Gene methylation analysis methods, products and uses

Abstract

The present invention relates to gene methylation analysis methods, products, and uses. A DNA methylation analysis method is provided, comprising: 1) simultaneously contacting a biological sample containing DNA with a carrier capable of adsorbing nucleic acids and a lysis solution to enrich the DNA on the carrier; 2) directly contacting the DNA-adsorbed carrier obtained in step 1) with a conversion reagent to convert at least one unmethylated cytosine base in the DNA enriched on the carrier to uracil or another base different from cytosine that is detectable by hybridization; and 3) treating the mixed solution obtained in step 2) with a binding solution to re-enrich the converted DNA on the carrier. The present invention also provides corresponding gene methylation detection methods, kits, and uses.

Description

Technical Field

[0001] The present invention belongs to the field of gene analysis. Specifically, the present invention relates to gene methylation detection methods, products and uses. Background Art

[0002] DNA methylation is one of the most important epigenetic modifications in gene transcription regulation. This modification plays an important role in biological processes related to development and disease. As a molecular marker for early cancer diagnosis and monitoring disease progression, various methods have been designed in recent years to detect and distinguish methylated sequences in normal and cancer samples. These methods are generally based on nucleic acid extraction, bisulfite conversion, and recovery of the resulting bisDNA (bisulfite-converted DNA).

[0003] Currently, there are a variety of commercial products on the market, including Qiagen's extraction kits and ZYMO's conversion and purification kits. The current extraction kits mainly include the following steps: sample pretreatment; surfactants, proteinase K, etc. to lyse cells and digest proteins to release DNA from nuclear proteins; DNA enrichment using centrifugal columns or magnetic beads; multiple washes to remove impurities such as proteins and polysaccharides; and finally, eluting the DNA from the solid phase carrier. The conversion and purification kits mainly include the following steps: bisulfite conversion of the DNA obtained by the above extraction; desulfonation using a strong base (such as sodium hydroxide); multiple washes; and finally, eluting the DNA from the solid phase carrier for detection. The entire process requires multiple washes and replacement of reaction tubes, which is cumbersome, and results in high DNA loss and a high probability of contamination.

[0004] While the two steps described above in commercial kits represent significant improvements over traditional methods, they still require nearly eight hours to obtain a template for testing. Furthermore, for samples with relatively scarce tumor-specific circulating DNA, the complex handling process can significantly increase nucleic acid loss, posing a challenge to existing DNA extraction and conversion processing technologies. Summary of the Invention

[0005] Through extensive research, the inventors have developed a single-tube method and kit for simultaneous sample lysis and DNA enrichment on a carrier capable of nucleic acid adsorption, followed by direct DNA conversion, re-enrichment, and recovery for detection on the carrier. This method and kit allow the entire process, from biological sample collection to sample preparation for testing, to be completed within three hours. This streamlines the process, minimizing tedious steps such as pretreatment, rinsing, elution, conversion, and re-elution, and reduces DNA degradation and loss during the process, achieving superior detection results compared to traditional methylation detection methods. Furthermore, the reagents of this invention offer increased stability and facilitate automation.

[0006] In some embodiments, the present invention provides a method for analyzing DNA methylation, comprising: 1) simultaneously contacting a biological sample containing DNA with a carrier having nucleic acid adsorption capacity and a lysis solution to enrich the DNA on the carrier; 2) directly contacting the DNA-adsorbed carrier obtained in step 1) with a conversion reagent to convert at least one unmethylated cytosine base in the DNA enriched on the carrier into uracil or other bases different from cytosine that can be detected in hybridization; and 3) treating the mixed solution obtained in step 2) with a binding solution to re-enrich the converted DNA on the carrier.

[0007] In some embodiments, step 2) of the present invention is performed immediately after step 1), i.e., no other treatment, such as washing, may be performed between step 1) and step 2). In some embodiments, step 1) of the method further comprises adding a nucleic acid binding enhancer, so that the biological sample containing methylated DNA is simultaneously contacted with a carrier having nucleic acid adsorption capacity, the nucleic acid binding enhancer, and a lysis buffer, thereby enriching the nucleic acid containing methylated DNA on the carrier.

[0008] In some embodiments, the nucleic acid binding promoter in the method of the present invention includes any agent that promotes the binding of nucleic acids in the lysate to a carrier with nucleic acid adsorption capacity, for example, it can be a suitable organic solvent and / or wetting agent. In some embodiments, the nucleic acid binding promoter includes, for example, one or more of isopropanol, isobutanol, n-butanol, acetone, pyridine, acetonitrile, methyl formate, ethyl acetate, propylene glycol, glycerol, dimethyl sulfoxide, polyethylene glycol, alkyl sulfates, sulfonates or esters, polyol surfactants (e.g., Spans, Tweens), and polyoxyethylene surfactants.

[0009] In some embodiments, the lysate that can be used in the method of the present invention is not particularly limited, and appropriate lysates that have been used in the art can be used. In some embodiments, the lysate comprises a guanidine salt, such as guanidine hydrochloride, guanidine isothiocyanate. In some embodiments, the lysate may also comprise one or more detergents such as SDS, metal ion chelators such as EDTA, metal salts such as NaCl, etc. In some embodiments, the lysate can be formulated into a buffer solution, such as tris buffer, citric acid buffer. In some embodiments, the lysis step of the present invention may also include a suitable reagent that promotes the release and separation of nucleic acids from the sample, for example, 1) of the method of the present invention may further include contacting the biological sample containing methylated DNA with a protease such as proteinase K.

[0010] In some embodiments, the reagent for converting DNA is not particularly limited, and any suitable reagent known in the art that converts at least one unmethylated cytosine base in DNA into uracil or other bases detectable by hybridization that are different from cytosine can be used. In some embodiments, the conversion reagent can include bisulfite, metabisulfite, or a combination thereof, such as one or more of sodium bisulfite, magnesium bisulfite, ammonium bisulfite, potassium metabisulfite, sodium sulfite, and sodium metabisulfite. In some embodiments, the conversion reagent can include a mixed solution of sodium bisulfite and sodium sulfite.

[0011] In some embodiments, any reagent that promotes DNA conversion and / or DNA protective agent can be added during the conversion process of the method of the present invention, such as a free radical trap with DNA protective effect, including but not limited to: an organic solution of one or more of hydroquinone, 6-hydroxy-2,5,7,8-tetramethylchroman-2-carboxylic acid, and 6-hydroxy-2,5,7,8-tetramethylchroman-2-carboxylic acid, such as a diethylene glycol dimethyl ether solution, a chloroform solution, or a tetrahydrofuran solution.

[0012] In some embodiments, any suitable carrier capable of adsorbing nucleic acids may be used in the methods of the present invention, including but not limited to magnetic beads, non-magnetic microspheres, adsorption membranes, and the like. In some embodiments, carriers capable of adsorbing nucleic acids include but are not limited to hydroxyl magnetic beads, carboxyl magnetic beads, streptavidin immunomagnetic beads, ion exchange resins, and the like. In some embodiments, the shell of the magnetic beads is preferably silica or agarose. In some embodiments, the magnetic beads are preferably superparamagnetic beads. In some embodiments, the particle size of the magnetic beads may range from 100 nm to 3 μm, preferably 500-1 μm.

[0013] In some embodiments, the conversion process of step 2) of the method of the present invention further includes a post-conversion treatment step to prepare a sample suitable for direct subsequent methylation detection, wherein the post-conversion treatment step includes a step of using a cleaning solution to clean the nucleic acids enriched on the magnetic beads after conversion, optionally the cleaning solution includes a tris(hydroxymethyl)aminomethane solution, a tris(hydroxymethyl)aminomethane-hydrochloric acid solution, or an ethanol solution, optionally the pH of the cleaning solution is in the range of 6 to 10, preferably a pH of 8 to 10, and preferably the post-conversion treatment does not include desulfonation. In some embodiments, the inventors have found that the conversion sample prepared by the method of the present invention does not require desulfonation, thereby greatly saving experimental time. For example, the sample after cleaning in the method of the present invention can be directly subjected to subsequent methylation detection, such as PCR detection, sequencing detection, etc. In some embodiments, when the conversion sample prepared by the method of the present invention is not subjected to desulfonation, a period of high temperature treatment can be added during the pre-denaturation stage of the PCR detection process, such as 10-20 minutes (e.g., 15 minutes) at 90-98°C (e.g., 95°C) for high temperature treatment for desulfonation.

[0014] In some embodiments, the source of the biological sample in the method of the present invention is not particularly limited. For example, the biological sample is selected from any sample containing DNA, including but not limited to: a cell-free sample or a cell-derived sample; optionally including but not limited to: blood, urine, feces, cell culture fluid, tissue, sputum, pleural effusion, ascites, cerebrospinal fluid and processed products of these samples. Optionally, the biological sample can be from a healthy subject and / or a patient, such as a cancer patient.

[0015] In some embodiments, the DNA methylation analysis method of the present invention includes a method for analyzing the presence and / or pattern of DNA methylation in a genome. In some embodiments, the DNA methylation analysis method of the present invention includes analyzing naturally occurring and / or abnormal DNA methylation in a genome (e.g., whether DNA methylation exists and / or whether the DNA methylation state changes), which can be used to study the effects of DNA methylation on gene expression, etc. In some embodiments, the DNA methylation analysis method of the present invention includes a method for preparing a sample suitable for DNA methylation detection, for example, including the steps of extracting, enriching, transforming, and re-enriching and detecting methylated DNA. In some embodiments, the DNA methylation analysis method of the present invention includes a method for detecting the DNA methylation state, which includes the step of performing DNA methylation detection on the prepared sample. In some embodiments, DNA methylation detection can be performed using any appropriate method, such as PCR analysis, probe analysis, sequencing analysis, etc. In some embodiments, the converted sample prepared by the method of the present invention is directly subjected to subsequent analysis without the need for additional processing steps. For example, the converted sample bound to the magnetic beads can be directly subjected to subsequent PCR analysis without elution.

[0016] In some embodiments, the binding solution used in the method of the present invention may include a chaotropic salt, such as a guanidine salt, for example, guanidine hydrochloride, guanidine isothiocyanate, and optionally a buffer solution, such as a citric acid buffer, may be added to the binding solution.

[0017] In some embodiments, the present invention provides a DNA methylation analysis kit suitable for the method of the present invention, the instructions for use of which include the method described in the present invention, and which includes one or more containers, wherein the containers include reagents used in the method described in the present invention. In some embodiments, the kit includes a carrier and a lysis solution with nucleic acid adsorption capacity, and optionally a nucleic acid binding promoter. In some embodiments, the kit further includes a conversion reagent. In some embodiments, the kit includes an appropriate buffer. In some embodiments, the kit further includes reagents for detecting methylated DNA treated after conversion, such as PCR reagents, probes, and sequencing reagents.

[0018] The methods and kits provided by the present invention can be used for various analyses of DNA methylation. In some embodiments, the methods and kits provided by the present invention can be used to study various biological functions of DNA methylation, such as regulating gene expression.

[0019] The present invention provides a simple and efficient DNA processing method and a corresponding reagent kit, which can complete DNA enrichment, transformation and re-enrichment on the same set of magnetic beads in one go.

[0020] In some embodiments, the present invention provides a DNA processing method in a DNA methylation analysis method, which includes the steps of releasing DNA from a biological sample with a lysis solution while simultaneously enriching nucleic acids using a carrier with nucleic acid adsorption function, such as magnetic beads; directly transforming the DNA on the carrier, and then enriching the DNA onto the same set of magnetic beads.

[0021] In some embodiments, in the method of the present invention, the lysis solution includes but is not limited to salt solutions such as guanidine hydrochloride and guanidine isothiocyanate, and includes but is not limited to organic solvents such as isopropanol, with or without proteinase K reagent.

[0022] In some embodiments, in the methods of the present invention, the surface of the magnetic beads may be modified with hydroxyl groups or carboxyl groups.

[0023] In some embodiments, in the methods of the present invention, the conversion reagent is a mixture of bisulfite, metabisulfite, and / or a DNA protectant. In some embodiments, the conversion reagent is preferably a mixed solution of sodium bisulfite and sodium metabisulfite, or a DNA protectant is further added. In some embodiments, the DNA protectant is a free radical trap with DNA protective effects, including but not limited to: an organic solution of one or more of hydroquinone, 6-hydroxy-2,5,7,8-tetramethylchroman-2-carboxylic acid, and 6-hydroxy-2,5,7,8-tetramethylchroman-2-carboxylic acid, such as a diethylene glycol dimethyl ether solution, a chloroform solution, or a tetrahydrofuran solution.

[0024] In some embodiments, the magnetic bead shell is preferably silica, agarose, etc. In some embodiments, when the bisulfite is ammonium bisulfite, the magnetic bead shell is preferably agarose.

[0025] In some embodiments, the method of the present invention further comprises a process of simultaneously performing sample lysis and nucleic acid adsorption on magnetic beads.

[0026] In some embodiments, the method of the present invention further comprises a process in which the enriched DNA is directly subjected to bisulfite conversion by magnetic beads.

[0027] In some embodiments, the method of the present invention further comprises a process in which the converted DNA can be subjected to PCR detection without the desulfonation step.

[0028] In some embodiments, the present invention also provides a kit for DNA methylation analysis, which includes a lysis solution, magnetic beads, a conversion reagent, a binding solution, a washing solution, a buffer solution, and the like.

[0029] In some embodiments, in the kit of the present invention, the lysis buffer is a mixture of guanidine salt and / or tris(hydroxymethylaminomethane) and / or ethylenediaminetetraacetic acid and / or sodium hydroxide and / or Tween and / or isopropanol and / or proteinase K.

[0030] In some embodiments, in the kit of the present invention, the surface of the carrier such as the magnetic beads can be modified with silanol groups. For example, the shell of the magnetic beads can be selected from silica, agarose, etc. The particle size of the magnetic beads is preferably selected but not limited to 100 nm to 2 μm.

[0031] In some embodiments, in the kits of the present invention, the conversion reagent is a mixture of bisulfite, metabisulfite, and / or a DNA protectant. In some embodiments, the conversion reagent is preferably a mixed solution of sodium bisulfite and sodium metabisulfite, or a DNA protectant is further added. In some embodiments, the DNA protectant is a DNA-protective free radical trap, including but not limited to an organic solution of one or more of hydroquinone, 6-hydroxy-2,5,7,8-tetramethylchroman-2-carboxylic acid, and 6-hydroxy-2,5,7,8-tetramethylchroman-2-carboxylic acid, such as a solution in diethylene glycol dimethyl ether, chloroform, or tetrahydrofuran. In some embodiments, the conversion temperature is 80-100°C, and the conversion time is 40-60 minutes.

[0032] In some embodiments, in the kit of the present invention, the binding solution includes but is not limited to chaotropic salts such as guanidine hydrochloride, guanidine isothiocyanate and other salt solutions.

[0033] In some embodiments, in the kit of the present invention, the pH of the cleaning solution may be in the range of 6-10, but preferably 8-10.

[0034] In some embodiments, in the kit of the present invention, the buffer includes but is not limited to purified water, TE buffer, and sodium hydroxide weak base solution.

[0035] In some embodiments, the present invention provides a method for detecting DNA methylation, comprising the following steps:

[0036] Concentration: Biological samples are lysed and nucleic acids are released under the action of lysis buffer. Magnetic beads come into contact with nucleic acids in the high-concentration and high-chaotropic salt environment of the lysis buffer to form magnetic bead-nucleic acid complexes. Sample lysis and magnetic bead adsorption of nucleic acids proceed simultaneously.

[0037] Transformation: treating the magnetic bead-nucleic acid complex obtained by concentration in step (1) with a transformation reagent to obtain transformed DNA;

[0038] Reconcentration: Under the action of the binding solution, the magnetic beads and the converted DNA are re-contacted to form a magnetic bead-nucleic acid complex. After washing with the cleaning solution and eluting with the buffer solution, the converted DNA is obtained;

[0039] Detection: Measure the transformed DNA obtained in step (3).

[0040] In some embodiments, the DNA methylation information obtained according to the above detection method can be directly used in laboratory and clinical research.

[0041] In the method of the present invention, in step (1), sample lysis and nucleic acid adsorption by magnetic beads are performed simultaneously;

[0042] In the method of the present invention, the transformed DNA in step (2) can be detected without desulfonation;

[0043] In the method of the present invention, the PCR detection in step (4) only needs to add 15 minutes of treatment to the pre-denaturation stage to omit the reagent desulfonation process.

[0044] In the method of the present invention, the method in step (4) can be a dye method or a probe method, and can be used for PCR qualitative detection or PCR quantitative detection, and the obtained PCR products can also be sequenced and analyzed.

[0045] In some embodiments, the biological sample can be selected from any sample containing DNA, including but not limited to: samples of cell-free origin or samples of cell origin; optionally including but not limited to: blood, urine, feces, cell culture fluid, tissue, sputum, pleural effusion, ascites, cerebrospinal fluid and processed products of these samples, optionally the biological sample can be from a healthy subject and / or a patient, such as a cancer patient.

[0046] The entire technical process of the method provided by the present invention realizes a single-tube reaction, is easy to operate, has a high nucleic acid recovery rate, and reduces the probability of contamination and error. The kit prepared based on the above method provided by the present invention makes the implementation of the above method even simpler and faster.

[0047] The present invention has been found that in the lysis step, magnetic beads and lysate can be added simultaneously to achieve synchronous lysis and magnetic bead binding, which will not affect the effect of lysis. In addition, a nucleic acid binding promoter can be added to enhance the hydrophobic interaction between the nucleic acid in the lysate and the surface of the magnetic beads. By processing in this way, the present invention can greatly simplify the step of nucleic acid extraction. It has also been found that by contacting a biological sample containing DNA with a carrier and a lysate having nucleic acid adsorption capacity at the same time, after DNA is enriched on the carrier, it is possible not to perform any other treatment (such as washing treatment), directly using the carrier adsorbed with DNA obtained by the above-mentioned treatment to contact with a conversion reagent, at least one unmethylated cytosine base of the DNA enriched on the carrier is converted into uracil or other bases different from cytosine that can be detected on hybridization. After the conversion step, the mixed solution obtained in the previous step can be processed with a binding solution again so that the converted DNA is enriched on the carrier again. When performing PCR detection, the converted DNA can be eluted from the carrier using an eluent, or the carrier rich in DNA can be directly resuspended using PCR buffer and directly amplified by PCR. As a result, DNA enrichment, conversion, recovery, and detection can all be completed in a single reagent tube containing magnetic beads, eliminating cumbersome elution and / or rinsing processes (such as elution and / or rinsing steps after lysis and / or conversion, and desulfurization steps after conversion, etc.), reducing DNA loss and improving nucleic acid recovery efficiency.

[0048] The methods and products of the present invention are simple to operate. For example, biological samples can be heated and incubated with or without proteinase K; extracted DNA can be directly methylated without separation from magnetic beads; and multiple rinses and sodium hydroxide desulfonation steps are eliminated. The entire process, from sample preparation to template preparation for the instrument, can be completed in a single reagent tube.

[0049] The method and product of the present invention save time. For example, the method and product of the present invention can complete the entire process of obtaining a sample for machine testing from a biological sample in just 3 hours. BRIEF DESCRIPTION OF THE DRAWINGS

[0050] Figure 1 The method of the present invention is used to distinguish between methylation and unmethylation of the Septin 9 gene: CpG in the Septin 9 gene is methylated in Hela cells and unmethylated in Jurkat cells.

[0051] Figure 2 It shows the effect of simultaneous cleavage and binding on nucleic acid concentration;

[0052] Figure 3 This shows that the DNA of the present invention can be directly transformed on magnetic beads.

[0053] Figure 4 The DNA sequence before transformation is shown.

[0054] Figure 5 The sequence of DNA after conversion at 80°C for 40 min is shown.

[0055] Figure 6 The sequence of DNA after conversion at 100°C for 60 minutes is shown.

[0056] Figure 7 Shown are the detection limits for conversion after DNA elution from the beads.

[0057] Figure 8 Shown are the detection limits for direct conversion of DNA onto magnetic beads.

[0058] Figure 9 It is shown that the present invention is suitable for the detection of urine DNA methylation.

[0059] Figure 10 It is shown that the present invention is suitable for the detection of fecal DNA methylation. DETAILED DESCRIPTION

[0060] The following examples describe in detail the method for concentrating and detecting methylated DNA provided by the present invention.

[0061] Example 1:

[0062] Detection of septin9 gene methylation

[0063] Sample pretreatment: Equal amounts of fragmented Hela and Jurkat cell DNA were added to 2 ml of healthy human plasma samples;

[0064] Concentration: Add 4 ml of lysis buffer (the lysis buffer consists of 5.22 M guanidine isothiocyanate, 0.42 M TE buffer and 17% Triton) and 40 μl of magnetic beads (Beaver, hydroxy magnetic beads), shake at room temperature for 10 minutes, adsorb the magnetic beads on a magnetic stand and discard the supernatant.

[0065] Transformation: Add 320 μL of transformation reagent (the transformation reagent is a mixture of 5.34 M ammonium bisulfite, 0.53 M sodium sulfite, and 0.05 M protective agent (the protective agent component is a solution of 6-hydroxy-2,5,7,8-tetramethylchroman-2-carboxylic acid in tetrahydrofuran)) to resuspend the magnetic beads and incubate the mixture at 85°C for 40 minutes.

[0066] Reconcentration: Add 1 ml of binding buffer (7 M guanidine hydrochloride) to the mixture and shake at room temperature for 10 minutes. Rinse the magnetic beads twice with washing buffer and then elute with 60 μl of buffer.

[0067] Detection: Design specific PCR primers and probes. Reference: Warren, Jorja D, et al., Septin9methylated DNA is a sensitive and specific blood test for colorectal cancer, BMC MEDICINE, 2011, 9:133.

[0068] The detection region of Septin9 gene is abnormally methylated in Hela cells and normal in Jurkat cells. In the above treated samples, the methylation status difference of the target region of the gene is distinguished, such as Figure 1 The results show that the method provided by the present invention can realize the extraction and simultaneous enrichment of DNA onto carrier magnetic beads, followed by direct conversion on the magnetic beads and further enrichment for detection. Figure 1 The results of gene methylation detection using the method of the present invention are shown.

[0069] Example 2:

[0070] In this example, the effects of adding the lysate and magnetic beads simultaneously and adding the lysate and magnetic beads sequentially were compared. The remaining steps of the method of adding the magnetic beads sequentially were the same as those in Example 1, and nucleic acid was concentrated on the same batch of samples. The obtained DNA was subjected to methylation detection using real-time fluorescence PCR, and the detection results are shown in Table 1. Figure 2 The results show that magnetic beads can be added to the sample simultaneously with the addition of lysis buffer, which will not affect the lysis and binding effects, allowing the binding and lysis steps to be completed simultaneously, thereby greatly simplifying the nucleic acid extraction steps. Figure 2 It shows that the simultaneous cleavage and binding have an impact on nucleic acid concentration.

[0071] Example 3:

[0072] DNA is enriched by magnetic beads and then directly transformed

[0073] Add the fragmented HeLa cell DNA to 2 mL of healthy human plasma sample. One portion was treated exactly as in Example 1, and the other portion was treated in step (1) as follows:

[0074] Concentration: Add 4 ml of lysis buffer and 40 μl of magnetic beads, shake at room temperature for 10 minutes, adsorb the magnetic beads on a magnetic rack, discard the supernatant, and then wash and elute with 100 μl of buffer (TE buffer, pH = 8.0).

[0075] Transformation: Add 220 μl of transformation reagent (the transformation reagent is a mixture of 7.8 M ammonium bisulfite, 0.78 M sodium sulfite, and 0.07 M DNA protectant (the protectant component is a solution of 6-hydroxy-2,5,7,8-tetramethylchroman-2-carboxylic acid in diethylene glycol dimethyl ether) and incubate the mixture at 85°C for 40 minutes.

[0076] Reconcentration: Add 1 ml of binding solution (7 M guanidine hydrochloride) and 40 μl of magnetic beads to the mixture and shake at room temperature for 10 minutes.

[0077] The magnetic beads were rinsed twice with a cleaning solution and then eluted with 60 μl of a buffer solution (TE buffer, pH=8.0). Finally, the difference between the two methods was detected by PCR.

[0078] like Figure 3 As shown in the figure, the DNA can be directly converted without being separated from the magnetic beads, and the nucleic acid recovery efficiency is improved after reducing the intermediate DNA elution step, indicating that after the magnetic beads are enriched with nucleic acids, the conversion process can be directly carried out without the intermediate DNA elution step, and no new magnetic beads need to be added during purification. Figure 3 The results show that the DNA of the present invention was directly transformed on magnetic beads.

[0079] Example 4:

[0080] DNA conversion temperature and conversion time

[0081] This example uses different conversion temperatures and conversion times, and the remaining steps follow the method of Example 1 to extract and convert nucleic acids from the same batch of samples. DNA obtained using different conversion temperatures and conversion times is then tested for methylation using real-time fluorescence PCR. The test results are shown in Table 1:

[0082] Table 1: Methylated DNA detection results after treatment with different transformation conditions

[0083]

[0084]

[0085] As can be seen, using real-time fluorescence PCR to detect methylation, the present conversion reagent performs normally at temperatures between 80°C and 100°C, with comparable Cp values ​​at each temperature. Furthermore, analysis of the effects of different incubation times on conversion efficiency under the same temperature conditions reveals that constant-temperature conversion using the present conversion reagent achieves excellent results in just 40 minutes, significantly reducing conversion time. Compared to traditional variable-temperature methods, the present constant-temperature method is simpler and easier to operate, enabling the use of inexpensive, convenient constant-temperature instruments for relevant experimental studies.

[0086] In addition, we evaluated the DNA conversion efficiency under the optimized conversion temperature and conversion time.

[0087] The method for determining conversion efficiency reported in the reference (Emily Eva Holmes, Maria Jung, Sebastian Meller, et al. Performance Evaluation of Kits for Bisulfite-Conversion of DNA from Tissues, Cell Lines, FFPE Tissues, Aspiraes, Lavages, Effusions, Plasma, Serum and Urine. PLOS ONE, 2014, 9(4): e93933.) is as follows: using the above-mentioned human genomic DNA as the research object, using universal primers that do not contain "C", the DNA before and after conversion is amplified, and the amplified region contains 30 "C". The DNA before conversion is amplified and sequenced to confirm the status of "C" in the original DNA sequence. The amplified products of the transformed DNA were subjected to TA cloning. Twenty transformants were randomly selected from each TA clone and sequenced. Among the 20 transformants, a total of 600 "C"s were contained. The number of "C"s converted to "T"s was counted. If it was N, the conversion efficiency was: (N / 600)×100%.

[0088] Sequencing results before and after transformation Figure 4-6 As shown: Figure 4 The DNA sequence before transformation is shown. Figure 5 The sequence of DNA after conversion at 80°C for 40 min is shown. Figure 6 The sequence of DNA after conversion at 100°C for 60 minutes is shown.

[0089] It can be seen that the C before conversion is basically converted to T; the sequencing results are statistically analyzed and the conversion efficiency is calculated. (1) Sample state before conversion: The state of the 30 "C" in the template: all are "C". (2) Sample state after conversion (80℃ conversion for 40min): 20 transformants: 598 of the 600 "C" were converted to "T" after conversion, and the conversion efficiency = 99.7%. (3) Sample state after conversion (100℃ conversion for 60min): 20 transformants: All of the 600 "C" were converted to "T" after conversion, and the conversion efficiency = 100%; In summary, the conversion efficiency of this kit is ≥99.7%.

[0090] Example 5:

[0091] 10 ng / mL of fragmented Hela and Jurkat cell DNA was added to 1 mL of healthy human plasma sample, where the ratio of Hela cell DNA to Jurkat cell DNA was 10%, 1%, 0.5%, 0.1%, 0.05%, and 0%, respectively. The treatment was carried out according to Example 1 and Example 2, respectively. The detection limit was compared, and the results were as follows: Figure 7-8 shown. Figure 7 Shown are the detection limits for conversion after DNA elution from the beads. Figure 8 Shown are the detection limits for direct conversion of DNA onto magnetic beads.

[0092] As can be seen from the figure, the detection limit of the traditional DNA conversion process after elution from the magnetic beads is 0.5%, while the detection limit of the method provided by the present invention reaches 0.05%, which is a significant effect.

[0093] Example 6: Detection of septin 9 gene methylation in intestinal plasma samples

[0094] Plasma samples from 30 colorectal cancer patients and 30 healthy individuals were tested. Sample pretreatment was performed using a control kit (EPI kit) and the method of the present invention. Septin 9 was detected in three wells using the PCR reagents and procedures in the EPI kit. The test results are summarized in the following table:

[0095] Table 2 Comparison results of the method of the present invention and the control method

[0096]

[0097]

[0098]

[0099] Table 3: Plasma DNA methylation detection results of the method of the present invention and the control method

[0100]

[0101]

[0102] As can be seen from the above table, the present invention has a great advantage in the number of positive detection wells of colorectal cancer samples, while the detection specificity is still maintained.

[0103] Example 7: Detection of NID2 gene methylation in urine samples

[0104] Whole urine samples from 10 patients with bladder cancer and 10 patients with benign urological diseases were tested. The specific steps of the method used in this example were the same as those in Example 1, except that the corresponding detection primers and the lysate components were different (the lysate consisted of 2.75 M guanidine thiocyanate, 0.22 M TE buffer, 9% Triton X-ray dilution, and 47% isopropanol). The resulting converted DNA was directly tested for NID2 methylation using the methylate method (Weisenberger DJ, Campan M, Long TI, Kim M, Woods C, et al. (2005) Analysis of repetitive element DNA methylation by MethyLight. Nucleic Acids Res 33: 6823-6836), demonstrating good discrimination. Figure 9 The results of urine DNA methylation detection using the method of the present invention are shown.

[0105] Example 8: Detection of BMP3 gene methylation in stool samples

[0106] The methylation status of the BMP3 gene was assessed in stool samples from 10 patients with colorectal cancer (CRC), 10 patients with advanced adenomas (AA), and 10 patients with non-advanced adenomas (NAA) using the ΔΔct method. The specific steps of the method used in this example were the same as those in Example 1. The resulting converted DNA was directly subjected to methylation analysis using the Methylate method (Weisenberger DJ, Campan M, Long TI, Kim M, Woods C, et al. (2005) Analysis of repetitive element DNA methylation by MethyLight. Nucleic Acids Res 33: 6823-6836), demonstrating good discrimination. Figure 10 The results of the fecal DNA methylation test are shown.

Claims

1. A method for processing DNA samples for DNA methylation analysis, comprising: 1) simultaneously contacting a biological sample containing DNA with a carrier having nucleic acid adsorption capacity and a lysis solution to enrich the DNA sample on the carrier; 2) directly contacting the carrier with the adsorbed DNA sample obtained in step 1) with a conversion reagent to convert at least one unmethylated cytosine base in the DNA sample enriched on the carrier to uracil or other bases different from cytosine that are detectable in hybridization; and 3) treating the mixed solution obtained in step 2) with a binding solution to re-enrich the converted DNA sample on the carrier.

2. The method according to claim 1, wherein step 1) further comprises adding a nucleic acid binding promoter, wherein the nucleic acid binding promoter comprises at least one of an organic solvent and / or a wetting agent that promotes the binding of nucleic acids in the lysate to the carrier with nucleic acid adsorption capacity.

3. The method of claim 2, wherein the nucleic acid binding promoter comprises at least one of the following: isopropanol, isobutanol, n-butanol, acetone, pyridine, acetonitrile, methyl formate, ethyl acetate, propylene glycol, glycerol, dimethyl sulfoxide, polyethylene glycol, alkyl sulfates, sulfonates or esters, polyol surfactants, and polyoxyethylene surfactants.

4. The method of claim 3, wherein the polyol surfactant comprises a Span or Tween surfactant.

5. The method of any one of claims 1 to 4, wherein the lysate comprises a guanidine salt. The method of claim 5 , wherein the guanidine salt comprises guanidine hydrochloride or guanidine isothiocyanate.

7. The method according to any one of claims 1 to 4, wherein the lysate further comprises a detergent, a metal ion chelator, or a metal salt.

8. The method of claim 7, wherein the detergent is SDS, the metal ion chelator is EDTA, and the metal salt is NaCl.

9. The method according to any one of claims 1 to 4, wherein the lysate is prepared as a buffer solution.

10. The method of claim 9, wherein the buffer solution comprises Tris buffer or citric acid buffer.

11. The method according to any one of claims 1 to 4, wherein step 1) further comprises contacting the biological sample containing DNA with a protease.

12. The method of claim 11, wherein the protease is proteinase K.

13. The method of any one of claims 1-4, wherein the conversion reagent comprises bisulfite, metabisulfite, or a combination thereof.

14. The method according to any one of claims 1 to 4, wherein the conversion reagent comprises one or more of sodium bisulfite, magnesium bisulfite, ammonium bisulfite, potassium metabisulfite, sodium metabisulfite, and sodium sulfite.

15. The method according to any one of claims 1 to 4, wherein the conversion reagent comprises a mixed solution of sodium bisulfite and sodium sulfite.

16. The method according to any one of claims 1 to 4, wherein a free radical trap with DNA protection function is added during the conversion process of step 2).

17. The method according to any one of claims 1 to 4, wherein an organic solution of one or more of hydroquinone, 6-hydroxy-2,5,7,8-tetramethylchroman-2-carboxylic acid, and 6-hydroxy-2,5,7,8-tetramethylchroman-2-carboxylic acid is further added during the conversion process in step 2).

18. The method according to any one of claims 1 to 4, wherein a diethylene glycol dimethyl ether solution, a chloroform solution or a tetrahydrofuran solution is further added during the conversion process in step 2).

19. The method according to any one of claims 1 to 4, wherein the carrier capable of adsorbing nucleic acids comprises magnetic beads, non-magnetic microspheres, or adsorption membranes.

20. The method of claim 19, wherein the carrier comprises hydroxy magnetic beads, carboxyl magnetic beads, streptavidin immunomagnetic beads, or ion exchange resin. The method of claim 19 , wherein the shell of the magnetic beads is silica or agarose.

22. The method of claim 19, wherein the magnetic beads are superparamagnetic beads.

23. The method according to claim 19, wherein the magnetic beads have a particle size of 100 nm to 3 μm.

24. The method of claim 19, wherein the magnetic beads have a particle size of 500 nm to 1 μm.

25. The method according to any one of claims 1 to 4, further comprising a post-conversion processing step to prepare a sample suitable for direct subsequent methylation detection, wherein the post-conversion processing step comprises the step of washing the converted nucleic acids enriched on the magnetic beads with a washing solution.

26. The method of claim 25, wherein the cleaning solution comprises a Tris solution, a Tris-hydrochloric acid solution, or an ethanol solution.

27. The method of claim 25, wherein the pH of the cleaning solution is in the range of 6 to 10.

28. The method of claim 25, wherein the pH of the cleaning solution is 8-10.

29. The method of claim 25, wherein the post-conversion treatment does not include desulfonation.

30. The method of any one of claims 1-4, wherein the biological sample is selected from any sample containing DNA.

31. The method of any one of claims 1-4, wherein the biological sample comprises a sample of acellular origin or a sample of cellular origin.

32. The method according to any one of claims 1 to 4, wherein the biological sample comprises blood, urine, feces, cell culture fluid, tissue, sputum, pleural effusion, ascites, cerebrospinal fluid, and processed products of these samples.

33. The method of any one of claims 1-4, wherein the biological sample is from a healthy subject and / or a patient.

34. The method of any one of claims 1-4, wherein the biological sample is from a cancer patient.

35. The method of any one of claims 1-4, wherein the binding solution comprises a chaotropic salt.

36. The method of any one of claims 1-4, wherein the binding solution comprises a guanidine salt.

37. The method of claim 36, wherein the guanidine salt comprises guanidine hydrochloride or guanidine isothiocyanate.

38. The method according to any one of claims 1 to 4, wherein a buffer solution can be added to the binding solution.

39. A DNA sample processing kit for DNA methylation analysis suitable for the method of any one of claims 1 to 38, comprising one or more containers, wherein the kit comprises: 1) A container containing a carrier with nucleic acid adsorption capacity and a lysis solution, which is used to simultaneously contact a biological sample containing DNA to enrich the DNA sample on the carrier. 2) a conversion reagent, used to directly contact the carrier adsorbed with the DNA sample obtained in 1) to convert at least one unmethylated cytosine base of the DNA sample enriched on the carrier into uracil or other bases different from cytosine that can be detected in hybridization, 3) Binding solution, used to treat the mixed solution obtained in 2) to re-enrich the transformed DNA sample on the carrier.

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