Method for preparing bisulfite DNA and use thereof

Abstract

Provided are a method for preparing Bisulfite DNA and a use thereof. In the method, the process is simplified and optimized, and the same reagent is used as a reagent having both lysis and transformation functions, thereby avoiding the conversion of multiple systems, and reducing the steps of washing, elution and the like of a DNA sample.

Description

Method for preparing bisulfite DNA and application thereof

[0001] The present invention claims priority to the patent application with application number CN 202411298070.X, filed on September 14, 2024; the entire content of which is incorporated herein. TECHNICAL FIELD

[0002] The present invention belongs to the field of epigenetic technology, and more specifically, the present invention relates to a method for preparing bisulfite DNA and application thereof. BACKGROUND

[0003] Epigenetics mainly includes biochemical processes such as DNA methylation, histone modification, and microRNA level changes; DNA methylation is an epigenetic mechanism that has been studied in depth in the art and has important application prospects in tumor clinical practice including diagnosis and treatment. DNA methylation refers to a process in which S-adenosyl methionine (SAM) is used as a methyl donor to transfer a methyl group to a specific base under the catalysis of DNA methyltransferase (DMT) in the body. DNA methylation can occur at the N-6 position of adenine, the N-4 position of cytosine, the N-7 position of guanine, or the C-5 position of cytosine. However, in mammals, DNA methylation mainly occurs at the C of 5'-CpG-3', generating 5-methylcytosine (5mC).

[0004] DNA methylation modification plays an important role in regulating gene expression, maintaining chromatin structure, and stabilizing cell genetics. In terms of gene expression regulation, DNA methylation can prevent the binding of transcription factors and lead to gene silencing. In addition, DNA methylation is also involved in the formation of spatial structure of chromatin and affects the rules of gene proximity.

[0005] DNA methylation plays an important role in the occurrence and development of diseases. Many studies have found that DNA methylation is abnormal in certain cancers, cardiovascular diseases, and nervous system diseases. For example, abnormal DNA methylation can lead to the inactivation of certain tumor suppressor genes, thereby promoting the occurrence of tumors. In addition, abnormal DNA methylation is also closely related to the occurrence and development of nervous system diseases such as autism, Parkinson's disease, and Alzheimer's disease.

[0006] With the development of high-throughput sequencing and proteomics technology in the field of biotechnology, the study of methylation modification has gradually expanded. Researchers not only comprehensively revealed the distribution pattern of methylation modification at the genomic and proteomic levels, but also explored the potential mechanisms of methylation modification in the occurrence of diseases.

[0007] At present, some kits for DNA methylation detection have been developed in the art to perform qualitative, quantitative or semi-quantitative methylation analysis. However, the existing detection methods still have problems such as relatively complicated process, long time consumption, relatively poor signal, insufficient sensitivity, etc.

[0008] In summary, there is an urgent need in the art to develop methylation modification analysis technology and tools to provide powerful tools and methods for methylation level detection. SUMMARY

[0009] The present application aims to provide a method for preparing Bisulfite DNA and its application.

[0010] In the first aspect of the present application, a method for preparing Bisulfite DNA is provided, which comprises treating a cell sample with a reagent containing only a sulfite (hydrogen) salt as a lysing conversion reagent to obtain a converted product, wherein the non-methylated modified cytosine in the DNA of the product is converted to uracil, which is Bisulfite DNA.

[0011] In one or more embodiments, the method comprises:

[0012] (a) providing a cell sample;

[0013] (b) treating the sample of (a) with a reagent containing a sulfite (hydrogen) salt as a lysing conversion reagent to convert the non-methylated modified cytosine in the sample to uracil to obtain a converted product;

[0014] provided that the cell sample in (a) is treated by a process selected from (1) or (2) as follows:

[0015] (1) without lysing the cell sample (without lysing with a conventional lysing solution, without lysing with a reagent other than a reagent containing a sulfite (hydrogen) salt), the cell is used in step (b); or

[0016] (2) lysing the cell to obtain a lysate, which is used in step (b) without washing or eluting the DNA sample (preferably, without isolating the DNA) so that the DNA extraction and conversion are performed in one step.

[0017] In one or more embodiments, the method converts the DNA in the cell sample to Bisulfite DNA, wherein the non-methylated modified cytosine in the Bisulfite DNA is converted to uracil (C→U).

[0018] In one or more embodiments, the cell sample comprises a cell-containing sample from a tissue.

[0019] In one or more embodiments, the (a)-(b) steps comprise or consist of the following steps: providing a cell sample, lysing and converting the sample with a (hydrogen)sulfite-containing reagent, and obtaining the converted product.

[0020] In one or more embodiments, the (a)-(b) steps comprise or consist of the following steps: providing a cell sample, lysing the cells, adsorbing the DNA onto a solid support, treating the adsorbed sample with a (hydrogen)sulfite, and obtaining the converted product.

[0021] In one or more embodiments, the solid support is selected from the group consisting of: magnetic beads, magnetic beads, polystyrene, glass.

[0022] In one or more embodiments, when the (a)-(b) steps are treated with a (hydrogen)sulfite-containing reagent, the (hydrogen)sulfite concentration is 15-75%, preferably 20-70%, more preferably 30-60%, and even more preferably 40-50%.

[0023] In one or more embodiments, when the (a)-(b) steps are treated with a (hydrogen)sulfite-containing reagent, the temperature is 60-99°C, preferably 65-98°C, more preferably 68-96°C, and even more preferably 70-94°C (e.g., 72°C, 75°C, 78°C, 80°C, 82°C, 85°C, 88°C, 90°C, 92°C).

[0024] In one or more embodiments, when the (a)-(b) steps are treated with a (hydrogen)sulfite-containing reagent, the treatment time (conversion time) is 5-40 min, preferably 8-35 min, and more preferably 10-30 min (e.g., 12, 15, 18, 20, 25, 28 min).

[0025] In one or more embodiments, the (hydrogen)sulfite comprises: ammonium bisulfite, ammonium sulfite, sodium bisulfite, sodium sulfite, or a combination thereof.

[0026] In one or more embodiments, after the (a) and (b) steps, further comprising: (c) desulfonating and purifying the converted DNA; preferably, the purifying comprises: washing and / or eluting.

[0027] In one or more embodiments, after the (a) and (b) steps and optionally the (c) step, the converted DNA can be used for amplification.

[0028] In another aspect of the present application, there is provided use of the method of any one of the preceding aspects in analyzing the DNA methylation level of a test cell sample.

[0029] In one or more embodiments, the method of analyzing the DNA methylation level of a test cell sample is not diagnostic and non-therapeutic, and is not directly aimed at obtaining a clinical disease diagnosis. For example, the method is used for scientific research and analysis on cells ex vivo, to determine the methylation profile of the cells.

[0030] In another aspect of the present application, there is provided a method of analyzing the DNA methylation level of a test cell sample, comprising: (i) providing a test cell sample, and preparing Bisulfite DNA by any of the methods described above; and (ii) analyzing the Bisulfite DNA for the presence of uracil, thereby determining the methylation level of the test sample.

[0031] In one or more embodiments, after step (i), the method further comprises a step of amplifying the Bisulfite DNA.

[0032] In another aspect of the present application, there is provided a kit for preparing Bisulfite DNA, the kit comprising: a (hydrogen)sulfite-containing reagent as the lysing-conversion reagent.

[0033] In one or more embodiments, the kit further comprises a reagent selected from the group consisting of: a DNA desulfonation reagent, a DNA washing reagent, a DNA elution reagent, and a solid support (e.g., magnetic beads).

[0034] In one or more embodiments, the kit does not comprise an additional (other than the (hydrogen)sulfite-containing reagent) cell lysing reagent.

[0035] In one or more embodiments, the kit further comprises instructions for using the (hydrogen)sulfite-containing reagent as the lysing-conversion reagent to obtain a conversion product, wherein the DNA of the conversion product has non-methylated cytosine converted to uracil; and preferably, the instructions further comprise instructions for processing the cell sample by a method selected from (1) or (2) as follows: (1) without lysing the cell sample (without lysing the cell sample using a reagent other than the (hydrogen)sulfite-containing reagent), and using the cell sample for the conversion step; or (2) lysing the cell sample, and obtaining a lysate without performing a step of washing or eluting the DNA sample (and preferably, without performing a step of isolating the DNA) for the conversion step, wherein the DNA extraction and conversion are performed in one step.

[0036] In one or more embodiments, the instructions further specify that when the sample is treated with the sulfite-containing reagent: the sulfite concentration is 15-75%, preferably 20-70%, more preferably 30-60%, even more preferably 40-50%; and / or, the temperature is 60-99°C, preferably 65-98°C, more preferably 68-96°C, even more preferably 70-94°C (e.g., 72°C, 75°C, 78°C, 80°C, 82°C, 85°C, 88°C, 90°C, 92°C); and / or, the treatment time (conversion time) is 5-40 min, preferably 8-35 min, more preferably 10-30 min (e.g., 12, 15, 18, 20, 25, 28 min).

[0037] In another aspect of the present application, there is provided use of the kit for analyzing the DNA methylation level of a test cell sample, or for preparing a detection system for analyzing the DNA methylation level of a test cell sample.

[0038] In another aspect of the present application, there is provided use of the sulfite-containing reagent for preparing a cell sample lysis conversion reagent; preferably, the sulfite concentration is 15-75%; preferably, the sulfite treatment temperature is 60-99°C.

[0039] In another aspect of the present application, there is provided a cell sample lysis method, comprising the following steps: (a) providing a cell sample; and, (b) treating the sample of (a) with a sulfite-containing reagent, at a temperature of 60-99°C (preferably 70-98°C) for a time period of 5-40 min (preferably 5-30 min).

[0040] Other aspects of the present application will be apparent to those skilled in the art from consideration of the disclosure herein. BRIEF DESCRIPTION OF DRAWINGS

[0041] Figure 1. Results of qPCR detection of Bisulfite DNA prepared by the fast conversion method (using high concentration of ammonium bisulfite solution, concentration 60%-70%) and the traditional conversion method.

[0042] Figure 2. Analysis of different conversion temperature and time conditions in the fast conversion method.

[0043] Figure 3. Amplification results of the extraction-conversion one-step method, with the extraction and traditional conversion method, and the extraction and fast conversion method as controls.

[0044] Figure 4. Amplification results of the lysis-free conversion method, compared with the extraction-conversion one-step method, with the non-conversion group as a control.

[0045] Figure 5, analysis of the transformation effect of the transformation reagent (ammonium bisulfite or in combination with other reagents) at a concentration of 40% or above.

[0046] Figure 6, analysis of the transformation effect of the transformation reagent (ammonium bisulfite) at a concentration of 50% or below.

[0047] Figure 7, analysis of the transformation effect of the transformation temperature time in the sample lysate-free transformation method.

[0048] Figure 8, analysis of the transformation effect of the lysate-free transformation method for detecting HeLa cell (methanol-fixed) samples.

[0049] Figure 9, analysis of the transformation effect of the lysate-free transformation method for detecting fresh brain tissue samples.

[0050] Figure 10, analysis of the transformation effect of the lysate-free transformation method for detecting HPV samples.

[0051] Figure 11, analysis of the transformation effect of the lysate-free transformation method for detecting TCT samples.

[0052] Figure 12, verification of the ammonium bisulfite lysis effect under different conditions. DETAILED DESCRIPTION

[0053] In view of the problems of complicated process and poor controllability in the DNA methylation detection technology, the application discloses an optimized method for preparing Bisulfite DNA, which is optimized by simplifying the process, uses the same reagent as the reagent with lysis and transformation functions, realizes lysis and transformation at a relatively high temperature at the same time, avoids the conversion of multiple systems, reduces the steps of DNA sample washing and elution, is time-saving, and has excellent transformation effect.

[0054] The bisulfite needs to be processed under specific conditions (such as temperature), and the general DNA sample acquisition, lysis, purification, etc. are usually different from the processing conditions of bisulfite, so that the technical personnel have not found that bisulfite can be used as a lysis and transformation reagent so far, and the application changes this situation and first proposes a reagent containing bisulfite as a lysis and transformation reagent.

[0055] In the present invention, "sample" or "specimen" includes a substance obtained from an individual (e.g., a human) or an isolated tissue, cell or body fluid (e.g., plasma or serum) that is subjected to methylation detection. In a preferred manner, the specimen is a cell sample or a tissue sample that is treated with a bisulfite-containing reagent at a relatively high temperature to release DNA and simultaneously convert it without lysis by a conventional lysis reagent. "Sample" or "specimen" includes cells that are ex vivo and propagated in vitro. For example, the specimen can include, but is not limited to, a tissue sample, a blood sample, a pleural effusion sample, and a lung lavage sample, ascites, lavage sample, bile sample, stool sample, urine sample, saliva sample, cerebrospinal fluid sample, cell smear sample, cervical scrape or brush sample, tissue and cell biopsy sample.

[0056] In the present invention, "bisulfite" is a reagent that deaminates the amino group of unmodified cytosine to distinguish it from modified cytosine, which facilitates distinguishing between regions of DNA that have a modified state and regions of DNA that do not have a modified state. The bisulfite can include ammonium bisulfite, ammonium sulfite, sodium bisulfite, sodium sulfite, or a combination thereof. Analogues and functional substitutes thereof are also included in the present invention.

[0057] In the present invention, "conversion" refers to the conversion of non-methylated modified cytosine to uracil.

[0058] In the present invention, "lysis-free" means that an additional, separate "lysis" step is not required, and that a conventional lysis reagent other than "bisulfite" is not required. In the present invention, "bisulfite" is used for both lysis and conversion for the first time as a lysis-conversion reagent. The conventional lysis reagent is, for example, proteinase K.

[0059] That is, "lysis-free" in the present invention does not mean that lysis of the cell does not occur, but rather that a "conventional lysis reagent" is not used or a "conventional lysis step" is not used, and that a bisulfite-containing reagent is used as a lysis-conversion reagent to replace the lysis function of the conventional lysis reagent.

[0060] In the present invention, "isolated," "purified," or "recovered" are used interchangeably and generally refer to the removal of one or more components from a mixture (e.g., a sample) to separate it from one or more other components of the mixture. Sample components include nucleic acids, and the sample components can include cell fragments, proteins, carbohydrates, lipids, and other compounds.

[0061] In the present invention, "elution" generally refers to the act of removing biological material from a substrate or solution. In some aspects, the removal can be performed using a liquid or fluid, such as a suitable buffer system.

[0062] In the present invention, "treatment" or "contacting" generally refers to bringing two or more components into at least partial contact with each other. Contacting can be achieved by mixing the components in a fluid or semi-fluid mixture. Contacting can also be achieved when one or more components come into physical contact with one or more other components on a solid surface, such as a solid tissue section or a substrate.

[0063] In the present invention, "solid support" generally refers to a substrate that is insoluble in the reaction system of the present invention, for example, it is a substrate whose surface is capable of interacting with the phosphate groups of the nucleic acid backbone. The solid support can take the form of a porous or non-porous particle, a powdered particle or a fiber. In a preferred mode, the solid support is a magnetic bead.

[0064] In the art, for a cell sample obtained from a tissue, the DNA sample is usually extracted with an extraction reagent, and then the DNA is transformed. The present inventors have unexpectedly found that some steps in the conventional procedure can be simplified, and a desirable technical effect is obtained.

[0065] Based on the new findings of the present inventors, a method for preparing Bisulfite DNA is provided, comprising: (a) providing a cell sample; (b) treating the sample of (a) with a reagent containing bisulfite, converting the non-methylated modified cytosine in the sample into uracil, to obtain a transformed product. Compared with the conventional method, the present method omits an essential step, and the present inventors have omitted one step selected from the following group:

[0066] (1) The cells are not required to be treated with an additional (other than the reagent containing bisulfite) cell lysis solution before being used in step (b); or

[0067] (2) The cells are lysed, and the lysate is used in step (b) without being subjected to a step of washing or eluting the DNA sample (preferably, the DNA is also not isolated), and the DNA extraction and transformation are performed in one step.

[0068] As a preferred embodiment, the essential steps of the technical solution of the present invention include: providing a cell sample, treating (lysing and transforming) the cells with a reagent containing bisulfite, to obtain a transformed product.

[0069] As another preferred embodiment, the essential steps of the technical solution of the present invention include: providing a cell sample, lysing the cells, adsorbing the DNA on a solid support, treating the adsorbed sample with a reagent containing bisulfite, to obtain a transformed product.

[0070] The method for preparing Bisulfite DNA of the present invention, starting from a cell sample, is compared with the conventional method as shown in Table 1.

[0071] Table 1

[0072] In some embodiments of the application, the method can further comprise a desulfonation treatment of the converted product to obtain a desulfonated product. This process can be performed using desulfonation reagents known in the art. For example, the converted product can be treated with a desulfonation buffer.

[0073] The method of the application can further comprise a step of purifying the Bisulfite DNA from the converted product obtained. In some embodiments of the application, the purification can comprise contacting the reaction mixture with a solid support prior to the conversion and / or contacting the converted product with a solid support. For example, the solid support can be selected from the group consisting of magnetic beads, polystyrene, glass.

[0074] In some embodiments of the application, the purification can further comprise washing the converted product.

[0075] In some embodiments of the application, the purification can further comprise eluting the nucleic acid from the converted product that was contacted with the solid support from the solid support.

[0076] The application also provides a method for detecting the methylation level in a sample to be tested, the method comprising treating a cell sample using the method of the application to obtain the conversion of non-methylated modified cytosine to uracil, obtaining a converted product (Bisulfite DNA); and then analyzing the Bisulfite DNA for the presence of uracil, thereby determining the methylation level of the sample to be tested.

[0077] In some embodiments of the application, after obtaining the Bisulfite DNA, the method further comprises amplifying the nucleic acid in the cytosine conversion product, thereby obtaining sufficient nucleic acid product for analyzing the methylation level.

[0078] The application also provides a kit comprising: a bisulfite reagent; and preferably further comprising a reagent selected from the group consisting of: a DNA desulfonation reagent, a DNA washing reagent, a DNA elution reagent, a solid support (such as magnetic beads); and preferably wherein the kit does not comprise an additional (other than the bisulfite reagent) cell lysis reagent.

[0079] In a preferred embodiment, the kit further comprises a manual, which describes the treatment of the cell sample selected from (1) or (2) as follows: (1) without lysing the cell sample, the cell is used for the transformation step; or (2) the cell is lysed, and the lysate is directly used for the transformation without washing or eluting the DNA sample (preferably, without isolating the DNA). Preferably, the manual further describes the preferred conditions such as the concentration, temperature, time, etc. for the treatment with the reagent containing the sulfite. Preferably, the manual further describes the preferred methods for desulfonation, purification, washing, etc. after the transformation. The form of the manual is not particularly limited, and can be in a physical form and / or in an electronic form readable by a computer.

[0080] The main advantages of the method for preparing Bisulfite DNA according to the present application include:

[0081] (1) The transformation method is simple, and the transformation time is only 10-15 min from the cell sample to the completion of the DNA transformation. The ammonium bisulfite at an appropriate concentration plays the role of cell lysis and DNA transformation, and no protease K or other protein solubilizer, protein denaturant is needed, which greatly reduces the time and cost;

[0082] (2) The extraction and transformation are performed in one step. When adsorbed by a solid carrier, the elution step after the adsorption of the solid carrier (such as magnetic beads) to the DNA is reduced;

[0083] (3) The overall extraction and transformation efficiency is higher than that of the commercial reagent DNA Methylation-Gold kit.

[0084] The present application will be further described in conjunction with specific examples. It should be understood that these examples are only used to illustrate the present application and are not used to limit the scope of the present application. The experimental methods in the following examples, if not otherwise specified, are usually carried out according to the conventional conditions described in J. Sambrook et al., Molecular Cloning: A Laboratory Manual, Science Press, or according to the conditions suggested by the manufacturer.

[0085] Example 1, Rapid Transformation of DNA Sulfite

[0086] 1, Preparation of Sample DNA

[0087] Human white blood cells were used, and the nucleic acid extraction reagent (A02) produced by Shanghai Yipu Jinding Biotechnology Co., Ltd. was used to extract the cell DNA to obtain the DNA.

[0088] 2, Sulfite Transformation

[0089] Take 200 ng of DNA, according to the volume ratio of DNA to ammonium bisulfate 1:9, add high concentration ammonium bisulfate solution (60%-70% (mass fraction, i.e. 60g / 100g), conversion reagent), bisulfite treatment, 94℃ for 15 min.

[0090] Another commercial reagent, Zymo Research kit group (EZ-96 DNA Methylation-Gold kit, item number D5006) conversion reagent, according to its instructions for conversion treatment, as a conversion control (i.e.: traditional conversion method), the overall test process is about 3h.

[0091] 3, desulfurization and purification of DNA after sulfite conversion

[0092] Use Zymo Research kit, according to the instructions to complete the subsequent DNA desulfurization and purification. The specific steps are as follows:

[0093] (1) washing: add 300uL binding buffer (Binding Buffer) to the adsorption column, add 75uL sample treated by bisulfite to the adsorption column, mix by hand for 10 times, centrifuge at 12000g for 30s, discard the liquid; add 200uL washing buffer (Washing Buffer), 12,000g, room temperature for 30s, discard the waste liquid.

[0094] (2) desulfurization: add 100uL desulfurization buffer (Desulphonation Buffer) to the adsorption column, stand at room temperature for 20min, centrifuge at 12000g for 30s.

[0095] (3) washing: add 200uL washing buffer, 12,000g, centrifuge at room temperature for 30s, repeat this step (repeat 4 times); 12000g room temperature empty for 2min; transfer to 1.5ml EP tube, room temperature dry for 2min.

[0096] (4) elution: add 44uL 65℃ preheated TE to the center of the filter membrane, stand for 3min; centrifuge at 12000g for 30s, recover bisulfite DNA.

[0097] 4, qPCR detection to verify the conversion effect

[0098] Detection of GAPDH gene, the primer and probe sequence of GAPDH gene is shown in Table 2.

[0099] Table 2

[0100] The reaction system is shown in Table 3.

[0101] Table 3

[0102] As shown in FIG. 1, the rapid conversion method (using a high concentration of ammonium bisulfite solution, concentration 60%-70%) presents a significantly superior fluorescence signal intensity, indicating that the conversion effect of high concentration of sulfite is not inferior to the existing commercial reagent, but the conversion time is greatly reduced.

[0103] Therefore, the present embodiment provides a DNA sulfite rapid conversion reagent and method, which can realize rapid (15 min) completion of DNA sulfite conversion, and has high conversion efficiency.

[0104] Example 2, analysis of conversion temperature and time conditions

[0105] The method as described in Example 1 (rapid conversion method) is operated, and the difference lies in the adjustment of the conversion temperature and time, as shown in Table 4.

[0106] Table 4

[0107] As shown in FIG. 2, 90℃-98℃, 10min-15min has good conversion effect, among which 94℃ 15min and 98℃ 15min have smaller CT values, and 98℃ 15min has the lowest CT value.

[0108] Example 3, one-step method of DNA extraction and conversion

[0109] In the present embodiment, the optimization of DNA extraction and conversion for tissue cell samples is carried out. Through further research analysis and experiments, the inventors omit the steps and implement the one-step method of extraction and conversion.

[0110] 1, sample preparation

[0111] Methylation-negative cervical exfoliated cell samples are obtained.

[0112] 2, DNA extraction and sulfite conversion

[0113] (1) one-step method of extraction and conversion

[0114] Take 800uL of the above sample, extract the cell DNA by using the nucleic acid extraction reagent (A02), which specifically includes centrifuging the sample, removing the supernatant, adding 20uL of protease K and 300uL of digestion solution A, vortexing, 65℃ water bath for 2hr, adding 300uL of lysis solution L and 300uL of isopropanol, vortexing, instantaneous separation, and adding magnetic bead suspension. Without washing and eluting the DNA, the subsequent steps are directly performed.

[0115] The magnetic beads combined with DNA were instantaneously separated, the tube was placed on a magnetic stand for 1 min, the supernatant was absorbed (without touching the magnetic beads), 180 uL of 60%-70% bisulfite solution was added, and 94°C was maintained for 15 min.

[0116] The recovery of bisulfite DNA was completed according to step 3 of Example 1.

[0117] (2) Traditional method control group (test one)

[0118] 800 uL of sample was taken, DNA was extracted using nucleic acid extraction reagent (A02), and the volume was adjusted, and DNA transformation was completed according to the instructions of the Zymo Research kit.

[0119] (3) Rapid extraction method control group (test two)

[0120] 800 uL of sample was taken, DNA was extracted using nucleic acid extraction reagent (A02), and the volume was adjusted, and DNA transformation was completed according to steps 2-4 of Example 1.

[0121] 3. qPCR detection verifies the transformation effect

[0122] PCR amplification was performed according to step 4 of Example 1, and detection was performed for the GAPDH gene.

[0123] The results are shown in Figure 3. The CT value of the one-step extraction and transformation method is lower than that of the other two groups, indicating that the one-step extraction and transformation method has better DNA transformation effect. Since the DNA washing and elution steps are omitted in the DNA extraction process, the DNA loss is reduced.

[0124] According to the above, the method not only greatly shortens the transformation time, but also the DNA adsorbed by the magnetic beads does not need to be separated and purified, and can be directly transformed, that is, the one-step extraction and transformation method, which simplifies the operation steps.

[0125] Example 4, lysis-free transformation method

[0126] In this embodiment, the optimization of DNA extraction and transformation for tissue cell samples is carried out. Through further analysis and experiment, the inventors further simplify the steps on the basis of the one-step extraction and transformation method, and carry out lysis-free transformation.

[0127] The steps include:

[0128] 1. Take 800 uL of methylation-negative cervical exfoliated cell sample, centrifuge, remove the supernatant, and add 180 uL of 60%-70% bisulfite solution to the sample, and maintain 94°C for 15 min.

[0129] 2. Purification and amplification of transformed DNA were completed according to steps 3 and 4 of Example 1.

[0130] Results are shown in Figure 4. Compared with the extraction and conversion one-step method, the lysis-free conversion method can further improve the fluorescence signal value. According to the CT value, the lysis-free conversion method curve starts earlier, indicating that the conversion efficiency is higher than the extraction and conversion one-step method. In this experiment, the high concentration of ammonium bisulfite solution is a cell lysate and also a conversion solution, which is a lysis and conversion reagent.

[0131] Therefore, for the DNA lysis-free conversion method of the cell sample, the traditional extraction reagent does not need to be added, and the bisulfite conversion (basically complete conversion) can be directly completed.

[0132] Example 5, analysis of conversion reagents in the lysis-free method conversion of samples

[0133] 1. Analysis of conversion reagents (ammonium bisulfite or combination with other reagents) with a concentration of 40% or above

[0134] The method described in Example 4 is operated, and the difference is that the conversion reagent and the concentration are adjusted, as shown in Table 5.

[0135] Table 5

[0136] Results are shown in Figure 5. Bisulfite with a concentration of 40%-70% and commonly used bisulfite can achieve lysis-free and conversion. Bisulfite with a concentration of 40-50% has the relatively most ideal effect, and the fluorescence signal value is the best. According to the CT value, the curve starts the earliest, and the relative conversion efficiency is the highest.

[0137] 2. Analysis of conversion reagents (ammonium bisulfite) with a concentration of 50% or below

[0138] The method described in Example 4 is operated, and the difference is that the conversion reagent and the concentration are adjusted, as shown in Table 6.

[0139] Table 6

[0140] Results are shown in Figure 6. Bisulfite has good conversion effect under the condition of 20-50%. When the concentration is lower than 12%, the conversion effect decreases obviously.

[0141] Example 6, analysis of conversion temperature and time in the lysis-free method conversion of samples

[0142] The method described in Example 4 is operated, and the difference is that the conversion temperature and time are adjusted, as shown in Table 7. Ammonium bisulfite is used with a concentration of 40-50%.

[0143] Table 7

[0144] Results as shown in Figure 7, the CT value is the lowest under the condition of 70°C for 30 minutes, indicating the best lysis and conversion effect. The time required is shortened with the increase of temperature.

[0145] Example 7, lysis-free conversion detection of different tissue or cell samples

[0146] 1. HeLa cells (methanol fixed) as test samples

[0147] Sample acquisition and processing: the method described in Example 4 was performed, with the difference that HeLa cells were detected. The lysis and conversion reagent was ammonium bisulfite, 40-50% concentration, 70°C for 30 min.

[0148] Results as shown in Figure 8, it can be seen that lysis-free conversion method can achieve efficient amplification, indicating high DNA conversion efficiency (basically complete conversion).

[0149] 2. Fresh tissue as test sample

[0150] Sample acquisition and processing: the method described in Example 4 was performed, with the difference that fresh brain tissue was detected, which was used after grinding. The lysis and conversion reagent was ammonium bisulfite, 40-50% concentration, 70°C for 30 min.

[0151] Results as shown in Figure 9, it can be seen that lysis-free conversion method has amplification curve, early take-off, ideal signal, indicating good conversion efficiency for fresh tissue (basically complete conversion).

[0152] 3. HPV sample as test sample

[0153] Sample acquisition and processing: the method described in Example 4 was performed, with the difference that HPV sample (cervical exfoliated cells) was detected. The lysis and conversion reagent was 60%-70% ammonium bisulfite solution, 94°C for 15 min.

[0154] Results as shown in Figure 10, it can be seen that lysis-free conversion method has amplification curve, early take-off, ideal signal, indicating good conversion efficiency for HPV sample (basically complete conversion).

[0155] 4. TCT sample as test sample

[0156] Sample acquisition and processing: the method described in Example 4 was performed, with the difference that methylation-negative cervical exfoliated cells (TCT) were detected. The lysis and conversion reagent was 60%-70% ammonium bisulfite solution, 94°C for 15 min.

[0157] The results are shown in Figure 11. It can be seen that the non-lysis transformation method has an amplification curve, early take-off and ideal signal, indicating that it has good transformation efficiency (substantially complete transformation) for TCT samples.

[0158] Example 8. Verification of the effect of bisulfite lysis

[0159] The method described in Example 4 was used, except that the transformation temperature and time were 37°C for 10 min and 50°C for 10 min, respectively, and 40-50% ammonium bisulfite was used. The untransformed and transformed sequences of the GAPDH gene were detected simultaneously. The transformed DNA was detected according to Example 1, and the reagents for detecting the untransformed sequence were as follows:

[0160] F: CAATTCCCCATCTCAGTC (SEQ ID NO: 4);

[0161] R: GTGATCGGTGCTGGTTCC (SEQ ID NO: 5);

[0162] Probe: CTTGACTCCCTAGTGTCC (SEQ ID NO: 6).

[0163] The results are shown in Figure 12. It can be seen that an amplification signal can be detected at 37°C for 10 min, indicating that the cells have been lysed but the DNA has not been transformed under this condition.

[0164] Therefore, bisulfite needs to be treated under specific conditions (such as temperature, etc.), and the general DNA sample acquisition, lysis, purification, etc. are usually different from the treatment conditions of bisulfite, which is one of the factors why the skilled person in the art has not found that bisulfite can be used as a lysing and transforming reagent at the same time.

[0165] The above examples only express several embodiments of the present application, and the description is more specific and detailed, but it should not be understood as limiting the scope of the patent of the present application. It should be noted that for ordinary skilled persons in the art, several modifications and improvements can be made without departing from the concept of the present application, and these all belong to the protection scope of the present application. Therefore, the protection scope of the patent of the present application should be subject to the appended claims. At the same time, all the documents mentioned in the present application are cited as references in the present application, just as each document is cited as a reference.

Claims

1. A method for preparing Bisulfite DNA, comprising: treating a cell sample with a reagent containing bisulfite as a lysing and converting reagent to obtain a converted product, wherein non-methylated modified cytosine in the DNA of the product is converted to uracil and to thymine in a subsequent amplification process, and the product is Bisulfite DNA.

2. The method of claim 1, wherein, comprising: (a) providing a cell sample; (b) treating the sample of (a) with a reagent containing bisulfite as a lysing and converting reagent to convert non-methylated modified cytosine in the sample to uracil to obtain a converted product; with the proviso that the cell sample in (a) is treated with a process selected from (1) or (2): (1) without lysing the cell sample, the cell is used in step (b); or (2) lysing the cell to obtain a lysate, the lysate is used in step (b) without a step of washing or eluting the DNA sample, and the DNA extraction and conversion are performed in one step.

3. The method of claim 2, wherein, said steps (a)-(b) comprise or consist of the following steps: providing a cell sample, lysing and converting the sample with a reagent containing bisulfite to obtain a converted product; or providing a cell sample, lysing the cell, adsorbing the DNA on a solid support, treating the adsorbed sample with a reagent containing bisulfite to obtain a converted product.

4. The method according to any one of claims 1 to 3, characterized in that when treating with a reagent containing bisulfite, the concentration of bisulfite is 15-75%, preferably 20-70%, more preferably 30-60%, and further more preferably 40-50%.

5. The method according to any one of claims 1 to 3, characterized in that when treating with a reagent containing bisulfite, the temperature is 60-99°C, preferably 65-98°C, more preferably 68-96°C, and further more preferably 70-94°C.

6. The method of any one of claims 1 to 3, wherein, the treatment time is 5-40 min, preferably 8-35 min, and more preferably 10-30 min.

7. The method of any one of claims 1 to 3, wherein the bisulfite includes ammonium bisulfite, ammonium sulfite, sodium bisulfite, sodium sulfite, or a combination thereof.

8. The method according to claims 1 to 3, characterized in that after steps (a) and (b), further comprising: (c) desulfonating and purifying the DNA after the conversion is completed, and preferably the purification comprises washing and / or eluting.

9. Use of the method of any one of claims 1-6 in analyzing the DNA methylation level of a test cell sample.

10. A method for analyzing the DNA methylation level of a test cell sample, comprising: (i) providing a test cell sample, and preparing Bisulfite DNA from the test cell sample by the method of any one of claims 1-6; (ii) analyzing the presence of uracil in the Bisulfite DNA to determine the methylation level of the test sample.

11. The method of claim 10, wherein, after step (i), further comprising a step of amplifying the Bisulfite DNA.

12. A kit for preparing Bisulfite DNA, characterized in that, the kit comprises a reagent containing bisulfite as a lysing and converting reagent, and preferably further comprises a reagent selected from the group consisting of a DNA desulfonation reagent, a DNA washing reagent, a DNA eluting reagent, and a solid support, and preferably does not comprise an additional cell lysing reagent. Preferably, the kit further comprises an instruction for using the reagent containing a (hydrogen) sulphite to obtain a converted product, wherein the non-methylated cytosine in the DNA of the product is converted to uracil; more preferably, the instruction is for treating the cell sample by a method selected from (1) or (2): (1) without lysing the cell sample, the cell is used for the conversion step; or (2) lysing the cell to obtain a lysate, the lysate is used for the conversion step without washing or eluting the DNA sample, and the DNA extraction and conversion are performed in one step. Preferably, the instruction further indicates that the reagent containing a (hydrogen) sulphite is used at a concentration of 15-75%, preferably 20-70%, more preferably 30-60%, and even more preferably 40-50%; and / or at a temperature of 60-99°C, preferably 65-98°C, more preferably 68-96°C, and even more preferably 70-94°C; and / or for a time period of 5-40 min, preferably 8-35 min, and more preferably 10-30 min.

13. Use of the kit of claim 12 in analyzing the DNA methylation level of a cell sample to be tested, or in preparing a detection system for analyzing the DNA methylation level of a cell sample to be tested.

14. Use of a reagent containing a (hydrogen) sulphite in preparing a lysate conversion reagent for a cell sample; preferably, the (hydrogen) sulphite is used at a concentration of 15-75%; and preferably, the (hydrogen) sulphite is used at a temperature of 60-99°C.

15. A method for lysing a cell sample, comprising: The following steps are performed: (a) providing a cell sample; and (b) treating the sample of (a) with a reagent containing a (hydrogen) sulphite at a temperature of 60-99°C for a time period of 5-40 min.

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