CN114507719A - Quantitative analysis method for DNA methylation monitoring - Google Patents
Quantitative analysis method for DNA methylation monitoring Download PDFInfo
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- CN114507719A CN114507719A CN202210168358.XA CN202210168358A CN114507719A CN 114507719 A CN114507719 A CN 114507719A CN 202210168358 A CN202210168358 A CN 202210168358A CN 114507719 A CN114507719 A CN 114507719A
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Abstract
The invention relates to the technical field of DNA methylation quantitative analysis, in particular to a quantitative analysis method aiming at DNA methylation monitoring, S1, firstly extracting DNA in various samples, carrying out bisulfite modification on genome DNA, and taking the modified DNA as a template; s2, taking normal human peripheral blood leukocyte DNA as negative control, redistilled water as blank control, and T-24 tumor cell DNA as positive control; s3, when detecting, firstly, processing the DNA to be detected by HSO 3-; s4, after PCR amplification, taking two parts of amplification products; two digoxin-labeled oligonucleotide probes respectively complementary with methylated and unmethylated PCR products are designed and synthesized, then the two probes are respectively hybridized with the PCR products in a light-emitting tube coated by streptavidin, chemiluminescence detection is carried out, and the methylation degree in sample DNA is determined according to the ratio of the hybridization signal intensities of the two probes, so that the quantitative analysis effect and the efficiency of DNA methylation monitoring are better.
Description
Technical Field
The invention relates to the technical field of DNA methylation quantitative analysis, in particular to a quantitative analysis method aiming at DNA methylation monitoring.
Background
DNA methylation refers to the transfer of a methyl group from adenosylmethionine to adenine or cytosine nucleotide of DNA with a special sequence under the catalytic action of methyltransferase, which is commonly seen in the sequences of genes 5 '-CpG-3', 5 '-G-A-T-C-3', and the like, and forms 5 '-mCPG-3' and 5 '-G-mA-T-C-3' respectively after methylation. DNA methylation was one of the earliest discovered gene modification pathways in epigenetic studies. Methylation of DNA protects genes in prokaryotes/eukaryotes and affects mammalian gene transcription and replication, as well as embryo formation and development. In human, DNA methylation aberration inhibits DNA transcription, resulting in uncontrolled DNA expression and a series of pathological changes in the body. Therefore, monitoring of DNA methylation has great clinical significance for early diagnosis and adjuvant treatment of cancers related to the DNA methylation.
Various detection means have been used for detecting and monitoring the DNA methylation level, but the existing monitoring quantitative analysis method has not ideal effect, so that a quantitative analysis method aiming at DNA methylation monitoring is provided.
Disclosure of Invention
The present invention is directed to a quantitative analysis method for monitoring DNA methylation, which solves the above problems of the background art.
In order to achieve the purpose, the invention provides the following technical scheme:
a quantitative analysis method for DNA methylation monitoring, comprising the steps of:
s1, firstly, extracting DNA from various samples, carrying out bisulfite modification on genome DNA, and amplifying a 273bp target fragment in a promoter region of a p16 gene by taking the modified DNA as a template;
s2, taking normal human peripheral blood leukocyte DNA as negative control, redistilled water as blank control, and T-24 tumor cell DNA as positive control;
s3, for detection, HSO is used first3 -Treating DNA to be detected, converting unmethylated cytosine into uracil, and then performing PCR amplification, wherein a primer is designed in a sequence which does not contain CpG sites but contains cytosine residues in the original sequence;
s4, after PCR amplification is finished, taking two amplification products, adding 1.5pmol of each of two detection probes into 5 mul of each amplification product, supplementing a hybridization buffer solution to a final volume of 30 mul, uniformly mixing, denaturing at 95 ℃ for 10min, cooling to 48 ℃, adding 20 mul into a streptavidin-coated light-emitting tube, supplementing the hybridization buffer solution to a final volume of 200 mul, incubating for 40min at 48 ℃, pouring out the hybridization buffer solution, washing twice by using a washing buffer solution at 48 ℃ and room temperature, then adding 200 mul of an anti-digoxin-peroxidase compound, incubating for 40min at 25 ℃, washing for 5 times by PBS, adding 200 mul of a luminescence detection substrate, and standing for 2 min;
in a preferred embodiment of the present invention, the primer sequence of S1 does not contain CpG sites, so that the pair of primers can simultaneously amplify bisulfite-modified, methylated and unmethylated target fragments, and both have the same amplification efficiency.
As a preferred embodiment of the present invention, the S2 is designed with two digoxin labeled oligonucleotide probes complementary to the methylated and unmethylated PCR products, respectively, and the two primers are different in 3 positions and correspond to 3 CpG site probe sequences, respectively.
As a preferred embodiment of the present invention, the S3 is used to purify PCR products, perform restriction enzyme digestion, perform polyacrylamide gel electrophoresis separation, and finally perform hybridization detection, wherein the original DNA methylation level can be represented by the relative amount of digested and undigested PCR products.
In a preferred embodiment of the present invention, in S4, the value of the integrated 6 luminescence is measured by an ultra-weak luminescence detector, and the percentage methylation-Rm/(Rm + Ru) x 100% of a blank tube, Rm: methylated PC: relative luminescence of methylated CR product.
Compared with the prior art, the invention has the beneficial effects that:
in the invention, through a methylation detection method based on sodium bisulfite treatment, the change of the DNA sequence can cause the generation of new methylation-dependent restriction enzyme cutting sites or can cause the loss of the existing methylation-dependent restriction enzyme cutting sites, and two digoxin-labeled oligonucleotide probes respectively complementary to methylated and unmethylated PCR products are designed and synthesized, and then the two probes are respectively hybridized with the PCR products in a streptavidin-coated luminous tube for chemiluminescence detection, and the methylation degree in the sample DNA is determined according to the ratio of the hybridization signal intensities of the two probes, so that the quantitative analysis effect and the efficiency of DNA methylation monitoring are better.
Detailed Description
The technical solutions in the embodiments of the present invention will be clearly and completely described below with reference to the embodiments of the present invention, and it is obvious that the described embodiments are only a part of the embodiments of the present invention, rather than all embodiments, and all other embodiments obtained by a person of ordinary skill in the art without any creative work based on the embodiments of the present invention belong to the protection scope of the present invention.
The present invention will now be described more fully hereinafter with reference to the accompanying drawings, in order to facilitate an understanding of the invention, but the invention may be embodied in many different forms and is not limited to the embodiments described herein; rather, these embodiments are provided so that this disclosure will be thorough and complete.
Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs, and the terms used herein in the specification of the present invention are for the purpose of describing particular embodiments only and are not intended to limit the present invention, and the term "and/or" as used herein includes any and all combinations of one or more of the associated listed items.
The invention provides a technical scheme that:
a quantitative analysis method for DNA methylation monitoring, comprising the steps of:
s1, firstly, extracting DNA from various samples, carrying out bisulfite modification on genome DNA, and amplifying a 273bp target fragment in a promoter region of a p16 gene by taking the modified DNA as a template;
s2, taking normal human peripheral blood leukocyte DNA as negative control, redistilled water as blank control, and T-24 tumor cell DNA as positive control;
s3, for detection, HSO is used first3 -Treating DNA to be detected, converting unmethylated cytosine into uracil, and then performing PCR amplification, wherein a primer is designed in a sequence which does not contain CpG sites but contains cytosine residues in the original sequence;
s4, after PCR amplification is finished, taking two amplification products, adding 1.5pmol of each of two detection probes into 5 mul of each amplification product, supplementing a hybridization buffer solution to a final volume of 30 mul, uniformly mixing, denaturing at 95 ℃ for 10min, cooling to 48 ℃, adding 20 mul into a streptavidin-coated light-emitting tube, supplementing the hybridization buffer solution to a final volume of 200 mul, incubating for 40min at 48 ℃, pouring out the hybridization buffer solution, washing twice by using a washing buffer solution at 48 ℃ and room temperature, then adding 200 mul of an anti-digoxin-peroxidase compound, incubating for 40min at 25 ℃, washing for 5 times by PBS, adding 200 mul of a luminescence detection substrate, and standing for 2 min;
s1, because the primer sequence does not contain CpG sites, the pair of primers can simultaneously amplify the bisulfite modified, methylated and unmethylated target fragments, and the amplification efficiency of the two primers is the same.
S2 two digoxin-labeled oligonucleotide probes were designed, complementary to the methylated and unmethylated PCR products, respectively, with 3 different primers, corresponding to 3 CpG site probe sequences, respectively.
S3 PCR products are purified, restriction enzyme digestion, polyacrylamide gel electrophoresis separation, and finally hybridization detection, and the original DNA methylation level can be represented by the relative amount of digested and undigested PCR products.
S4 measuring the 6 luminous integral value by using an ultra-weak luminous detector, and simultaneously measuring the methylation percentage-Rm/(Rm + Ru) x 100% of a blank tube, Rm: methylated PC: relative luminescence of methylated CR product;
the methylation detection method based on sodium bisulfite treatment can lead to the generation of new methylation-dependent restriction enzyme cutting sites or can lead to the loss of the original existing methylation-dependent restriction enzyme cutting sites, and two digoxin-labeled oligonucleotide probes which are respectively complementary with methylated and non-methylated PCR products are designed and synthesized, then the two probes are respectively hybridized with the PCR products in a light-emitting tube coated by streptavidin, chemiluminescence detection is carried out, the degree of methylation in the DNA sample is determined according to the ratio of the hybridization signal intensity of the two probes, and the quantitative analysis effect and the efficiency of DNA methylation monitoring are better.
Example (b): firstly, extracting DNA in various samples, carrying out bisulfite modification on genomic DNA, amplifying a target fragment with the length of 273bp in a promoter region of a p16 gene by using the modified DNA as a template, wherein because a primer sequence does not contain CpG sites, the pair of primers can simultaneously amplify the target fragments which are modified by bisulfite, methylated and unmethylated, and the two have the same amplification efficiency;
meanwhile, taking normal human peripheral blood leukocyte DNA as negative control, redistilled water as blank control, and T-24 tumor cell DNA as positive control, designing two digoxin labeled oligonucleotide probes which are respectively complementary with methylated and unmethylated PCR products, wherein the two primers have 3 different positions and respectively correspond to 3 CpG site probe sequences;
during detection, HSO is firstly used3 -Processing DNA to be detected, converting unmethylated cytosine into uracil, carrying out PCR amplification, designing a primer in a sequence which does not contain CpG sites but contains cytosine residues in the original sequence, purifying a PCR product, carrying out restriction enzyme digestion, carrying out polyacrylamide gel electrophoresis separation, and finally carrying out hybridization detection, wherein the methylation level of the original DNA can be represented by the relative quantity of digested and undigested PCR products;
after PCR amplification, taking two amplification products, each 5 mu l, respectively adding 1.5pmol of two detection probes, supplementing a hybridization buffer solution to a final volume of 30 mu l, uniformly mixing, denaturing at 95 ℃ for 10min, cooling to 48 ℃, adding 20 mu l into a streptavidin-coated light-emitting tube, supplementing a hybridization buffer solution to a final volume of 200 mu l, incubating at 48 ℃ for 40min, pouring out the hybridization buffer solution, washing twice with a washing buffer solution at 48 ℃ and room temperature, then adding 200 mu l of an anti-digoxin-peroxidase complex, incubating at 25 ℃ for 40min, washing 5 times with PBS, adding 200 mu l of a luminescence detection substrate, standing for 2min, measuring a 6 luminescence integral value with an ultra-weak luminescence detector, and simultaneously measuring a blank tube, wherein the methylation percentage is-Rm/(Rm + Ru) x 100%, Rm: methylated PC: relative luminescence of methylated CR product;
the methylation detection method based on sodium bisulfite treatment can lead to the generation of new methylation-dependent restriction enzyme cutting sites or can cause the loss of the existing methylation-dependent restriction enzyme cutting sites, and two digoxin-labeled oligonucleotide probes respectively complementary to methylated and unmethylated PCR products are designed and synthesized, then the two probes are respectively hybridized with the PCR products in a luminous tube coated by streptavidin, chemiluminescence detection is carried out, the degree of methylation in the sample DNA is determined according to the ratio of the hybridization signal intensity of the two probes, and the quantitative analysis effect and the efficiency of DNA methylation monitoring are better.
The above description is only for the preferred embodiment of the present invention, but the scope of the present invention is not limited thereto, and any person skilled in the art should be considered to be within the technical scope of the present invention, and the technical solutions and the inventive concepts thereof according to the present invention should be equivalent or changed within the scope of the present invention.
Claims (5)
1. A quantitative analysis method for DNA methylation monitoring, comprising the steps of:
s1, firstly, extracting DNA from various samples, carrying out bisulfite modification on genome DNA, and amplifying a 273bp target fragment in a promoter region of a p16 gene by taking the modified DNA as a template;
s2, taking normal human peripheral blood leukocyte DNA as negative control, redistilled water as blank control, and T-24 tumor cell DNA as positive control;
s3, for detection, HSO is used first3 -Treating DNA to be detected, converting unmethylated cytosine into uracil, and then performing PCR amplification, wherein a primer is designed in a sequence which does not contain CpG sites but contains cytosine residues in the original sequence;
s4, after PCR amplification, taking two amplification products, each 5 mu l, respectively adding 1.5pmol of each of two detection probes, supplementing hybridization buffer solution to a final volume of 30 mu l, mixing uniformly, denaturing at 95 ℃ for 10min, cooling to 48 ℃, adding 20 mu l into a streptavidin-coated light-emitting tube, supplementing hybridization buffer solution to a final volume of 200 mu l, incubating at 48 ℃ for 40min, pouring out hybridization buffer solution, washing twice with washing buffer solution at 48 ℃ and room temperature, then adding 200 mu l of anti-digoxin-peroxidase complex, incubating at 25 ℃ for 40min, washing 5 times with PBS, adding 200 mu l of luminescence detection substrate, and standing for 2 min.
2. The quantitative analysis method for monitoring DNA methylation according to claim 1, wherein the primer sequence of S1 does not contain CpG sites, so that the pair of primers can simultaneously amplify bisulfite modified, methylated and unmethylated target fragments, and both have the same amplification efficiency.
3. The quantitative analysis method for DNA methylation monitoring according to claim 1, wherein S2 designs two digoxin labeled oligonucleotide probes complementary to methylated and unmethylated PCR products, respectively, and the two primers are different by 3 positions and correspond to 3 CpG site probe sequences, respectively.
4. The quantitative analysis method for monitoring DNA methylation according to claim 1, wherein S3 is used for purifying PCR products, performing restriction enzyme digestion, performing polyacrylamide gel electrophoresis separation, and finally performing hybridization detection, and the original DNA methylation level can be represented by the relative amount of digested and undigested PCR products.
5. The quantitative analysis method for monitoring DNA methylation according to claim 1, wherein the S4 is characterized in that an ultra-weak luminescence detector is used for measuring the 6 luminescence integral value, and a blank tube is simultaneously measured, wherein the methylation percentage is-Rm/(Rm + Ru) x 100%, Rm: methylated PC: relative luminescence of methylated CR product.
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| CN107592885A (en) * | 2015-02-24 | 2018-01-16 | 兹莫研究公司 | Determine the analytic approach of DNA methylation and the DNA methylation mark of cancer |
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Patent Citations (5)
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| US20070269801A1 (en) * | 2000-02-07 | 2007-11-22 | Jian-Bing Fan | Multiplexed Methylation Detection Methods |
| CN1539023A (en) * | 2001-03-09 | 2004-10-20 | EPI������ɷݹ�˾ | Method for detecting cytosine methylation patterns having high sensitivity |
| CA2616601A1 (en) * | 2005-07-28 | 2007-02-08 | Oncomethylome Sciences | Methylation markers for prognosis and treatment of cancers |
| CN105408494A (en) * | 2012-05-11 | 2016-03-16 | 独立行政法人国立癌症研究中心 | Method for predicting prognosis of renal cell carcinoma |
| CN107592885A (en) * | 2015-02-24 | 2018-01-16 | 兹莫研究公司 | Determine the analytic approach of DNA methylation and the DNA methylation mark of cancer |
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