Method for enriching DNA methylated fragments based on immunoprecipitation technology

Abstract

The invention relates to a method for enriching DNA methylated fragments based on an immunoprecipitation technology. According to the method, a magnetic bead-antibody-nucleic acid binding environment is optimized, a proper amount of modified lambda DNA is added as a DNA methylation protective agent, a competitive binding mode is adopted, non-specific binding caused by self reasons of the antibody is reduced, washing steps are optimized, magnetic bead antibody nucleic acid conjugates are washed by adopting different salt ion concentration gradients, and the detection accuracy is improved. Non-specific enrichment is reduced, and the binding efficiency of methylated genes is effectively improved.

Description

Technical Field

[0001] The present invention relates to the field of biomedicine, and more specifically, to a method for enriching DNA methylation fragments based on immunoprecipitation technology. Background Art

[0002] DNA methylation generally refers to the methylation status of the 5th carbon position of cytosine in genomic CpG. As an important epigenetic regulatory mechanism, it plays an important role in development and disease progression. Abnormal DNA methylation is an important early event in the occurrence and development of cancer.

[0003] In normal human DNA, 2% to 7% of cytosine is methylated, among which CpG is the most important methylation site. It is unevenly distributed in the genome, with high methylation, low methylation and non-methylation regions. In certain regions of the genome, such as the promoter region, 5'UTR (untranslated region) and the first exon (exon), the sequence density of CpG is very high. In general, high DNA methylation in the promoter region is associated with gene silencing, while low methylation is associated with gene activation. A large number of studies have shown that the inactivation of tumor suppressor genes is directly related to the excessive methylation of CpG islands in the promoter region of the gene, which leads to the occurrence of cancer.

[0004] For genomic methylation analysis, the main method is currently sulfite treatment. The sample DNA is treated with bisulfite to convert the unmethylated cytosine in the DNA into uracil, which is further converted into thymine after PCR amplification, while the originally methylated C remains unchanged, thereby effectively converting epigenetic variation into sequence differences. However, the process of DNA treatment by bisulfite conversion includes steps such as denaturation, deamination and desulfonation. The DNA is first denatured into a single strand, and then subjected to high temperature, high salt, acidic and alkaline environments, encountering two extremes of ice and fire. The morphology of the converted DNA obtained is mainly single-stranded, double-stranded, mixed, fragment nicks, gap damage, and uracil state nucleotides. This process generally causes more than 90% of the DNA template to be lost, and a large amount of methylation information cannot be detected by subsequent processes. At the same time, this method has incomplete or excessive sequence conversion during base conversion processing, resulting in artificial bias, which will be further amplified by subsequent PCR amplification, resulting in a large amount of data waste. In order to reduce the occurrence of bias, it is necessary to invest more DNA, reduce the number of PCR cycles and optimize the efficiency of PCR amplification enzymes.

[0005] The principle of methylated DNA immunoprecipitation (MeDIP) is that since mammalian methylation usually occurs on the 5th carbon atom (5mc) of cytosine in CpG, highly methylated fragments can be enriched by using 5-methylcytosine antibodies that can specifically bind to 5mc. Moreover, since 5-methylcytosine antibodies can also recognize 5mc on non-CpG sites, they have higher specificity. This method avoids the disadvantages of traditional methods such as large losses, high input, and data waste. However, there are no corresponding kits in China at present, and they are all imported and expensive. Therefore, finding optimized or alternative methods is the research focus of this field. Summary of the invention

[0006] The present invention provides a method for enriching DNA methylation fragments based on immunoprecipitation technology, comprising the following steps:

[0007] S1. adding λDNA as a DNA methylation protective agent to the sample to be tested to obtain a sample with added λDNA;

[0008] S2. Mix the immunoprecipitation buffer and the sample with added λDNA, incubate at 90-95°C for 5-20 minutes, and then cool rapidly to obtain a mixed solution;

[0009] S3. Add the antibody and magnetic beads to the mixture obtained in step S2 and incubate at 2-8°C for 4-16 hours;

[0010] S4. After instant centrifugation, place on a pre-cooled magnetic rack and discard the supernatant; wash the magnetic beads with washing solution;

[0011] S5. Elute the DNA with elution buffer at 50-60℃ / 15min, 95-100℃ / 15min.

[0012] Preferably, in step S1, in the DNA methylation protective agent, the mass ratio of methylated λDNA:unmethylated λDNA is 1:1;

[0013] Preferably, in the sample to which λDNA is added, the mass ratio of cfDNA to DNA methylation protectant is 1:10-1:40, wherein the concentration of cfDNA is not less than 0.01 ng / μL.

[0014] Preferably, in step S2, during mixing, the volume ratio of the immunoprecipitation buffer and the sample to which λDNA is added is (30-35) μL:(55-60) μL.

[0015] Preferably, in step S2, the immunoprecipitation buffer comprises 0.1-0.2 M sodium phosphate solution, 1-2 M sodium chloride solution, and 1%-5% Triton X-100.

[0016] Preferably, step S3 further includes a step of washing the magnetic beads and a step of diluting the antibody:

[0017] Washing step of magnetic beads: take 2-6 μL of 10 mg / mL magnetic bead stock solution and wash the magnetic beads with ten-fold diluted immunoprecipitation buffer, wash at least twice, and resuspend with 20 μL of ten-fold diluted immunoprecipitation buffer for the last time to obtain a magnetic bead suspension with a concentration of 1-3 mg / mL;

[0018] Antibody dilution step: Dilute the 5-methylcytosine antibody at a concentration of 1 mg / mL by 10-100 times using ten-fold diluted immunoprecipitation buffer to obtain a solution with an antibody concentration of 0.01-0.1 mg / mL.

[0019] Preferably, in step S3, the antibody, magnetic beads, and the mixed solution obtained in step S2 are mixed in the following volume ratio: 1:1:(15-20).

[0020] Preferably, in step S3, the magnetic beads are IgG magnetic beads.

[0021] Preferably, in step S3, the antibody is a 5-methylcytosine antibody.

[0022] Preferably, in step S4, the washing solution comprises a ten-fold diluted immunoprecipitation buffer and an immunoprecipitation buffer with a high sodium chloride salt concentration; the immunoprecipitation buffer with a high sodium chloride salt concentration comprises 0.01-0.02M sodium phosphate solution, 1-4M sodium chloride solution, and 0.1%-0.5% Triton X-100.

[0023] Preferably, in step S5, the elution solution comprises 0.01-0.1 M Tris-HCl, 0.01-0.1 M EDTA, 0.001-0.1% SDS, and 0.1-1 mg / mL proteinase K.

[0024] Beneficial Effects

[0025] The present invention aims at the low starting amount of human peripheral blood free DNA, optimizes the binding environment and adds an appropriate amount of modified λDNA as a DNA methylation protectant, adopts a competitive binding method, reduces the nonspecific binding caused by the antibody itself, improves the specificity of MeDIP binding, and reduces nonspecificity. Compared with the BS method, the binding efficiency of methylated genes is effectively improved. In addition to the significant effect of the efficient and specific methylated fragment enrichment of peripheral blood free DNA, the present invention is also applicable to the methylation enrichment of genomic DNA in clinical tissue samples such as paraffin sections, cancer tissues, etc. BRIEF DESCRIPTION OF THE DRAWINGS

[0026] Figure 1The figure shows the comparison of methylation recovery efficiency with the Diagenode commercial kit based on the same principle.

[0027] Figure 2 The results are shown in comparison with the traditional method bisulfite conversion (BS) in the detection of methylation levels.

[0028] Figure 3 The results are shown in comparison with the methylation recovery efficiency of Takara's commercial kit based on the MBD enrichment principle.

[0029] Figure 4 Comparative results of the binding efficiency of methylated genes (meDNA) and unmethylated genes (unDNA) are shown. DETAILED DESCRIPTION

[0030] The present invention provides a non-bisulfite conversion, non-enzymatic conversion, 5mC detection method for enriching and purifying human peripheral blood free DNA methylation, which uses antibodies to capture methylated DNA and uses the antibodies to immunoprecipitate the methylated DNA. The method can separate specific DNA from a sample and requires that the antibody be coupled to a fixed matrix (such as magnetic beads) within a required time range to separate the DNA, comprising the following steps:

[0031] S1. Add Escherichia coli phage DNA (λDNA) as a DNA methylation protective agent to the sample to be tested, that is, obtain a sample with added λDNA by quantitatively adding methylated λDNA and non-methylated λDNA as protective agents for competitive binding.

[0032] The starting mass of cfDNA samples is generally not less than 0.5ng; the mass ratio of cfDNA to DNA methylation protectant is in the range of 1:10-1:40. The volume after mixing has little effect on the subsequent reaction, and technicians in this field can adjust the total volume of the sample mixture to which λDNA is added according to actual conditions.

[0033] For example, the configuration of the sample with added λDNA is as follows: the mass of the cfDNA sample starts at 0.5ng, the volume of the cfDNA sample is 42μL, the concentration of the DNA methylation protectant is 50ng / μL, the volume of the added DNA methylation protectant is 0.1-15μL, so that the mass ratio of cfDNA to DNA methylation protectant is in the range of 1:10-1:40, and water is added to make the total volume of the sample mixture with added λDNA reach the selected volume, for example, 55-60μL.

[0034] Preferably, in the sample to which λDNA is added, the mass ratio of cfDNA template to DNA methylation protectant (λDNA) is 1:10-1:40.

[0035] Preferably, the mass ratio of methylated λDNA:unmethylated λDNA is 1:1.

[0036] S2. Mix the immunoprecipitation buffer and the sample to which λDNA is added (for example, by gently pipetting and mixing), incubate at 90-95°C for 5-20 minutes, and then cool rapidly to obtain a mixed solution.

[0037] Preferably, during mixing, the volume ratio of the immunoprecipitation buffer and the sample to which λDNA is added is (30-35) μL:(55-60) μL.

[0038] S3. Add the antibody and magnetic beads to the mixed solution obtained in step S2, and incubate at 2-8°C for 4-16 hours, for example, at 4°C for 8 hours.

[0039] Preferably, this step S3 also includes pre-treatment steps: a magnetic bead washing step and an antibody dilution step.

[0040] S031. Magnetic bead washing step: Pipette 2-6 μL of magnetic bead stock solution (10 mg / mL) and wash the magnetic beads with ten-fold diluted immunoprecipitation buffer, wash at least twice, and resuspend with 20 μL of ten-fold diluted immunoprecipitation buffer for the last time to obtain a magnetic bead suspension with a concentration of 1-3 mg / mL, preferably a 2.5 mg / mL magnetic bead suspension.

[0041] S032. Antibody dilution step: dilute the 5-methylcytosine antibody with a stock concentration of 1 mg / mL by 10-100 times using a ten-fold diluted immunoprecipitation buffer to obtain a solution with an antibody concentration of 0.01-0.1 mg / mL, preferably 0.02-0.05 mg / mL.

[0042] Preferably, the antibody, magnetic beads, and the mixed solution obtained in step S2 are mixed in the following ratio: 1 μL antibody: 1 μL magnetic beads: 15 μL mixed solution obtained in step S2.

[0043] Preferably, the magnetic beads are 2.8 μm superparamagnetic microbeads with affinity-purified IgG covalently coupled to their surfaces. For example, Dynabeads TM M-280 goat anti-mouse IgG magnetic beads (Thermo Fisher Scientific (USA), catalog number: 11202D), DiaMag anti-mouse IgG coated magnetic beads (Diagenode (Belgium), catalog number: C03010022-220), IgGMagBeads Goat anti-Mouse (Yisun Biotechnology (Shanghai) Co., Ltd., catalog number: 47502ES03).

[0044] Preferably, the antibody is a 5-methylcytosine antibody (monoclonal antibody, MeDIP / CHIP grade), for example, mouse monoclonal antibody [33D3] to 5-methylcytosine (5-mC) (product name: Anti-5-methylcytosine (5-mC) antibody [33D3], Abcam (Shanghai) Trading Co., Ltd., catalog number: ab10805), 5-Methylcytosine (5-mC) Monoclonal Antibody [33D3] (Epigentek, catalog number: A-1014-010), 5-methylcytosine (5-mC) Antibody-clone 33D3 (Diagenode, catalog number: C15200003).

[0045] S4. After brief centrifugation, place on a pre-cooled magnetic rack and discard the supernatant; wash the magnetic beads with washing solution.

[0046] Preferably, at least two washing solutions (such as immunoprecipitation buffer, 1-4M NaCl solution, etc.) are used to wash the magnetic bead precipitate, and the washing is performed at least four times, and the residual liquid is discarded each time by centrifugation. More preferably, one of the washing solutions is: a ten-fold diluted immunoprecipitation buffer; the other washing solution is a ten-fold diluted immunoprecipitation buffer with a high sodium chloride salt concentration, and its components are 0.01-0.02M (mol / L) sodium phosphate solution (PH=7.0), 1-4M sodium chloride solution, 0.1%-0.5% Triton X-100; preferably, the components are 0.02M sodium phosphate solution, 2M sodium chloride solution, and 0.5% Triton X-100.

[0047] More preferably, two washing solutions with different salt ion concentrations are combined and gradient dilutions with different salt ion concentrations are performed to further reduce non-specific amplification. Specifically, the cells are first washed twice with a ten-fold diluted immunoprecipitation buffer with a low sodium chloride salt concentration, and then washed twice with a ten-fold diluted immunoprecipitation buffer with a high sodium chloride salt concentration.

[0048] In each of the above steps, the ten-fold diluted immunoprecipitation buffer comprises a 0.01-0.02M (mol / L) sodium phosphate solution (PH=7.0), a 0.1-0.2M sodium chloride solution, and 0.1%-0.5% Triton X-100; preferably, the components are 0.02M sodium phosphate solution, 0.2M sodium chloride solution, and 0.5% Triton X-100.

[0049] The operation method is to dilute the immunoprecipitation buffer tenfold. Those skilled in the art can perform the operation according to conventional procedures, for example, mixing the immunoprecipitation buffer with TE solution (10mM Tris-HCl 1mM EDTA PH=8.0) (Biyuntian, R0225) or nuclease-free water in a volume ratio of 1:9.

[0050] Ten-fold diluted immunoprecipitation means the immunoprecipitation buffer was diluted ten-fold.

[0051] S5. Elute the DNA with an elution buffer at 50-60°C / 15 min, 95-100°C / 15 min, for example, at 55°C / 15 min, 100°C / 15 min.

[0052] The present invention does not limit the eluent, and those skilled in the art can select commercial products or prepare them by themselves according to their needs.

[0053] Preferably, the elution solution comprises 0.01-0.1 M Tris-HCl (pH 8.0), 0.01-0.1 M EDTA, 0.001-0.1% SDS, and 0.1-1 mg / mL proteinase K.

[0054] The test data was analyzed and the recovery efficiency was used as the evaluation index. The formula is as follows:

[0055] Recovery efficiency (%) = 2^(Ct 10％input -Ct 样本 -3.32)×100

[0056] During the test, 10% of the sample was aspirated as the 10% input sample, and the remaining sample was processed later. The two were amplified together by qPCR, and Ct 10％input Indicates the Ct value of 10% input sample amplification; Ct 样本 Indicates the Ct value of the amplified sample.

[0057] The principle of the above method is: based on the specific binding of antigen and antibody, the buffer in step S2 provides the ionic environment required for the reaction, the temperature of the reaction denatures the DNA, and then in step S3, 5mc specifically binds to the IgG coated on the magnetic beads to enrich the DNA methylation fragments. The preferred binding environment and the purity quality of the 5mc antibody will affect the enrichment recovery rate. Steps S4 and S5 are elution steps. After washing the magnetic beads with a washing solution, proteinase K is used to digest the 5mc antibody and the IgG protein coated on the magnetic beads to release the captured methylated DNA. The eluent and temperature mainly provide the action environment of proteinase K so that proteinase K can play a role. The reagents used in this method are basically ionic reagents, which reduces the experimental cost, and compared with the traditional BS method, the damage to the methylated DNA fragments is small and the recovery rate is high, so as to more accurately identify cancer-related signals and greatly improve the detection accuracy.

[0058] By optimizing the binding environment and adding an appropriate amount of modified λDNA (50-200ng, methylated λDNA: unmethylated λDNA=1:1) as a DNA methylation protectant, the nonspecific binding caused by insufficient antibody purity, weak specificity, etc. is reduced through competitive binding, thereby improving the specificity of MeDIP binding and reducing nonspecificity.

[0059] Example 1

[0060] Follow the steps below to complete the enrichment of methylated DNA.

[0061] S1. Add 50-200 ng of λDNA, for example, 120 ng of λDNA, to a cfDNA sample with a mass of 5 ng, wherein the mass ratio of methylated λDNA to unmethylated λDNA is 1:1. In this embodiment, 0.1-15 μL of a DNA methylation protective agent with a concentration of 50 ng / μL is added to a sample to be tested with a mass of cfDNA of not less than 0.5 ng, so that the mass ratio of cfDNA to DNA methylation protective agent is in the range of 1:10-1:40, and the volume of the sample to which λDNA is added is supplemented to 57 μL.

[0062] S2. Mix 33 μL of immunoprecipitation buffer and 57 μL of sample with λDNA added, incubate at 95℃ for 10 min, and cool on ice for 15 min. Then take 7.5 μL of the reaction solution as input and store at 2-8℃. Take 75 μL of the remaining solution for subsequent experiments.

[0063] S3. Washing the magnetic beads: Pipette 2-6 μL of the magnetic bead stock solution (10 mg / mL) and wash the magnetic beads twice with ten-fold diluted immunoprecipitation buffer, and resuspend them for the last time with 20 μL of ten-fold diluted immunoprecipitation buffer to obtain a magnetic bead suspension with a concentration of 2.5-3 mg / mL.

[0064] Dilute the antibody: Dilute the 5-methylcytosine antibody with a stock concentration of 1 mg / mL by 10-100 times using ten-fold diluted immunoprecipitation buffer to obtain an antibody solution with a concentration of 0.03-0.05 mg / mL.

[0065] Add 5 μL of antibody solution and 5 μL of magnetic bead suspension to 75 μL of the mixture obtained in step S2, and incubate at 4°C with rotation for 4-16 hours.

[0066] S4. Add 100 μL of ten-fold diluted immunoprecipitation buffer in an ice bath, invert to resuspend the magnetic beads, rotate and incubate at 4°C for 5 min (40 rpm), centrifuge immediately after the end, place on a pre-cooled magnetic stand, wait for 1 min, and discard the supernatant. Repeat the same steps at least once.

[0067] Add 100 μL of immunoprecipitation buffer with high sodium chloride concentration in an ice bath, invert and resuspend the magnetic beads, rotate and incubate at 4°C for 5 minutes (40 rpm), centrifuge immediately after the end, place on a pre-cooled magnetic stand, wait for 1 minute, and discard the supernatant. Repeat the same steps at least once.

[0068] The components of the immunoprecipitation buffer with high sodium chloride salt concentration are: 0.02M sodium phosphate solution, 2M sodium chloride solution, and 0.5% Triton X-100.

[0069] S5. Elute the DNA with elution buffer at 55°C / 15 min and 100°C / 15 min.

[0070] The components of the elution buffer are: 0.05 M Tris-HCl, 0.01 M EDTA, 0.001% SDS, and 0.4 mg / mL proteinase K.

[0071] Comparative Example 1

[0072] After extracting free DNA from human peripheral blood, the same sample was taken for enrichment according to the method in Example 1 and for methylated fragment antibody enrichment using a methylated DNA enrichment kit (eg, MagMeDIP kit, Cat. No. C02010021, Diagenode).

[0073] The results are as follows Figure 1As shown in Table 1, using the quality control products meDNA (methylation quality control product) and unDNA (unmethylation quality control product) in the Diagenode kit, the results show that the recovery rate of the methylation quality control product of the present invention is 66.5%, and the recovery rate of the methylation quality control product of Diagenode is 52.6%. The coefficient of variation CV value of the recovery rate of Diagenode is 22%, and the coefficient of variation CV value of the recovery rate of the present invention is 5%, which shows that the recovery rate of the present invention is relatively stable, the method has good reproducibility, and the result is highly reliable.

[0074] Table 1: Stability evaluation of methylation recovery rate compared with Diagenode commercial kit

[0075]

[0076] Comparative Example 2

[0077] After free DNA was extracted from human peripheral blood, the same sample was enriched according to the method in Example 1 and the bisulfite conversion method (BS method), and then sequenced by qPCR.

[0078] The sample detection data were analyzed. The amplification curves of the MeDIP method and the BS method are shown in the attached figure. Figure 2 Attached Figure 2 The qPCR test results of the four target genes of the same colorectal cancer positive sample using both the MeDIP and BS methods are shown. The results showed that the CT value of the MeDIP method in the SEPTIN9 gene was 30.7, and the BS method was 35.4, indicating that the methylation enrichment level of the MeDIP method was 26.0 times that of the BS. The detection results of the SDC2 gene were 31.1 for the MeDIP method and 36.1 for the BS method, indicating that the methylation enrichment level of the MeDIP method was 32 times that of the BS (methylation enrichment factor = 2 (CtBS-CtMeDIP) ). The detection results of ITGA4 gene showed that the CT value of MeDIP method was 32.5, and that of BS method was 36.8, indicating that the methylation enrichment level of MeDIP method was 19.7 times that of BS. The detection results of IRF4 gene showed that the CT value of MeDIP method was 34.5, and that of BS method was 38.1, indicating that the methylation enrichment level of MeDIP method was 12.1 times that of BS. It can be seen that the methylation enrichment efficiency of MeDIP is better than that of the traditional BS detection method, and its enrichment efficiency is more than 10 times that of the traditional BS method.

[0079] Comparative Example 3

[0080] According to the method of Example 1, only the components of the immunoprecipitation buffer were adjusted without changing the steps, and the optimization effect was observed by detecting the Ct values ​​of the TSH2B gene and the GAPDH gene.

[0081] The different components of the immunoprecipitation buffer were adjusted to obtain the different combinations shown in Table 2.

[0082] Table 2

[0083]

[0084] The corresponding immunoprecipitation buffer component concentrations are shown in Table 3.

[0085] Table 3

[0086]

[0087] The test results are shown in Table 4, where P1 and P2 are colorectal cancer positive samples, N1 and N2 are normal negative samples, PC and NC are positive quality control (commercial human fully methylated genomic DNA) and negative quality control (commercial human non-methylated genomic DNA), respectively.

[0088] Table 4

[0089]

[0090] The dosage of sodium phosphate was optimized through combination 1, combination 2, and combination 3, the dosage of sodium chloride was optimized through combination 1, combination 4, and combination 5, the dosage of Trition X-100 was optimized through combination 1, combination 6, and combination 7, and finally the dosage of each component was optimized through combination 8 and combination 9. In the test results, the smaller the Ct value of TSH2B, the more methylated fragments were enriched, and the better the result, and the larger the Ct value of GAPDH, the less nonspecific enrichment, and the better the result. It can be seen that the enrichment effect of all combinations meets the requirements; among them, combination 9 has the least nonspecific detection and a high total enrichment rate. Combination 9, i.e., a combination with a sodium phosphate concentration of 0.2M, a sodium chloride concentration of 2M, and a Triton X-100 concentration of 5%, is preferred.

[0091] Comparative Example 4

[0092] In addition to the Diagenode product, the EpiXplore Methylated DNA Enrichment Kit (Takara (Japan), Catalog No. 631963) based on the MBD (methylated CpG binding domain) protein enrichment principle was also compared. The results showed that the enrichment efficiency of the present invention based on the MeDIP principle was 1.47 times that of the Takara enrichment efficiency based on the MBD principle. The cv value of Takara was 0.15, and the cv value of the present invention was 0.04, which showed that the recovery rate of the present invention was more stable ( Figure 3 ).

[0093] Comparative Example 5

[0094] According to the method of Example 1, the following conditions are changed without changing the steps:

[0095] Control group: 1) No λDNA was added; 2) The concentrations of the components of the methylation immunoprecipitation buffer were: 0.15M sodium phosphate, 1.5M sodium chloride, and 3% Triton X-100; 3) The washing process was washed 3 times with a ten-fold dilution of the immunoprecipitation buffer with a low sodium chloride salt concentration.

[0096] Example 1: 1) Add 120ng of modified λDNA (methylated λDNA: unmethylated λDNA=1:1) as a protective agent for methylated genes, 2) The concentrations of the components of the methylation immunoprecipitation buffer are: 0.2M sodium phosphate, 2M sodium chloride, and 5% Triton X-100; 3) Washing process: first wash twice with a ten-fold diluted immunoprecipitation buffer with a low sodium chloride salt concentration, and then wash twice with an immunoprecipitation buffer with a high sodium chloride salt concentration.

[0097] The results are as follows Figure 4 As shown, the average methylation gene binding efficiency of the control group is 43.8%, and the average methylation gene binding efficiency of Example 1 is 66.74%.

[0098] The above specific implementations do not constitute a limitation on the protection scope of the present invention. It should be understood by those skilled in the art that various modifications, combinations, sub-combinations and substitutions can be made according to design requirements and other factors. Any modification, equivalent substitution and improvement made within the spirit and principle of the present invention should be included in the protection scope of the present invention.

Claims

1. A method for enriching DNA methylation fragments based on immunoprecipitation technology, comprising the following steps: S1. adding λDNA as a DNA methylation protective agent to the sample to be tested to obtain a sample with added λDNA; S2. Mix the immunoprecipitation buffer and the sample with added λDNA, incubate at 90-95°C for 5-20 minutes, and then cool rapidly to obtain a mixed solution; S3. Add the antibody and magnetic beads to the mixture obtained in step S2 and incubate at 2-8°C for 4-16 hours; S4. After instant centrifugation, place on a pre-cooled magnetic rack and discard the supernatant; wash the magnetic beads with washing solution; S5. Elute the DNA with elution buffer at 50-60℃ / 15min, 95-100℃ / 15min.

2. The method according to claim 1, characterized in that In step S1, in the DNA methylation protective agent, the mass ratio of methylated λDNA:unmethylated λDNA is 1:1; In the samples with added λDNA, the concentration ratio of cfDNA to DNA methylation protectant is 1:10-1:40, and the concentration of cfDNA is not less than 0.01 ng / μL.

3. The method according to claim 2, characterized in that In step S2, during mixing, the volume ratio of the immunoprecipitation buffer and the sample to which λDNA is added is (30-35) μL:(55-60) μL.

4. The method according to claim 1, characterized in that Step S3 also includes a magnetic bead washing step and an antibody dilution step: Washing step of magnetic beads: take 2-6 μL of 10 mg / mL magnetic bead stock solution and wash the magnetic beads with ten-fold diluted immunoprecipitation buffer, wash at least twice, and resuspend with 20 μL of ten-fold diluted immunoprecipitation buffer for the last time to obtain a magnetic bead suspension with a concentration of 1-3 mg / mL; Antibody dilution step: Dilute the 5-methylcytosine antibody at a concentration of 1 mg / mL by 10-100 times using ten-fold diluted immunoprecipitation buffer to obtain a solution with an antibody concentration of 0.01-0.1 mg / mL.

5. The method according to claim 4, characterized in that In step S3, the antibody, magnetic beads, and the mixed solution obtained in step S2 are mixed according to the following volume ratio: 1:1:(15-20).

6. The method according to claim 4, characterized in that In step S3, the magnetic beads are IgG magnetic beads.

7. The method according to claim 4, characterized in that In step S3, the antibody is a 5-methylcytosine antibody.

8. The method according to any one of claims 1 to 7, characterized in that The immunoprecipitation buffer includes 0.1-0.2 M sodium phosphate solution, 1-2 M sodium chloride solution and 1%-5% Triton X-100; In step S4, the washing solution includes a ten-fold diluted immunoprecipitation buffer and an immunoprecipitation buffer with a high sodium chloride salt concentration; the immunoprecipitation buffer with a high sodium chloride salt concentration comprises 0.01-0.02M sodium phosphate solution, 1-4M sodium chloride solution and 0.1%-0.5% Triton X-100.

9. The method according to claim 1, characterized in that In step S5, the elution solution includes 0.01-0.1 M Tris-HCl, 0.01-0.1 M EDTA, 0.001-0.1% SDS and 0.1-1 mg / mL proteinase K.

10. The method according to claim 1, characterized in that include: S1. Add 0.1-15 μL of a DNA methylation protectant at a concentration of 50 ng / μL to the sample to be tested with a cfDNA mass of not less than 0.5 ng, so that the mass ratio of cfDNA to DNA methylation protectant is in the range of 1:10-1:40, and add water until the volume of the sample to which λDNA is added reaches 57 μL; S2. Mix 33 μL of immunoprecipitation buffer and 57 μL of sample with added λDNA, incubate at 95°C for 10 min, and then cool on ice for 15 min. S3. Washing the magnetic beads: Pipette 2-6 μL of 10 mg / mL magnetic bead stock solution and wash the magnetic beads twice with ten-fold diluted immunoprecipitation buffer, and resuspend them with 20 μL of ten-fold diluted immunoprecipitation buffer for the last time to obtain a magnetic bead suspension with a concentration of 2.5-3 mg / mL; Dilute the antibody: dilute the 5-methylcytosine antibody with a stock concentration of 1 mg / mL by 10-100 times using a ten-fold diluted immunoprecipitation buffer to obtain an antibody solution with a concentration of 0.03-0.05 mg / mL; Add 5 μL of antibody solution and 5 μL of magnetic bead suspension to 75 μL of the mixture obtained in step S2, and incubate at 4°C with rotation for 4-16 hours; S4. Add 100 μL of ten-fold diluted immunoprecipitation buffer in an ice bath, invert and resuspend the magnetic beads, rotate and incubate at 4°C for 5 minutes, centrifuge immediately after the end, place on a pre-cooled magnetic stand, wait for 1 minute, and discard the supernatant; repeat the same steps at least once; Add 100 μL of immunoprecipitation buffer with high sodium chloride concentration in an ice bath, invert and resuspend the magnetic beads, rotate and incubate at 4°C for 5 minutes, centrifuge immediately after the end, place on a pre-cooled magnetic stand, wait for 1 minute and discard the supernatant; repeat the same steps at least once; The components of the immunoprecipitation buffer with high sodium chloride salt concentration are: 0.02M sodium phosphate solution, 2M sodium chloride solution and 0.5% Triton X-100; S5. Elute DNA with elution buffer at 55°C / 15min and 100°C / 15min; The elution buffer included 0.05 M Tris-HCl, 0.01 M EDTA, 0.001% SDS and 0.4 mg / mL proteinase K.

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