CN110643702A - Method for determining methylation level of DNA of specific site in biological sample and application thereof - Google Patents
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- C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
- C12Q1/6876—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes
- C12Q1/6883—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes for diseases caused by alterations of genetic material
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- G—PHYSICS
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- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N21/00—Investigating or analysing materials by the use of optical means, i.e. using sub-millimetre waves, infrared, visible or ultraviolet light
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N21/00—Investigating or analysing materials by the use of optical means, i.e. using sub-millimetre waves, infrared, visible or ultraviolet light
- G01N21/62—Systems in which the material investigated is excited whereby it emits light or causes a change in wavelength of the incident light
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- C12Q2600/00—Oligonucleotides characterized by their use
- C12Q2600/154—Methylation markers
Abstract
The invention discloses a method for measuring the methylation level of DNA of a specific site in a biological sample and application thereof. The method comprises the following steps: obtaining a biological sample, and extracting a DNA fragment to be detected in the biological sample; modifying a DNA segment to be detected, and converting unmethylated cytosine deamination in the DNA segment to be detected into uracil; taking the modified DNA fragment as a template DNA fragment, and respectively carrying out PCR amplification on the template DNA fragment from two sides of the template DNA fragment by using a first primer and a second primer; processing the PCR product after PCR amplification, removing redundant first primers, second primers and dNTPs, and purifying and recovering; detecting methylation and demethylation fluorescence polarization values of CpG sites of the DNA fragment to be detected; and calculating the methylation level of the DNA fragment to be detected according to the methylation and demethylation fluorescence polarization values of the CpG sites of the DNA fragment to be detected. The method has the advantages of simplicity, convenience, rapidness, high flux, low cost, no need of separation and purification and the like, and is suitable for detecting methylation of a large amount of clinically precious low-concentration DNA samples.
Description
Technical Field
The invention relates to the technical field of early disease detection, in particular to a method for determining the methylation level of DNA (deoxyribonucleic acid) at a specific site in a biological sample and application thereof.
Background
DNA methylation, an important epigenetic modification, is predominantly represented by 5-methylcytosine (5mC,5-methyl-Cytosine) in the mammalian genome and occurs mostly at the C of two adjacent cytosines (C) and guanines (G) dinucleotides (CpG). 60% -90% of CpG in mammal genome is methylated, a plurality of CpG form a CpG island, a core sequence and transcription initiation point of a structural gene promoter are positioned, and the CpG island is involved in the expression regulation of genes through the change of methylation level. Recent researches find that DNA methylation abnormality is related to a plurality of serious diseases such as tumors and mental diseases, and the high and low methylation degree of CpG sites of some genes can be potential markers of the diseases. The rapid detection of the methylation state of CpG sites of these disease-associated genes is very important for the timely discovery and treatment of these diseases.
At present, methylation detection aiming at gene specific CpG sites is mostly carried out on the basis of sodium bisulfite conversion and PCR amplification. After treatment of genomic DNA with sodium bisulfite, methylated C remained unchanged, whereas unmethylated C converted Uracil (Uracil, U) and PCR amplified it turned into T. Thus, the C position for an incompletely methylated CpG site becomes a C/T mixture, and the quantification of C reflects the methylation level of the CpG site. Most of the conventional quantitative methods are based on single nucleotide extension markers (Ms-SnuPE) of methylation specific primers, and then separation and quantification are carried out according to different markers, although methylation of a plurality of sites can be determined, the operation is complex, expensive instruments and equipment are needed, and the cost is high; the method based on restriction endonuclease or the method based on isotope labeling requires separation by DNA gel and then quantification, although the cost is relatively low, complicated separation or transfer steps are required, which causes great deviation of the quantification result, or radioactive labeling is involved; detection methods based on biotin labeling or fluorescence labeling energy transfer (FRET) and the like, although sensitivity is increased, require steps of labeling, purification, separation and the like, and increase complexity of operation; real-time quantitative PCR technology based on fluorescent probes improves the detection sensitivity, but can not detect all sites due to high requirements on the length and specificity of the probes. The existing method has defects, which limits the wide application of the method in the clinical and scientific research fields.
Disclosure of Invention
The invention mainly aims to provide a simple and rapid method for determining specific site CpG methylation, and aims to solve the technical problems of complex operation and inconvenience in wide application in the clinical and scientific research fields in the prior art.
To achieve the above object, the present invention provides a method for determining the methylation level of DNA at a specific site in a biological sample, comprising the steps of:
obtaining a biological sample, and extracting a DNA fragment to be detected in the biological sample;
modifying a DNA fragment to be detected by using sodium bisulfite, and converting unmethylated cytosine deamination in the DNA fragment to be detected into uracil;
taking a DNA fragment modified by sodium bisulfite as a template DNA fragment, and respectively carrying out PCR amplification on the template DNA fragment from two sides of the template DNA fragment by using a first primer and a second primer, wherein the template DNA fragments amplified by the first primer and the second primer comprise CpG sites of the DNA fragment to be detected;
treating the PCR product after PCR amplification by alkaline phosphatase, removing redundant first primers, second primers and dNTPs, and purifying and recovering;
respectively taking the PCR products after the equal purification, respectively carrying out single base extension labeling reaction guided by a methylation specific third primer in two independent reaction systems, and detecting the methylation and demethylation fluorescence polarization values of the CpG sites of the DNA fragments to be detected;
and calculating the methylation level of the DNA fragment to be detected according to the methylation and demethylation fluorescence polarization values of the CpG sites of the DNA fragment to be detected.
Preferably, the step of performing methylation specific third primer-directed single base extension labeling reactions in two separate reaction systems comprises:
firstly, detecting methylation of CpG sites of a DNA fragment to be detected by adopting fluorescein dCTP;
secondly, detecting demethylation of CpG sites of the DNA fragment to be detected by adopting fluorescein dUTP;
the step of calculating the methylation level of the DNA fragment to be detected according to the methylation and demethylation fluorescence polarization values of the CpG sites of the DNA fragment to be detected comprises the following steps:
and calculating the methylation level of the DNA fragment to be detected according to the dCTP fluorescence polarization value and the dUTP fluorescence polarization value.
Preferably, the distance between the first primer and the CpG site of the DNA fragment to be detected and the second primer is larger than the length of the third primer.
Preferably, the length of the first primer and the second primer is 20-30 bp, and the GC content is 55-60%.
Preferably, the sequence of the third primer is specifically combined with a corresponding complementary sequence of 20-30 bp sequence at the upstream of the CpG locus of the DNA fragment to be detected.
Preferably, the third primer has a length of 22-25bp and does not contain CpG sites.
Preferably, the DNA fragment to be tested is derived from a blood sample, a pathological sample, an organ or tissue cell sample, and recombinant DNA or synthetic DNA.
Preferably, the pathological sample is a tumor tissue sample.
Preferably, the tumor tissue sample is a lung cancer, gastric cancer or liver cancer tissue sample.
The invention also provides application of the method for determining the methylation level of the DNA of the specific site in the biological sample in detecting the methylation of the target DNA.
Compared with the prior art, the method for determining the methylation level of the DNA of the specific site in the biological sample and the application thereof adopt the technical scheme, and the following technical effects are achieved: the methylation state of the target CpG is determined by measuring the fluorescence polarization quantity of the marker, the defects that the existing DNA methylation detection technology needs separation and purification and is complex in operation in electrophoretic analysis are overcome, and the method has the advantages of simplicity, convenience, rapidness, high flux, low cost, no need of separation and purification and the like, and is suitable for detecting the methylation of a large number of clinically precious low-concentration DNA samples.
Drawings
FIG. 1 is a schematic flow chart of a preferred embodiment of the method of the present invention for determining the level of DNA methylation at a specific site in a biological sample.
The objects, features and advantages of the present invention will be further explained with reference to the accompanying drawings.
Detailed Description
To further illustrate the technical means and effects of the present invention adopted to achieve the above objects, the following detailed description of the preferred embodiments is provided to illustrate the present invention. It should be understood that the specific embodiments described herein are merely illustrative of the invention and are not intended to limit the invention.
Referring to FIG. 1, FIG. 1 is a schematic flow chart of a preferred embodiment of the method for determining the methylation level of site-specific DNA in a biological sample according to the present invention.
The experimental procedures in the following examples are conventional unless otherwise specified. In the following examples, the methylation of CpG sites in part of the promoter region was examined using LINE1 element (NCBI sequence number X58075, with the specific sequence shown in SEQ ID NO.1) in the human genome as the subject.
The specific sequence of LINE1 element in human genome (SEQ ID NO.1) is:
as other examples, the DNA fragment to be tested may be derived from a pathological sample, an organ or tissue cell sample, and recombinant DNA or synthetic DNA. The pathological sample is preferably a tumor tissue sample, and the tumor tissue sample is preferably a lung cancer, gastric cancer or liver cancer tissue sample.
The embodiment of the invention is explained by taking the application of the method in the detection of human peripheral blood genome DNA methylation as an example, and the method comprises the following steps:
step S1: obtaining a biological sample, and extracting a DNA fragment to be detected in the biological sample;
in this example, human peripheral blood genomic DNA was extracted according to the conventional phenol/chloroform method.
Step S2: modifying a DNA fragment to be detected by using sodium bisulfite, and converting unmethylated cytosine deamination in the DNA fragment to be detected into uracil;
in this example, 1. mu.g of human peripheral blood genomic DNA was treated with sodium bisulfite to deaminate unmethylated cytosine to uracil in the test DNA, and stored at-20 ℃.
The reaction parameters of the sodium bisulfite for treating the DNA to be detected are as follows:
(1)1 μ g of DNA to be tested was incubated with 0.3M NaOH at 42 ℃ for 20 minutes, then at 95 ℃ for 3 minutes and at 0 ℃ for 1 minute;
(2) followed by incubation with sodium bisulfite at pH 5.0 (final concentration 5.0M) and hydroquinone (final concentration 0.5mM) at 55 ℃ for 16 hours, kept protected from light;
(3) the treated DNA was purified using Promega Wizard DNA Clean System, dissolved in 0.3M NaOH, incubated at 37 ℃ for 15 minutes and then neutralized to pH7.0 with 3M ammonium acetate;
(4) the neutralized product was precipitated with 75% ethanol and dissolved in 20. mu.l TE and stored at-20 ℃.
Step S3: taking a DNA fragment modified by sodium bisulfite as a template DNA fragment, and respectively carrying out PCR amplification on the template DNA fragment from two sides of the template DNA fragment by using a first primer and a second primer, wherein the template DNA fragments amplified by the first primer and the second primer comprise CpG sites of the DNA fragment to be detected;
in the present embodiment, it is preferred that,
a first primer: TTTTTTGAGTTAGGTGTGTGGG (SEQ ID NO. 2);
a second primer: CATCTCTAAAAAATACCAAACAA (SEQ ID NO. 3);
and (3) PCR reaction conditions: the 15 microliter reaction system comprises: 75nM of first and second primers, 50nM of dNTPs, 1XPCR buffer, and 1 unit of Taq DNA polymerase; after the mixture was left to react at 95 ℃ for 1 minute, the following 30 cycles were carried out: heating at 95 ℃ for 1 minute, 60 ℃ for 1 minute, and 72 ℃ for 1 minute.
Step S4: treating the PCR product after PCR amplification by alkaline phosphatase, removing redundant first primers, second primers and dNTPs, and purifying and recovering;
in this example, the PCR product was digested to remove unreacted primers and dNTPs. The enzyme digestion system contains: mu.l 10 Xalkaline phosphatase reaction buffer, 3.4 units alkaline phosphatase, 2.3 units exonuclease and 13. mu.l PCR product were made up to 37.5. mu.l with deionized water. The mixture was left at 37 ℃ for 45 minutes and heated at 85 ℃ for 15 minutes to inactivate the enzyme.
Step S5: respectively taking the PCR products after the equal purification, respectively carrying out single base extension labeling reaction guided by a methylation specific third primer in two independent reaction systems, and detecting the methylation and demethylation fluorescence polarization values of the CpG sites of the DNA fragments to be detected;
the total volume of the extension labeling reaction was 15. mu.l, containing 1.5. mu.l of the enzyme digested PCR product, 0.75 units of Tag polymerase, 1XPCR Buffer, 3.75. mu.M of the third primer, 1.125. mu.M fluorescein dCTP or 0.5625. mu.M fluorescein dUTP. Reaction conditions are as follows: after denaturation at 95 ℃ for 3 min, the following 30 cycles were performed: incubate at 95 ℃ for 30 seconds and 55 ℃ for 30 seconds.
A third primer: GGTGGGAGTGATT (SEQ ID NO. 4).
The step of performing methylation-specific third primer-directed single-base extension labeling reactions in two separate reaction systems, respectively, comprises:
firstly, detecting methylation of CpG sites of a DNA fragment to be detected by adopting fluorescein dCTP;
and secondly, detecting the demethylation of the CpG sites of the DNA fragment to be detected by adopting fluorescein dUTP.
Step S6: and calculating the methylation level of the DNA fragment to be detected according to the methylation and demethylation fluorescence polarization values of the CpG sites of the DNA fragment to be detected.
In this embodiment, the step of calculating the methylation level of the test DNA fragment according to the methylation and demethylation fluorescence polarization values of the CpG sites of the test DNA fragment comprises: and calculating the methylation level of the DNA fragment to be detected according to the dCTP fluorescence polarization value and the dUTP fluorescence polarization value. The product after extension labeling is placed on an FP (fluorescence polarization) detector to directly measure the fluorescence polarization value, the excitation wavelength is 544nm, and the emission wavelength is 595 nm.
After subtracting the FP value of a zero-concentration blank point from the FP value of each DNA concentration point, estimating the methylation level of the site to be detected according to the following formula:
methylation level%dCTP/(FPdCTP+FPdUTP)；
Experiments show that the methylation level detection of the third primer pair CpG1 shows good stability in the range of 20-100ng, and the values are as follows: 57.13 +/-0.26%
In a preferred embodiment, the first primer and the second primer are separated from the CpG site of the test DNA fragment by a distance greater than the length of the third primer. The length of the first primer and the second primer is 20-30 bp, and the GC content is 55-60%. And the sequence of the third primer is specifically combined with the corresponding complementary sequence of the 20-30 bp sequence at the upstream of the CpG locus of the DNA fragment to be detected. The third primer is 22-25bp in length and does not contain CpG sites.
The invention also provides application of the method for determining the methylation level of the DNA of the specific site in the biological sample in detecting the methylation of the target DNA.
The method for determining the methylation level of the DNA of the specific site in the biological sample, provided by the invention, determines the methylation state of the target CpG by determining the fluorescence polarization amount of the marker, overcomes the defects that the existing DNA methylation detection technology such as electrophoretic analysis needs separation and purification and is complex to operate, has the advantages of simplicity, convenience, rapidness, high flux, low cost, no need of separation and purification and the like, and is suitable for detecting the methylation of a large amount of clinically precious low-concentration DNA samples.
The above description is only a preferred embodiment of the present invention, and not intended to limit the scope of the present invention, and all modifications of equivalent structures and equivalent functions made by the present specification and drawings, or directly or indirectly applied to other related technical fields, are included in the scope of the present invention.
Sequence listing
<110> Shenzhen Shengbizhi science and technology development Limited
<120> method for determining methylation level of DNA at specific site in biological sample and application thereof
<130> 2018
<160> 4
<170> SIPOSequenceListing 1.0
<210> 1
<211> 250
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<213> humo.
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ttttttgagt taggtgtggg atatagtttc gtggtgcgtc gttttttaag tcggtttgaa 60
aagcgtaata ttcgggtggg agtgattcga ttttttaggt gcgttcgtta tttttttttt 120
tgattcggaa agggaatttt ttgatttttt gcgtttttta ggtgaggtaa tgtttcgttt 180
tgtttcggtt cgcgtacggt gcgcgtatat agtggtttgc gtttattgtt tggtattttt 240
tagtgagatg 250
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<212> DNA
<213> Artificial sequence (unknown)
<400> 2
tttttgagtt aggtgtgtgg g 21
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<212> DNA
<213> Artificial sequence (unknown)
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catctctaaa aaataccaaa caa 23
<210> 4
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<212> DNA
<213> Artificial sequence (unknown)
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ggtgggagtg att 13
Claims (10)
1. A method for determining the level of DNA methylation at a specific site in a biological sample, the method comprising the steps of:
obtaining a biological sample, and extracting a DNA fragment to be detected in the biological sample;
modifying a DNA fragment to be detected by using sodium bisulfite, and converting unmethylated cytosine deamination in the DNA fragment to be detected into uracil;
taking a DNA fragment modified by sodium bisulfite as a template DNA fragment, and respectively carrying out PCR amplification on the template DNA fragment from two sides of the template DNA fragment by using a first primer and a second primer, wherein the template DNA fragments amplified by the first primer and the second primer comprise CpG sites of the DNA fragment to be detected;
treating the PCR product after PCR amplification by alkaline phosphatase, removing redundant first primers, second primers and dNTPs, and purifying and recovering;
respectively taking the PCR products after the equal purification, respectively carrying out single base extension labeling reaction guided by a methylation specific third primer in two independent reaction systems, and detecting the methylation and demethylation fluorescence polarization values of the CpG sites of the DNA fragments to be detected;
and calculating the methylation level of the DNA fragment to be detected according to the methylation and demethylation fluorescence polarization values of the CpG sites of the DNA fragment to be detected.
2. The method of claim 1, wherein the step of performing methylation-specific third primer-directed single-base extension labeling reactions in two separate reaction systems comprises:
detecting methylation of CpG sites of the DNA fragment to be detected by adopting fluorescein dCTP; detecting demethylation of CpG sites of the DNA fragment to be detected by adopting fluorescein dUTP;
the step of calculating the methylation level of the DNA fragment to be detected according to the methylation and demethylation fluorescence polarization values of the CpG sites of the DNA fragment to be detected comprises the following steps:
and calculating the methylation level of the DNA fragment to be detected according to the dCTP fluorescence polarization value and the dUTP fluorescence polarization value.
3. The method of claim 1, wherein the first primer and the second primer are separated from the CpG site of the test DNA fragment by a distance greater than the length of the third primer.
4. The method for determining the methylation level of DNA at a specific site in a biological sample according to claim 3, wherein the first primer and the second primer have a length of 20-30 bp and a GC content of 55-60%.
5. The method according to claim 3, wherein the sequence of the third primer specifically binds to a complementary sequence 20-30 bp upstream of the CpG site of the test DNA fragment.
6. The method of claim 5, wherein the third primer is 22-25bp long and does not contain CpG sites.
7. The method of claim 1, wherein the DNA fragments to be tested are derived from blood samples, pathological samples, organ or tissue cell samples, recombinant DNA or synthetic DNA.
8. The method of claim 7, wherein the pathological sample is a tumor tissue sample.
9. The method of claim 8, wherein the tumor tissue sample is a lung cancer, gastric cancer or liver cancer tissue sample.
10. Use of the method of any one of claims 1 to 9 for determining the level of site-specific DNA methylation in a biological sample for detecting DNA methylation of interest.
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| CN101644708A (en) * | 2009-09-10 | 2010-02-10 | 中国科学院生态环境研究中心 | Fluorescence polarization enhancing immune capillary electrophoresis analysis |
| CN101736083A (en) * | 2009-09-25 | 2010-06-16 | 中国人民解放军第四军医大学 | Gene methylation detection method suitable for fluorescence polarization technique |
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Application publication date: 20200103 |