WO2020228306A1 - 基因甲基化分析方法、产品和用途 - Google Patents

基因甲基化分析方法、产品和用途 Download PDF

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WO2020228306A1
WO2020228306A1 PCT/CN2019/123433 CN2019123433W WO2020228306A1 WO 2020228306 A1 WO2020228306 A1 WO 2020228306A1 CN 2019123433 W CN2019123433 W CN 2019123433W WO 2020228306 A1 WO2020228306 A1 WO 2020228306A1
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dna
solution
nucleic acid
magnetic beads
conversion
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PCT/CN2019/123433
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English (en)
French (fr)
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王弢
张田田
王冬华
巴兆粉
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江苏为真生物医药技术股份有限公司
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    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
    • C12Q1/6806—Preparing nucleic acids for analysis, e.g. for polymerase chain reaction [PCR] assay

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  • the invention belongs to the field of gene analysis. Specifically, the present invention relates to gene methylation detection methods, products and uses.
  • 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. Gene methylation is a molecular marker for early cancer diagnosis and monitoring of disease progression. In recent years, a variety of methods have been designed to detect and distinguish methylated sequences in normal and cancer samples. These methods are basically based on nucleic acid extraction, bisulfite conversion, and recovered bisDNA (bisulfite converted DNA) for detection.
  • the current extraction kits mainly include the following steps: sample pretreatment; surfactants, proteinase K, etc. lyse cells to digest proteins, and release DNA from nucleoproteins; use spin columns or magnetic beads to enrich DNA; more The second elution removes impurities such as protein and polysaccharide; finally the DNA is eluted from the solid phase carrier.
  • the conversion purification kit mainly includes the following steps: the DNA obtained by the above extraction is converted into bisulfite; the sulfonic acid group is removed with a strong base (such as sodium hydroxide); multiple leaching; and finally the DNA is removed from the solid phase carrier Elute down for detection.
  • a strong base such as sodium hydroxide
  • multiple leaching and finally the DNA is removed from the solid phase carrier Elute down for detection.
  • the whole process requires multiple rinsing and replacement of the reaction tube, which is cumbersome to operate, and the DNA loss is large and the probability of contamination is high.
  • the method and kit of the present invention allow the whole process from the biological sample to the on-machine test sample to be completed within 3 hours; the operation process is simplified, and the pre-processing, rinsing, elution, conversion, and reelution are minimized. Steps reduce the degradation and loss of DNA in the process, and achieve a detection effect superior to the traditional methylation detection processing method.
  • the reagent of the present invention has higher stability and is easier to realize automatic operation.
  • the present invention provides a DNA methylation analysis method, which includes 1) simultaneously contacting a biological sample containing DNA with a carrier with nucleic acid adsorption capacity and a lysis solution to enrich the DNA on the carrier; 2) The DNA-adsorbed vector obtained in step 1) is directly contacted with a transformation reagent to convert at least one unmethylated cytosine base of the DNA enriched on the vector into uracil or detectably different from the cytosine on the hybridization. For other bases of pyrimidine, 3) treat the mixed solution obtained in step 2) with the binding solution to enrich the transformed DNA on the carrier.
  • step 2) of the present invention is performed immediately after step 1), that is, any other treatments, such as washing treatments, may not be performed between step 1) and step 2).
  • step 1) of the method further includes adding a nucleic acid binding promoter, so that the biological sample containing methylated DNA is in contact with the carrier capable of nucleic acid adsorption, the nucleic acid binding promoter, and the lysis solution at the same time.
  • the nucleic acid of methylated DNA is enriched on the carrier.
  • the nucleic acid binding promoter in the method of the present invention includes any agent that promotes the binding of nucleic acid in the lysate to a carrier with nucleic acid adsorption ability, for example, it may be a suitable organic solvent and/or wetting agent.
  • the nucleic acid binding promoter includes, for example, isopropanol, isobutanol, n-butanol, acetone, pyridine, acetonitrile, methyl formate, ethyl acetate, propylene glycol, glycerol, dimethyl sulfoxide, poly One or more of ethylene glycol, alkyl sulfate, sulfonate or ester, polyol surfactant (for example, Span type, Tween type surfactant), and polyoxyethylene surfactant.
  • polyol surfactant for example, Span type, Tween type surfactant
  • the lysis solution that can be used in the method of the present invention is not particularly limited, and an appropriate lysis solution that has been used in the art can be used.
  • the lysis solution contains a guanidine salt, such as guanidine hydrochloride, guanidine isothiocyanate.
  • the lysis solution may also contain one or more of detergents such as SDS, metal ion chelating agents such as EDTA, metal salts such as NaCl and the like.
  • the lysate can be formulated into a buffer solution, such as tris buffer, citrate buffer.
  • the lysis step of the present invention may also include appropriate reagents that promote the release and separation of nucleic acids from the sample.
  • 1) of the method of the present invention may further include combining the biological sample containing methylated DNA with Protease such as proteinase K contact.
  • the reagent for transforming DNA is not particularly limited, and any reagent in the art that can convert at least one unmethylated cytosine base in DNA to uracil or other detectable hybridization is different from cytosine.
  • the appropriate reagent for the base may include bisulfite, metabisulfite, or a combination thereof, such as sodium bisulfite, magnesium bisulfite, ammonium bisulfite, potassium metabisulfite, sodium sulfite, sodium metabisulfite One or more of.
  • the conversion reagent may include a mixed solution of sodium bisulfite and sodium sulfite.
  • any reagents and/or DNA protective agents that promote DNA conversion can be added during the conversion process of the method of the present invention, such as free radical traps with DNA protection, including but not limited to: hydroquinone, 6-hydroxyl -2,5,7,8-tetramethylchroman-2-carboxylic acid, 6-hydroxy-2,5,7,8-tetramethylchroman-2-carboxylic acid or Various organic solutions, such as diethylene glycol dimethyl ether solution, chloroform solution or tetrahydrofuran solution.
  • any suitable carrier capable of nucleic acid adsorption can be used in the method of the present invention, including but not limited to: magnetic beads, non-magnetic microspheres, adsorption membranes, and the like.
  • carriers with nucleic acid adsorption capabilities include but are not limited to: hydroxyl magnetic beads, carboxyl magnetic beads, streptavidin immunomagnetic beads, ion exchange resins, and the like.
  • the shell layer of the magnetic beads is preferably silica or agarose.
  • the magnetic beads are preferably superparamagnetic beads.
  • the particle size of the magnetic beads may be 100 nm to 3 ⁇ m, preferably 500 to 1 ⁇ m.
  • the conversion process in step 2) of the method of the present invention further includes a post-conversion treatment step, so as to prepare a sample suitable for direct subsequent methylation detection.
  • the post-conversion treatment step includes using a cleaning solution to In the step of cleaning the nucleic acid enriched on the magnetic beads, optionally the cleaning solution includes a tris solution, a tris-hydrochloric acid solution, an ethanol solution, and optionally the cleaning solution
  • the pH is in the range of 6-10, preferably the pH is 8-10.
  • the post-conversion treatment does not include desulfurization treatment.
  • the inventors have found that the transformed sample prepared by the method of the present invention does not need to be desulfonated, which greatly saves experimental time.
  • the cleaned sample in the method of the present invention can be directly subjected to subsequent methylation. Detection, such as PCR detection, sequencing detection, etc.
  • a period of high temperature treatment may be added during the pre-denaturation stage of the PCR detection process, such as 10-20 minutes (eg 15 minutes) 90- 98°C (for example, 95°C) high temperature treatment for desulfurization.
  • the source of the biological sample in the method of the present invention is not particularly limited.
  • 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 Examples include but are not limited to: blood, urine, stool, cell culture fluid, tissue, sputum, pleural fluid, ascites, cerebrospinal fluid, and processed products of these samples.
  • the biological sample may be from a healthy subject and/or Patients, such as cancer patients.
  • the DNA methylation analysis method of the present invention includes a method for analyzing the presence and/or pattern of DNA methylation in the genome.
  • the DNA methylation analysis method of the present invention includes detection of naturally occurring and/or abnormal DNA methylation in the genome (for example, whether there is DNA methylation and/or whether the DNA methylation status has changed ) For analysis, which can be used to study the effect of DNA methylation on gene expression.
  • the DNA methylation analysis method of the present invention includes a method of preparing samples suitable for DNA methylation detection, for example, including the steps of extracting, enriching and transforming methylated DNA, and re-enriching and detecting methylated DNA. .
  • the DNA methylation analysis method of the present invention includes a method for detecting the DNA methylation status, which includes the step of subjecting the prepared sample to DNA methylation detection.
  • DNA methylation detection can be performed by any appropriate method, such as PCR analysis, probe analysis, sequencing analysis, and the like.
  • the transformed sample prepared by the method of the present invention is directly subjected to subsequent analysis without additional processing steps. For example, the transformed sample bound to the magnetic beads may not be eluted, but directly subjected to subsequent analysis. PCR analysis.
  • the binding solution used in the method of the present invention may include chaotropic salts, such as guanidine salts, such as guanidine hydrochloride and guanidine isothiocyanate.
  • the binding solution may Add a buffer solution, such as citrate buffer.
  • the present invention provides a DNA methylation analysis kit suitable for the method of the present invention
  • the instructions for use include the method described in the present invention, which contains one or more containers, and the container contains the present invention.
  • the kit includes a carrier with nucleic acid adsorption capacity and a lysate, and optionally a nucleic acid binding promoter.
  • the kit further includes conversion reagents.
  • the kit contains an appropriate buffer.
  • the kit further includes reagents for detecting methylated DNA processed after transformation, such as PCR reagents, probes, and sequencing reagents.
  • the method and kit provided by the present invention can be used for various analyses of DNA methylation.
  • 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.
  • the invention provides a simple and high-efficiency DNA processing method and a corresponding kit that can complete the DNA enrichment, transformation and re-enrichment on the same set of magnetic beads in sequence at one time.
  • the present invention provides a DNA processing method in a DNA methylation analysis method, which includes releasing DNA in a biological sample from a lysis solution and simultaneously enriching nucleic acid with a carrier with nucleic acid adsorption function, such as magnetic beads; The DNA is directly transformed on the vector, and the DNA is then enriched to the same set of magnetic beads.
  • the lysis solution includes, but is not limited to, guanidine hydrochloride, guanidine isothiocyanate and other salt solutions, including but not limited to organic solvents such as isopropanol, with or without proteinase K reagent Can be.
  • the surface of the magnetic beads may be modified with hydroxyl or carboxyl groups.
  • the transforming agent is a mixture of bisulfite, metasulfite, and/or DNA protective agent.
  • the conversion reagent is preferably: a mixed solution of sodium bisulfite and sodium metabisulfite, or a DNA protective agent is further added.
  • the DNA protective agent is a free radical trap with DNA protection, including but not limited to: hydroquinone, 6-hydroxy-2,5,7,8-tetramethylchroman An organic solution of one or more of -2-carboxylic acid and 6-hydroxy-2,5,7,8-tetramethylchroman-2-carboxylic acid, such as diethylene glycol dimethyl ether solution and chloroform solution Or tetrahydrofuran solution.
  • the magnetic bead shell layer is preferably silica, agarose, or the like. In some embodiments, when the bisulfite is ammonium bisulfite, the magnetic bead shell layer is preferably agarose.
  • the method of the present invention further includes a process of simultaneous sample lysis and magnetic bead adsorption of nucleic acids.
  • the method of the present invention further includes the process of directly carrying out the bisulfite conversion process with the enriched DNA carried by the magnetic beads.
  • the method of the present invention further includes a process in which the transformed DNA can be subjected to PCR detection without a desulphonation step.
  • the present invention also provides a kit for DNA methylation analysis, which includes a lysis solution, magnetic beads, conversion reagents, binding solutions, washing solutions, buffers, and the like.
  • the lysis solution is guanidine salt and/or tris and/or ethylenediaminetetraacetic acid and/or sodium hydroxide and/or Tween and/or iso Mixture of propanol and/or proteinase K.
  • the surface of the carrier such as magnetic beads can be modified with silanol groups.
  • the shell layer of the magnetic beads can be selected from silica, agarose, etc., and the particle size of the magnetic beads is best selected But it is not limited to 100nm ⁇ 2 ⁇ m.
  • the conversion reagent in the kit of the present invention, is a mixture of bisulfite, metasulfite, and/or DNA protective agent. In some embodiments, the conversion reagent is preferably: a mixed solution of sodium bisulfite and sodium metabisulfite, or a DNA protective agent is further added.
  • the DNA protective agent is a free radical trap with DNA protection, including but not limited to: hydroquinone, 6-hydroxy-2,5,7,8-tetramethylchroman An organic solution of one or more of -2-carboxylic acid and 6-hydroxy-2,5,7,8-tetramethylchroman-2-carboxylic acid, such as diethylene glycol dimethyl ether solution and chloroform solution Or tetrahydrofuran solution.
  • the conversion temperature is 80-100°C and the conversion time is 40-60 min.
  • the binding solution includes, but is not limited to, chaotropic salts such as guanidine hydrochloride, guanidine isothiocyanate and other salt solutions.
  • the pH of the cleaning solution may be in the range of 6-10, but 8-10 is preferred.
  • the buffer includes, but is not limited to, purified water, TE buffer, and weak alkali solution of sodium hydroxide.
  • the present invention provides a DNA methylation detection method, which includes the following steps:
  • the biological sample is lysed and nucleic acid released under the action of the lysis solution.
  • the magnetic beads are in contact with the nucleic acid under the high concentration and chaotropic salt environment of the lysis solution to form a magnetic bead-nucleic acid complex, and the sample lysis is performed simultaneously with the adsorption of nucleic acid by the magnetic beads;
  • Transformation treat the magnetic bead-nucleic acid complex obtained in step (1) with a transforming reagent to obtain transformed DNA;
  • Re-concentration under the action of the binding solution, the magnetic beads and the transformed DNA are re-contacted to form a magnetic bead-nucleic acid complex, which is washed with the washing solution and then eluted with a buffer to obtain the transformed DNA;
  • Detection Determine the transformed DNA obtained in step (3).
  • the DNA methylation information obtained according to the above detection method can be directly used in laboratory and clinical research.
  • the sample lysis in step (1) is performed synchronously with the magnetic beads for nucleic acid adsorption;
  • the transformed DNA in step (2) can be detected without desulphurization treatment
  • the PCR detection in step (4) only needs to add 15 minutes of processing in the pre-denaturation stage to omit the reagent desulphurization process.
  • the method in step (4) can be a dye method or a probe method, PCR qualitative detection or PCR quantitative detection can be performed, and the obtained PCR product can also be sequenced and analyzed.
  • the biological sample can be 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 fluid, ascites, cerebrospinal fluid and processed products of these samples.
  • the biological sample may be from healthy subjects and/or patients, such as cancer patients.
  • the entire technical process of the method provided by the present invention realizes a single-tube reaction, has simple operation, high nucleic acid recovery rate, and reduces pollution and error probability.
  • the kit prepared based on the above method provided by the present invention makes the implementation of the above method easier and faster.
  • the present invention has discovered that in the lysis step, magnetic beads and lysis solution can be added at the same time to realize simultaneous cleavage and magnetic bead combination without affecting the effect of lysis.
  • a nucleic acid binding promoter can be added to enhance the hydrophobic interaction between the nucleic acid in the lysis solution and the surface of the magnetic beads.
  • the present invention can greatly simplify the nucleic acid extraction steps. It was also found that by contacting a biological sample containing DNA with a carrier with nucleic acid adsorption capacity and a lysis solution at the same time, after the DNA is enriched on the carrier, it can be directly used without any other treatment (such as washing treatment).
  • the DNA-adsorbed carrier is contacted with a conversion reagent to convert at least one unmethylated cytosine base of the DNA enriched on the carrier into uracil or other bases different from cytosine detectable on hybridization.
  • the mixed solution obtained in the previous step can be treated with the binding solution to re-enrich the transformed DNA on the carrier.
  • the transformed DNA can be eluted from the vector with the eluent for detection, or PCR buffer can be used to directly resuspend the DNA-rich vector for direct PCR amplification.
  • the method and product of the invention are easy to operate.
  • biological samples can be incubated with or without proteinase K; the extracted DNA can be directly subjected to methylation treatment without being separated from the magnetic beads; multiple rinsing and sodium hydroxide desulphurization processes are omitted.
  • the entire process from the sample to the template on the machine can be completed in one reagent tube.
  • the method and product of the invention save time.
  • the method and product of the present invention can complete the whole process of obtaining samples for on-board testing from biological samples in only 3 hours.
  • FIG. 1 The method of the present invention is used to distinguish between methylation and unmethylation of Septin9 gene: CpG in Septin9 gene is methylated in Hela cells and unmethylated in Jurkat cells.
  • Figure 2 shows the effect of simultaneous lysis and binding on the nucleic acid concentration effect
  • Figure 3 shows that the DNA of the present invention can be directly transformed on magnetic beads.
  • Figure 4 shows the DNA sequence before transformation.
  • Figure 5 shows the sequence of DNA transformed at 80°C for 40 min.
  • Figure 6 shows the sequence of DNA transformed at 100°C for 60 min.
  • Figure 7 shows the detection limit of DNA transformation after elution from magnetic beads.
  • Figure 8 shows the detection limit of direct DNA conversion on magnetic beads.
  • Figure 9 shows that the present invention is suitable for the detection of DNA methylation in urine.
  • Figure 10 shows that the present invention is suitable for detection of fecal DNA methylation.
  • Sample pretreatment put equal amounts of interrupted Hela and Jurkat cell DNA into 2 ml of healthy human plasma samples;
  • lysis buffer the lysis buffer is composed of 5.22M guanidine isothiocyanate, 0.42M TE buffer and 17% Triton
  • magnetic beads beaver, hydroxy magnetic beads
  • conversion reagent is 5.34M ammonium bisulfite, 0.53M sodium sulfite, 0.05M protective agent (protective component 6-hydroxy-2,5,7,8-tetramethyl
  • protective agent protecting component 6-hydroxy-2,5,7,8-tetramethyl
  • the detection area of Septin9 gene is abnormally methylated in Hela cells, and it is normal in Jurkat cells.
  • the difference in the methyl status of the target region of the gene can be distinguished, as shown in Figure 1.
  • Figure 1 shows the results of gene methylation detection performed by the method of the present invention.
  • DNA is directly transformed after being enriched by magnetic beads
  • step (1) Put interrupted Hela cell DNA into 2mL healthy human plasma sample.
  • One part was processed completely in accordance with Example 1, and one part of step (1) was processed according to the following steps:
  • conversion reagent add 220 microliters of conversion reagent (the conversion reagent is 7.8M ammonium bisulfite, 0.78M sodium sulfite, 0.07M DNA protectant (protective component 6-hydroxy-2,5,7,8) -A mixture of tetramethylchroman-2-carboxylic acid in diethylene glycol dimethyl ether), the above mixture is kept at 85°C for 40 minutes.
  • the DNA can be transformed directly without being separated from the magnetic beads, and after reducing the intermediate DNA elution step, the nucleic acid recovery efficiency is improved, indicating that the magnetic beads do not need to perform the intermediate DNA elution step after the nucleic acid is enriched.
  • the conversion process is carried out directly, and there is no need to add new magnetic beads during purification.
  • Figure 3 shows the result of direct transformation of the DNA of the present invention on magnetic beads.
  • the real-time fluorescent PCR method is used to detect methylation.
  • This conversion reagent normally transforms at 80 ⁇ 100°C, and the Cp value detected at each temperature is equivalent; at the same time, the conversion effect is improved by different incubation times under the same temperature conditions. It can be seen from the analysis of the influence that the conversion reagent of the present invention is used for constant temperature conversion, and good results can be obtained in only 40 minutes, which greatly saves the conversion time.
  • the method of adopting constant temperature in the present invention is simpler and easy to operate, and can use cheap and convenient constant temperature instruments for related experimental research.
  • the amplified products of the transformed DNA were subjected to TA cloning.
  • Each TA cloned product randomly picked 20 transformants and sequenced them. Among the 20 transformants, there were a total of 600 “C”s.
  • the statistical "C” was transformed into " If the number of T" is N, the conversion efficiency is: (N/600) ⁇ 100%.
  • Figure 4 shows the DNA sequence before transformation.
  • Figure 5 shows the sequence of DNA transformed at 80°C for 40 min.
  • Figure 6 shows the sequence of DNA transformed at 100°C for 60 min.
  • Example 6 Detection of methylation of septin9 gene in intestinal plasma samples
  • test result statistics are as follows:
  • the present invention has a greater advantage in the number of positive detection holes of colorectal cancer samples, while the detection specificity is still maintained.
  • the specific steps of the method used in this example are the same as those in Example 1, except that the corresponding detection primers are different, and the lysate components are different.
  • the lysate consists of 2.75M guanidine isothiocyanate, 0.22M TE buffer , 9% Triton and 47% isopropanol).
  • the finally obtained transformed DNA is directly processed by 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) NID2 methylation detection shows that they have good discrimination.
  • Figure 9 shows the detection result of urine DNA methylation by the method of the present invention.
  • the BMP3 gene methylation in stool samples of 10 cases of colorectal cancer (CRC), 10 cases of advanced adenoma (AA) and 10 cases of non-advanced adenoma (NAA) were detected and evaluated by the ⁇ ct method.
  • the specific steps of the method used in this example are the same as those in Example 1.
  • the finally obtained transformed DNA directly uses the Methylate method (Weisenberger DJ, Campan M, Long TI, Kim M, Woods C, et al. (2005) Analysis of reactive element DNA methylation by MethyLight. Nucleic Acids Res 33: 6823-6836) for methylation detection, all showed good discrimination.
  • Figure 10 shows the detection result of fecal DNA methylation in the method of the present invention.

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Abstract

提供一种基因甲基化分析方法、产品和用途，所述DNA甲基化分析方法包括1）使含有DNA的生物样品与有核酸吸附能力的载体、裂解液同时接触，将DNA富集于载体上；2）直接使步骤1）获得的吸附有DNA的载体与转化试剂接触从而将载体上富集的DNA的至少一个未甲基化的胞嘧啶碱基转化为尿嘧啶或在杂交上可检测的不同于胞嘧啶的其它碱基，3）再用结合液处理步骤2）得到的混合液，使转化后的DNA再富集于载体上。还提供相应基因甲基化检测方法、试剂盒和用途。

Description

基因甲基化分析方法、产品和用途 技术领域
本发明属于基因分析领域。具体而言，本发明涉及基因甲基化检测方法、产品和用途。
背景技术
DNA甲基化是基因转录调控中最重要的表观遗传学修饰之一。这种修饰在与发育和疾病相关的生物学过程中起着重要作用。基因甲基化作为早癌诊断和监测疾病进展的分子标记物，近年来已经设计了多种方法来检测和区分正常和癌症样本中的甲基化序列。这些方法基本都基于核酸提取、亚硫酸氢盐转化、回收后得到的bisDNA(亚硫酸氢盐转化后的DNA)进行检测。
当前市面上已出现了多种多样的商业化产品，包括凯杰公司的提取试剂盒、ZYMO公司的转化纯化试剂盒等。当前提取试剂盒主要包括以下几个步骤：样本的前处理；表面活性剂、蛋白酶K等裂解细胞消化蛋白，将DNA从核蛋白上释放出来；采用离心柱或磁珠的方法富集DNA；多次淋洗去除蛋白、多糖等杂质；最后将DNA从固相载体上洗脱下来。转化纯化试剂盒主要包括以下几个步骤：上述提取得到的DNA进行亚硫酸氢盐转化；采用强碱(如氢氧化钠)脱磺酸基；多次淋洗；最后将DNA从固相载体上洗脱下来进行检测。整个过程需要多次淋洗、更换反应管，操作繁琐，且DNA损耗大、污染概率高。
商业化试剂盒的上述两个过程相比传统方法有了很大的改进，然而仍需近8个小时的时间才能获得上机检测模板；此外，对于一些肿瘤特异性循环DNA相对稀缺性的样本复杂的操作过程会大大提高核酸的损耗。这对现有的DNA提取和转化处理技术提出了挑战。
发明内容
发明人通过大量研究，提出一种样品裂解及DNA在有核酸吸附能力的载体上进行富集同步进行、有核酸吸附能力的载体上直接进行DNA转化、再富集及回收检测的单管处理的方法和试剂盒。本发明的方法和试剂盒允许在3小时内完成从生物样品到获得上机检测样品的全过程；简化了操作过程，最大程度减少了前处理、漂洗、洗脱、转化、再洗脱等繁琐步骤，减少了过程中DNA的降解和丢失，达到优于传统的甲基化检测处理方法的检测效果。此外，本发明的试剂稳定性更高，且更易实现自动化操作。
在一些实施方案中，本发明提供一种DNA甲基化分析方法，包括1)使含有DNA的生物样品与有核酸吸附能力的载体、裂解液同时接触，将DNA富集于载体上；2)直接使步骤1)获得的吸附有DNA的载体与转化试剂接触从而将载体上富集的DNA的至少一个未甲基化的胞嘧啶碱基转化为尿嘧啶或在杂交上可检测的不同于胞嘧啶的其它碱基，3)再用结合液处理步骤2)得到的混合液，使转化后的DNA再富集于载体上。
在一些实施方案中，本发明的步骤2)紧接步骤1)进行，即步骤1)和步骤2)之 间可以不进行任何其他处理，如洗涤处理等。在一些实施方案中，所述方法的步骤1)还包括添加核酸结合促进剂，使得含有甲基化DNA的生物样品与有核酸吸附能力的载体、核酸结合促进剂和裂解液同时接触，将含有甲基化DNA的核酸富集于载体上。
在一些实施方案中，本发明的方法中的核酸结合促进剂包括促进裂解液中的核酸与有核酸吸附能力的载体结合的任何试剂，例如其可以是适当的有机溶剂和/或润湿剂。在一些实施方案中，所述核酸结合促进剂包括例如异丙醇、异丁醇、正丁醇、丙酮、吡啶、乙腈、甲酸甲酯、乙酸乙酯、丙二醇、甘油、二甲亚砜、聚乙二醇、烷基硫酸盐、磺酸盐或酯、多元醇表面活性剂(例如司盘类、吐温类表面活性剂)、聚氧乙烯表面活性剂中的一种或多种。
在一些实施方案中，可以在本发明的方法中使用的裂解液没有特别限制，可以使用本领域中已经采用的适当裂解液。在一些实施方案中，所述裂解液包含胍盐，如盐酸胍、异硫氰酸胍。在一些实施方案中，所述裂解液还可以包含去垢剂如SDS，金属离子螯合剂如EDTA，金属盐如NaCl等中的一种或多种。在一些实施方案中，所述裂解液可以配制成缓冲溶液，如三羟甲基氨基甲烷缓冲液、柠檬酸缓冲液。在一些实施方案中，本发明的裂解步骤中还可以包括适当的促进核酸从样品释放和分离的试剂，例如本发明的方法的1)可以进一步包括使所述含有甲基化DNA的生物样品与蛋白酶如蛋白酶K接触。
在一些实施方案中，转化DNA的试剂没有特别限制，可以使用本领域任何使DNA中至少一个未甲基化的胞嘧啶碱基转化为尿嘧啶或在杂交上可检测的不同于胞嘧啶的其它碱基的适当的试剂。在一些实施方案中，所述转化试剂可以包括亚硫酸氢盐、焦亚硫酸盐或其组合，例如亚硫酸氢钠、亚硫酸氢镁、亚硫酸氢铵、焦亚硫酸钾、亚硫酸钠、焦亚硫酸钠中的一种或多种。在一些实施方案中，所述转化试剂可以包括亚硫酸氢钠、亚硫酸钠的混合溶液。
在一些实施方案中，本发明的方法的转化过程中可以添加任何促进DNA转化的试剂和/或DNA保护剂，例如有DNA保护作用的自由基阱，包括但不限于：氢醌、6-羟基-2，5，7，8-四甲基苯并二氢吡喃-2-羧酸、6-羟基-2，5，7，8-四甲基色满-2-甲酸中的一种或多种的有机溶液，如二乙二醇二甲醚溶液、氯仿溶液或四氢呋喃溶液。
在一些实施方案中，本发明的方法中可以采用任何适当的有核酸吸附能力的载体，包括但不限于：磁珠、非磁性的微球、吸附膜等。在一些实施方案中，有核酸吸附能力的载体包括但不限于：羟基磁珠、羧基磁珠、链霉亲和素免疫磁珠、离子交换树脂等。在一些实施方案中，磁珠的壳层优选氧化硅或琼脂糖。在一些实施方案中，所述磁珠优选超顺磁珠。在一些实施方案中，磁珠粒径可以为100nm～3μm，优选500-1μm。
在一些实施方案中，本发明方法的步骤2)的转化过程还包括转化后处理步骤，从而制备适于直接进行后续甲基化检测的样品，所述转化后处理步骤 包括使用清洗液对转化后的富集于磁珠上的核酸进行清洗的步骤，任选地所述清洗液包括三羟甲基氨基甲烷溶液、三羟甲基氨基甲烷-盐酸溶液、乙醇溶液，任选地所述清洗液的pH在6～10范围内，优选pH为8～10，优选地所述转化后处理不包括脱磺处理。在一些实施方案中，发明人已经发现本发明的方法制备的转化样品不需要进行脱磺处理，由此大大节省了实验时间，例如本发明的方法中清洗后的样品可以直接进行后续甲基化检测，例如PCR检测、测序检测等。在一些实施方案中，本发明的方法制备的转化样品不进行脱磺处理时，可以在PCR检测过程中的预变性阶段增加一段时间的高温处理，例如10-20分钟(例如15分钟)90-98℃(例如95℃)高温处理进行脱磺。
在一些实施方案中，本发明方法中的生物样品的来源没有特别限制，例如所述生物样品选自任何含有DNA的样品，包括但不限于：无细胞来源的样品或细胞来源的样品；任选的包括但不限于：血液、尿液、粪便、细胞培养液、组织、痰液、胸水、腹水、脑脊液以及这些样品的处理品，任选地所述生物样品可以来自健康受试者和/或患者，例如癌症患者。
在一些实施方案中，本发明的DNA甲基化分析方法包括对基因组中DNA甲基化的有无和/或模式进行分析的方法。在一些实施方案中，本发明的DNA甲基化分析方法包括对基因组中天然存在的和/或异常的DNA甲基化(例如是否存在DNA甲基化和/或DNA甲基化状态是否发生改变)进行分析，可以用于研究DNA甲基化对基因表达的影响等。在一些实施方案中，本发明的DNA甲基化分析方法包括制备适合DNA甲基化检测的样品的方法，例如包括对甲基化DNA进行提取、富集和转化和再富集、检测的步骤。在一些实施方案中，本发明的DNA甲基化分析方法包括对DNA甲基化状态检测的方法，其包括将制备的样品进行DNA甲基化检测的步骤。在一些实施方案中，DNA甲基化检测可以采用任何适当的方法进行，例如PCR分析，探针分析，测序分析等。在一些实施方案中，将本发明的方法制备的转化后样品直接进行后续分析，而不需要另外的处理步骤，例如转化后的结合在磁珠上的样品可以不进行洗脱，而直接进行后续PCR分析。
在一些实施方案中，本发明的方法中采用的结合液可以包括离液序列高的盐(Chaotropic salt)，如胍盐，例如盐酸胍、异硫氰酸胍，任选地所述结合液可以加入缓冲溶液，如柠檬酸缓冲液。
在一些实施方案中，本发明提供一种适于本发明方法的DNA甲基化分析试剂盒，其使用说明中包含本发明描述的方法，其包含一个或多个容器，所述容器中包含本发明描述的方法中使用的试剂。在一些实施方案中，所述试剂盒包括有核酸吸附能力的载体和裂解液，以及任选地核酸结合促进剂。在一些实施方案中，所述试剂盒进一步包括转化试剂。在一些实施方案中，所述试剂盒包含适当的缓冲液。在一些实施方案中，试剂盒中进一步包括对转化后处理的甲基化DNA进行检测的试剂，例如PCR试剂，探针，测序试剂。
本发明提供的方法和试剂盒可以用于DNA甲基化的各种分析。在一些实施方案中，本发明提供的方法和试剂盒可以用于研究DNA甲基化的各种生物 功能，例如调节基因表达等。
本发明提供一种简单、高效，可将DNA富集、转化、再富集在同一套磁珠上顺序一次性完成的DNA简便处理方法和相应的试剂盒。
在一些实施方案中，本发明提供一种DNA甲基化分析方法中的DNA处理方法，该方法包括裂解液释放生物样品中DNA的同时用具有核酸吸附功能的载体如磁珠同步富集核酸；DNA在载体上直接进行转化、再富集处理后DNA到同一套磁珠上的步骤。
在一些实施方案中，在本发明的方法中，裂解液包括但不限于盐酸胍、异硫氰酸胍等盐类溶液，包括但不限于异丙醇等有机溶剂，加或不加蛋白酶K试剂均可。
在一些实施方案中，在本发明的方法中，磁珠表面可以修饰有羟基或羧基。
在一些实施方案中，在本发明的方法中，转化试剂是亚硫酸氢盐、焦亚硫酸盐和/或DNA保护剂的混合物。在一些实施方案中，转化试剂优选为：亚硫酸氢钠、焦亚硫酸钠的混合溶液，或者进一步的添加DNA保护剂。在一些实施方案中，所述DNA保护剂为有DNA保护作用的自由基阱，包括但不限于：氢醌、6-羟基-2，5，7，8-四甲基苯并二氢吡喃-2-羧酸、6-羟基-2，5，7，8-四甲基色满-2-甲酸中的一种或多种的有机溶液，如二乙二醇二甲醚溶液、氯仿溶液或四氢呋喃溶液。
在一些实施方案中，磁珠壳层优选氧化硅、琼脂糖等。在一些实施方案中，亚硫酸氢盐是亚硫酸氢铵时，磁珠壳层优选琼脂糖。
在一些实施方案中，本发明的方法还包括样本裂解与磁珠吸附核酸同步进行的过程。
在一些实施方案中，本发明的方法还包括磁珠携带富集的DNA直接进行亚硫酸氢盐转化的过程。
在一些实施方案中，本发明的方法还包括转化的DNA无需脱磺步骤即可进行PCR检测的过程。
在一些实施方案中，本发明还提供DNA甲基化分析的试剂盒，该试剂盒包括裂解液、磁珠、转化试剂、结合液、清洗液和缓冲液等。
在一些实施方案中，在本发明的试剂盒中，裂解液是胍盐和/或三羟甲基氨基甲烷和/或乙二胺四乙酸和/或氢氧化钠和/或吐温和/或异丙醇和/或蛋白酶K的混合物。
在一些实施方案中，在本发明的试剂盒中，载体例如磁珠表面可以修饰有硅烷醇基团，例如磁珠的壳层可以选自氧化硅、琼脂糖等，磁珠粒径最好选择但不限于100nm～2μm。
在一些实施方案中，在本发明的试剂盒中，转化试剂是亚硫酸氢盐、焦亚硫酸盐和/或DNA保护剂的混合物。在一些实施方案中，转化试剂优选为：亚硫酸氢钠、焦亚硫酸钠的混合溶液，或者进一步的添加DNA保护剂。在一些实施方案中，所述DNA保护剂为有DNA保护作用的自由基阱，包括但不限 于：氢醌、6-羟基-2，5，7，8-四甲基苯并二氢吡喃-2-羧酸、6-羟基-2，5，7，8-四甲基色满-2-甲酸中的一种或多种的有机溶液，如二乙二醇二甲醚溶液、氯仿溶液或四氢呋喃溶液。在一些实施方案中，转化温度为80～100℃，转化时间为40～60min。
在一些实施方案中，在本发明的试剂盒中，结合液包括但不限于离液序列高的盐如盐酸胍、异硫氰酸胍等盐类溶液。
在一些实施方案中，在本发明的试剂盒中，清洗液的pH在6～10范围内均可，但优选8～10。
在一些实施方案中，在本发明的试剂盒中，缓冲液包括但不限于纯化水、TE缓冲液、氢氧化钠弱碱溶液。
在一些实施方案中，本发明提供一种DNA甲基化检测方法，该方法包括以下步骤：
浓缩：生物样品在裂解液作用下细胞裂解、核酸释放，磁珠在裂解液高浓度高离液盐环境下与核酸接触，形成磁珠-核酸复合物，样本裂解与磁珠吸附核酸同步进行；
转化：使用转化试剂处理步骤(1)浓缩得到的磁珠-核酸复合物，得到转化后DNA；
再浓缩：在结合液作用下，磁珠与转化后DNA重新接触，形成磁珠-核酸复合物，清洗液淋洗后再经缓冲液洗脱得到转化后DNA；
检测：测定步骤(3)得到的转化后DNA。
在一些实施方案中，根据上述检测方法得到的DNA甲基化信息可以直接用于实验室和临床研究。
在本发明的方法中，步骤(1)中样本裂解与磁珠吸附核酸同步进行；
在本发明的方法中，步骤(2)中的转化后DNA无需脱磺处理即可进行检测；
在本发明的方法中，步骤(4)中的PCR检测只需在预变性阶段增加15分钟处理即可省去试剂脱磺过程。
在本发明的方法中，步骤(4)中的方法可以是染料法也可以是探针法，可以进行PCR定性检测也可以进行PCR定量检测，所得PCR产物也可以进行测序分析。
在一些实施方案中，生物样品可以选自任何含有DNA的样品，包括但不限于：无细胞来源的样品或细胞来源的样品；任选的包括但不限于：血液、尿液、粪便、细胞培养液、组织、痰液、胸水、腹水、脑脊液以及这些样品的处理品，任选地所述生物样品可以来自健康受试者和/或患者，例如癌症患者。
本发明所提供的方法的整个技术过程实现单管反应，操作简便，核酸回收率高，降低污染和出错概率。基于本发明所提供的上述方法而制备的试剂盒使得上述方法的实施更加简便快捷。
本发明已经发现，在裂解步骤中可以同时添加磁珠和裂解液实现同步裂 解和磁珠结合，不会影响裂解的效果。另外，可以添加核酸结合促进剂，增强裂解液中的核酸与磁珠表面的疏水作用。通过这样处理，本发明可以在极大程度上简化核酸提取的步骤。还发现，通过使含有DNA的生物样品与有核酸吸附能力的载体、裂解液同时接触，将DNA富集于载体上后，能够不进行任何其他处理(如洗涤处理)，直接使用上述处理获得的吸附有DNA的载体与转化试剂接触，将载体上富集的DNA的至少一个未甲基化的胞嘧啶碱基转化为尿嘧啶或在杂交上可检测的不同于胞嘧啶的其它碱基。在转化步骤后，可以再用结合液处理前步得到的混合液，使转化后的DNA再富集于载体上。在进行PCR检测时，可以采用洗脱液将转化后的DNA从载体上洗脱后检测，也可以使用PCR缓冲液直接重悬富含DNA的载体直接进行PCR扩增。由此，DNA的富集、转化、回收、检测等均可以在含有磁珠的单个试剂管中完成，省去了繁琐的洗脱和/或漂洗过程(例如裂解后和/或转化后的洗脱和/或漂洗步骤，以及转化后脱磺处理步骤等)，降低了DNA的损耗，提高了核酸回收效率。
本发明的方法和产品的操作简便。例如，生物样本可加或不加蛋白酶K加热孵育；提取得到的DNA无需从磁珠上分离即可直接进行甲二基化处理；省去了多次漂洗和氢氧化钠脱磺过程。从样本到获得上机的模板整个过程在1个试剂管中即可完成。
本发明的方法和产品节省时间。例如，本发明的方法和产品仅需3h即可完成从生物样品获得上机检测样品的全过程。
附图说明
图1本发明的方法用于区分Septin9基因的甲基化和未甲基化：Septin9基因中的CpG在Hela细胞中是甲基化的，在Jurkat细胞中是未甲基化的。
图2显示裂解与结合同步进行对对核酸浓缩效果影响；
图3显示本发明DNA可在磁珠上直接转化。
图4显示转化前DNA序列。
图5显示DNA 80℃转化40min后序列。
图6显示DNA 100℃转化60min后序列。
图7显示DNA从磁珠上洗脱后进行转化的检测限。
图8显示DNA在磁珠上直接转化的检测限。
图9显示本发明适于尿液DNA甲基化的检测。
图10显示本发明适于粪便DNA甲基化的检测。
具体实施方式
下面通过实施例对本发明所提供的甲基化DNA的浓缩和检测方法进行详细说明。
实施例1：
septin9基因甲基化的检测
样品的预处理：在2毫升健康人血浆样本中分别投入等量的打断的Hela和Jurkat细胞DNA；
浓缩：加入4毫升裂解液(所述裂解液由5.22M异硫氰酸胍、0.42M的TE缓冲液和17％的曲拉通组成)和40微升磁珠(海狸，羟基磁珠)，室温振摇10分钟，磁力架吸附磁珠后弃上清。
转化：加入320微升转化试剂(所述转化试剂为5.34M的亚硫酸氢铵、0.53M的亚硫酸钠、0.05M保护剂(保护剂组分6-羟基-2，5，7，8-四甲基苯并二氢吡喃-2-羧酸的四氢呋喃溶液的混合液))重悬磁珠，上述混合物在85℃恒温孵育40分钟。
再浓缩：在混合物中加入1毫升的结合液(7M盐酸胍)，室温振摇10分钟。磁珠经清洗液漂洗2次，再经60微升缓冲液洗脱。
检测：设计特异性的PCR引物和探针参考文献：Warren，Jorja D，et al.，Septin9methylated DNA is a sensitive and specific blood test for colorectal cancer，BMC MEDICINE，2011，9：133中设计的引物、探针及PCR扩增程序方法。
Septin9基因的检测区域在Hela细胞中是异常甲基化的，在Jurkat细胞中是甲基化状态是正常的。在上述处理过的样本中，区分出该基因靶区域的甲基状态差异，如图1所示。该结果表明，利用本发明提供的方法可以实现DNA的提取及同步富集到载体磁珠上，后续直接在磁珠上转化，进而再富集后用于检测。图1显示本发明的方法进行基因甲基化检测的结果。
实施例2：
在本实施例中，比较了裂解液与磁珠同步加入和裂解液与磁珠依次加入的效果。磁珠依次加入方法的其余步骤与实施例1步骤相同，对同一批次样本进行核酸浓缩。得到DNA采用实时荧光PCR进行甲基化检测，检测结果见图2。该结果表明，样本中加入裂解液的同时可以同步加入磁珠，不会影响裂解、结合的效果，使结合的步骤与裂解的步骤同时完成，从而极大程度上简化了核酸提取的步骤。图2显示裂解与结合同步进行对对核酸浓缩效果影响。
实施例3：
DNA被磁珠富集后直接进行转化
在2mL健康人血浆样本中投入打断的Hela细胞DNA。1份完全按照实施例1进行处理，1份步骤(1)按照下述步骤处理：
浓缩：加入4毫升裂解液和40微升磁珠，室温振摇10分钟，磁力架吸附磁珠后弃上清，再经淋洗和100微升缓冲液(TE缓冲液，pH＝8.0)洗脱。
转化：加入220微升的转化试剂(所述转化试剂为7.8M的亚硫酸氢铵、0.78M的亚硫酸钠、0.07M的DNA保护剂(保护剂组分6-羟基-2，5，7，8-四甲基苯并二氢吡喃-2-羧酸的二乙二醇二甲醚溶液的混合液)，上述混合物在85℃保温40分钟。
再浓缩：在混合物中加入1毫升的结合液(7M盐酸胍)和40微升磁珠，室温振摇10分钟。
磁珠经清洗液漂洗2次，再经60微升缓冲液(TE缓冲液，pH＝8.0)洗脱最后，PCR检测两种方式差异。
如图3所示，DNA不从磁珠上分离下来可以直接进行转化，而且减少中间DNA洗脱步骤后，核酸回收效率得到提高，表明磁珠富集核酸后无需进行中间DNA洗脱环节即可直接进行转化过程，且纯化时无需再加入新的磁珠。图3显示本发明DNA在磁珠上直接转化的结果。
实施例4：
DNA转化温度及转化时间
本实施例采用不同组别的转化温度及转化时间，其余步骤均采用实施例1的使用方法，对同一批次样本进行核酸提取转化。通过不同转化温度及转化时间方法得到DNA采用实时荧光PCR进行甲基化检测，检测结果见表1：
表1：经不同转化条件处理后的甲基化DNA检测结果
Figure PCTCN2019123433-appb-000001
可以看出，采用实时荧光PCR方法检测甲基化情况，本转化试剂在80～100℃下均正常进行转化，各温度检测Cp值相当；同时，通过对相同温度条件下不同孵育时间对转化效果的影响分析可知，采用本发明的转化试剂进行恒温转化，仅需40分钟即可得到很好的效果，大大节省了转化时间。相对于传统的变温方法而言，本发明采用恒温的方法更简单，易操作，能使用便宜、简便恒温仪器进行相关实验研究。
此外，我们对优化得出的转化温度、转化时间下的DNA转化效率进行评价。
参考文献(Emily Eva Holmes，MariaJung，Sebastian Meller，etal.Performance Evaluation of Kits for Bisulfite-Conversion of DNA from Tissues，Cell Lines，FFPE Tissues，Aspiraes，Lavages，Effusions，Plasma，Serum and Urine.PLOSONE，2014，9(4)：e93933.)报道中的测定转化效率的方法：以上述人基因组DNA为研究对象，使用不含“C”的通用引物，对转化前和转化后的DNA进行扩增，扩增区域内含有30个“C”。其中，转化前DNA扩增后进行测序，以确认DNA原始序列中“C”的状态。转化后DNA的扩增产物进行TA克隆，每个TA克隆产物随机挑20个转化子，对其进行测序，其中20个转化子中，共含有“C”600个，统计“C”转化成“T”的个数，若为N，则转化效率为：(N/600)×100％。
转化前后测序结果如图4-6所示：图4显示转化前DNA序列。图5显示DNA80℃转化40min后序列。图6显示DNA 100℃转化60min后序列。
从中可以看出，转化前的C基本转化为T；统计测序结果，计算转化效率。(1)样品转化前状态：模板中30个“C”的状态：均为“C”。(2)样品转化后状态(80℃转化40min)：20个转化子：600个“C”中共有598个“C”在转化后转化成“T”，转化效率＝99.7％。(3)样品转化后状态(100℃转化60min)：20个转化子：600个“C”在转化后全部转化为“T”，转化效率＝100％；综上所述，在本试剂盒的转化效率≥99.7％。
实施例5：
在1mL健康人血浆样本中投入10ng/mL的打断的Hela和Jurkat细胞DNA，其中Hela细胞DNA与Jurkat细胞掺比分别为10％、1％、0.5％、0.1％、0.05％、0％。分别按照实施例1和实施例2进行处理。对比检测下限，结果如图7-8所示。图7显示DNA从磁珠上洗脱后进行转化的检测限。图8显示DNA在磁珠上直接转化的检测限。
从图中我们可以看出，传统的DNA从磁珠上洗脱后再进行转化处理过程检测下限到0.5％，而本发明提供的方法检测下限达到0.05％，具有显著效果。
实施例6：肠血浆样本中septin9基因甲基化的检测
检测30例结直肠癌、30例健康人血浆样本。分别采用对照试剂盒(EPI公司试剂盒)和本发明的方法进行样本前处理，采用EPI试剂盒中PCR试剂、程序进行Septin9三孔检测。检测结果统计如下表：
表2本发明的方法和对照方法的比较结果
Figure PCTCN2019123433-appb-000002
Figure PCTCN2019123433-appb-000003
Figure PCTCN2019123433-appb-000004
表3：本发明方法和对照方法的血浆DNA甲基化检测结果
Figure PCTCN2019123433-appb-000005
从上表可以看出，本发明在结直肠癌样本的阳性检出孔数上具有较大优势，同时检测特异性仍得以保持。
实施例7：尿液样本中NID2基因甲基化的检测
检测10例膀胱癌和10例泌尿系统良性疾病全尿样本。本实施例采用方法的具体步骤同实施例1，不同之处在于，相应的检测引物不同，裂解液组分不同，(所述裂解液由2.75M异硫氰酸胍、0.22M的TE缓冲液、9％的曲拉通和47％异丙醇组成)。最终得到的转化的DNA直接用Methylate方法(Weisenberger DJ，Campan M，Long TI，Kim M，Woods C，et al.(2005)Analysisof repetitive element DNA methylation by MethyLight.Nucleic Acids Res 33：6823-6836)进行NID2甲基化检测，显示均具有较好的区分度。图9显示本发明方法尿液DNA甲基化的检测结果。
实施例8：粪便样本中BMP3基因甲基化的检测
检测10例结直肠癌(CRC)、10例进展期腺瘤(AA)和10例非进展期腺瘤(NAA)粪便样本BMP3基因甲基化情况，采用ΔΔct方法评价。本实施例采用方法的具体步骤同实施例1，最终得到的转化的DNA直接用Methylate方法(Weisenberger DJ，Campan M，Long TI，Kim M，Woods C，et al.(2005)Analysisof repetitive element DNA methylation by MethyLight.Nucleic Acids Res 33：6823-6836)进行甲基化检测，显示均具有较好的区分度。图10显示本发明方法粪便DNA甲基化的检测结果。

Claims (12)

  1. 一种DNA甲基化分析方法，包括1)使含有DNA的生物样品与有核酸吸附能力的载体、裂解液同时接触，将DNA富集于载体上；2)直接使步骤1)获得的吸附有DNA的载体与转化试剂接触从而将载体上富集的DNA的至少一个未甲基化的胞嘧啶碱基转化为尿嘧啶或在杂交上可检测的不同于胞嘧啶的其它碱基，3)再用结合液处理步骤2)得到的混合液，使转化后的DNA再富集于载体上。
  2. 权利要求1所述的方法，其中步骤1)还包括添加核酸结合促进剂，所述核酸结合促进剂包括促进裂解液中的核酸与有核酸吸附能力的载体结合的有机溶剂和/或润湿剂中的至少一种，例如异丙醇、异丁醇、正丁醇、丙酮、吡啶、乙腈、甲酸甲酯、乙酸乙酯、丙二醇、甘油、二甲亚砜、聚乙二醇、烷基硫酸盐、磺酸盐或酯、多元醇表面活性剂(例如司盘类、吐温类表面活性剂)、聚氧乙烯表面活性剂。
  3. 权利要求1或2所述的方法，其中所述裂解液包含胍盐，如盐酸胍、异硫氰酸胍，任选地还包含去垢剂如SDS，金属离子螯合剂如EDTA，金属盐如NaCl，任选地所述裂解液配制成缓冲溶液，如三羟甲基氨基甲烷缓冲液、柠檬酸缓冲液，任选地步骤1)进一步包括使所述含有DNA的生物样品与蛋白酶如蛋白酶K接触。
  4. 权利要求1-3任一项所述的方法，其中所述转化试剂包括亚硫酸氢盐、焦亚硫酸盐或其组合，例如亚硫酸氢钠、亚硫酸氢镁、亚硫酸氢铵、焦亚硫酸钾、焦亚硫酸钠、亚硫酸钠中的一种或多种，例如亚硫酸氢钠、亚硫酸钠的混合溶液。
  5. 权利要求1-4任一项所述的方法，其中步骤2)的转化过程中还添加有DNA保护作用的自由基阱，包括但不限于：氢醌、6-羟基-2，5，7，8-四甲基苯并二氢吡喃-2-羧酸、6-羟基-2，5，7，8-四甲基色满-2-甲酸中的一种或多种的有机溶液，如二乙二醇二甲醚溶液、氯仿溶液或四氢呋喃溶液。
  6. 权利要求1-5任一项所述的方法，其中有核酸吸附能力的载体包括但不限于：磁珠、非磁性的微球、吸附膜。
  7. 权利要求6所述的方法，其中所述载体包括但不限于：羟基磁珠、羧基磁珠、链霉亲和素免疫磁珠、离子交换树脂，磁珠的壳层优选氧化硅或琼脂糖，磁珠优选超顺磁珠，磁珠粒径为100nm～3μm，优选500-1μm。
  8. 权利要求1-7任一项所述的方法，还包括转化后处理步骤，从而制备适于直接进行后续甲基化检测的样品，所述转化后处理步骤包括使用清洗液对转化后的富集于磁珠上的核酸进行清洗的步骤，任选地所述清洗液包括三羟甲基氨基甲烷溶液、三羟甲基氨基甲烷-盐酸溶液、乙醇溶液，任选地所述清洗液的pH在6～10范围内，优选pH为8～10，优选地所述转化后处理不包括脱磺处理。
  9. 权利要求1-8任一项所述的方法，其中所述生物样品选自任何含有DNA的样品，包括但不限于：无细胞来源的样品或细胞来源的样品；任选的包括但不限于：血液、尿液、粪便、细胞培养液、组织、痰液、胸水、腹水、脑脊液以及这些样品的处理品，任选地所述生物样品可以来自健康受试者和/或患者，例如癌症患者。
  10. 权利要求1-9任一项所述的方法，其中所述结合液包括离液序列高的盐，如胍盐，例如盐酸胍、异硫氰酸胍，任选地所述结合液可以加入缓冲溶液。
  11. 适合于权利要求1-10任一种方法的用于DNA甲基化分析的试剂盒，其使用说明中包含权利要求1-10任一项所述的方法，其包含一个或多个容器，所述容器中包含权利要求1-10中任一项中限定的试剂。
  12. 权利要求11所述的试剂盒，所述试剂盒中进一步包括对转化处理后的DNA进行检测的试剂，例如PCR试剂，探针，测序试剂。
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