CN113755592A - Detection method of extrachromosomal circular DNA - Google Patents
Detection method of extrachromosomal circular DNA Download PDFInfo
- Publication number
- CN113755592A CN113755592A CN202111095137.6A CN202111095137A CN113755592A CN 113755592 A CN113755592 A CN 113755592A CN 202111095137 A CN202111095137 A CN 202111095137A CN 113755592 A CN113755592 A CN 113755592A
- Authority
- CN
- China
- Prior art keywords
- eccdna
- circular dna
- detecting
- dna
- extrachromosomal circular
- 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
Images
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
- 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
- C12Q2600/00—Oligonucleotides characterized by their use
- C12Q2600/158—Expression markers
-
- 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
- C12Q2600/00—Oligonucleotides characterized by their use
- C12Q2600/178—Oligonucleotides characterized by their use miRNA, siRNA or ncRNA
Abstract
The invention provides a detection method of extrachromosomal circular DNA, which takes eccDNA as a liquid biopsy marker of a tumor patient, and completes identification and analysis of the eccDNA through removal of linear DNA, purification of the eccDNA, library construction, sequencing and identification of a reverse cleavage site (junctionalsite) by biological information analysis.
Description
Technical Field
The invention belongs to the technical field of biology, and relates to a detection method of extrachromosomal circular DNA for identification and analysis of eccDNA.
Background
Extrachromosomal circular DNA (eccDNA) is short, and the eccDNA is derived from genome DNA and does not carry telomeres and centromere, and the size of the eccDNA varies from 100bp to millions of bp. Leading-edge studies have shown that the generation and amplification of eccDNA is a marker event for tumor cells, and is a source of high expression and high mutation of protooncogenes in tumor cells. Therefore, the identification of eccDNA against tumor cells is a key technical approach in the field. However, there is a lack of a mature, stable method for extraction and isolation from blood in the current field. Research shows that both tumor tissue and normal tissue release eccDNA into the circulatory system, the biological stability of the eccDNA in the circulatory system is superior to that of linear DNA, and the eccDNA has a unique molecular structure and can be used as a liquid biopsy detection target.
Disclosure of Invention
The invention aims to provide a detection method of extrachromosomal circular DNA, which takes the eccDNA as a liquid biopsy marker of a tumor patient, and completes the identification and analysis of the eccDNA through the removal of linear DNA, the purification of the eccDNA, the library establishment, the sequencing and the identification of a reverse cleavage site (ligation site) by bioinformatics analysis.
In order to achieve the purpose, the invention adopts the following technical scheme:
the detection method uses the eccDNA as a liquid detection target, and completes the identification and analysis of the eccDNA by removing linear DNA, purifying and establishing a library of the ECCDNA, identifying a trans-splicing site through sequencing and biological information analysis.
As a preferable aspect of the present invention, the detection method includes the steps of:
1) obtaining a sample, and enriching free DNA in the sample;
2) removing linear DNA in the free DNA in the step 1) by enzyme digestion, and reserving eccDNA;
3) breaking the circular DNA in the step 2) by using transposase, inserting into an adaptor, amplifying and establishing a library;
4) sequencing the library of step 3) and identifying the number of relevant eccDNA and chromatin regions.
In a preferred embodiment of the present invention, in step 2), the enzyme is cleaved with exonuclease.
As a preferred embodiment of the present invention, in step 3), the transposase is selected from one or a combination of any more of Tn1, Tn2, Tn3, Tn4, Tn5, Tn6, Tn7, Tn9, and Tn 10.
In a preferred embodiment of the present invention, in the step 3), the amplification includes a first round of PCR amplification, a qPCR amplification and a second round of PCR amplification.
In a preferred embodiment of the present invention, the number of cycles of the second round of PCR amplification is determined by the qPCR amplification result.
In a preferred embodiment of the present invention, the sample comprises a peripheral blood sample of a tumor patient.
As a preferred embodiment of the present invention, the sequencing in step 4) is based on a second generation sequencing platform.
The detection method of the extrachromosomal circular DNA is applied to monitoring the medication of tumor patients.
The method for detecting the extrachromosomal circular DNA is applied to monitoring prognosis eccDNA of a tumor patient.
Compared with the prior art, the invention has the following beneficial effects:
the invention provides a detection method of extrachromosomal circular DNA, which takes eccDNA as a liquid biopsy marker of a tumor patient, and completes identification and analysis of the eccDNA through removal of linear DNA, purification of the eccDNA, library establishment, sequencing and identification of a reverse cleavage site (junction site) by biological information analysis.
Drawings
FIG. 1 is a graph showing a fragment distribution of an eccDNA library for detecting free DNA of sample numbers EA _001 to EA _003 of the present invention;
FIG. 2 is a graph showing a fragment distribution of an eccDNA library for detecting free DNA of sample numbers EA _004 to EA _006 according to the present invention;
FIG. 3 is a graph showing a fragment distribution of an eccDNA library for detecting free DNA of sample Nos. EA _007 to EA _009 according to the present invention;
FIG. 4 is a graph showing a fragment distribution of an eccDNA library for detecting free DNA of sample numbers EA _010 to EA _012 according to the present invention;
FIG. 5 is a graph showing a fragment distribution of an eccDNA library for detecting free DNA of sample numbers EA _013 to EA _015 according to the present invention;
FIG. 6 is a graph showing the fragment distribution of the eccDNA library for detecting free DNA of sample numbers EA _016 to EA _018 according to the present invention;
FIG. 7 is a distribution diagram of fragments of a test eccDNA library of the invention after construction of sample numbers EA _001 to EA _ 003;
FIG. 8 is a distribution diagram of fragments of a detected eccDNA library after construction of libraries with sample numbers EA _004 to EA _006 according to the present invention;
FIG. 9 is a graph showing a fragment distribution of a test eccDNA library of the present invention after constructing the libraries with sample numbers EA _007 to EA _ 009;
FIG. 10 is a graph showing a fragment distribution of an eccDNA library after construction of libraries with sample numbers EA _010 to EA _012 according to the present invention;
FIG. 11 is a graph showing a fragment distribution of a detected eccDNA library after construction of libraries with sample numbers EA _013 to EA _015 according to the present invention;
FIG. 12 is a distribution diagram of fragments of a test eccDNA library of the invention after construction of the sample numbers EA _016 to EA _018 libraries;
FIG. 13 is a flow chart of the present invention.
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, and not all of the embodiments. All other embodiments, which can be derived by a person skilled in the art from the embodiments given herein without making any creative effort, shall fall within the protection scope of the present invention.
Referring to fig. 13, the present invention provides a method for detecting extrachromosomal circular DNA, wherein the method comprises the steps of taking the eccDNA as a liquid detection target, removing linear DNA, purifying and banking eccDNA, identifying a trans-cleavage site by sequencing and bioinformatic analysis, and identifying and analyzing the eccDNA, wherein the method comprises the following steps:
1) obtaining a sample, and enriching free DNA in the sample;
2) removing linear DNA in the free DNA in the step 1) by enzyme digestion, and reserving eccDNA;
3) breaking the circular DNA in the step 2) by using transposase, inserting into an adaptor, amplifying and establishing a library;
4) sequencing the library of step 3) and identifying the number of relevant eccDNA and chromatin regions.
Examples
The invention uses peripheral blood of a subject as a test sample to identify the number of relevant eccDNA.
1. Blood collection, preservation and free DNA extraction
1.1. Sample preparation
1.1.1 sample acquisition requirements:
the EDTAK2 anticoagulation tube collects 10mL of peripheral blood, and the tube is slightly inverted 8-10 times immediately after the blood sample is collected and mixed (mixing is not violent, and hemolysis is prevented).
1.1.2 sample preservation and transport:
after the specimen is collected, the specimen is stored at 4 ℃; after the blood sampling is finished, the blood reaches an inspection laboratory within 4 hours, the ice bag is subjected to low-temperature shockproof transportation in the transportation process, and the ice bag cannot be in direct contact with the blood sample.
1.1.3 sample State:
after the specimen arrives at the laboratory, the appearance of the specimen should be observed to be free of obvious hemolysis and coagulation.
1.2.1 plasma Collection
(1) Centrifuging whole blood at 25 deg.C for 10min at 500g, and transferring upper plasma to new EP tube;
(2) the plasma was centrifuged at 1600g, 4 ℃ for 10min and the supernatant plasma was transferred to a new EP tube;
(3) the plasma was again 16000g, centrifuged at 4 ℃ for 10min, and the upper plasma transferred to a new EP tube and frozen.
1.3 free nucleic acid (cfDNA) extraction
Free DNA was extracted using 4mL plasma according to QIAamp Circulating Nucleic Acid Kit (Qiagen) instructions, and 18 clinical samples were currently collected (sample information as in table 1):
TABLE 1 sample information
After extraction of free DNA, the fragment size distribution of the free DNA was examined using an Agilent2100 instrument, and the results are shown in FIGS. 1 to 6.
1.4 Primary reagents and instruments
Reagent: QIAamp Circulating Nucleic Acid Kit (55114), NEB Exonuclease V (M0345S), MinElute Reaction clean Kit (28206), Vazyme Tn5(TD501-2), NEBnext 2xHifi PCR mix, Agilent2100, and Qubit4.0 reagents.
The instrument comprises the following steps: applied Biosystems ProFlexTM3X 32-well PCR System, Roche-LightCycler-480, Agilent2100, Qubit4.0.
Enrichment of eccDNA
(1) The volume of the eccDNA enrichment (digestion of linear DNA) reaction is shown in Table 2:
TABLE 2 reaction System
(2) Placing into a PCR instrument, and incubating at 37 deg.C for 30 min;
(3) add 1.1. mu.L of EDTA (0.5M) into the EP tube;
(4) placing into a PCR instrument, and incubating at 70 deg.C for 30 min;
(5) samples were purified according to the MinElute Reaction Cleanup Kit.
(6) And (3) measuring the DNA quantity (determining the OD260 value by the NanoDrop) by an ultraviolet spectrophotometer method, if the cfDNA has genome pollution, theoretically, repeating the steps for a plurality of times for digestion until the OD260 value is unchanged.
3. Construction of an eccDNA library based on the Tn5 enzyme
(1) The 50 μ L reaction system is shown in table 3:
TABLE 3 reaction System
(2) Fragmentation product purification
a. The corresponding reagents in the Qiagen Minelute PCR Purification Kit were removed.
b. Add 250. mu.L PB buffer, mix well with Tn5 enzyme mix, load onto column, centrifuge at 17900g for 1 min.
c. The tube was discarded, 750. mu.LPE, 17900g was added and centrifuged for 1 min.
d. The tube was discarded, 17900g and left empty for 1 min.
e. Replace the new EP tube, add 22. mu.L buffer EB, stand at room temperature for 5min, centrifuge at 17900g for 1min, this step may be suspended and the product stored at-20 ℃.
(3) PCR amplification, see Table 4;
TABLE 4 PCR amplification System and reaction procedure
(4) qPCR amplification (determining the number of continued PCR cycles), see table 5;
TABLE 5 qPCR amplification System and reaction procedure
And after qPCR is finished, analyzing the result, and determining the amplification cycle number when half of the maximum fluorescence value of each sample is determined, wherein the amplification cycle number is N, and N is the cycle number of the residual PCR product for continuous PCR amplification.
(5) Continued PCR amplification
The remaining 45. mu.l of product was subjected to a subsequent PCR, reaction procedure: 30s at 98 ℃, 10s at 98 ℃, 30s at 63 ℃ and 1min at 72 ℃ N cycles; hold (N is determined by qPCR results) at 4 ℃.
(6) PCR product purification
a. The corresponding reagents in the Qiagen Minelute PCR Purification Kit were removed.
b. Add 250. mu.L PB buffer, homogenize PCR product, load onto column, and centrifuge at 17900g for 1 min.
c. The tube was discarded, 750. mu.L PE, 17900g was added, and the mixture was centrifuged for 1 min.
d. The tube was discarded, 17900g and left empty for 1 min.
e. Replace the new EP tube, add 25. mu.L buffer EB, stand at room temperature for 5min, centrifuge at 17900g for 1 min.
f. mu.L of each was taken for Agilent2100 assay of library fragment size and Qubit concentration, the remainder was stored at-20 ℃.
Experimental results of the eccDNA library
At present, 18 cases of clinical sample eccDNA high-throughput sequencing libraries are constructed, from the view of the concentration, the total amount and the fragment distribution of the libraries, a main peak exists around 180bp, small peaks exist around 300bp and 500bp respectively, the quality control of the libraries is qualified, and all Bailey and Kangsu sequencing companies are sent to sequence the eccDNA libraries.
The specific experimental results are shown in table 6 and fig. 7 to 12:
TABLE 6 sample Experimental results
Therefore, the method for detecting the extrachromosomal circular DNA can provide effective information for accurate medical schemes such as drug administration and prognosis of tumor patients in the future by taking the eccDNA as a liquid biopsy marker of the tumor patients, and identifying and analyzing the eccDNA through linear DNA removal, eccDNA purification, library construction, sequencing and bioinformatics analysis on the recognition of a trans-splicing site (junction site).
While the invention has been described with respect to a preferred embodiment, it will be understood by those skilled in the art that the foregoing and other changes, omissions and deviations in the form and detail thereof may be made without departing from the scope of this invention. Those skilled in the art can make various changes, modifications and equivalent arrangements, which are equivalent to the embodiments of the present invention, without departing from the spirit and scope of the present invention, and which may be made by utilizing the techniques disclosed above; meanwhile, any changes, modifications and variations of the above-described embodiments, which are equivalent to those of the technical spirit of the present invention, are within the scope of the technical solution of the present invention.
Claims (10)
1. The method for detecting the extrachromosomal circular DNA is characterized in that the detection method takes the eccDNA as a liquid detection target, and finishes the identification and analysis of the eccDNA by removing linear DNA, purifying and establishing an ECCDNA, identifying a trans-splicing site through sequencing and biological information analysis.
2. The method for detecting the extrachromosomal circular DNA as claimed in claim 1, wherein the method comprises the steps of:
1) obtaining a sample, and enriching free DNA in the sample;
2) removing linear DNA in the free DNA in the step 1) by enzyme digestion, and reserving eccDNA;
3) breaking the circular DNA in the step 2) by using transposase, inserting into an adaptor, amplifying and establishing a library;
4) sequencing the library of step 3) and identifying the number of relevant eccDNA and chromatin regions.
3. The method for detecting the extrachromosomal circular DNA as claimed in claim 2, wherein in the step 2), the enzyme digestion is performed by exonuclease.
4. The method for detecting extrachromosomal circular DNA as claimed in claim 2, wherein in step 3), said transposase is selected from one or a combination of any of Tn1, Tn2, Tn3, Tn4, Tn5, Tn6, Tn7, Tn9 and Tn 10.
5. The method for detecting extrachromosomal circular DNA as claimed in claim 4, wherein in step 3), the amplification comprises a first round of PCR amplification, a qPCR amplification and a second round of PCR amplification.
6. The method of claim 5, wherein the number of cycles of the second round of PCR amplification is determined by the qPCR amplification result.
7. The method for detecting extrachromosomal circular DNA as claimed in any one of claims 2 to 6, wherein the sample comprises a peripheral blood sample from a patient with a tumor.
8. The method for detecting extrachromosomal circular DNA of claim 7, wherein the sequencing in step 4) is based on a next-generation sequencing platform.
9. Use of the method of detecting extrachromosomal circular DNA as claimed in any one of claims 1 to 8 for monitoring the administration of a drug to a patient suffering from a tumour.
10. Use of the method of detecting extrachromosomal circular DNA according to any one of claims 1 to 8 for monitoring prognosis of eccDNA in a patient with a tumour.
Priority Applications (2)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN202111095137.6A CN113755592A (en) | 2021-09-17 | 2021-09-17 | Detection method of extrachromosomal circular DNA |
| PCT/CN2022/119489 WO2023041077A1 (en) | 2021-09-17 | 2022-09-17 | Eccdna-related marker, use, system, and method |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN202111095137.6A CN113755592A (en) | 2021-09-17 | 2021-09-17 | Detection method of extrachromosomal circular DNA |
Publications (1)
| Publication Number | Publication Date |
|---|---|
| CN113755592A true CN113755592A (en) | 2021-12-07 |
Family
ID=78796319
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN202111095137.6A Pending CN113755592A (en) | 2021-09-17 | 2021-09-17 | Detection method of extrachromosomal circular DNA |
Country Status (2)
| Country | Link |
|---|---|
| CN (1) | CN113755592A (en) |
| WO (1) | WO2023041077A1 (en) |
Cited By (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| WO2023041077A1 (en) * | 2021-09-17 | 2023-03-23 | 杭州瀚因生命科技有限公司 | Eccdna-related marker, use, system, and method |
Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| WO2020210802A1 (en) * | 2019-04-11 | 2020-10-15 | University Of Virginia Patent Foundation | Tagmentation to open up circles of dna and detect extrachromosomal circles of dna for diagnosis |
| US20200407799A1 (en) * | 2019-03-25 | 2020-12-31 | The Chinese University Of Hong Kong | Determining linear and circular forms of circulating nucleic acids |
Family Cites Families (5)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US5500356A (en) * | 1993-08-10 | 1996-03-19 | Life Technologies, Inc. | Method of nucleic acid sequence selection |
| WO2005108608A1 (en) * | 2004-05-10 | 2005-11-17 | Kabushiki Kaisha Dnaform | Method for isolating nucleic acid isoforms |
| US20090233291A1 (en) * | 2005-06-06 | 2009-09-17 | 454 Life Sciences Corporation | Paired end sequencing |
| US9080211B2 (en) * | 2008-10-24 | 2015-07-14 | Epicentre Technologies Corporation | Transposon end compositions and methods for modifying nucleic acids |
| CN113755592A (en) * | 2021-09-17 | 2021-12-07 | 杭州瀚因生命科技有限公司 | Detection method of extrachromosomal circular DNA |
-
2021
- 2021-09-17 CN CN202111095137.6A patent/CN113755592A/en active Pending
-
2022
- 2022-09-17 WO PCT/CN2022/119489 patent/WO2023041077A1/en unknown
Patent Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US20200407799A1 (en) * | 2019-03-25 | 2020-12-31 | The Chinese University Of Hong Kong | Determining linear and circular forms of circulating nucleic acids |
| WO2020210802A1 (en) * | 2019-04-11 | 2020-10-15 | University Of Virginia Patent Foundation | Tagmentation to open up circles of dna and detect extrachromosomal circles of dna for diagnosis |
Non-Patent Citations (1)
| Title |
|---|
| JASON BUENROSTRO等: "ATAC-seq: A Method for Assaying Chromatin Accessibility Genome-Wide", CURRENT PROTOCOLS IN MOLECULAR BIOLOGY, pages 4 - 5 * |
Cited By (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| WO2023041077A1 (en) * | 2021-09-17 | 2023-03-23 | 杭州瀚因生命科技有限公司 | Eccdna-related marker, use, system, and method |
Also Published As
| Publication number | Publication date |
|---|---|
| WO2023041077A1 (en) | 2023-03-23 |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| US20220228197A1 (en) | Method for determining copy number variations | |
| US20220205038A1 (en) | Single-molecule sequencing of plasma dna | |
| CN103003447B (en) | Method for determining the presence or absence of different aneuploidies in a sample | |
| CN106906211B (en) | Molecular joint and application thereof | |
| CN103468818B (en) | A kind of test kit and method detecting CYP2C19 gene pleiomorphism | |
| CN103298955A (en) | Method for construction of plasma DNA sequencing library and kit thereof | |
| CN103080336A (en) | Kits, devices and methods for detecting chromosome copy number of embryo or tumor | |
| US10227587B2 (en) | Method for constructing a plasma DNA sequencing library | |
| CN108463559A (en) | The deep sequencing profile analysis of tumour | |
| CN108753954B (en) | Capture probe set of dementia-related gene, kit, library construction method and application | |
| CN107893116A (en) | For detecting primer pair combination, kit and the method for building library of gene mutation | |
| US20240052396A1 (en) | Selective protection of nucleic acids | |
| CN110760936A (en) | Method for constructing DNA methylation library and application thereof | |
| CN107893260A (en) | Efficiently remove the method and kit of the structure transcript profile sequencing library of rRNA | |
| EP3719182B1 (en) | Method for constructing library of cell-free dnas in body fluids and application thereof | |
| WO2015196752A1 (en) | A method and a kit for quickly constructing a plasma dna sequencing library | |
| Lee et al. | Single-cell toolkits opening a new era for cell engineering | |
| CN113755592A (en) | Detection method of extrachromosomal circular DNA | |
| CN108728522A (en) | Drug Discovery detection method | |
| CN110438220A (en) | The motionless syndrome gene panel kit of cilium and its application | |
| CN113604540A (en) | Method for rapidly constructing RRBS sequencing library by using blood circulation tumor DNA | |
| CN115011695A (en) | Multiple cancer species identification marker based on free circular DNA gene, kit and application | |
| CN107446998A (en) | MTHFR C677T, rs1801133 single nucleotide polymorphisms quick detections primer, molecular beacon, kit and its detection method | |
| CN112680794A (en) | Ultramicro nucleic acid sample library building method applied to NGS platform | |
| JPWO2019009431A1 (en) | Highly accurate method for identifying mutations in tumor cells |
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 |