Methylated DNA standard substance as well as preparation method and application thereof

Abstract

The invention discloses a methylated DNA standard substance as well as a preparation method and application thereof. A preparation method of a completely demethylated DNA standard substance comprises the following steps: S1, combining single-stranded DNA of a human genome DNA standard substance NA12878 with a random heptamer primer to obtain single-stranded DNA combined with the primer; s2, carrying out amplification on the single-stranded DNA obtained in the step S1 by using Equi-Phi29 DNA polymerase, so as to obtain an amplification product; s3, performing enzyme digestion on the amplification product obtained in the step S2 by using mung bean nuclease to obtain an amplification product subjected to enzyme digestion; and purifying the amplification product subjected to enzyme digestion by using a nucleic acid purification column to obtain the completely demethylated DNA standard substance. According to the method for preparing the DNA standard substance, the experiment efficiency is improved, the operation is simple and convenient, standardization is easy, pollution is reduced, the high-purity DNA standard substance is obtained, the concentration and the total amount are better, the quality is high, and the accuracy and the reliability of an experiment result are ensured.

Description

Technical Field

[0001] The present invention relates to the field of biotechnology, and in particular to a methylated DNA standard and a preparation method and application thereof. Background Art

[0002] DNA methylation is an important epigenetic modification that is widely involved in biological processes such as gene expression regulation, genome stability maintenance, and embryonic development. With the deepening of molecular biology and medical research, DNA methylation plays an increasingly important role in disease diagnosis, prognosis assessment, and drug development, especially in the early detection and precision treatment of major diseases such as cancer. Methylation markers have become one of the research hotspots. However, when conducting methylation detection and research, accurate and stable methylated DNA standards are essential.

[0003] The method of obtaining methylated DNA based on cell lines requires culturing cells for a long time to obtain DNA with a specific methylation level. However, this method has obvious shortcomings: on the one hand, the culture cycle of the cell line is long, usually taking several weeks or even months, which is time-consuming and inefficient for researchers; on the other hand, the cell culture process is easily affected by external factors, such as culture medium components, environmental conditions, etc., resulting in a certain variability in the obtained DNA methylation level. In addition, the cost of this method is relatively high, especially when preparing standards on a large scale, the cost will increase significantly. Therefore, the method of obtaining methylated DNA based on cell lines cannot meet the experimental requirements of high efficiency and low cost.

[0004] Another method for preparing methylated DNA standards is chemical synthesis, which has the advantage of being able to precisely control the methylation state of DNA molecules through chemical means. However, chemical synthesis also faces many challenges: first, the chemical synthesis process usually involves complex processes, especially when synthesizing long-chain DNA molecules, which is more difficult. The complexity of this process not only increases the difficulty of experimental operation, but also increases the cost of the entire preparation process; secondly, chemical synthesis is also difficult to flexibly adjust the methylation level of DNA in practical applications, which to a certain extent limits its applicability in different experimental scenarios. Therefore, although chemical synthesis can theoretically provide accurate methylation standards, there are still many difficulties in practical applications.

[0005] The method for preparing methylated DNA by simulating cfDNA (circulating DNA) is to obtain a completely unmethylated DNA sample by whole-genome amplification of the genomic DNA of a methyltransferase-deficient cell line. The sample is then sheared and purified by magnetic bead sorting to obtain a completely unmethylated simulated cfDNA sample with a fragment length distribution similar to that of natural cfDNA. Next, these samples are treated with methyltransferase to obtain a completely methylated simulated cfDNA sample. Finally, the unmethylated and methylated samples are mixed in proportion to obtain a series of simulated cfDNA methylation standards with different methylation levels.

[0006] The preparation method of simulated cfDNA methylation standards is usually based on methyltransferase-deficient cell lines. This process first requires the successful preparation or acquisition of this special cell line. Since such cell lines are not conventional products in commercial cell banks, researchers may need to use gene editing technologies such as CRISPR / Cas9 to knock out specific methyltransferase genes to create these cell lines. The gene editing process itself requires sophisticated design and professional operation skills. In order to ensure the knockout efficiency and accuracy of the target gene in the cell line, a series of verification tests must also be carried out. Usually including molecular biology and genetic methods, such as polymerase chain reaction (PCR), Western blot, immunofluorescence staining or RNA interference technology, to evaluate the changes in expression levels after gene knockout, detect whether the mRNA and protein expression of the target gene are inhibited, and thus verify whether the knockout is successful.

[0007] When constructing unmethylated DNA samples, this preparation method requires the DNA product after whole genome amplification to undergo gel cutting and recovery steps. This step requires precise positioning of the gel area containing the target DNA fragment, and the use of manual or automated equipment for cutting and recovery. This process is very prone to cross contamination, so it also has certain technical requirements for operators. Summary of the invention

[0008] The purpose of the present invention is to overcome the above-mentioned deficiencies in the prior art and to provide a methylated DNA standard and a preparation method and application thereof.

[0009] The present invention amplifies the human genome DNA standard NA12878, and obtains a completely demethylated DNA standard by enzyme digestion and adsorption column purification technology. On this basis, the completely demethylated DNA standard is methylated by methyltransferase to obtain a completely methylated DNA standard. Finally, a series of DNA standards with different methylation levels are prepared by mixing the completely demethylated DNA standard and the completely methylated DNA standard in different proportions to meet diverse experimental needs.

[0010] The first object of the present invention is to provide a method for preparing a completely demethylated DNA standard.

[0011] The second object of the present invention is to provide a completely demethylated DNA standard.

[0012] The third object of the present invention is to provide the use of the completely demethylated DNA standard in the preparation of a completely methylated DNA standard.

[0013] The fourth object of the present invention is to provide a completely methylated DNA standard.

[0014] The fifth object of the present invention is to provide a method for preparing the fully methylated DNA standard.

[0015] The sixth object of the present invention is to provide the use of the completely demethylated DNA standard and / or the completely methylated DNA standard in the preparation of DNA standards with different methylation levels.

[0016] The seventh object of the present invention is to provide a method for preparing DNA standards with different methylation levels.

[0017] The eighth object of the present invention is to provide a DNA standard with different methylation levels.

[0018] In order to achieve the above object, the present invention is implemented by the following scheme:

[0019] The present invention provides a method for preparing a completely demethylated DNA standard, comprising the following steps:

[0020] S1: Combine the single-stranded DNA of human genomic DNA standard NA12878 with random heptamer primers to obtain single-stranded DNA bound to the primers;

[0021] S2: Amplify the single-stranded DNA in step S1 using Equi-Phi29 DNA polymerase to obtain an amplified product;

[0022] S3: digest the amplified product of S2 with mung bean nuclease to obtain an amplified product after digestion; purify the amplified product after digestion with a nucleic acid purification column to obtain a completely demethylated DNA standard.

[0023] The present invention uses high temperature denaturation of human genome DNA standard NA12878 to obtain single-stranded DNA; the single-stranded DNA is combined with a random heptamer primer with a 5' end blocked to obtain single-stranded DNA combined with the primer. The sticky ends and short fragments in the amplified product of step S2 are digested by mung bean nuclease.

[0024] The random heptamer primers in step S1 have the best effect compared to random hexamers and random octamers. The efficiency of primer binding to template DNA and the efficiency of amplification reaction have both reached the optimal state.

[0025] In a specific embodiment of the present invention, step S1 is used for denaturation, and the components of the configured denaturation system are: 30-50 ng of human genomic DNA standard NA12878, 1× reaction buffer and 100-150 μM random heptamer primers, and the total volume of the denaturation system is supplemented with enzyme-free nucleic acid water to 10 μL.

[0026] The preferred components of the denaturation system are: 30 ng of human genomic DNA standard NA12878, 1× reaction buffer and 100 μM random heptamer primers, and the total volume of the denaturation system is made up to 10 μL with enzyme-free nucleic acid water.

[0027] The 1× reaction buffer comprises: 33-40 mM tris(hydroxymethyl)aminomethane acetate (pH 7.9, 37° C.), 10-15 nM magnesium acetate, 66-75 mM potassium acetate, 0.1%-0.3% (v / v) Tween 20 and 1-2.5 mM dithiothreitol, and the solvent is water.

[0028] Preferably, the components of 1× reaction buffer are: 33 mM tris(hydroxymethyl)aminomethane acetate (pH 7.9, 37° C.), 10 nM magnesium acetate, 66 mM potassium acetate, 0.1% (v / v) Tween 20 and 1 mM dithiothreitol, and the solvent is water.

[0029] The denaturation condition is high temperature denaturation: denature the denaturation system at 95°C for 3 minutes.

[0030] Furthermore, the ratio of the Equi-Phi29 DNA polymerase in step S2 to the human genomic DNA standard NA12878 in step S1 is 30 ng: 10-30 U, preferably 20 U, and the concentration and total amount of the amplified product are better and the quality is the highest.

[0031] In a specific embodiment of the present invention, when the Equi-Phi29 DNA polymerase of step S2 is selected from Thermo Company, with a product number of A39392, the concentration and total amount of the amplified product are better and the quality is the highest.

[0032] In a specific embodiment of the present invention, step S2 performs single-stranded DNA amplification, and the components of the configured single-stranded DNA amplification system are: 10 μL of the denaturation system after the denaturation reaction in step S1, 50-150 mM dithiothreitol, 50-200 mM dNTP and 10-30 U Equi-Phi29 DNA polymerase, and the total volume of the single-stranded DNA amplification system is supplemented with enzyme-free nucleic acid water to 50 μL.

[0033] The components of the preferred single-stranded DNA amplification system are: 10 μL of the denaturation system after the denaturation reaction in step S1, 50 mM dithiothreitol, 50 mM dNTP and 20 U Equi-Phi29 DNA polymerase, and the total volume of the single-stranded DNA amplification system is supplemented with enzyme-free nucleic acid water to 50 μL.

[0034] The dNTPs include dATP, dGTP, dTTP and dCTP, all of which are 50-200 mM, preferably 50 mM.

[0035] The single-stranded DNA amplification conditions are as follows: subject the single-stranded DNA amplification system to PCR reaction, 30°C for 16 hours; 65°C for 10 minutes; and 4°C for hold.

[0036] In a specific embodiment of the present invention, step S3 performs an enzyme digestion reaction, and the components of the configured enzyme digestion reaction system are: 50 μL of the amplification product of step S2, 1× mung bean nuclease reaction buffer and 3-5U of mung bean nuclease, preferably 3U.

[0037] The 1× mung bean nuclease reaction buffer comprises: 50-60 mM sodium acetate (pH 5.0, 25° C.), 25-35 mM potassium chloride and 1-1.5 mM zinc sulfate, and the solvent is water.

[0038] Preferably, the components of 1× mung bean nuclease reaction buffer are: 50 mM sodium acetate (pH 5.0, 25° C.), 30 mM potassium chloride and 1 mM zinc sulfate, and the solvent is water.

[0039] The enzyme digestion reaction conditions are: incubate the enzyme digestion reaction system at 30°C for 30 minutes.

[0040] After the incubation, 0.5-1% (v / v) sodium dodecyl sulfate is added to terminate the enzyme digestion reaction, vortexed and mixed, and centrifuged briefly to obtain the enzyme digestion product. The preferred concentration of sodium dodecyl sulfate is 1% (v / v).

[0041] In a specific embodiment of the present invention, the enzyme digestion product can be purified using a nucleic acid purification column using a QIAquick PCR Purification Kit.

[0042] The invention provides a completely demethylated DNA standard product, which is prepared by the preparation method.

[0043] The present invention provides the use of the completely demethylated DNA standard in the preparation of a completely methylated DNA standard.

[0044] The present invention provides a completely methylated DNA standard product, which is prepared from the completely demethylated DNA standard product.

[0045] The present invention provides a method for preparing the completely methylated DNA standard product, wherein the completely demethylated DNA standard product is methylated by using CpG methyltransferase to obtain methylated DNA.

[0046] Take 2.5 μg of the fully methylated DNA standard, and prepare the following components of the methylation reaction system: 2.5 μg of fully methylated DNA, 1×NEB Buffer 2, 1×S-adenosylmethionine, 10-15 U CpG methyltransferase, and add enzyme-free nucleic acid water to make the total volume of the denaturation system 10 μL.

[0047] The preferred components of the methylation reaction system are: 2.5 μg of fully methylated DNA, 1×NEB Buffer 2, 1×S-adenosylmethionine, 10 U CpG methyltransferase, and the total volume of the denaturation system is made up to 10 μL with enzyme-free nucleic acid water.

[0048] The 1×NEB Buffer 2 comprises: 50-60 mM sodium chloride, 10-15 mM tris(hydroxymethyl)aminomethane hydrochloride, 10-20 mM magnesium chloride and 1-1.5 mM dithiothreitol, and the solvent is water (pH 7.9, 25° C.).

[0049] Preferably, the components of 1×NEB Buffer 2 are: 50 mM sodium chloride, 10 mM tris(hydroxymethyl)aminomethane hydrochloride, 10 mM magnesium chloride and 1 mM dithiothreitol, and the solvent is water (pH 7.9, 25° C.).

[0050] The methylation reaction conditions are as follows: subject the methylation reaction system to PCR reaction, 30°C for 4 to 16 hours; 65°C for 20 minutes; 4°C hold.

[0051] When the CpG methyltransferase was selected from Zymo Company, the CpG methyltransferase with product number E2010 was obtained, and the fully methylated DNA standard product had the highest quality.

[0052] Furthermore, the methylated DNA is purified using a nucleic acid purification column to obtain a completely methylated DNA standard.

[0053] In a specific embodiment of the present invention, the methylated DNA can be purified using a nucleic acid purification column using a QIAquick PCR Purification Kit.

[0054] The present invention provides the use of the completely demethylated DNA standard and / or the completely methylated DNA standard in preparing DNA standards with different methylation levels.

[0055] The present invention provides a method for preparing DNA standards with different methylation levels, wherein the completely demethylated DNA standard and the completely methylated DNA standard are mixed in different proportions to obtain DNA standards with different methylation levels;

[0056] Methylation level = mass of fully methylated DNA standard / (mass of fully demethylated DNA standard + mass of fully methylated DNA standard).

[0057] The invention provides DNA standards with different methylation levels, which are prepared by the preparation method.

[0058] In a specific embodiment of the present invention, a 100% methylation level DNA standard is completely methylated DNA; a 0% methylation level DNA standard is completely demethylated DNA;

[0059] In the 75% methylation level DNA standard, the mass ratio of the fully methylated DNA standard to the fully demethylated DNA standard is 3:1;

[0060] In the 50% methylation level DNA standard, the mass ratio of the fully methylated DNA standard to the fully demethylated DNA standard is 1:1;

[0061] In the 25% methylation level DNA standard, the mass ratio of the fully methylated DNA standard to the fully demethylated DNA standard is 1:3.

[0062] In the 10% methylation level DNA standard, the mass ratio of the fully methylated DNA standard to the fully demethylated DNA standard is 1:9.

[0063] In the 5% methylation level DNA standard, the mass ratio of the fully methylated DNA standard to the fully demethylated DNA standard is 1:19.

[0064] In the 1% methylation level DNA standard, the mass ratio of the fully methylated DNA standard to the fully demethylated DNA standard is 1:99.

[0065] In the 0.5% methylation level DNA standard, the mass ratio of the fully methylated DNA standard to the fully demethylated DNA standard is 1:199.

[0066] Compared with the prior art, the present invention has the following beneficial effects:

[0067] The present invention is directly based on the human genome DNA standard NA12878 to prepare completely demethylated DNA standards and completely methylated DNA standards. Compared with the preparation method of the simulated cfDNA methylation standard, the cell culture step and the gel cutting and DNA recovery step are omitted, which greatly simplifies the experimental process and improves the experimental efficiency. In the construction process of the demethylated DNA standard, the present invention purifies the amplified product using an adsorption column purification and recovery technology. Compared with the gel cutting and recovery step, this technology has the advantages of simple operation, easy standardization and reduced pollution. The adsorption column purification and recovery technology can effectively separate and purify the target DNA fragments, while removing non-target fragments and impurities, and obtaining a high-purity demethylated DNA standard with better concentration and total amount, high quality, ensuring the accuracy and reliability of the experimental results, and laying a good experimental foundation for DNA methylation research. BRIEF DESCRIPTION OF THE DRAWINGS

[0068] Figure 1 Flow chart for the preparation of methylated DNA standards. DETAILED DESCRIPTION

[0069] The present invention is further described in detail below in conjunction with the accompanying drawings and specific examples of the specification. The examples are only used to explain the present invention and are not used to limit the scope of the present invention. The test methods used in the following examples are conventional methods unless otherwise specified; the materials and reagents used are reagents and materials that can be obtained from commercial channels unless otherwise specified.

[0070] Example 1 Preparation of Completely Demethylated DNA Standards

[0071] like Figure 1 Shown is a flow chart for the preparation of methylated DNA standards.

[0072] 1. Preparation of single-stranded DNA

[0073] (1) NA12878 DNA sample (human genomic DNA standard), 10× buffer solution and random heptamer primers were thawed at room temperature, vortexed and briefly centrifuged to prepare a DNA denaturation mixture (Table 1).

[0074] Table 1

[0075] Components content NA12878DNA 30ng 10× reaction buffer 1μL 500 μM random heptamer primers 2μL Enzyme-free Nuclease Water Make up to a total volume of 10 μL DNA denaturation mixture

[0076] The components of 10× reaction buffer are: 330 mM tris(hydroxymethyl)aminomethane acetate (pH 7.9, 37° C.), 100 nM magnesium acetate, 660 mM potassium acetate, 1% (v / v) Tween 20 and 10 mM dithiothreitol, and the solvent is water.

[0077] (2) Use a pipette to gently pipette and briefly centrifuge to mix the DNA denaturation mixture. Perform PCR reaction and denature at 95°C for 3 min to obtain the denatured DNA denaturation mixture, which is then placed on ice for later use.

[0078] 2. Constant temperature amplification

[0079] After thawing the reagent components in Table 2, gently invert the Equi-Phi29 DNA polymerase (manufacturer: Thermo, catalog number: A39392) upside down to mix and briefly centrifuge. Vortex and briefly centrifuge the reagents in Table 2 to obtain a constant temperature amplification solution. Use a pipette to gently blow and mix. Briefly centrifuge to mix the constant temperature amplification solution. Perform PCR reaction. The PCR reaction conditions are: 30°C for 16h; 65°C for 10min; 4°C hold to obtain an amplified product.

[0080] Table 2

[0081]

[0082] 3. Enzymatic digestion

[0083] Prepare the enzyme digestion reaction system according to Table 3, mix it by gently blowing with a pipette, briefly centrifuge the enzyme digestion reaction system, incubate it at 30°C for 30 min using a PCR instrument to obtain the incubated enzyme digestion reaction system, add 0.6 μL of 1% (v / v) sodium dodecyl sulfate to the incubated enzyme digestion reaction system, terminate the reaction, vortex mix it, briefly centrifuge it, and obtain the enzyme digestion product.

[0084] Table 3

[0085] Components content Amplification products 50μL 10× Mung Bean Nuclease Reaction Buffer 5.9μL Mung Bean Nuclease 3μL

[0086] The amount of mung bean nuclease (manufacturer: NEB, catalog number: M0250) in the restriction reaction system is 1U / μg NA12878DNA sample (10U=1μl of mung bean nuclease is added to every 10μg DNA sample). When the amount of DNA input changes, the amount of enzyme input and the mung bean nuclease reaction buffer should be changed accordingly (so that the final concentration of the mung bean nuclease reaction buffer in the restriction reaction system is 1×).

[0087] The components of 10× mung bean nuclease reaction buffer are: 500 mM sodium acetate (pH 5.0, 25° C.), 300 mM potassium chloride and 10 mM zinc sulfate, and the solvent is water.

[0088] 4. Column purification

[0089] The enzyme cleavage product was purified and recovered using QIAquick PCR Purification Kit (purchased from QIAGEN, catalog number: 28104) according to the instructions to obtain a completely demethylated DNA standard.

[0090] The fully demethylated DNA standard was diluted 10-fold and then analyzed by Thermo Fisher's Qubit TM The dsDNA HSAssay Kit was used to detect the DNA concentration and convert it into the concentration of the completely demethylated DNA standard before dilution 10 times.

[0091] Example 2 Preparation of fully methylated DNA standards

[0092] 1. Methylation

[0093] Take 2.5 μg of the completely demethylated sample prepared in Example 1, dilute it to 40 μL with enzyme-free nucleic acid water to obtain the DNA to be methylated.

[0094] The methylation reaction system (total volume 50 μL) was prepared as shown in Table 4.

[0095] Table 4

[0096] Components content 10×NEB Buffer2 5μL 20×SAM (S-adenosylmethionine) 2.5μL CpG methyltransferase (M.SssI) (4U / μl) 2.5μL DNA to be methylated 40

[0097] The components of 10×NEB Buffer 2 are: 500 mM sodium chloride, 100 mM tris(hydroxymethyl)aminomethane hydrochloride, 100 mM magnesium chloride and 10 mM dithiothreitol, and the solvent is water (pH 7.9, 25° C.).

[0098] The prepared methylation reaction system was gently pipetted and mixed, and then centrifuged briefly to mix, and then PCR reaction was performed. The PCR reaction conditions were: 30°C for 4 to 16 h; 65°C for 20 min; 4°C hold to obtain methylated DNA.

[0099] The manufacturer of CpG methyltransferase (M.SssI enzyme) is Zymo Company, and the product number is E2010.

[0100] The methylated DNA was purified by column method according to the method of Example 1 to obtain a completely methylated DNA standard.

[0101] Example 3 Preparation and verification of DNA standards with different methylation levels

[0102] 1. Experimental Methods

[0103] 1. Preparation of DNA standards with different methylation levels

[0104] The completely demethylated DNA standard (0% methylation level) of Example 1 and the completely methylated DNA standard (100% methylation level) of Example 2 are mixed at different mass (weight) ratios as required to prepare DNA standards with different methylation levels. Table 5 shows some commonly used mixing ratios.

[0105] Table 5

[0106]

[0107] 2. Verify the methylation levels of DNA standards with different methylation levels

[0108] The library was constructed using the Hieff NGS dsDNAMethyl Library PrepKit for Illumina (Cat. No.: 12214ES) and Hieff Mag DNA Methylation Bisulfite Kit (Cat. No.: 12223ES) kits of Yisheng Biotechnology (Shanghai) Co., Ltd., and high-throughput sequencing analysis was performed using Illumina NovaSeq 6000 to verify the actual methylation status of each expected simulated DNA standard methylation level in Table 5.

[0109] 2. Experimental Results

[0110] The results of high-throughput sequencing analysis of different expected simulated DNA standard methylation levels are shown in Table 6, indicating that DNA standards with different methylation levels were successfully prepared.

[0111] Table 6

[0112]

[0113]

[0114] Example 4 Effects of different types of phi29 enzymes and addition amounts on the preparation of methylated DNA standards

[0115] 1. Experimental Methods

[0116] 1. Different types and addition amounts of phi29 DNA polymerase (10U, 20U and 30U) were set in Example 1. According to the methods of Example 1 and Example 2, completely demethylated DNA standards (0% methylation level) and completely methylated DNA standards (100% methylation level) were prepared. Three replicate wells were prepared for each sample. Thermo Fisher's Qubit TM The concentration of the obtained DNA standard was detected by dsDNA HSAssay Kit.

[0117] 2. The amount of phi29 DNA polymerase added was set to 20 U, and a DNA standard with a methylation level of 10% and a mass ratio of completely methylated DNA to completely demethylated DNA of 1:9 was prepared according to the methods of Examples 1 to 3.

[0118] Using completely demethylated DNA standards and DNA standards with a methylation level of 10% as templates, β-actin as an internal reference gene, and SDC2 gene for qPCR verification, the ΔCT value of the CT value after qPCR reaction of DNA standards with a methylation level of 10% and completely demethylated DNA standards was used to evaluate the quality of DNA standards prepared by different enzymes, thereby judging the effect of Phi29 enzymes from different manufacturers.

[0119] The high-fidelity enzyme (Cat. No.: F530S) from Thermo was used for amplification, and primers and fluorescent probes designed for the SDC2 gene and the β-actin internal reference gene were used for dual fluorescent PCR detection, with three replicates for each sample.

[0120] PCR reaction system: template 5ng, 1×Phusion HF buffer (a matching reagent for high-fidelity enzyme), dNTPs200μM, high-fidelity enzyme 0.2U, ROX dye 50nM, DMSO (dimethyl sulfoxide) mass concentration 3%, SDC2 gene primers 400μM (upstream and downstream primers 400μM each), SDC2 gene probe 200μM, β-actin internal reference gene primers 300μM (upstream and downstream primers 300μM each), β-actin internal reference gene probe 150μM, and the total PCR reaction system volume is supplemented with enzyme-free nucleic acid water to 20μL.

[0121] Primers and probe for SDC2 gene: forward primer 5′-GGAGTTTGGGGAGTTTTAGT-3′, reverse primer 3′-CCCTCAAAACCCCTAAAACT-5′, probe 5′-FAM-TAAGTGAGAGGG CGTCGCGTTTTCG-BHQ1-3′;

[0122] β-actin internal reference gene primer pair and probe: forward primer 5'-CCTGGCACCCAGCACAAT-3', reverse primer 5'-AAGGAAGGCTGGAAGAGTGC-3', probe 5'-VIC-CCGAGCGT GGCCATCCGCAAAG-TAMRA-3'.

[0123] The PCR reaction conditions were: 98°C for 5 min; 98°C for 15 s, 60°C for 30 s, 72°C for 30 s, 45 cycles; 72°C for 1 min.

[0124] In this embodiment, the manufacturers of phi29 DNA polymerase are: (1) phi29 enzyme of Thermo Company, catalog number A39392; (2) phi29 enzyme of NEB Company, catalog number #M0269S; (3) phi29 enzyme of ABclonal Company, catalog number RK21002.

[0125] 2. Experimental Results

[0126] 1. After processing the data of the three replicate samples, the concentration and yield of the amplified products are shown in Table 7. There are significant differences in the total amount of amplified products of different phi29 DNA polymerases. The total amount of amplified products of phi29 DNA polymerases from Thermo, NEB, and ABclonal are 45μg to 64.7μg, 39.7μg to 46.5μg, and 38.3μg to 46.0μg, respectively. Therefore, the amplification efficiency is ranked as: Thermo>NEB>ABclonal. In addition, when the template amount is 30ng, the addition amount of 20U of phi29 DNA polymerase is most suitable.

[0127] Table 7

[0128] Serial number Enzyme source Enzyme addition amount Amplification product concentration Total amount of amplification product 1 Thermo 10 U 226.7 ng / μL 45.34 μg 2 Thermo 20U 323.5 ng / μL 64.70 μg 3 Thermo 30U 277.8 ng / μL 55.56μg 4 NEB 10U 198.4 ng / μL 39.68μg 5 NEB 20U 232.6 ng / μL 46.52 μg 6 NEB 30U 216.0ng / μL 43.20μg 7 ABclonal 10U 191.0ng / μL 38.25 μg 8 ABclonal 20U 229.9 ng / μL 45.98μg 9 ABclonal 30U 200.2 ng / μL 40.05 μg

[0129] As shown in Table 8, the larger the ΔCT, the better the quality of the prepared DNA standard, and the lowest degree of fragmentation, indicating that the DNA prepared using the Thermo phi29 enzyme has the highest quality.

[0130] Table 8

[0131]

[0132]

[0133] Example 5 Effect of random primers on the preparation of completely demethylated DNA standards

[0134] 1. Experimental Methods

[0135] According to the method of Example 1, completely demethylated DNA standards were prepared using different random primers. The random primers used were: N*N*N*N*N*N*N* (random hexamer primer), N*N*N*N*N*N*N* (random heptamer primer) and N*N*N*N*N*N*N*N*N* (random octamer primer). Three replicate wells were set to obtain completely demethylated DNA standards. After diluting 10 times, the standard was diluted with Qubit DNA from Thermo Fisher. TM The dsDNA HS Assay Kit detects the concentration of completely demethylated DNA standards.

[0136] 2. Experimental Results

[0137] As shown in Table 9, the PCR amplification effect was best when using random heptamer primers. The total amount of amplified products under this condition was significantly higher than that of other experimental groups, and the yield was the highest. This indicates that the binding efficiency of the primers with the template DNA and the efficiency of the amplification reaction have reached the optimal state.

[0138] Table 9

[0139]

[0140]

[0141] Example 6 Effects of different M.SssI enzymes on the preparation of methylated DNA standards

[0142] 1. Experimental Methods

[0143] According to the method of Example 1, a completely demethylated DNA standard was prepared. According to the method of Example 2, a completely methylated DNA standard was prepared using different CpG methyltransferases (M.SssI enzymes). According to the method of Example 3, a DNA standard with a 10% methylation level was prepared.

[0144] Using completely demethylated DNA standards and DNA standards with a methylation level of 10% as templates, β-actin as the internal reference gene, and SDC2 gene for qPCR verification, the quality of DNA standards prepared by different M.SssI enzymes was evaluated by the ΔCT value of the CT value of the DNA standards with a methylation level of 10% and the completely demethylated DNA standards, thereby judging the effect of M.SssI enzymes from different manufacturers.

[0145] The high-fidelity enzyme (Cat. No.: F530S) from Thermo was used for amplification, and primers and fluorescent probes designed for the SDC2 gene and the β-actin internal reference gene were used for dual fluorescent PCR detection, with three replicates for each sample.

[0146] PCR reaction system: template 5ng, 1×Phusion HF buffer (a matching reagent for high-fidelity enzyme), dNTPs200μM, high-fidelity enzyme 0.2U, ROX 50nM, DMSO (dimethyl sulfoxide) mass concentration 3%, SDC2 gene primers 400μM (400μM each for upstream and downstream primers), SDC2 gene probe 200μM, β-actin internal reference gene primers 300μM (300μM each for upstream and downstream primers), β-actin internal reference gene probe 150μM, and the volume was made up to 20μL with enzyme-free nucleic acid water.

[0147] Primers and probe for SDC2 gene: forward primer 5′-GGAGTTTGGGGAGTTTTAGT-3′, reverse primer 3′-CCCTCAAAACCCCTAAAACT-5′, probe 5′-FAM-TAAGTGAGAGGG CGTCGCGTTTTCG-BHQ1-3′;

[0148] β-actin internal reference gene primer pair and probe: forward primer 5'-CCTGGCACCCAGCACAAT-3', reverse primer 5'-AAGGAAGGCTGGAAGAGTGC-3', probe 5'-VIC-CCGAGCGT GGCCATCCGCAAAG-TAMRA-3'.

[0149] PCR amplification conditions were: 98°C for 5 min; 98°C for 15 s, 60°C for 30 s, 72°C for 30 s, 45 cycles; 72°C for 1 min.

[0150] In this example, the manufacturers of the M.SssI enzyme are: (1) Zymo's M.SssI enzyme, catalog number E2010; (2) NEB's M.SssI enzyme, catalog number M0226; and (3) Thermo's M.SssI enzyme, catalog number EM0821.

[0151] 2. Experimental Results

[0152] As shown in Table 10, the larger the ΔCT, the better the quality of the prepared DNA standard and the lowest degree of fragmentation, indicating that the DNA prepared using Zymo's M.SssI enzyme has the highest quality.

[0153] Table 10

[0154] Serial number Enzyme source ΔCT (CT_10% methylation-CT_0% methylation) 1 Zymo 3.47 2 Zymo 3.50 3 Zymo 3.21 4 NEB 2.46 5 NEB 2.74 6 NEB 2.36 7 Thermo 2.19 8 Thermo 2.61 9 Thermo 2.53

[0155] Finally, it should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention rather than to limit the protection scope of the present invention. For ordinary technicians in this field, other different forms of changes or modifications can be made based on the above descriptions and ideas. It is not necessary and impossible to list all the implementation methods here. Any modifications, equivalent substitutions and improvements made within the spirit and principles of the present invention should be included in the protection scope of the claims of the present invention.

Claims

1. A method for preparing a completely demethylated DNA standard, characterized in that: The following steps are involved: S1: Combine the single-stranded DNA of human genomic DNA standard NA12878 with random heptamer primers to obtain single-stranded DNA bound to the primers; S2: Amplify the single-stranded DNA in step S1 using Equi-Phi29 DNA polymerase to obtain an amplified product; S3: digesting the amplified product of step S2 with mung bean nuclease to obtain an amplified product after digestion; purifying the amplified product after digestion with a nucleic acid purification column to obtain a completely demethylated DNA standard.

2. A completely demethylated DNA standard, characterized in that: The method is prepared according to claim 1.

3. Use of the completely demethylated DNA standard according to claim 2 in the preparation of a completely methylated DNA standard.

4. A fully methylated DNA standard, characterized in that: Prepared from the completely demethylated DNA standard according to claim 2.

5. The method for preparing the fully methylated DNA standard according to claim 4, characterized in that: The completely demethylated DNA standard according to claim 2 is methylated using CpG methyltransferase to obtain methylated DNA.

6. The preparation method according to claim 5, characterized in that: The methylated DNA is purified using a nucleic acid purification column to obtain a fully methylated DNA standard.

7. Use of the completely demethylated DNA standard product according to claim 2 and / or the completely methylated DNA standard product according to claim 4 in the preparation of DNA standards of different methylation levels.

8. A method for preparing DNA standards with different methylation levels, characterized in that: The completely demethylated DNA standard product of claim 2 and the completely methylated DNA standard product of claim 4 are mixed in different proportions to obtain DNA standards of different methylation levels; Methylation level = mass of fully methylated DNA standard / (mass of fully demethylated DNA standard + mass of fully methylated DNA standard).

9. A DNA standard with different methylation levels, characterized in that: The product is prepared by the preparation method described in claim 8.

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