CN113584168A - Lung cancer detection method based on methylation immunoprecipitation high-throughput sequencing technology - Google Patents
Lung cancer detection method based on methylation immunoprecipitation high-throughput sequencing technology Download PDFInfo
- Publication number
- CN113584168A CN113584168A CN202110815235.6A CN202110815235A CN113584168A CN 113584168 A CN113584168 A CN 113584168A CN 202110815235 A CN202110815235 A CN 202110815235A CN 113584168 A CN113584168 A CN 113584168A
- Authority
- CN
- China
- Prior art keywords
- dna
- sequencing
- fragments
- methylated
- lung cancer
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
- Pending
Links
Classifications
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
- C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
- 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
- C12Q1/6886—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes for diseases caused by alterations of genetic material for cancer
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
- C12N15/09—Recombinant DNA-technology
- C12N15/10—Processes for the isolation, preparation or purification of DNA or RNA
- C12N15/1003—Extracting or separating nucleic acids from biological samples, e.g. pure separation or isolation methods; Conditions, buffers or apparatuses therefor
- C12N15/1006—Extracting or separating nucleic acids from biological samples, e.g. pure separation or isolation methods; Conditions, buffers or apparatuses therefor by means of a solid support carrier, e.g. particles, polymers
- C12N15/1013—Extracting or separating nucleic acids from biological samples, e.g. pure separation or isolation methods; Conditions, buffers or apparatuses therefor by means of a solid support carrier, e.g. particles, polymers by using magnetic beads
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
- C12N15/09—Recombinant DNA-technology
- C12N15/10—Processes for the isolation, preparation or purification of DNA or RNA
- C12N15/1034—Isolating an individual clone by screening libraries
- C12N15/1093—General methods of preparing gene libraries, not provided for in other subgroups
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
- C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
- C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
- C12Q1/6806—Preparing nucleic acids for analysis, e.g. for polymerase chain reaction [PCR] assay
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
- C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
- C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
- C12Q1/6869—Methods for sequencing
-
- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
- G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
- G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
- G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
- G01N33/536—Immunoassay; Biospecific binding assay; Materials therefor with immune complex formed in liquid phase
- G01N33/537—Immunoassay; Biospecific binding assay; Materials therefor with immune complex formed in liquid phase with separation of immune complex from unbound antigen or antibody
- G01N33/539—Immunoassay; Biospecific binding assay; Materials therefor with immune complex formed in liquid phase with separation of immune complex from unbound antigen or antibody involving precipitating reagent, e.g. ammonium sulfate
Abstract
The invention relates to the technical field of lung cancer detection, in particular to a lung cancer detection method based on a methylation immunoprecipitation high-throughput sequencing technology. The invention discloses a detection method for carrying out ctDNA5mC high-throughput sequencing by using a targeted capture technology, and realizes prediction of immune inspection site inhibitor response through ctDNA multi-epigenetics by a machine learning related algorithm. The non-invasive and real-time detection method provides an important molecular target for the response of the immunodetection site inhibitor, and is convenient for subsequent research and diagnosis.
Description
Technical Field
The invention relates to the technical field of lung cancer detection, in particular to a lung cancer detection method based on a methylation immunoprecipitation high-throughput sequencing technology.
Background
Currently, the biomarkers used are mainly the expression level of PD-L1 and the tumor mutation load (TMB). The former is the use of immunohistochemical detection of tissue samples for PD-L1 protein expression, but this evaluation index is not absolute, and PD-L1 positive cancer patients respond 36% to 100% to PD-1 drugs, but PD-L1 negative cancer patients also respond to treatment with a response rate of 0% to 17%. However, the positive standard of PD-L1 expression is difficult to define, and sometimes PD-L1 is expressed not only in cancer cells but also in non-cancer cells in the microenvironment surrounding the lesion, which will certainly cause certain interference. The last point is that the detection of PD-L1 is only suitable for the prediction of PD-1 or PD-L1 drugs, and the prediction of the curative effect of other immune drugs is ineffective.
TMB is the number of mutations per megabase in tumor tissue, and this indicator is typically detected by tissue samples, and currently blood-based TMB (btmb) is also detected. Tumor Mutational Burden (TMB) appears as a potential biomarker. The number of somatic mutations in different cancers varies from 0.01 mutation/Mb to over 400 mutations/Mb. Some of these mutations transcribe and express polypeptide epitopes or tumor neoantigens. Early studies on TMB analyzed tumor DNA and control germline DNA using Whole Exon (WES) sequencing. Although TMB is currently approved by the FDA for use in the guidance of drugs for immunodetection site inhibitors, it still has many drawbacks, such as differences in Cut-off values due to differences in the detected gene panel with different detection platforms, different algorithms affecting the prediction value of TMB, because the algorithms vary greatly among different gene detection panels, and the mutation types used to evaluate TMB also vary among the various detection methods. In many assays that employ WES, TMB contains only missense mutations, and no other mutation types are incorporated. In particular, differences in the method of processing formalin-fixed paraffin-embedded samples can also greatly affect the value of TMB of 10. In addition, different algorithms also influence the predictive value of TMB, since the algorithms vary greatly among different gene detection panels, and the mutation types used to evaluate TMB also vary among the various detection methods. In many assays that employ WES, TMB contains only missense mutations, and no other mutation types are incorporated. In particular, differences in the method of processing formalin-fixed paraffin-embedded samples can also greatly affect the value of TMB of 10.
The epigenetic modification of DNA is closely related to the occurrence and development of tumors, the DNA epigenetic modification of tumors is different from that of healthy people, the DNA epigenetic modification of different types of tumors is also different, and the difference of the tumors of the same type is also large in different periods. cfDNA is a DNA fragment released by cells into the blood circulation system, and cfDNA from tumors is called ctDNA, and these fragments retain information such as epigenetic modifications of tumor DNA. Theoretically, detection of cfDNA epigenetic modifications by liquid biopsy techniques can be used for the prediction of the response efficiency of immune check site inhibitors.
With the progress of immunotherapy research and clinical application of immunosuppressive agents, immunodiagnostic site inhibitors have largely changed the treatment modalities of non-small cell lung cancer (NSCLC). Immunotherapy comprising PD-1/PD-L1 and CTLA4 is the trend of tumor therapy, but not all tumor patients are suitable for immunotherapy, and researches show that the response efficiency of PD-1 single drug is only about 25 percent in non-small cell lung cancer, so that how to predict the response of immunoassay site inhibitors such as PD-1 by using appropriate tumor standards is an urgent problem to be solved.
Disclosure of Invention
Aiming at the problem that a proper tumor standard substance is needed to predict the response of an immunodetection site inhibitor such as PD-1 in the background technology, a lung cancer detection method based on a methylation immunoprecipitation high-throughput sequencing technology is provided. The invention discloses a detection method for performing ctDNA5mC high-throughput sequencing by using a targeted capture technology, and realizes prediction of immune inspection site inhibitor response through ctDNA multi-epigenetics by a machine learning related algorithm. The non-invasive and real-time detection method provides an important molecular target for the response of the immunodetection site inhibitor, and is convenient for subsequent research and diagnosis.
The invention provides a lung cancer detection method based on a methylation immunoprecipitation high-throughput sequencing technology, which comprises the following steps:
step 1: extracting plasma free dna (cfdna);
step 2: performing end repair and completion on the cfDNA fragments;
and step 3: connecting the DNA with the filled end with a sequencing joint to obtain a connection product;
and 4, step 4: adding unmethylated fragments with length similar to that of free DNA fragments in the DNA library and methylated fragments with different degrees, ensuring constant ratio of the antibody and the original free DNA, helping to keep similar immunoprecipitation efficiency among samples, and reducing nonspecific binding of the antibody and binding of the DNA and plastic products;
and 5: adding standard methylated and unmethylated samples;
step 6: heat treatment to melt the double stranded DNA and enrich for hypermethylated DNA fragments by adding 5-hydroxymethylcytosine antibodies that specifically bind methylated cytosine as DNA methylation occurs at the cytosine 5-position carbon atom;
and 7: adding magnetic beads to capture methylated DNA fragments in a targeted mode;
and 8: using methylated purified magnetic beads, washing for multiple times to remove unbound DNA fragments;
and step 9: performing PCR amplification on the methylated fragments obtained by immunoprecipitation to prepare a sequencing library; the preparation process of the sequencing library comprises a plurality of purification steps, and a magnetic bead method is selected for purification;
step 10: performing quality inspection on the sequencing library;
step 11: uniformly mixing libraries containing different barcode according to the same molar concentration, and performing on-machine sequencing according to a second-generation sequencing instrument by using a standard method to obtain a sequencing result;
step 12: 5mC biomarker selection and model construction;
step 13: and (6) analyzing the data.
Preferably, in step 2, the extracted cfDNA is first subjected to fragment length detection; and performing end repair and completion on the screened cfDNA.
Compared with the prior art, the invention has the following beneficial technical effects:
the invention discloses a detection method for carrying out ctDNA5mC high-throughput sequencing by using a targeted capture technology, and realizes prediction of immune inspection site inhibitor response through ctDNA multi-epigenetics by a machine learning related algorithm. The non-invasive and real-time detection method provides an important molecular target for the response of the immunodetection site inhibitor, and is convenient for subsequent research and diagnosis.
Detailed Description
The invention provides a free DNA methylation immunoprecipitation high-throughput sequencing technical scheme, which comprises the following steps:
s1, extracting cfDNA: 10ng of plasma cfDNA was extracted from 108 stage III and stage IV lung cancer patient samples, by any method known to those skilled in the art to be suitable for extracting plasma cfDNA.
S2, detecting the fragment length of the extracted cfDNA, repairing and filling the tail end, and connecting the DNA with the filled tail end with a sequencing adaptor to obtain a connection product, wherein the method comprises the following steps:
according to the Vazyme DNA Library Prep Kit instructions, a system containing 10ng cfDNA, 15. mu.L of End-Prep mix 4, 1. mu.L spike in and a total volume of 50. mu.L supplemented with nucleic-free water was prepared in a PCR tube, incubated at 20 ℃ for 30 minutes and then at 65 ℃ for 15 minutes; to the reaction mixture were added 25. mu.L of Rapid Ligation buffer2, 5. mu.L of Rapid DNA Ligase, 1. mu.L of adapter, and a system supplemented with nucleic-free water to a total volume of 100. mu.L, incubated at 20 ℃ for 15 minutes, and then maintained at 4 ℃. The reaction product was purified using AmpureXP beads and eluted with 10. mu.L of nucleic-free water to obtain the final DNA ligation sample.
S3, taking the primary library connected with the sequencing linker as initial DNA, carrying out methylation immunoprecipitation enrichment capture to obtain a methylation DNA enrichment product, wherein the method comprises the following steps:
according to the Magnetic Methylated DNA Immunoprecitation Kit instructions, a system of 10ng of the initial library DNA, 24. mu.L of Buffer A, 6. mu.L of Buffer B and nucleic-free water supplemented to 90. mu.L in a PCR tube was prepared, incubated at 95 ℃ for 10 minutes and then on ice for 10 minutes; in a 1.5ml low adsorption tube, 75. mu.L of the reaction mixture was added with methylation specific antibody and Magnetic beads supplied in Magnetic Methylated DNA immunization Kit, and incubated at 4 ℃ for 17 hours with rotation; purification was performed using wash buffers I and II provided in the Magnetic Methylated DNA Immunoprecipitation Kit in this order to remove DNA not bound to the Magnetic beads.
S4, purifying the enriched methylated DNA to obtain a purified product, wherein the method comprises the following steps:
elution Buffer was prepared using Buffer A and Buffer B from Magnetic IPure kit v2, and the enriched product was eluted in two runs, each with 50. mu.L; adding 10 μ L of purified Magnetic beads from Magnetic IPure kit v2 and 100 μ L of isopropanol, performing rotary incubation at room temperature for 10 min, and removing the supernatant; washing with wash buffer I and II in Magnetic IPure kit v2 in sequence; finally, 24. mu.L of buffer C from Magnetic IPure kit v2 was added to elute the methylated DNA bound to the purified Magnetic beads.
S5, PCR amplification was performed using the following DNA removed as a template to prepare a sequencing library:
a reaction system containing 25. mu.L of VAHTS HiFi amplification Mix, 2. mu.L of PCR Primer Mix 3for Illumina, and 23. mu.L of the above-mentioned eluted DNA was prepared in a total volume of 50. mu.L, and amplified according to the PCR reaction conditions of Table 1:
TABLE 1
The amplification product was purified using AmpureXP beads to obtain the final sequencing library.
S6, performing high-throughput sequencing after quality inspection on the sequencing library, wherein the method comprises the following steps:
the obtained sequencing library was subjected to concentration determination using Qubit and the size content of the library DNA fragments was determined using LabChip GX Touch. The sequencing library by quality inspection can be used for high-throughput sequencing, a certain number (1-96) of libraries containing different barcode are uniformly mixed according to the same concentration, and the on-machine sequencing is carried out by using a standard method according to a second-generation sequencing instrument.
Selection and model construction of S7, 5mC biomarker:
rpackage glmnet (version 2.0-18) was chosen for feature selection and prediction model construction, relying on elastic net regularization on a logistic linear regression model. 70% of respondents (n-48) and 70% of non-responders (n-22) were randomly divided into training and validation sets. To avoid overfitting, the training set was randomly divided into 5-fold cross-validation. 4 folds were selected for each cross as the cross training set and the cross test set on the left. DMPs between responders and non-responders in the Cross-training set (p-values less than 0.001 and log)2The FoldChange absolute value is greater than 0.5 and still serves as a candidate. 100 replicates were then performed to further select markers that appeared above 95%. Finally, a final predictive model was built using the final markers observed in at least 5 hybridization combinations.
S8, data analysis:
the 11 5mC biomarkers were subjected to elastic net model training and prediction. The EI-score for each sample was calculated from the biomarker numbers of the integrated model as follows:
EI-score (coef (k) × log2CPM (k)), (k representations the marker) using the R packet cut points to select the cut-off points for the EI score. To maximize sensitivity while satisfying sufficient specificity, we chose spec _ constraint as a metric.
Using the 11-feature 5mC model of the invention, it was possible to distinguish between non-responders and responders (AUC 0.79; sensitivity, 0.9; specificity, 0.6).
While the embodiments of the present invention have been described in detail, the present invention is not limited thereto, and various changes can be made without departing from the gist of the present invention within the knowledge of those skilled in the art.
Claims (2)
1. The lung cancer detection method based on the methylation immunoprecipitation high-throughput sequencing technology is characterized by comprising the following steps:
step 1: extracting plasma free dna (cfdna);
step 2: performing end repair and completion on the cfDNA fragments;
and step 3: connecting the DNA with the filled end with a sequencing joint to obtain a connection product;
and 4, step 4: adding unmethylated fragments with length similar to that of free DNA fragments in the DNA library and methylated fragments with different degrees to ensure that the ratio of the antibody to the original free DNA is constant;
and 5: adding standard methylated and unmethylated samples;
step 6: heat treatment to melt the double stranded DNA and enrich for hypermethylated DNA fragments by adding 5-hydroxymethylcytosine antibodies that specifically bind methylated cytosine as DNA methylation occurs at the cytosine 5-position carbon atom;
and 7: adding magnetic beads to capture methylated DNA fragments in a targeted mode;
and 8: using methylated purified magnetic beads, washing for multiple times to remove unbound DNA fragments;
and step 9: performing PCR amplification on the methylated fragments obtained by immunoprecipitation to prepare a sequencing library; the preparation process of the sequencing library comprises a plurality of purification steps, and a magnetic bead method is selected for purification;
step 10: performing quality inspection on the sequencing library;
step 11: uniformly mixing libraries containing different barcode according to the same molar concentration, and performing on-machine sequencing according to a second-generation sequencing instrument by using a standard method to obtain a sequencing result;
step 12: 5mC biomarker selection and model construction;
step 13: and (6) analyzing the data.
2. The method for detecting lung cancer based on the methylation immunoprecipitation high-throughput sequencing technology according to claim 1, wherein in step 2, the extracted cfDNA is firstly subjected to fragment length detection; and performing end repair and completion on the screened cfDNA.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN202110815235.6A CN113584168A (en) | 2021-07-19 | 2021-07-19 | Lung cancer detection method based on methylation immunoprecipitation high-throughput sequencing technology |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN202110815235.6A CN113584168A (en) | 2021-07-19 | 2021-07-19 | Lung cancer detection method based on methylation immunoprecipitation high-throughput sequencing technology |
Publications (1)
| Publication Number | Publication Date |
|---|---|
| CN113584168A true CN113584168A (en) | 2021-11-02 |
Family
ID=78248175
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN202110815235.6A Pending CN113584168A (en) | 2021-07-19 | 2021-07-19 | Lung cancer detection method based on methylation immunoprecipitation high-throughput sequencing technology |
Country Status (1)
| Country | Link |
|---|---|
| CN (1) | CN113584168A (en) |
Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN111154846A (en) * | 2020-01-13 | 2020-05-15 | 四川大学华西医院 | Detection method of methylated nucleic acid |
| CN114045342A (en) * | 2021-12-01 | 2022-02-15 | 大连晶泰生物技术有限公司 | Detection method and kit for methylation mutation of free DNA (cfDNA) |
Citations (6)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| WO2009115313A1 (en) * | 2008-03-19 | 2009-09-24 | Oryzon Genomics, S.A. | Method and composition for methylation analysis |
| CN102971434A (en) * | 2010-08-11 | 2013-03-13 | 中国科学院心理研究所 | High-throughput sequencing method for methylated DNA and use thereof |
| CN104448001A (en) * | 2014-11-14 | 2015-03-25 | 杭州博谱医药科技有限公司 | Anti-5-methylcytosine monoclonal antibody and application thereof |
| CN107002125A (en) * | 2014-08-28 | 2017-08-01 | 西达-赛奈医疗中心 | Early stage of lung cancer detection is carried out by the DNA methylation Phenotype typing of saliva derived cell |
| CN107365845A (en) * | 2017-08-04 | 2017-11-21 | 上海易毕恩生物技术有限公司 | For detecting the gene marker, kit and lung cancer detection method of lung cancer |
| CN112941180A (en) * | 2021-02-25 | 2021-06-11 | 浙江大学医学院附属妇产科医院 | Group of lung cancer DNA methylation molecular markers and application thereof in preparation of lung cancer early diagnosis kit |
-
2021
- 2021-07-19 CN CN202110815235.6A patent/CN113584168A/en active Pending
Patent Citations (7)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| WO2009115313A1 (en) * | 2008-03-19 | 2009-09-24 | Oryzon Genomics, S.A. | Method and composition for methylation analysis |
| CN102971434A (en) * | 2010-08-11 | 2013-03-13 | 中国科学院心理研究所 | High-throughput sequencing method for methylated DNA and use thereof |
| CN107002125A (en) * | 2014-08-28 | 2017-08-01 | 西达-赛奈医疗中心 | Early stage of lung cancer detection is carried out by the DNA methylation Phenotype typing of saliva derived cell |
| US20170283881A1 (en) * | 2014-08-28 | 2017-10-05 | Cedars-Sinai Medical Center | Early lung cancer detection by dna methylation phenotyping of sputum-derived cells |
| CN104448001A (en) * | 2014-11-14 | 2015-03-25 | 杭州博谱医药科技有限公司 | Anti-5-methylcytosine monoclonal antibody and application thereof |
| CN107365845A (en) * | 2017-08-04 | 2017-11-21 | 上海易毕恩生物技术有限公司 | For detecting the gene marker, kit and lung cancer detection method of lung cancer |
| CN112941180A (en) * | 2021-02-25 | 2021-06-11 | 浙江大学医学院附属妇产科医院 | Group of lung cancer DNA methylation molecular markers and application thereof in preparation of lung cancer early diagnosis kit |
Cited By (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN111154846A (en) * | 2020-01-13 | 2020-05-15 | 四川大学华西医院 | Detection method of methylated nucleic acid |
| CN114045342A (en) * | 2021-12-01 | 2022-02-15 | 大连晶泰生物技术有限公司 | Detection method and kit for methylation mutation of free DNA (cfDNA) |
| WO2023098137A1 (en) * | 2021-12-01 | 2023-06-08 | 大连晶泰生物技术有限公司 | Method and kit for detecting methylation mutation of free dna |
| GB2620492A (en) * | 2021-12-01 | 2024-01-10 | Dalian Gentalker Bio Tech Co Ltd | Method and kit for detecting methylation mutation of free DNA |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| CN107475375B (en) | A kind of DNA probe library, detection method and kit hybridized for microsatellite locus related to microsatellite instability | |
| Rauluseviciute et al. | DNA hypermethylation associated with upregulated gene expression in prostate cancer demonstrates the diversity of epigenetic regulation | |
| Wang et al. | Dynamic monitoring of circulating tumor DNA to predict prognosis and efficacy of adjuvant chemotherapy after resection of colorectal liver metastases | |
| CN109852672B (en) | Method for screening acute myeloid leukemia DNA methylation prognosis marker | |
| JP6553085B2 (en) | Method of measuring a biomarker in gastrointestinal cancer | |
| CN112501293B (en) | Reagent combination for detecting liver cancer, kit and application thereof | |
| CN113584168A (en) | Lung cancer detection method based on methylation immunoprecipitation high-throughput sequencing technology | |
| CN112992354A (en) | Method and system for assessing colorectal cancer metastasis and recurrence risk and dynamically monitoring based on methyl marker combination | |
| CN112280865B (en) | Reagent combination for detecting liver cancer, kit and application thereof | |
| CN106845154B (en) | A device for FFPE sample copy number variation detects | |
| CN113430255A (en) | Lung cancer detection method based on 5hmC click chemistry high-throughput sequencing technology | |
| Yamaguchi et al. | Molecular and clinical features of the TP53 signature gene expression profile in early-stage breast cancer | |
| Capoluongo et al. | Main implications related to the switch to BRCA1/2 tumor testing in ovarian cancer patients: a proposal of a consensus | |
| Nørgaard et al. | Epigenetic silencing of MEIS2 in prostate cancer recurrence | |
| Hirotsu et al. | Microsatellite instability status is determined by targeted sequencing with MSIcall in 25 cancer types | |
| Fu et al. | Analysis of DNA methylation-driven genes for predicting the prognosis of patients with colorectal cancer | |
| Lau et al. | Single-molecule methylation profiles of cell-free DNA in cancer with nanopore sequencing | |
| Zhou et al. | Serial surveillance by circulating tumor DNA profiling after chimeric antigen receptor T therapy for the guidance of r/r diffuse large B cell lymphoma precise treatment | |
| US20190024184A1 (en) | Distinguishing metastatic-lethal prostate cancer from indolent prostate cancer using methylation status of epigenetic markers | |
| US20180066323A1 (en) | Biomarkers in Cancer, Methods, and Systems Related Thereto | |
| Zhang et al. | Identification of five cytotoxicity-related genes involved in the progression of triple-negative breast cancer | |
| Ahuno et al. | Circulating tumor DNA is readily detectable among Ghanaian breast cancer patients supporting non-invasive cancer genomic studies in Africa | |
| CN112011612B (en) | Biomarker and kit for prognosis classification of glioma patient survival prediction | |
| Szász et al. | Identification of liquid biopsy-based mutations in colorectal cancer by targeted sequencing assays | |
| CN112391474A (en) | Method for predicting esophageal squamous carcinoma metastasis based on fusobacterium nucleatum in tumor |
Legal Events
| Date | Code | Title | Description |
|---|---|---|---|
| PB01 | Publication | ||
| PB01 | Publication | ||
| SE01 | Entry into force of request for substantive examination | ||
| SE01 | Entry into force of request for substantive examination |