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

Abstract

The invention relates to a DNA methylation standard substance as well as a preparation method and application thereof. The DNA methylation standard substance comprises a 100% negative DNA methylation standard substance and a 100% positive DNA methylation standard substance, and the preparation method comprises the following steps: (1) carrying out first PCR amplification on a target gene, diluting an amplification product, and then carrying out second PCR amplification to obtain the 100% negative DNA methylation standard substance; and (2) carrying out methylation treatment on the 100% negative DNA methylation standard substance to obtain the 100% positive DNA methylation standard substance. According to the DNA methylation standard substance prepared by the invention, whether the MSP-qPCR primer has specificity or not can be judged, a primary basis is provided for establishment of an MSP-qPCR detection method, the sensitivity of the MSP-qPCR detection method can be evaluated, and a preparation scheme is simple and easy to implement.

Description

Technical Field

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

[0002] DNA methylation is a form of DNA chemical modification. It refers to the chemical modification process in which a methyl donor binds to DNA through a covalent bond under the catalysis of DNA methyltransferase, obtaining a methyl group at the C-5 position of cytosine, the N-6 position of adenine, and the G-7 position of guanine. In general, DNA methylation mainly refers to the methylation process of the 5th carbon atom on cytosine in CpG dinucleotides. Its product is called 5-methylcytosine (5-mC), which is the only form of DNA methylation in mammals. DNA methylation can cause changes in chromatin structure, DNA conformation, DNA stability, and the way DNA interacts with proteins without changing the DNA sequence, thereby controlling gene expression. It is an important epigenetic regulatory mechanism.

[0003] Gene silencing or overexpression caused by DNA methylation is closely related to the occurrence and development of many diseases, such as cardiovascular and cerebrovascular diseases, hereditary diseases, mental illness, autoimmune diseases, etc. Especially in the field of tumor diseases, DNA methylation has become the most promising biomarker for early diagnosis and prognostic evaluation of tumors. It is of great significance for tumor screening and risk assessment, early diagnosis, staging and typing, prognosis judgment and treatment detection.

[0004] The original methylation-specific PCR (MSP) combines PCR technology with the design of methylation-specific primers, which can qualitatively analyze the methylation level of DNA sequences.

[0005] With the establishment of the real-time fluorescence quantitative PCR technology platform, hydrolysis probes or dyes are used as indicator signals for the presence or absence of PCR amplification products, and a methylation-specific real-time fluorescence quantitative PCR (MSP-qPCR) detection method has been established. This method can both qualitatively and quantitatively analyze the methylation level of DNA sequences. After further simplification and improvement, at present, MSP-qPCR mostly uses methylation primers + housekeeping genes for simultaneous or separate PCR amplification to perform relative quantitative detection of the methylation level of the target gene, that is, housekeeping gene PCR amplification replaces non-methylation primer amplification.

[0006] The preparation of 100% negative DNA methylation standards is extremely complex and difficult. Natural completely unmethylated DNA standards are difficult to obtain. At present, artificial synthesis methods include using various methods to knock out certain methyltransferase genes in normal cell lines, obtain methyltransferase-deficient cell lines, and then extract DNA from methyltransferase-deficient cell lines as 100% negative DNA methylation standards. The above methods require biological system engineering to complete and are complex to operate; or extract DNA from human fetal cell lines to prepare 100% negative standards. Since embryo samples are very precious and difficult to obtain, this type of method involves multiple complex projects from medical ethics to biotechnology.

[0007] It can be seen that the current preparation methods of DNA methylation standards have the problems of complicated preparation process and difficulty in obtaining. Therefore, providing a DNA methylation standard that can be prepared in the laboratory, is simple and easy to operate, and can be used for quantitative analysis has become a research hotspot. Summary of the invention

[0008] In order to solve at least one of the above technical problems, the present disclosure provides a DNA methylation standard and a preparation method and application thereof.

[0009] In a first aspect, the present disclosure provides a method for preparing a DNA methylation standard, wherein the DNA methylation standard includes a 100% negative DNA methylation standard and a 100% positive DNA methylation standard, and the preparation method includes:

[0010] (1) performing a first PCR amplification on the target gene, diluting the amplified product and then performing a second PCR amplification to obtain the 100% negative DNA methylation standard;

[0011] (2) Performing methylation treatment on the 100% negative DNA methylation standard to obtain the 100% positive DNA methylation standard.

[0012] As a preferred technical solution of the present disclosure, the amplification reagents used in the first amplification and the second amplification do not include methylated bases.

[0013] As a preferred technical solution of the present disclosure, the preparation method also includes: mixing 100% negative DNA methylation standards and 100% positive DNA methylation standards of the same level in different proportions to obtain DNA methylation standards with different methylation rates.

[0014] As a preferred technical solution of the present disclosure, the method of adjusting the 100% negative DNA methylation standard and the 100% positive DNA methylation standard to the same level includes:

[0015] The 100% negative DNA methylation standard and the 100% positive DNA methylation standard were serially diluted, and the serially diluted samples were amplified by real-time fluorescence quantitative-PCR (qPCR);

[0016] Assign values ​​to the copy numbers of the gradient 100% negative DNA methylation standard or the gradient 100% positive DNA methylation standard, and establish an absolute quantitative standard curve;

[0017] The copy number of the 100% positive DNA methylation standard is read using the absolute quantitative standard curve of the assigned 100% negative DNA methylation standard; or the copy number of the 100% negative DNA methylation standard is read using the absolute quantitative standard curve of the assigned 100% positive DNA methylation standard;

[0018] The 100% negative DNA methylation standard and the 100% positive DNA methylation standard were adjusted to the same level using copy number.

[0019] As a preferred technical solution of the present disclosure, the different methylation rates include 50% methylation rate, 25% methylation rate, 12.5% ​​methylation rate, 6.25% methylation rate or 3.125% methylation rate.

[0020] As a preferred technical solution of the present disclosure, the dilution multiple is 10 5 -10 8 times.

[0021] As a preferred technical solution of the present disclosure, the preparation method further comprises: diluting the second PCR amplification sample again to obtain the 100% negative DNA methylation standard.

[0022] In a second aspect, the present disclosure provides a DNA methylation standard obtained using the preparation method described in the first aspect.

[0023] In a third aspect, the present disclosure provides an application of the DNA methylation standard described in the second aspect in evaluating a methylation specificity-real-time fluorescence quantitative PCR detection method.

[0024] As a preferred technical solution of the present disclosure, the application includes the evaluation of primer specificity in a methylation-specific real-time fluorescence quantitative PCR detection method.

[0025] The technical solution provided by the embodiments of the present disclosure has the following advantages:

[0026] (1) The preparation method of the DNA methylation standard provided by the present disclosure is simple and easy, and does not require complicated steps such as gene knockout. It only needs to synthesize corresponding primers and probes for the target gene to achieve the preparation of the DNA methylation standard, especially the preparation of 100% negative DNA methylation standard, which solves the problem of complex preparation methods of DNA methylation standards;

[0027] (2) The DNA methylation standard prepared by the present disclosure can determine whether the MSP-qPCR primers are specific, providing the primary basis for the establishment of the MSP-qPCR detection method;

[0028] (3) The DNA methylation standards prepared by the present disclosure can not only achieve qualitative evaluation of the detection efficiency of the MSP-qPCR detection method, but also can quantitatively evaluate it through the obtained DNA methylation standards with different methylation rates;

[0029] (4) The preparation method provided in the present disclosure can prepare DNA methylation standards for different target genes and has wide applicability. BRIEF DESCRIPTION OF THE DRAWINGS

[0030] The accompanying drawings, which are incorporated in and constitute a part of this specification, illustrate embodiments consistent with the present disclosure and, together with the description, serve to explain the principles of the present disclosure.

[0031] In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings required for use in the embodiments or the description of the prior art will be briefly introduced below. Obviously, for ordinary technicians in this field, other drawings can be obtained based on these drawings without paying any creative labor.

[0032] Figure 1 This is a schematic diagram of the process of preparing the DNA methylation standard according to the embodiment of the present disclosure;

[0033] Figure 2 This is a diagram showing the correspondence between the amplification primer pair sequence and the TERT gene (5′-3′) sequence in the first PCR amplification of Example 1 of the present disclosure;

[0034] Figure 3 This is a comparison diagram of agarose gel electrophoresis of the first PCR amplification product and the second PCR amplification after dilution of the amplification product in Example 1 of the present disclosure;

[0035] Figure 4 It is a sequence correspondence diagram of the upstream primer sequence, the downstream primer sequence, the d-probe sequence and the TERT gene (5′-3′) in Example 1 of the present disclosure;

[0036] Figure 5 It is a comparison chart of qPCR amplification curves of the 100% negative DNA methylation standard (i.e., unmethylated standard) and the 100% positive DNA methylation standard (i.e., methylated standard) of Example 1 of the present disclosure;

[0037] Figure 6 This is a comparison chart of standard regression curves of the 100% negative DNA methylation standard and the 100% positive DNA methylation standard of Example 1 of the present disclosure;

[0038] Figure 7 It is a correspondence diagram of the DNA methylation upstream primer sequence, the DNA methylation downstream primer sequence, the M-probe sequence and the sequence of the methylated TERT gene (5′-3′) after sulfite conversion of the MSP-qPCR in Example 2 of the present disclosure;

[0039] Figure 8 This is an amplification curve diagram of the MSP-qPCR verification of the methylation rate standard in Example 2 of the present disclosure;

[0040] Fig. 9 This is a standard regression curve diagram of the MSP-qPCR validation methylation rate standard in Example 2 of the present disclosure. DETAILED DESCRIPTION

[0041] In order to more clearly understand the above-mentioned objectives, features and advantages of the present disclosure, the scheme of the present disclosure will be further described below. It should be noted that the embodiments of the present disclosure and the features in the embodiments can be combined with each other without conflict.

[0042] In the following description, many specific details are set forth to facilitate a full understanding of the present disclosure, but the present disclosure may also be implemented in other ways different from those described herein; it is obvious that the embodiments in the specification are only part of the embodiments of the present disclosure, rather than all of the embodiments.

[0043] The detection principle of the methylation-specific real-time fluorescence quantitative PCR (MSP-qPCR) detection method is as follows: first, the genomic DNA is modified and treated with sodium bisulfite. The purpose of this step is to convert unmethylated cytosine (C) into thymine (T), while methylated cytosine remains unchanged. This conversion allows specific primers to be designed for PCR amplification according to the methylation status; that is, two different sets of primer pairs are designed for the treated DNA sequences, methylated primers are designed for methylated DNA sequences, and non-methylated primers are designed for non-methylated DNA sequences. PCR amplification is performed simultaneously, and the amplified products are analyzed by real-time fluorescence quantitative PCR (Quantitative Real-time PCR). The methylation status of the DNA sequence complementary to the primer is determined based on the presence or absence of amplified products, thereby achieving the purposes of tumor screening and risk assessment, early diagnosis, staging and typing, prognosis judgment and treatment detection.

[0044] However, since methylation is an epigenetic modification, it is difficult to artificially control the methylation level. Therefore, in the development of corresponding MSP-qPCR detection methods, there is a lack of DNA methylation standards with controllable methylation levels and simple preparation methods.

[0045] To this end, the present disclosure provides a DNA methylation standard and a preparation method and application thereof, such as Figure 1 As shown, the DNA methylation standard includes a 100% negative DNA methylation standard and a 100% positive DNA methylation standard, and may also include a series of standards with different methylation rates. The preparation method includes the following steps:

[0046] (1) Extract DNA to obtain the target gene;

[0047] (2) Preparation of 100% negative DNA methylation standards, including:

[0048] The target gene is used as an amplification template to perform the first PCR amplification, and the first amplification product is diluted for the first time;

[0049] Using the product after the first dilution as a PCR amplification template, performing a second PCR amplification, and performing a second dilution on the second amplification product to obtain the 100% negative DNA methylation standard;

[0050] (3) Preparation of 100% positive DNA methylation standards

[0051] Methylating and purifying the 100% negative DNA methylation standard to obtain the 100% positive DNA methylation standard;

[0052] (4) qPCR calibration (100% negative DNA methylation standard and 100% positive DNA methylation standard were calibrated to the same level)

[0053] The 100% negative DNA methylation standard and the 100% positive DNA methylation standard were serially diluted, and the serially diluted samples were amplified by qPCR;

[0054] Assign values ​​to the copy numbers of the gradient 100% negative DNA methylation standard or the gradient 100% positive DNA methylation standard, and establish an absolute quantitative standard curve;

[0055] The copy number of the 100% positive DNA methylation standard is read using the absolute quantitative standard curve of the assigned 100% negative DNA methylation standard; or the copy number of the 100% negative DNA methylation standard is read using the absolute quantitative standard curve of the assigned 100% positive DNA methylation standard;

[0056] The 100% negative DNA methylation standard and the 100% positive DNA methylation standard were adjusted to the same level using copy number;

[0057] (5) Prepare standards with different methylation rates

[0058] The 100% negative DNA methylation standard and the 100% positive DNA methylation standard of the same level were mixed in different proportions to obtain DNA methylation standards with different methylation rates.

[0059] The DNA methylation standards provided in the present invention include 100% negative DNA standards and 100% positive DNA standards, both of which can realize the evaluation of the qualitative detection efficiency of MSP-qPCR, and can qualitatively evaluate whether the primers and probes designed for MSP-qPCR are specific, thus establishing the primary foundation for the establishment of the MSP-qPCR detection method.

[0060] The DNA methylation standards provided in the present disclosure can not only qualitatively evaluate the detection efficiency of MSP-qPCR, but also can be configured into a series of standards with different methylation rates to achieve quantitative evaluation of the detection efficiency of MSP-qPCR, such as sensitivity evaluation.

[0061] The preparation method of the DNA methylation standard provided by the present invention is simple and easy, and does not require complicated steps such as gene knockout. The preparation of the DNA methylation standard can be achieved by only synthesizing the corresponding primers and probes for the target gene, especially the preparation of 100% negative DNA methylation standards, which solves the problem of the complexity of the preparation method of the DNA methylation standard.

[0062] In the present disclosure, for step (1), the DNA specimen can be a specimen from various sources, for extracting DNA, such as peripheral blood genomic DNA, tissue DNA, plasma free DNA, etc. The present disclosure does not limit the extraction method, and any method that can achieve the purpose of "DNA extraction" described in the present disclosure can be adopted, for example, DNA extraction can be performed using a DNA extraction kit. The target gene described in the present disclosure can be any gene of interest. In the subsequent preparation process, it is only necessary to design an introduction and probe for the gene of interest, and perform ordinary PCR amplification to obtain the 100% negative DNA methylation standard described in the present disclosure, and complete the preparation of other subsequent standards.

[0063] In the present disclosure, for step (2), as a preferred technical solution of the present disclosure, the amplification reagents used in the first amplification and the second amplification do not include methylated bases.

[0064] In the preparation method provided by the present disclosure, by taking advantage of the characteristics that the A, T, C, and G bases in the PCR reagent are all non-methylated bases, the target gene is amplified by ordinary PCR. After the first PCR amplification, since the bases in the PCR product are derived from the A, T, C, and G in the PCR reagent, all of which are non-methylated bases, the first PCR amplification product is almost entirely non-methylated product, in which only a very small amount of the original DNA template is mixed, which can be called a primary non-methylated product. After the primary non-methylated product is diluted and amplified again, since its amplification template is the product after the primary non-methylated product is diluted, and at the same time the bases are also derived from the non-methylated bases A, T, C, and G in the PCR reagent, therefore, the second amplification product (diluted or not diluted) finally obtained can be used as a completely negative DNA methylation standard, that is, a 100% negative DNA methylation standard is obtained.

[0065] In the present disclosure, the target gene and the primers and probes used for PCR amplification are not limited. As long as two PCR amplifications can be achieved for a specific target gene to obtain a 100% negative DNA methylation standard, the present disclosure emphasizes that both PCR amplifications are ordinary PCR amplifications, and the PCR reagents to be used must not include methylated bases.

[0066] In the preparation method provided in the present disclosure, by performing two ordinary PCR amplifications on the target gene and combining the dilution steps, the target gene can be substantially not contained in the amplified product, and thus, a 100% negative DNA standard can be successfully obtained.

[0067] Since the prepared 100% negative DNA methylation standard is determined to have no methylated bases, through MSP-qPCR amplification, it is possible to judge whether the designed primers are specific based on the presence or absence of an amplification curve. Therefore, the 100% negative DNA methylation standard provided by the present invention can provide the primary basis for the establishment of a specific MSP-qPCR detection method.

[0068] As a preferred technical solution of the present disclosure, the first dilution multiple is 10 5 -10 8 times, for example 10 6 , 10 7 wait.

[0069] As a preferred technical solution of the present disclosure, the second dilution multiple is 10 2 -10 5 times, for example 10 3 , 10 4 wait.

[0070] In the present disclosure, for step (3), the 100% negative DNA standard is treated with methyltransferase and then purified to obtain a 100% positive DNA standard.

[0071] The present disclosure does not limit the use of any methyltransferase, as long as the methylation reaction can be achieved and combined with a purification step, a 100% positive DNA standard can be obtained.

[0072] In the present disclosure, for step (4), as a preferred technical solution of the present disclosure, the method of adjusting the 100% negative DNA methylation standard and the 100% positive DNA methylation standard to the same level comprises:

[0073] Performing gradient dilutions on the 100% negative DNA methylation standard and the 100% positive DNA methylation standard, for example, 10-fold dilution, at least 4 gradients, and performing PCR amplification using the gradient dilution samples as PCR amplification templates;

[0074] The amplified products of the gradient 100% negative DNA methylation standard were assigned values ​​in sequence, such as 1000, 100, 10, 1 copy number / mL, etc., and an absolute quantitative standard curve was established;

[0075] The copy number of the 100% positive DNA methylation standard was read by the absolute quantification standard curve;

[0076] The copy number was used to adjust the 100% negative DNA methylation standard and the 100% positive DNA methylation standard to the same level, that is, the same copy number was the same level.

[0077] The present invention adjusts 100% negative DNA methylation standards and 100% positive DNA methylation standards to the same level by calibrating the copy number of methylation standards through PCR, which is more accurate than simply adjusting by concentration detection.

[0078] In the present disclosure, for step (5), the present disclosure can utilize 100% negative DNA methylation standards and 100% positive DNA methylation standards of the same level to configure different standards with methylation rates of 0-100%. The present disclosure does not limit the specific methylation rate, and the configuration can be performed according to the actual situation during actual application.

[0079] As a preferred technical solution of the present disclosure, the different methylation rates include 50% methylation rate, 25% methylation rate, 12.5% ​​methylation rate, 6.25% methylation rate or 3.125% methylation rate.

[0080] In the preparation method described in the present disclosure, the present disclosure may also include verification of DNA methylation standards, which may be verified using the MSP-qPCR detection method, including:

[0081] The standard can be modified by using sodium bisulfite;

[0082] After treatment, purification and recovery are performed;

[0083] MSP-qPCR primers and probes were used for amplification detection;

[0084] The amplified products were analyzed by real-time fluorescence quantitative PCR.

[0085] In a second aspect, the present disclosure provides a DNA methylation standard obtained using the preparation method described in the first aspect.

[0086] In a third aspect, the present disclosure provides an application of the DNA methylation standard described in the second aspect in evaluating a methylation specificity-real-time fluorescence quantitative PCR detection method.

[0087] As a preferred technical solution of the present disclosure, the application includes the evaluation of primer specificity in a methylation-specific real-time fluorescence quantitative PCR detection method.

[0088] In order to further illustrate the effects of the present disclosure, specific embodiments are used for illustration below.

[0089] This disclosure takes the preparation of standards, evaluation of primer specificity and evaluation of detection performance of the MSP-qPCR detection method established for five methylation sites of the TERT gene (DNA sequence number: Chr5:1295471-1295504, GRCh38.p14) as an example to explain:

[0090] The relevant information of the TERT gene (5′-3′) is as follows:

[0091] Original sequence (Chr5:1295394-1295577, GRCh38.p14):

[0092]

[0093]

[0094] The bold "cg" in the sequence is a CpG site, and the " cg " are the 5 CpG sites to be detected.

[0095] The TERT gene (5′-3′) sulfite conversion sequence includes the following two types:

[0096] One is the methylation sequence, as follows:

[0097]

[0098] The other is the unmethylated sequence, as follows:

[0099]

[0100] The purpose of this disclosure is not to protect a certain extraction or detection kit, but to provide a method for preparing a standard product and the standard product and application obtained therefrom. Therefore, for the preparation method taking TERT as an example, the kit involved is described as follows:

[0101] The “DNA extraction kit” used for “extracting target genes” can be any commercial DNA extraction kit, and the DNA of the pre-test sample can be extracted according to the operating instructions of the kit.

[0102] The "PCR premix kit" used in PCR amplification can be any commercial conventional PCR premix kit, and PCR amplification is performed according to the operating instructions of the kit. Unless otherwise specified, the "T, A, C, G" bases in conventional PCR reagents are all non-methylated bases.

[0103] The "DNA purification and recovery kit" used in "purification and recovery of amplification products" can be any commercial DNA purification and recovery kit, and the DNA can be purified and recovered according to the operating instructions.

[0104] The "DNA methylation modification kit" used in the "methylation treatment" can be any commercial DNA methylation modification kit, and the methylation treatment is performed according to the operating instructions.

[0105] The "fluorescence quantitative PCR premix kit" used in the "DNA methylation standard DNA copy number calibration" can be any commercial fluorescence quantitative PCR premix kit, which can be used according to the operating instructions.

[0106] Example 1 Preparation of DNA methylation standards

[0107] The preparation method of DNA methylation standard for TERT gene is as follows:

[0108] (1) Extraction of target gene TERT

[0109] A commercial DNA extraction kit was used to extract genomic DNA from the peripheral blood of apparently healthy subjects in the pre-test samples according to the operating instructions of the kit.

[0110] (2) Preparation of 100% negative DNA methylation standards, including:

[0111] (2-1) Perform the first PCR amplification and the first dilution of the target gene

[0112] The genomic DNA of apparently healthy human peripheral blood extracted in step (1) was used as a PCR template to perform the first PCR amplification on the original sequence of the target gene TERT gene (5′-3′) (Chr5:1295394-1295577, GRCh38.p14), wherein the primers used for PCR amplification are specifically shown in Table 1:

[0113] Table 1

[0114]

[0115] Note: The underlined "C" and "G" bases in the primers and probes are methylation sites.

[0116] The composition of the reaction system for the first PCR amplification is shown in Table 2.

[0117] Table 2

[0118]

[0119]

[0120] in:

[0121] A. Master mix: 2×GC-Rich PCR premix reagent (Tiangen Biochemical Technology (Beijing) Co., Ltd., catalog number: KT206).

[0122] B. Amplification primer pair: The amplification primer pair includes an upstream amplification primer and a downstream amplification primer; the sequence of the upstream amplification primer is shown in SEQ ID NO.1 in Table 1, and the sequence of the downstream amplification primer is shown in SEQ ID NO.2 in Table 1. The corresponding relationship between the amplification primer pair sequence and the TERT gene (5′-3′) sequence is as follows: Figure 2 shown.

[0123] C. DNA template: TERT gene (5′-3′) in the genomic DNA of peripheral blood of apparently healthy people.

[0124] D. Cycling conditions: 98℃ for 3 min; 98℃ for 15 sec, 60℃ for 20 sec, 72℃ for 30 s, 40 cycles; 72℃ for 5 min.

[0125] E. Result judgment: Use 1.5% agarose gel for electrophoresis test. Judge whether the target fragment is amplified based on the presence or absence of electrophoresis bands and their electrophoresis positions; judge whether it is specific amplification based on whether there is a single electrophoresis band.

[0126] The agarose gel electrophoresis of the first PCR amplification product obtained by the first PCR amplification is shown in Figure 3 shown.

[0127] according to Figure 3 The following conclusions can be drawn from the agarose gel electrophoresis of the first PCR amplification product:

[0128] A. The first PCR amplification yielded the amplified fragment of the target gene TERT gene (5′-3′);

[0129] B. The first PCR amplification is specific amplification.

[0130] Use sterile water for injection as diluent to dilute the first PCR amplification product for the first time, and the dilution factor is 10 5 times to obtain a diluted product.

[0131] (2-2) Second PCR amplification, purification and second dilution

[0132] In the diluted first PCR amplification product, the generated first PCR amplification product all comes from the "T, A, C, G" bases in the PCR premix kit, that is, the first PCR amplification product is almost entirely unmethylated DNA sequence, but a very small portion of the original sample DNA still remains, and it is uncertain whether the residual DNA is methylated, so a second PCR amplification is required.

[0133] The diluted first PCR amplification product is used as a PCR amplification template for the second PCR amplification, and amplification is performed using the primer pair of the first PCR amplification.

[0134] The composition of the reaction system for the second PCR amplification is the same as that for the first PCR amplification, the only difference being the different DNA templates. The amounts of the reaction components are shown in Table 2.

[0135] in:

[0136] A. Master mix: 2×GC-Rich PCR premix reagent (Tiangen Biochemical Technology (Beijing) Co., Ltd., catalog number: KT206).

[0137] B. Amplification primer pair: The amplification primer pair includes an upstream amplification primer and a downstream amplification primer; the sequence of the upstream amplification primer is shown in SEQ ID NO.1 in Table 1, and the sequence of the downstream amplification primer is shown in SEQ ID NO.2 in Table 1.

[0138] C. DNA template: diluted first PCR amplification product.

[0139] D. Cycling conditions: 98℃ for 3 min; 98℃ for 15 sec, 60℃ for 20 sec, 72℃ for 30 s, 40 cycles; 72℃ for 5 min.

[0140] E. Result judgment: Use 1.5% agarose gel for electrophoresis test. Judge whether the target fragment is amplified based on the presence or absence of electrophoresis bands and their electrophoresis positions; judge whether it is specific amplification based on whether there is a single electrophoresis band.

[0141] The agarose gel electrophoresis of the second PCR amplification product obtained by the second PCR amplification is shown in Figure 3 shown.

[0142] according to Figure 3 The agarose gel electrophoresis of the second PCR amplification product in the figure can draw the following conclusions:

[0143] A. The second PCR amplification obtained the target gene TERT gene (5′-3′) fragment;

[0144] B. The second PCR amplification is specific amplification.

[0145] DNA was purified and recovered using a DNA purification kit (spin column method, catalog number: DP204) (Tiangen Biochemical Technology (Beijing) Co., Ltd.) according to the operating instructions;

[0146] The purified DNA was diluted a second time using sterile water for injection as diluent, with a dilution factor of 10 3 times to obtain a 100% negative DNA methylation standard.

[0147] In the 100% negative DNA methylation standard, the newly generated second PCR amplification products are all "T, A, C, G" bases from the PCR premix kit, and the DNA of the original sample (ie, the TERT gene in the peripheral blood genomic DNA of apparently healthy people) has no residue after two dilutions. Therefore, the product is almost entirely unmethylated DNA sequences and can be used as a 100% negative DNA methylation standard.

[0148] (3) Preparation of 100% positive DNA methylation standards

[0149] The obtained 100% negative DNA methylation standard solution was subjected to DNA methylation modification using the Thermo Fisher Scientific DNA Methylation Modification Kit (Thermo Fisher Scientific Inc, USA, catalog number: EM0821), and after purification and recovery, a 100% positive DNA methylation standard was obtained;

[0150] The composition of the reaction system for DNA methylation modification is shown in Table 3.

[0151] Table 3

[0152] name Dosage 10X M.SssI Buffer 2μL 50X SAM 0.4μL M.SssI 1μL DNA 17.5μL total 20μL

[0153] in:

[0154] A, 10X M.SssI Buffer, 50X SAM and M.SssI: DNA methylation modification kit reagents.

[0155] B. DNA: 100% negative methylation standard.

[0156] C. Reaction conditions: Incubate at 37°C for 15 minutes.

[0157] D. Reaction termination conditions: Heat at 65°C for 20 minutes to stop the reaction.

[0158] E. After the reaction is terminated, DNA is purified and recovered: DNA purification kit (spin column method, catalog number: DP204) (Tiangen Biochemical Technology (Beijing) Co., Ltd.) is used to purify and recover DNA according to the operating instructions.

[0159] (4) Calibration

[0160] Real-time fluorescence quantitative PCR (qPCR) was used for copy number calibration. The composition of the qPCR reaction system is shown in Table 4.

[0161] Table 4

[0162] name Dosage (final concentration) Master mix 10μL(-) TERT-dF 1μL (0.25μM) TERT-dR 1μL (0.25μM) d-probe 0.6μL(0.15μM) Sterile water for injection 5.4μL DNA template 2μL total 20μL

[0163] in:

[0164] A. Master mix: Super Real Fluorescence Quantitation Premix Kit (Probe Method, Catalog Number: FP206-02) (Tiangen Biochemical Technology (Beijing) Co., Ltd.).

[0165] B. Primers and probes

[0166] The sequence of the upstream primer is shown in SEQ ID NO.3 in Table 1; the sequence of the downstream primer is shown in SEQ ID NO.4 in Table 1; and the sequence of the first probe is shown in SEQ ID NO.5 in Table 1. The corresponding relationship among the sequence of the upstream primer, the sequence of the downstream primer, the sequence of the first probe and the sequence of the TERT gene (5′-3′) is shown in Figure 4 shown.

[0167] C. DNA template

[0168] The 100% negative DNA methylation standard was diluted 4 times by 10 (i.e., 4 dilutions were performed successively, and the dilution factor of each dilution was 10), and qPCR amplification was performed simultaneously; the 100% negative DNA methylation standard was named uM1, uM2, uM3 and uM4 respectively;

[0169] The 100% positive DNA methylation standard was diluted 10 times in four steps (ie, diluted 4 times in succession, each time with a dilution factor of 10), and qPCR amplification was performed simultaneously; the 100% positive DNA methylation standard was named M1, M2, M3, and M4, respectively.

[0170] D. Cycling conditions: 98°C for 10 min; 98°C for 15 sec, 60°C for 35 sec, 40 cycles.

[0171] E. DNA copy number results: The gradient of 100% negative DNA methylation standard uM1, uM2, uM3 and uM4 were assigned 1000, 100, 10 and 1 copy number / mL respectively. After the absolute quantitative standard curve was established, the copy number of 100% positive DNA methylation standard M1, M2, M3, M4 was obtained by reading the standard curve, that is, the 100% negative DNA methylation standard and the 100% positive DNA methylation standard were assigned.

[0172] The obtained 100% negative DNA methylation standard (unmethylated standard in the figure) and 100% positive DNA methylation standard (methylated standard in the figure) were tested for amplification curves. The test results are shown in FIG. Figure 5 shown.

[0173] The obtained 100% negative DNA methylation standard and 100% positive DNA methylation standard were tested for standard regression curves. The test results are as follows: Figure 6 shown.

[0174] Using sterile water for injection, dilute the uM1 copy number of the 100% negative methylation standard to the same copy number as the 100% positive methylation standard M1.

[0175] (5) Preparation of DNA methylation standards with different methylation rates

[0176] The gradient of 100% negative methylation standard after copy number calibration was uM1 (1000 copies / mL) as diluent, and 100% positive methylation standard M1 as test sample to prepare 100%, 50%, 25%, 12.5%, 6.25%, 3.125% and 0% methylation rate standards.

[0177] Example 2: Verification

[0178] 1. Conversion

[0179] Using ZYMO RESEARCH's EZ DNA Methylation-Direct TM Kit (USA, catalog number: D5005) was used to convert the DNA methylation rate standard. The details are as follows:

[0180] (1) Reaction system: 10uL DNA + 150uL CT Conversion Reagent;

[0181] (2) Conversion reaction: 98°C for 10 minutes; 64°C for 2.5 hours; 4°C for no more than 20 hours;

[0182] (3) ZymoSpin TM Add 600 μL of M-Binding Buffer to the IC column and place the column in the collection tube provided;

[0183] (4) Load the sample obtained after the conversion reaction in step (2) into a ZymoSpin containing M-binding buffer. TM IC column. Cover with lid and invert the column several times to mix;

[0184] (5) Centrifuge step (4) at full speed (12000 r / min) for 30 seconds and discard the liquid;

[0185] (6) Add 100 μL of M-wash buffer to the column in step (5) and centrifuge at full speed (12000 r / min) for 30 seconds;

[0186] (7) Add 200 μL of M-Desulphonation Buffer to the column obtained in step (6) and incubate at room temperature (20-30°C) for 15-20 minutes. After incubation, centrifuge at full speed (12000 r / min) for 30 seconds;

[0187] (8) Add 200 μL of M-wash buffer to the column obtained in step (7) and centrifuge at full speed (12000 r / min) for 30 seconds. Add another 200 μL of M-wash buffer and centrifuge for another 30 seconds;

[0188] (9) Place the chromatographic column obtained in step (8) into a 1.5 mL microcentrifuge tube, add 10 μL of M-Elution Buffer directly into the column matrix, and centrifuge at full speed (12,000 r / min) for 30 seconds to elute the DNA.

[0189] (10) Collect the eluted DNA liquid obtained in step (9) as the template to be tested.

[0190] 2. Verification

[0191] The obtained DNA methylation standards were verified using the methylation primers and probes in MSP-qPCR for the five CpG sites in the TERT gene of the sequence shown in SEQ ID NO. 9. The composition of the MSP-qPCR reaction system is shown in Table 5.

[0192] Table 5

[0193]

[0194]

[0195] in:

[0196] (1) Master mix: Super Real Fluorescence Quantitation Premix Kit (probe method, catalog number: FP206-02) (Tiangen Biochemical Technology (Beijing) Co., Ltd.).

[0197] (2) DNA methylation primers and DNA methylation probes:

[0198] The sequence of the DNA methylation upstream primer is shown in SEQ ID NO.6 in Table 1; the sequence of the DNA methylation downstream primer is shown in SEQ ID NO.7 in Table 1; and the sequence of the DNA methylation probe is shown in SEQ ID NO.8 in Table 1. The corresponding relationship between the DNA methylation upstream primer sequence, the DNA methylation downstream primer sequence, the DNA methylation probe sequence and the sequence of the TERT gene (5′-3′) is shown in Figure 7 shown.

[0199] (3) DNA template

[0200] The methylation rate standards of 100%, 50%, 25%, 12.5%, 6.25%, 3.125% and 0% obtained in Example 1 were tested twice in duplicate.

[0201] (4) Cycling conditions: 98°C for 10 min; 98°C for 15 sec, 60°C for 35 sec, 40 cycles.

[0202] (5) Test results: Test results are as follows: Figure 8 and Fig. 9 As shown. Among them, Figure 8 This is the amplification curve of the MSP-qPCR validation methylation rate standard (measured twice simultaneously); Fig. 9 This is the standard regression curve for MSP-qPCR validation of methylation rate standards.

[0203] according to Figure 8 and Fig. 9 From the data analysis, we can draw the following conclusions:

[0204] (1) There is no amplification curve for the 0% methylation rate standard, indicating that the methylation level of the 100% negative methylation standard is zero, which also shows that the primer specificity of the MSP-qPCR is high;

[0205] (2) For 100%, 50%, 25%, 12.5%, 6.25%, and 3.125% positive standards, a good amplification curve was presented, and the methylation rate regression curve was: Y = -3.428x + 35.373 (R2 = 0.996, amplification efficiency (%) = 95.775), indicating that the detection performance of the MSP-qPCR was excellent.

[0206] In summary, the DNA methylation standards and preparation methods and applications provided in the embodiments of the present disclosure have the following characteristics:

[0207] The purity of the DNA methylation standard is high, wherein the purity of the prepared 100% negative DNA methylation standard and 100% positive DNA methylation standard can almost reach 100%.

[0208] The preparation method of DNA methylation standard is simple, and only the corresponding primers and probes of the target gene need to be synthesized to prepare the DNA methylation standard. In particular, the preparation of 100% negative DNA methylation standard solves the problem of complex preparation of the standard.

[0209] The specificity of MSP-qPCR primers is clear and intuitive. Since there is no methylation in the prepared 100% negative DNA methylation standard, the specificity of the MSP-qPCR primers can be clearly judged based on the presence or absence of an amplification curve through MSP-qPCR amplification, which provides the necessary basis for the establishment of a specific MSP-qPCR detection method.

[0210] Strong application and promotion potential. Since MSP-qPCR methylation detection has been widely recognized and applied in the industry, the method for preparing DNA methylation standards disclosed in the present invention has the advantage of strong application and promotion potential.

[0211] It should be noted that, in this article, relational terms such as "first" and "second" are only used to distinguish one entity or operation from another entity or operation, and do not necessarily require or imply any such actual relationship or order between these entities or operations. Moreover, the terms "include", "comprise" or any other variants thereof are intended to cover non-exclusive inclusion, so that a process, method, article or device including a series of elements includes not only those elements, but also other elements not explicitly listed, or also includes elements inherent to such process, method, article or device. In the absence of further restrictions, the elements defined by the sentence "comprise a ..." do not exclude the existence of other identical elements in the process, method, article or device including the elements.

[0212] The above description is only a specific embodiment of the present disclosure, so that those skilled in the art can understand or implement the present disclosure. Various modifications to these embodiments will be apparent to those skilled in the art, and the general principles defined herein may be implemented in other embodiments without departing from the spirit or scope of the present disclosure. Therefore, the present disclosure will not be limited to the embodiments described herein, but will conform to the widest scope consistent with the principles and novel features disclosed herein.

Claims

1. A method for preparing a DNA methylation standard, characterized in that: The DNA methylation standard includes a 100% negative DNA methylation standard and a 100% positive DNA methylation standard, and the preparation method includes: (1) performing a first PCR amplification on the target gene, diluting the amplified product and then performing a second PCR amplification to obtain the 100% negative DNA methylation standard; (2) Performing methylation treatment on the 100% negative DNA methylation standard to obtain the 100% positive DNA methylation standard.

2. The preparation method according to claim 1, characterized in that: The amplification reagents used in the first amplification and the second amplification do not include methylated bases.

3. The preparation method according to claim 1, characterized in that: The preparation method further comprises: mixing 100% negative DNA methylation standards and 100% positive DNA methylation standards of the same level in different proportions to obtain DNA methylation standards with different methylation rates.

4. The preparation method according to claim 3, characterized in that: Methods for adjusting the 100% negative DNA methylation standard and the 100% positive DNA methylation standard to the same level include: The 100% negative DNA methylation standard and the 100% positive DNA methylation standard were serially diluted, and the serially diluted samples were amplified by real-time fluorescence quantitative PCR; Assign values ​​to the copy numbers of the gradient 100% negative DNA methylation standard or the gradient 100% positive DNA methylation standard, and establish an absolute quantitative standard curve; The copy number of the 100% positive DNA methylation standard is read using the absolute quantitative standard curve of the assigned 100% negative DNA methylation standard; or the copy number of the 100% negative DNA methylation standard is read using the absolute quantitative standard curve of the assigned 100% positive DNA methylation standard; The 100% negative DNA methylation standard and the 100% positive DNA methylation standard were adjusted to the same level using copy number.

5. The preparation method according to claim 3, characterized in that: The different methylation rates include a 50% methylation rate, a 25% methylation rate, a 12.5% ​​methylation rate, a 6.25% methylation rate or a 3.125% methylation rate.

6. The preparation method according to any one of claims 1 to 5, characterized in that: The dilution factor is 10 5 -10 8 times.

7. The preparation method according to any one of claims 1 to 5, characterized in that The preparation method further comprises: diluting the second PCR amplification sample again to obtain the 100% negative DNA methylation standard.

8. A DNA methylation standard obtained by the preparation method according to any one of claims 1 to 7.

9. Use of the DNA methylation standard according to claim 8 in evaluating a methylation specific real-time fluorescence quantitative PCR detection method.

10. The use according to claim 9, characterized in that: The application includes the evaluation of primer specificity in a methylation-specific real-time fluorescence quantitative PCR detection method.

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