and the results show that the DNA methylated DNA transformation kit 3 and the corresponding transformation method in example 3 have slightly higher transformation efficiency than those of company Z.
As can be seen, the DNA methylation transformation kit of the invention has slightly higher transformation efficiency than that of Z company, and the transformation time is shorter.
4. Test example 4 and kit comprising combinations of different Components (where the combination of different Components refers primarily to the different Components of the methylation transformation reagent and the DNA protective agent)
After the DNA methylation transformation kit 4 and other kits of example 4 were selected to perform methylation transformation on 6 genes (ACTB, CALCA, GRIK2, HOXA9, HISTAH3A, SHOX), the methylation transformation DNA was used as a template to amplify 6 methylation transformation DNAs by a quantitative PCR probe method, and CT values of the genes were compared, with the results shown in table 3 (wherein water is a blank control, tissue DNA near cancer is a negative control, and a DNA of a549 cell line of human non-small cell lung cancer is a positive control). Meanwhile, a plurality of methylation-specific PCR reactions were carried out using the methylated and converted DNA as a template, and the results of the detection of the amplification by agarose gel electrophoresis at a concentration of 2.5% are shown in FIG. 4 (lane M: DNA Ladder; lane 1: blank control; lane 2: negative control; lane 3: positive control; lane 4: result of using the methylated and converted DNA obtained by conversion with the reagent in A as a template amplification gene; lane 5: result of using the methylated and converted DNA obtained by conversion with the reagent in B as a template amplification gene; lane 6: result of using the methylated and converted DNA obtained by conversion with the reagent in C as a template amplification gene; lane 7: result of using the methylated and converted DNA obtained by conversion with the reagent in D as a template amplification gene; and lane 8: result of using the methylated and converted DNA obtained by conversion with the reagent in E as a template amplification gene).
Electrophoresis results show that brighter bands and more product can be obtained using the kit containing the B counterpart (methylation conversion reagents (sodium bisulfite and sodium hydroxide), and including a DNA protectant (hydroquinone)).
TABLE 3
Note: a-comparative example (methylation conversion reagent of DNA methylation conversion kit of Z company);
b-methylation conversion reagents of example 4 (DNA methylation conversion kit 4 (sodium bisulfite and sodium hydroxide), and including DNA protectant (hydroquinone));
c-includes sodium hydroxide in the methylation conversion reagent, excluding the DNA protectant (hydroquinone));
d-excluding sodium hydroxide in the methylation conversion reagent, including the DNA protectant (hydroquinone));
e-does not include sodium hydroxide in the methylation conversion reagent and does not include a DNA protectant (hydroquinone)).
Table 3 shows that the kits with different combinations of components compare CT values generated by each gene in the qPCR reaction, and the kit corresponding to B generates a lower CT value, indicating that the conversion efficiency is higher. Also in conjunction with the gel electrophoresis of FIG. 4, the kit 4 of example 4, which includes the methylation conversion reagent (with sodium hydroxide added) and the DNA protectant hydroquinone (B, corresponding to the results of 2 in Table), shows better methylation conversion for each gene than the conversion efficiencies of the kit and the Z company kit (A, corresponding to the results of 1 in Table) without the addition of the DNA protectant hydroquinone (C, corresponding to the results of 3 in Table), without the addition of sodium hydroxide (D, corresponding to the results of 4 in Table), without the addition of sodium hydroxide to the methylation conversion reagent, and without the addition of the DNA protectant hydroquinone (E, corresponding to the results of 5 in Table). Overall, the kit 4 of example 4 includes both the methylation conversion reagent (with sodium hydroxide added) and the DNA protecting agent (hydroquinone), which is the best conversion.
The embodiments of the present invention have been described in detail, but the present invention is not limited to the embodiments, and various changes can be made without departing from the gist of the present invention within the knowledge of those skilled in the art. Furthermore, the embodiments of the present invention and the features of the embodiments may be combined with each other without conflict.
Claims (10)
1. A method of DNA methylation transformation, comprising the steps of:
reacting DNA with a methylation conversion reagent, wherein the methylation conversion reagent deaminates unmethylated cytosine in the DNA into uracil to obtain methylation conversion DNA;
wherein the methylation conversion reagent comprises at least one of pyrosulfite and bisulfite and sodium hydroxide.
2. The method of claim 1, wherein in the methylation conversion reagent, the metabisulfite comprises at least one of sodium metabisulfite, potassium metabisulfite, or magnesium metabisulfite;
preferably, the bisulfite salt includes at least one of sodium bisulfite, magnesium bisulfite, or ammonium bisulfite.
3. The method according to claim 1, wherein the concentration of the pyrosulfite in the methylation conversion reagent is 1 to 1.2mol/L, the concentration of the bisulfite is 0.5 to 1.2mol/L, and the concentration of the sodium hydroxide is 0.005 to 0.2mol/L.
4. The method according to claim 1, wherein a DNA protecting agent is further added when the DNA reacts with the methylation conversion reagent; preferably, the DNA protectant includes at least one of hydroquinone, trehalose solution, agarose solution, dimethyl sulfoxide, ethylene glycol dimethyl ether, or 6-hydroxy-2,5,7,8-tetramethylchromane-2-carboxylic acid.
5. The method of claim 4, wherein a desulfurizing agent is added after the reaction of the DNA with the methylation conversion reagent to obtain the methylation conversion DNA; preferably, the desulfurizing agent comprises alkali liquor, sodium chloride solution or alcohol solution; more preferably, the base solution comprises one of sodium hydroxide or Tris base.
6. The method of claim 1, wherein said methylated conversion DNA is obtained and purified; preferably, the purification method comprises any one of magnetic bead method, silica gel chromatography column method, penetrating fluid purification and precipitation purification; more preferably, the method of purification is selected from the magnetic bead method.
7. A DNA methylation conversion kit is characterized by comprising a methylation conversion reagent, wherein the methylation conversion reagent deaminates unmethylated cytosine in DNA into uracil to obtain methylation conversion DNA; the methylation conversion reagent comprises at least one of pyrosulfite and bisulfite and sodium hydroxide.
8. The kit of claim 7, wherein the kit further comprises a DNA protectant; preferably, the kit further comprises a desulfurizing agent.
9. The kit of claim 8, further comprising a suspension of magnetic beads, a binding solution, a wash agent, and an elution solution.
10. Use of the method for the methylation transformation of DNA according to any one of claims 1 to 6 or the kit for the methylation transformation of DNA according to any one of claims 7 to 9 for the preparation of a methylation marker of a DNA sample to be tested.
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