CN116426624A - Standardized DNA methylation detection standard, preparation method, fixed value method and application thereof - Google Patents
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Abstract
The embodiment of the specification discloses a standardized DNA methylation detection standard substance, a preparation method, a value fixing method and application thereof. The DNA methylation detection standard is prepared from an original DNA sample and a methylated DNA sample obtained by methylation of the original DNA sample, wherein the original DNA sample comprises a target gene fragment, a first nucleotide fragment and a second nucleotide fragment, the first nucleotide fragment is a nucleotide sequence shown as SEQ ID No.1, the second nucleotide fragment is a nucleotide sequence shown as SEQ ID No.2, the methylated DNA sample comprises a target gene fragment after methylation, a third nucleotide fragment after methylation of the first nucleotide fragment and a fourth nucleotide fragment after methylation of the second nucleotide fragment, the third nucleotide fragment is a nucleotide sequence shown as SEQ ID No.3, and the fourth nucleotide fragment is a nucleotide sequence shown as SEQ ID No. 4.
Description
Technical Field
The application relates to the field of biotechnology, in particular to a standardized DNA methylation detection standard substance, a preparation method, a value fixing method and application thereof.
Background
DNA methylation plays an important regulatory role in the growth, development, pattern of gene expression, and stability of the genome of individuals, and is closely related to the occurrence of certain tumors and genetic diseases. In human genome, about 70% -80% of CpG dinucleotides are in a DNA methylation state, unmethylated CpG is unevenly distributed, and a plurality of areas with higher GC content and relatively aggregated CpG dinucleotides are formed, namely CpG islands, and abnormal methylation of the CpG islands is often accompanied with diseases such as cancers and the like. In mammalian genomes, DNA methylation occurs at the 5-carbon atom of cytosine in CpG dinucleotides, and tumor occurrence is manifested by a decrease in the overall methylation level of the genome and an abnormally elevated local methylation level of CpG islands. Thus, changes in the overall methylation level (i.e., methylation profile) of DNA and the methylation level of a particular gene can be used as a diagnostic indicator of a tumor.
In recent years, with the continuous development of high-throughput detection technology, the genome DNA methylation detection method is mature, and the method becomes one of important means for molecular diagnosis of related tumors and genetic diseases in the future. In the prior art, cells positive for methylation of a target sequence are found through searching a literature, and the modified bisDNA is used as a positive control, so that the DNA methylation level cannot be accurately estimated. With the continuous perfection and development of biotechnology, DNA methylation detection lacks a unified detection standard which can be accurately quantified. In addition, the existing fluorescent quantitative fixed value method for the DNA methylation detection standard substance has higher sequencing cost, higher sample quality requirement, can not give the methylation degree of a single gene, and can only simply carry out primary evaluation on the high-medium-low methylation level of the sample.
Therefore, it is necessary to provide a standard substance and a preparation and determination method thereof, which make the DNA methylation detection result more reliable and accurate.
Disclosure of Invention
According to an aspect of the present application, there is provided a standardized DNA methylation detection standard, which is prepared from an original DNA sample and a methylated DNA sample obtained by methylation of the original DNA sample, wherein the original DNA sample includes a target gene fragment, a first nucleotide fragment and a second nucleotide fragment, the first nucleotide fragment is a nucleotide sequence shown as SEQ ID No.1, the second nucleotide fragment is a nucleotide sequence shown as SEQ ID No.2, the methylated DNA sample includes a target gene fragment after methylation, a third nucleotide fragment obtained by methylation of the first nucleotide fragment, and a fourth nucleotide fragment obtained by methylation of the second nucleotide fragment, the third nucleotide fragment is a nucleotide sequence shown as SEQ ID No.3, and the fourth nucleotide fragment is a nucleotide sequence shown as SEQ ID No. 4.
According to another aspect of the present application, there is provided a method for preparing a standardized DNA methylation detection standard, comprising the steps of: synthesizing the original DNA sample; methylating the original DNA sample by using methyltransferase to obtain the methylated DNA sample; based on the original DNA sample and the methylated DNA sample, DNA methylation detection standards of different methylation duty cycles are configured.
According to another aspect of the present application, there is provided a method of quantifying a standardized DNA methylation detection standard, comprising the steps of: performing bisulfite conversion on the DNA methylation detection standard substance to obtain a vulcanized standard substance; performing PCR on the vulcanized standard substance by adopting a primer probe combination aiming at the third nucleotide fragment and a primer probe combination aiming at the fourth nucleotide fragment, and respectively determining the total DNA amount and the methylated DNA amount in the DNA methylation detection standard substance; determining a methylation duty cycle in the DNA methylation detection standard based on the total DNA amount and the amount of DNA that is subject to methylation. In some embodiments, the primer probe combination for the third nucleotide fragment comprises a forward primer as shown in SEQ ID No.5, a reverse primer as shown in SEQ ID No.6 and a probe as shown in SEQ ID No.7, and the primer probe combination for the fourth nucleotide fragment comprises a forward primer as shown in SEQ ID No.8, a reverse primer as shown in SEQ ID No.9 and a probe as shown in SEQ ID No. 10.
According to yet another aspect of the present application, there is provided the use of a standardized DNA methylation detection standard for the preparation of a methylation detection kit.
Drawings
The present application will be further illustrated by way of example embodiments, which will be described in detail with reference to the accompanying drawings. These embodiments are not limiting, wherein:
FIG. 1 is a schematic representation of methylation sample quantitation results according to some embodiments of the present disclosure.
Detailed Description
In order to more clearly illustrate the technical solutions of the embodiments of the present application, the drawings that are required to be used in the description of the embodiments will be briefly described below. It is apparent that the drawings in the following description are only some examples or embodiments of the present application, and it is obvious to those skilled in the art that the present application may be applied to other similar situations according to the drawings without inventive effort. Unless otherwise apparent from the context of the language or otherwise specified, like reference numerals in the figures refer to like structures or operations.
As used in this application and in the claims, the terms "a," "an," "the," and/or "the" are not specific to the singular, but may include the plural, unless the context clearly dictates otherwise. In general, the terms "comprises" and "comprising" merely indicate that the steps and elements are explicitly identified, and they do not constitute an exclusive list, as other steps or elements may be included in a method or apparatus.
Flowcharts are used in this application to describe the operations performed by systems according to embodiments of the present application. It should be appreciated that the preceding or following operations are not necessarily performed in order precisely. Rather, the steps may be processed in reverse order or simultaneously. Also, other operations may be added to or removed from these processes.
The following are definitions of some terms used in this application.
The term "DNA methylation" refers to a chemical modification process in which active methyl groups are transferred to specific bases in a DNA strand under the catalysis of a DNA methyltransferase using S-adenosylmethionine as a methyl donor. The term includes cytosine methylation at the C5 or N4 position of cytosine, the N6 position of adenine, or other types of nucleic acid methylation. "unmethylated DNA" or "methylated DNA" can refer to amplified DNA whose original template is unmethylated or methylated, respectively.
The term "CpG island" refers to a contiguous region of genomic DNA having a high density of CpG.
The term "forward primer" generally refers to the sense strand. The DNA molecule is double-stranded, wherein the 5'-3' strand is called the positive strand and the 3'-5' strand is called the negative strand. The forward primer may synthesize a strand that is identical to the sequence of the forward strand. The strand synthesized by the forward primer may be complementary to the sequence of the negative strand.
The term "reverse primer" generally refers to an antisense strand, and the strand synthesized by the reverse primer may be identical to the sequence of the negative strand. The strand synthesized by the reverse primer may be complementary to the sequence of the forward strand.
The term "probe" refers to any molecule capable of selectively binding to a target biomolecule (e.g., a nucleic acid sequence that hybridizes to the probe). In some embodiments, the probe is detectably labeled, for example with a fluorescent group and a quenching group.
The term "Polymerase Chain Reaction (PCR)" is a molecular biological technique for amplifying specific DNA fragments.
The term "real-time fluorescent quantitative PCR (Quantitative Real-time PCR)" may also be referred to as qPCR, RT-PCR. qPCR is a method of detecting the total amount of products after each Polymerase Chain Reaction (PCR) cycle by fluorescent chemicals in DNA amplification reaction, and can quantitatively analyze specific DNA sequences in a sample to be detected by internal or external reference methods.
The term "sample" refers to any composition containing nucleic acid isolated from a subject. The term may include purified or isolated cells, tissues, blood, urine, components of saliva, e.g., DNA or cell lysates.
The term "gene of interest" refers to a gene that may be mutated and can be used as a tumor marker for the prevention and detection of risk of disease. Illustratively, the genes of interest may include, but are not limited to, one or more of septin9, SHOX2, RASSF1A, RNF, SDC2, MGMT, BMP3, NDRG 4.
DNA methylation is an important component of epigenetic science, playing an important role in maintaining normal cellular function, genetic imprinting, embryo development, and human tumorigenesis. Methylation of DNA is the conversion of cytosine at the 5' end of CpG dinucleotides to methylcytosine by the action of DNA methyltransferase. This way of DNA modification regulates the expression of the gene. CpG sequences occur only 1% frequently in the genome, much less frequently than other dinucleotide sequences in the genome. However, in some regions of the genome, the density of CpG sequences is very high, which can be more than 5 times the mean, and become an enrichment region of guanine and cytosine, forming CpG islands. CpG islands are typically located in the promoter region or first exon region of a gene. In the genome of a healthy person, cpG sites in CpG islands are usually in an unmethylated state, whereas CpG sites outside CpG islands are usually methylated. When a tumor occurs, the degree of unmethylation of CpG sequences other than the CpG island of the cancer suppressor gene increases, and the CpG in the CpG island is in a highly methylated state, so that the degree of chromosome helicity increases and the expression of the cancer suppressor gene is lost. Thus, changes in the overall methylation level (i.e., methylation profile) of DNA and the methylation level of a particular gene can be used as a diagnostic indicator of a tumor.
Abnormal gene methylation is one of the most common epigenetic changes in tumorigenesis, which is manifested by a decrease in the overall methylation level of the genome (oncogene) and an abnormal increase in the local methylation level of CpG islands (oncogene inhibitor). Current research on tumor methylation has focused mainly on oncogenes. This is because it was found that tumor occurrence may be associated with the turn-off of the oncogene due to methylation of CpG islands in the promoter region of the oncogene. Since the local hypermethylation of CpG islands is earlier than the malignant proliferation of cells, diagnosis of methylation can be used for early prediction of tumorigenesis, and hypomethylation of the whole genome also occurs with tumorigenesis, and it is significant with increasing malignancy of tumors, so detection of methylation can be used for grading of tumors.
In recent years, with the continuous development of high-throughput detection technology, the genome DNA methylation detection method is mature, and the method becomes one of important means for molecular diagnosis of related tumors and genetic diseases in the future. Methylation detection can now be broadly divided into two categories: methylation detection at specific sites and methylation analysis of the whole genome, the latter also known as methylation profile analysis. Methylation detection of specific sites includes methylation-specific PCR (MS-PCR), bisulfite treatment+sequencing, restriction enzyme analysis with sodium bisulfite, fluorescent quantitation, methylation-sensitive high resolution melting curve analysis, pyrosequencing. However, with the continuous perfection and development of technology, DNA methylation detection has not yet had a unified detection standard capable of accurately quantifying, and has become a problem to be solved in the development process of methylation detection kits.
Several methylation detection standards are currently common in the art, which are prepared mainly by: (1) The target sequence methylation positive cells are found through searching the literature, the modified bisDNA is used as a positive control, the operation process is complicated, a large amount of literature investigation and subsequent verification work are required in the early stage, the method is not suitable for the research of polygene methylation or new target methylation projects, the methylation level cannot be accurately estimated, and in addition, the stability of the bisDNA is poor. (2) Commercial methylation positive controls were purchased, but were more expensive, commercial standards were fewer, methylation level data were not detailed, product customization cycles were longer, and large differences between methylation levels were observed.
Methylation-specific PCR is a classical method for detecting gene methylation. The principle is that the original genomic DNA is first modified with sodium bisulphite, all unmethylated cytosines (C) are converted to uracil (U), while methylated cytosines are unchanged. Primers for methylated and unmethylated sequences are then designed and amplified by Polymerase Chain Reaction (PCR), and the difference can be detected by using different primers for PCR, thereby determining whether the gene has CpG island methylation.
The invention provides a standardized DNA methylation standard substance and a preparation and value-determining method thereof by artificially synthesizing a segment of oligonucleotide sequence containing a target gene fragment, wherein the 5 'end and the 3' end of the sequence respectively comprise a segment of housekeeping gene oligonucleotide sequence (SEQ ID No.1 and SEQ ID No. 2) with a specific sequence, combining CpG methylation enzyme (M.SssI) treatment to obtain a methylated oligonucleotide sequence containing the target gene fragment, and combining a digital PCR technology to accurately quantify the concentration and the methylation level. In some embodiments of the present disclosure, sample DNA is modified by CpG methylase (m.sssi), which recognizes and methylates all cs in CpG to match the DNA status of the ideal biological genome, and mimics the modification pattern of the biological genome, thus being useful as a positive control. The positive control is used as a DNA methylation detection standard system, so that the reliability of a detection result can be ensured, the detection is quick and simple, the time and the detection cost are saved, and the subsequent quantitative detection of the methylation level of a detection site in a sample by combining a real-time quantitative PCR technology is facilitated.
In one aspect, the present application provides a standardized DNA methylation detection standard, which is prepared from an original DNA sample and a methylated DNA sample obtained by methylation of the original DNA sample, wherein the original DNA sample includes a target gene fragment, a first nucleotide fragment and a second nucleotide fragment, the first nucleotide fragment is a nucleotide sequence shown as SEQ ID No.1, the second nucleotide fragment is a nucleotide sequence shown as SEQ ID No.2, the methylated DNA sample includes a target gene fragment after methylation, a third nucleotide fragment after methylation of the first nucleotide fragment, and a fourth nucleotide fragment after methylation of the second nucleotide fragment, the third nucleotide fragment is a nucleotide sequence shown as SEQ ID No.3, and the fourth nucleotide fragment is a nucleotide sequence shown as SEQ ID No. 4.
In some embodiments, the first and third nucleotide fragments are human ACTB housekeeping gene nucleotide fragments that do not contain CG continuous bases, and the second and fourth nucleotide fragments are human ACTB housekeeping gene nucleotide fragments that contain multiple CG continuous bases. The original DNA sequence and methylated DNA sequence of the standard are shown in table 1.
Table 1 original DNA sequence and methylated DNA sequence of Standard
In some embodiments, the third nucleotide fragment is used to detect the total amount of DNA in the standard and the fourth nucleotide fragment is used to detect the amount of DNA methylated in the standard. Specifically, after denaturation and melting of double-stranded DNA, since methylation modification occurs only at C of CG continuous base structure in 5 'to 3' direction, and the third nucleotide fragment does not contain CG continuous base, the fourth nucleotide fragment contains CG continuous base, therefore, in HSO 3 - After the action, the third nucleotide fragment is not methylated, and the base CG in the sequence is changed into UG; methylation of the fourth nucleotide fragment maintains the base CG in the sequence as CG. Thus, the third nucleotide fragment can be used to detect the total amount of DNA in the standard and the fourth nucleotide fragment can be used to detect the amount of methylated DNA in the standard.
In the quality control of the methyltransferase modification efficiency, the DNA subjected to the M.SsssI treatment can be subjected to sulfuration purification treatment by adopting a bisulfite conversion kit to obtain bisM.SsssI DNA; the total bisDNA amount and bisDNA methylation amount are measured, and the methyltransferase enzyme modification efficiency is calculated, wherein the specific formula is as follows:
transfer efficiency= (amount of bisDNA methylation/amount of total bisDNA) ×100% (1)
In some embodiments, the methyltransferase enzyme transfer efficiency is not less than 90%.
In some embodiments, the gene of interest comprises one or more of septin9, SHOX2, RASSF1A, RNF180, SDC2, MGMT, BMP3, NDRG 4.
According to another aspect of the present application, there is provided a method for preparing a standardized DNA methylation detection standard, comprising the steps of: synthesizing an original DNA sample; methylating the original DNA sample by adopting methyltransferase to obtain a methylated DNA sample; based on the original DNA sample and the methylated DNA sample, DNA methylation detection standards of different methylation duty cycles were configured. The methylation duty cycle of the standard may be between 0% and 100%.
In some embodiments, the methyltransferase is a CpG methyltransferase.
In some embodiments, configuring DNA methylation detection standards of different methylation duty cycles based on the original DNA sample and the methylated DNA sample may include: purifying and recovering the methylated DNA sample, and calculating the recovery efficiency of the purified DNA sample; based on the original DNA sample and the purified methylated DNA sample, DNA methylation detection standards of different methylation duty cycles are configured. For example, a methylated DNA sample at a concentration of 1 ng/. Mu.L was mixed with the original DNA sample at a concentration of 1:19 (V: V), to prepare a standard having a methylation ratio of 5% at a concentration of 1 ng/. Mu.L. As another example, a methylated DNA sample at a concentration of 0.5 ng/. Mu.L was mixed with the original DNA sample at a concentration of 1:99 (V: V), to prepare a standard having a methylation ratio of 1% at a concentration of 0.5 ng/. Mu.L. In some embodiments, the methylated DNA sample can be purified using a PCR product purification kit, and the purification step can include: the eluent was added for research purposes and its theoretical DNA concentration was calculated, while the actual concentration was determined for the purified DNA with a qubit and its recovery efficiency during purification was calculated. In some embodiments, the recovery efficiency is not less than 90%.
In some embodiments, configuring DNA methylation detection standards of different methylation duty cycles based on the original DNA sample and the methylated DNA sample comprises: performing bisulfite conversion on the methylated DNA sample to obtain a vulcanized methylated DNA sample; and measuring the total vulcanized DNA amount and the vulcanized methylated DNA amount in the vulcanized methylated DNA sample, and calculating the transfer efficiency of the methyltransferase modification, wherein the transfer efficiency is not lower than 90%.
According to another aspect of the present application, there is provided a method of quantifying a standardized DNA methylation detection standard (i.e. determining the proportion of methylation in a DNA methylation detection standard), comprising the steps of: performing bisulfite conversion on the DNA methylation detection standard substance to obtain a vulcanized standard substance; performing PCR on the vulcanized standard substance by adopting a primer probe combination aiming at the third nucleotide fragment and a primer probe combination aiming at the fourth nucleotide fragment, and respectively determining the total DNA amount and the methylated DNA amount in the DNA methylation detection standard substance; determining a methylation duty cycle in the DNA methylation detection standard based on the total DNA amount and the amount of DNA that is subject to methylation. In some embodiments, the primer probe combination for the third nucleotide fragment comprises a forward primer as shown in SEQ ID No.5, a reverse primer as shown in SEQ ID No.6 and a probe as shown in SEQ ID No.7, and the primer probe combination for the fourth nucleotide fragment comprises a forward primer as shown in SEQ ID No.8, a reverse primer as shown in SEQ ID No.9 and a probe as shown in SEQ ID No. 10. The sequences of the primer probe sets are shown in Table 2. It should be noted that the fluorophores in the probes shown in SEQ ID No.7 and SEQ ID No.8 may be other fluorophores besides FAM, VIC (or HEX), MGB, e.g., HEX, TET, ROX, TAMRA, JOE, cy, cy5, etc.
TABLE 2 sequences of primer probe sets
According to yet another aspect of the present application, there is provided the use of a standardized DNA methylation detection standard for the preparation of a methylation detection kit. In some embodiments, the DNA methylation detection standard is stable for at least half a year in a low temperature environment, the low temperature environment being below-4 ℃. In some embodiments, the low temperature environment is from-4 ℃ to-80 ℃. In some embodiments, the low temperature environment is from-20 ℃ to-80 ℃. In some embodiments, the low temperature environment is from-40 ℃ to-80 ℃. In some embodiments, the low temperature environment is from-20 ℃ to-70 ℃. In some embodiments, the low temperature environment is from-40 ℃ to-70 ℃. Exemplary low temperature environments are, for example, -20 ℃, -40 ℃, -70 ℃, etc. The DNA methylation detection standards provided in the examples herein and methods of making and quantifying them are not limited to artificially constructed samples, cell lines, genomic DNA, hypomethylated samples.
The experimental methods in the following examples are conventional methods unless otherwise specified. The test materials used in the examples described below, unless otherwise specified, were purchased from conventional Biochemical reagent companies. The quantitative tests in the following examples were each set up for multiple replicates and the results averaged.
Examples
Example 1 preparation of DNA methylation detection Standard
1. Selection and determination of specific structures.
According to the requirement of experimental purposes, two sections of different characteristic regions of beta-actin are selected as quality control sequences. Wherein SEQ ID No.1 is a human ACTB housekeeping gene nucleotide fragment which does not contain CG continuous bases and is positioned at the position of chr7 (-) 5532083 ～ 5532223 of the human genome; SEQ ID No.2 is a nucleotide fragment of the human ACTB housekeeping gene containing multiple CG consecutive bases, located at the position chr7 (-) 5530404 ～ 5530580 of the human genome.
2. Design and synthesis of target gene segment with specific structure.
The specific structure mainly comprises: 5'-SEQ ID No.1+ target gene fragment+SEQ ID No.2-3' end or 5'-SEQ ID No.2+ target gene fragment+SEQ ID No.1-3' end.
3. Methyltransferase modification of a gene fragment of interest of a specific structure.
CH is added to the 5-position carbon atom of cytosine in CpG dinucleotide in standard gDNA by adopting a methyltransferase method 3 Methylation modification and purification were performed.
The methyltransferase modification process is as follows:
1) A synthetic DNA mother solution of a target gene fragment with a specific structure is prepared, and a qubit is used for determination, wherein the concentration of the DNA mother solution is more than or equal to 100ng/ul (the minimum concentration is not less than 50 ng/ul).
2) Methyltransferase modification systems were formulated as in table 3.
TABLE 3 methyltransferase modification System
3) And (3) performing DNA methylation modification according to the methyl transferase modification program in the following table to obtain a DNA methylation detection standard.
TABLE 4 methyltransferase modification procedure
| Temperature (temperature) | Time |
| 37℃ | 4 hours (10. Mu.L diluted SAM (1600. Mu.M) was added 2 hours of running) |
| 65℃ | 20 minutes |
Example 2 DNA methylation Standard fixed value
Bisulfite treatment: performing bisulfite treatment and purification on the DNA methylation detection standard in the step 4 in the example 1 to obtain bis DNA;
and (3) setting: diluting the treated bis DNA into different concentrations, and carrying out fixed value, wherein the specific operation is as follows:
1. taking out primer probe premix and sterilized water, standing at room temperature for 5-10 min or holding the reagent tube to make it fully melted, shaking, mixing and centrifuging for a short time. Then, 10 parts by xDigitalAmpPCR Master Mix were taken out to prepare a solution, which was prepared in accordance with the following Table 5.
Table 5 constant value formulation system
| Raw materials | Addition amount (mu L) |
| SEQ ID NO.5 (abbreviated as IC-F) | 1.5 |
| SEQ ID NO.6 (abbreviated as IC-R) | 1.5 |
| SEQ ID NO.7 (abbreviated as IC-P) | 0.75 |
| SEQ ID NO.8 (abbreviated ACTB-F) | 1.5 |
| SEQ ID NO.9 (abbreviated ACTB-R) | 1.5 |
| SEQ ID NO.10 (abbreviated ACTB-P) | 0.75 |
| Digital Amp PCR Master Mix(10X) | 2 |
| Ultrapure water | 9 |
| Template | 2 |
2. Transferring to a sample adding chamber, adding sample, shaking, vibrating, and shaking.
3. Chip loading: the operation was performed according to the digital PCR system operating manual.
4. And (3) spreading the packaged chips on a metal plate of a plate gene amplification instrument, and covering a hot cover to run a program.
TABLE 6 fixed value amplification procedure
5. The chip was transferred to a biochip reader for reading, see digital PCR System operating Manual.
6. And (5) data interpretation.
The methylation level was interpreted from the chip reading results, exemplary results are shown in FIG. 1: FAM channel is the number of methylation positive wells and HEX is the total number of wells of the methylation sample. Mutation frequency was measured to be 0.94%.
Example 3 methyltransferase transfer System optimization
Construction of a DNA methylation Standard preparation method.
1.1 optimization of the amount of methyltransferase.
Optimization of the methyltransferase modification system was performed taking as an example a healthy human gDNA sample, a cell line (specific gene hypomethylated cell line) and a plasmid a sample constructed according to the method in example 1.
(1) The design and synthesis of the target gene of a specific structure was performed according to example 1.
The sequence of the gene of interest for the preparation of plasmid A is shown in SEQ ID NO. 11:
CGCGGGGCCACTACTCACGCGCGCACTGCAGGCCTTTGCGCACGACGCCCCAGATGAAGTCGCCACAGAGGTCGCACCACGTGTGCGTGGCGGGCCCCGCGGGCTGGAAGCGGTGGCCACGGCCAGGGACCAGCTGCCG。
taking PUC57 as a construction vector, and adding SEQ ID NO.1 and SEQ ID NO.2 on a target gene fragment to obtain an original DNA sample sequence shown in SEQ ID NO. 12:
GGAGGAGGCTCAGCAAGTCTTCTGGACTGTGAACCTGTGTCTGCCACTGTGTGCTGGGTGGTGGTCATCTTTCCCACCAGGCTGTGGCCTCTGCAACCTTCAAGGGAGGAGCAGGTCCCATTGGCTGAGCACAGCCTTGTACGCGGGGCCACTACTCACGCGCGCACTGCAGGCCTTTGCGCACGACGCCCCAGATGAAGTCGCCACAGAGGTCGCACCACGTGTGCGTGGCGGGCCCCGCGGGCTGGAAGCGGTGGCCACGGCCAGGGACCAGCTGCCGCCGCGAGCACAGAGCCTCGCCTTTGCCGATCCGCCGCCCGTCCACACCCGCCGCCAGGTAAGCCCGGCCAGCCGACCGGGGCAGGCGGCTCACGGCCCGGCCGCAGGCGGCCGCGGCCCCTTCGCCCGTGCAGAGCCGCCGTCTGGGCCGCAGCGGGGGGCGCATGGGGGGGGAACC。
(2) The concentrations of plasmid A synthesized and of the extracted healthy human gDNA sample (abbreviated as H) and of cell line B (specific gene hypomethylated cell line) were determined as shown in Table 7 below.
TABLE 7 sample concentration information Table
| Sample of | Fragment Length | Qubit(ng/μL) |
| Plasmid A | 3167 | 171 |
| H | 3000000000 | 325 |
| Cell line B | 3000000000 | 178 |
Note that: and converting according to a unit conversion formula: copy/. Mu.L (copy/microliter) =6.02×10 23 *ng/μL*10 -9 /(660. Fragment length bp).
(3) methylationmodificationwasperformedontheabovesamplesinthemannerdescribedinstep3ofexample1,andthemethylation-modifiedsamplesweredesignatedasM-AandM-HandM-B.
The methyltransferase modification system was formulated as per table 8:
TABLE 8 methyltransferase modification System
(4) The modified sample was purified to a theoretical value of 5 ng/. Mu.L, and the Qubit concentration was measured, with the results shown in Table 9 below.
TABLE 9 sample concentration and recovery after modification with methyl enzyme
(5) The samples in Table 9 and the samples in Table 7 were treated with bisulfite, the DNA input was 500ng, and the treated samples were rated at 330 copy/. Mu.L.
Table 10 sample naming after bisulfite treatment
(6) The sample in (5) is synchronously detected by using a fluorescent quantitative PCR detection kit developed by the company and the constant value method of the invention, and the sample is subjected to gradient dilution for 4 gradients and detection, wherein the plasmid sample and the genomic DNA sample are subjected to conversion detection according to the plasmid sample and the genomic DNA sample, and the conversion detection result is as follows.
TABLE 11 fluorescent quantitative PCR reaction solution detection results
Analysis of results: according to the detection results shown in Table 10 and Table 11, it is shown that CH can be artificially added to the 5-position carbon atom of cytosine in CG island dinucleotide in a specific gene by a methyltransferase method 3 The recovery rate of the method in the DNA modification process can reach more than 90%, and the recovery rate of the method in the DNA modification process is better under the condition of 2 mu L of methyltransferase. Moreover, before methylation modification, samples are negative results, but after the samples pass through the methyltransferase process of the kit, the transfer efficiency can reach up to 100%, and only CG island is modified.
Example 4 optimization of the fixed value method
The existing fluorescence quantitative value-determining method needs to construct and optimize a system of each research target gene, is complex, and has limitation in usability; the NGS sequencing cost is high, the sample quality is high, the methylation degree of a single gene cannot be given, and the sample can be simply subjected to preliminary evaluation on the high, medium and low methylation levels; aiming at the phenomenon, the invention performs the flow development of the method for constructing the methylation standard DNA and the constant value by constructing the manual construction, and performs the optimization and design of the constant value method by combining the digital PCR chip platform by means of the characteristics of housekeeping genes.
Digital PCR was performed for 4 different concentrations of the samples in table 11, the assay being the same as in example 1.
Table 12 digital PCR constant value test data
Note that: the digital PCR chip is 20000 effective holes, and when the digital PCR chip is read, the effective holes of the chip are more than or equal to 12000, and the chip is qualified in manufacturing and the data are effective; wherein the digital PCR system is 20ul, the sample loading amount is 2ul, and the detection result of the digital PCR is 10-time diluted concentration data of the original concentration of the sample.
Analysis of results: the method is consistent with the fluorescence quantitative result, has no nonspecific fluorescence phenomenon, can accurately quantify the methylation level of a sample even at a low sample concentration of 0.33 copy/. Mu.L, and has sensitivity superior to that of fluorescence quantitative PCR.
Example 5 preparation of a Standard for standardized DNA methylation detection and application of the method for quantifying
(1) The methyltransferase modified samples of example 2 were diluted to 330copy/ul and mixed in the proportions shown in Table 13 below.
Table 13 sample formulations with different methylation duty cycles
(2) The invention is used for carrying out the value setting.
TABLE 14 sample constant value results at different methylation duty cycles
Analysis of results: the constant value data shows that the constant value method is feasible, and the measured actual methylation duty ratio accords with the theoretical duty ratio.
EXAMPLE 6 stability study of methyltransferase modified DNA
The DNA treated by the method is respectively stored in a refrigerator at-20 ℃ and a refrigerator at-70 ℃ for 6 months, and digital PCR (polymerase chain reaction) value determination is carried out on the DNA.
TABLE 15PCR fixed values
The current detection data show that the methylated DNA sample constructed by the kit can be stably stored in a refrigerator at-20 ℃ and-70 ℃.
The standardized DNA methylation detection standard disclosed in the embodiments of the present specification, and the preparation method, the rating method and the application thereof may have beneficial effects including but not limited to: (1) The 5-position carbon atom of cytosine in CG island dinucleotide in a specific gene can be artificially added with CH by a methyltransferase mode 3 The recovery rate of the method in the DNA modification process can reach more than 90%, the modification mode of the biological genome can be simulated, the reliability of the detection result is ensured, the method is quick and simple, time and detection cost are saved; (2) The prepared DNA methylation standard has good repeatability and stability, so that the DNA methylation standard can be used for a long time, the continuity and comparability among methylation detection batches are further ensured, and the detection accuracy is improved. (3) The DNA methylation detection standard product and the preparation and setting method thereof provided by the embodiment of the specification can quantitatively detect the methylation level of the detection site in the sample by combining a real-time quantitative PCR technology, further perfects the blank that the domestic DNA methylation detection lacks standard products or does not meet the accurate medical requirements, provides some data support for the methylation standard setting value, and promotes the standardization process of the methylation detection kit standard product.
It will be appreciated by those skilled in the art that the above examples are illustrative of the invention and are not to be construed as limiting the invention. Any modifications, equivalent substitutions and variations, etc., which are within the spirit and principles of the present invention, are intended to be included within the scope of the present invention.
Claims (10)
1. A standardized DNA methylation detection standard is characterized by being prepared from an original DNA sample and a methylated DNA sample obtained by methylation of the original DNA sample,
the original DNA sample comprises a target gene fragment, a first nucleotide fragment and a second nucleotide fragment, wherein the first nucleotide fragment is a nucleotide sequence shown as SEQ ID No.1, the second nucleotide fragment is a nucleotide sequence shown as SEQ ID No.2,
the methylated DNA sample comprises a target gene fragment after methylation, a third nucleotide fragment after methylation of the first nucleotide fragment and a fourth nucleotide fragment after methylation of the second nucleotide fragment, wherein the third nucleotide fragment is a nucleotide sequence shown as SEQ ID No.3, and the fourth nucleotide fragment is a nucleotide sequence shown as SEQ ID No. 4.
2. The standardized DNA methylation detection standard according to claim 1,
the third nucleotide fragment is used for detecting the total DNA amount in the standard,
the fourth nucleotide fragment is used for detecting the amount of methylated DNA in the standard.
3. A method of preparing the standardized DNA methylation detection standard of claim 1, comprising the steps of:
synthesizing the original DNA sample;
methylating the original DNA sample by using methyltransferase to obtain the methylated DNA sample;
based on the original DNA sample and the methylated DNA sample, DNA methylation detection standards of different methylation duty cycles are configured.
4. The method of claim 3, wherein the methyltransferase is a CpG methyltransferase.
5. The method of claim 3, wherein said configuring DNA methylation detection standards of different methylation duty cycles based on said raw DNA sample and said methylated DNA sample comprises:
purifying and recovering the methylated DNA sample, and calculating the recovery efficiency of the purified DNA sample, wherein the recovery efficiency is not lower than 90%;
based on the original DNA sample and the purified methylated DNA sample, DNA methylation detection standards of different methylation duty cycles are configured.
6. The method of any one of claims 3-5, wherein said configuring DNA methylation detection standards of different methylation duty cycles based on said raw DNA sample and said methylated DNA sample comprises:
performing bisulfite conversion on the methylated DNA sample to obtain a vulcanized methylated DNA sample;
and determining the total vulcanized DNA amount and the vulcanized methylated DNA amount in the vulcanized methylated DNA sample, and calculating the transfer efficiency of methyltransferase modification, wherein the transfer efficiency is not lower than 90%.
7. A method of quantifying the normalized DNA methylation detection standard of claim 1, comprising the steps of:
performing bisulfite conversion on the DNA methylation detection standard substance to obtain a vulcanized standard substance;
performing PCR on the vulcanized standard substance by adopting a primer probe combination aiming at the third nucleotide fragment and a primer probe combination aiming at the fourth nucleotide fragment, and respectively determining the total DNA amount and the methylated DNA amount in the DNA methylation detection standard substance;
determining a methylation duty cycle in the DNA methylation detection standard based on the total amount of DNA and the amount of DNA that undergoes methylation, wherein,
the primer probe combination for the third nucleotide fragment comprises a forward primer shown as SEQ ID No.5, a reverse primer shown as SEQ ID No.6 and a probe shown as SEQ ID No.7,
the primer probe combination for the fourth nucleotide fragment comprises a forward primer shown as SEQ ID No.8, a reverse primer shown as SEQ ID No.9 and a probe shown as SEQ ID No. 10.
8. The method of calibrating according to claim 7, wherein,
the nucleotide sequences shown in SEQ ID No.5, SEQ ID No.6, SEQ ID No.8 and SEQ ID No.9 are modified by phosphorothioate.
9. Use of a standardized DNA methylation detection standard according to claim 1 for the preparation of a methylation detection kit.
10. The use of claim 9, wherein the DNA methylation detection standard is stable for at least half a year in a low temperature environment, said low temperature environment being-4 ℃ or less.
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