CN117778578B - A primer combination for detecting the methylation degree of a target gene based on high-throughput sequencing and its application - Google Patents
A primer combination for detecting the methylation degree of a target gene based on high-throughput sequencing and its application Download PDFInfo
- Publication number
- CN117778578B CN117778578B CN202311862466.8A CN202311862466A CN117778578B CN 117778578 B CN117778578 B CN 117778578B CN 202311862466 A CN202311862466 A CN 202311862466A CN 117778578 B CN117778578 B CN 117778578B
- Authority
- CN
- China
- Prior art keywords
- primer
- seq
- nucleotide sequence
- gene
- library
- 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.)
- Active
Links
Landscapes
- Measuring Or Testing Involving Enzymes Or Micro-Organisms (AREA)
Abstract
The invention discloses a primer composition for detecting the methylation degree of a target gene based on high-throughput sequencing, and a detection system is constructed based on the primer composition, and the detection system can be used for amplifying a library by a one-step method aiming at a DNA template of a sample to be detected, so that the library establishment time is shortened; and the gene library of the target gene region of the sample to be detected obtained by the one-step amplification has excellent specificity, and the target product is single. When the detection system is used for detecting the methylation degree of MGMT or TWIST1 of a sample to be detected, other bands can not appear except the MGMT gene band or TWIST1 gene band in gel electrophoresis detection of the amplified gene library, and meanwhile, when the methylation degree analysis is carried out by combining the amplified gene library with targeted second-generation sequencing and bioinformatics, the detection accuracy reaches 100%.
Description
Technical Field
The invention relates to the technical field of molecular biology, in particular to a primer composition for detecting the methylation degree of a target gene based on high-throughput sequencing and application thereof.
Background
Methylation of CpG sites can lead to chromosome structural changes, inhibit the combination of promoters and transcription factors, and lead to silencing of genes so as to lose functions; gene methylation has a close relationship with the occurrence and development of tumors and cell canceration, and is mainly changed into the following mechanisms: (1) Cytosine in methylated CpG island dinucleotides deaminates with higher frequency to become thymine, so that gene mutation is caused; (2) The cancer suppressor gene and DNA repair gene are silenced due to hypermethylation; (3) activation by reduced methylation levels of oncogenes; (4) Reduced levels of global methylation of the genome activate transposons and repeat sequences resulting in reduced chromosomal stability.
Therefore, methylation can be used as a biomarker and a prognosis evaluation index for early diagnosis of tumors and the like, and has important significance for screening, risk evaluation, early diagnosis, classification, prognosis judgment and treatment monitoring of tumors.
O6-methylguanine-DNA methyltransferase (MGMT), which is a DNA repair enzyme located on chromosome 10q26, can rapidly repair DNA alkylation damage caused by alkylating agents, thereby maintaining the stability of genome in cells. The gene promoter for the MGMT protein comprises CpG islands rich in 97 CG dinucleotides (CpG sites), and in normal tissues, the CpG sites are in an unmethylated state generally; MGMT methylation suppresses MGMT activity, making tumor cells more sensitive to alkylating agents such as the chemotherapeutic drug Temozolomide (TMZ). The existing research shows that the MGMT gene is expressed in brain glioma tissues and is closely related to the drug resistance of tumor cells to nitrosurea drugs, and also is closely related to the chemotherapeutic effect of tumors and prognosis of tumor patients. Aiming at patients with brain glioma, the higher the methylation degree of MGMT gene is, the better the curative effect of temozolomide drug is; therefore, it is important to select a proper drug for treating the methylation degree of the gene of a tumor patient to achieve a good treatment effect.
TWIST1 is a highly conserved transcription factor in basic helix-loop-helix protein family, mRNA of which has highest expression level in placenta tissue, plays an important role in embryogenesis and development, and is also involved in regulation of epithelial-mesenchymal transition process, thereby affecting invasion and metastasis of tumor; therefore, detection of the methylation degree of TWIST1 of a tumor patient can realize accurate treatment and prognosis of the tumor patient.
The current technology for detecting methylation mainly comprises fluorescent PCR, digital PCR, pyrosequencing, gene chip, second generation sequencing and other technologies. Pyrosequencing is a currently accepted gold standard, but the detection method has low detection flux (1-5 CpG sites) and is realized by using chemical dyes, so that the sensitivity and the specificity are relatively poor; besides the defects, the most widely applied fluorescent PCR method in the market has the advantages that the detection result is only qualitative and can not be quantified; the digital PCR detection method realizes nucleic acid quantification by a single-molecule PCR method on the fluorescent PCR technology, and compared with the fluorescent PCR, the digital PCR detection method still depends on chemical dye, has low detection flux and poor specificity, and can realize absolute quantification.
The targeted second generation sequencing is to enrich single genes or genome regions related to clinic by using a molecular biological method, and sequencing with higher coverage depth is carried out on the enriched target region, so that rare variation is easier to find; and it only sequences for the enriched genomic region, which can reduce the amount of data and thus shorten the detection period. Currently targeted second generation sequencing generally has two techniques to construct libraries for specific genomic regions: hybrid capture techniques and amplicon-based library construction techniques. The amplicon-based library construction technique has various technical advantages over hybrid capture techniques, including higher target capture efficiency, higher detection sensitivity, and simpler and faster experimental procedures.
The amplicon-based library construction technology is a method of adding joints at two ends of a DNA fragment to be detected by utilizing a PCR reaction, the principle is relatively simple, a library of a target region can be obtained by only two rounds of PCR and two steps of purification, the first step is to combine a primer containing a general sequence with the target region, and the second step is to connect a sequencing joint through the PCR reaction, so that the constructed library can be used for on-machine sequencing; however, the existing two-step amplification library-building method has some problems when being applied: (1) At least two rounds of PCR amplification and two corresponding library purifications are needed, and the time required by the whole library construction process is long; (2) The product quantity of the first round of amplification is more, so that pollution is easy to cause in the secondary PCR; (3) The library construction process is complicated, and sample confusion is easy to cause. Although library construction can be achieved by one-step amplification by mixing the first and second step primers together, a large number of non-target libraries are generated.
Thus, there is a need for a primer composition for detecting the methylation level of a target gene based on high throughput sequencing, which is used to construct a gene library that only generates a single band, thereby shortening the library construction time and reducing the amount of sequencing data.
Disclosure of Invention
The invention aims to overcome the defects in the prior art and provides a primer composition for detecting the methylation degree of a target gene based on high-throughput sequencing and application thereof.
The first object of the present invention is to provide a primer composition for detecting the methylation degree of a target gene based on high throughput sequencing.
The second object of the invention is to provide a detection system for detecting the methylation degree of a target gene based on high-throughput sequencing.
The third object of the invention is to provide a construction method of a gene library for detecting the methylation degree of a target gene based on high-throughput sequencing.
The fourth object of the invention is to provide the application of the primer composition, the detection system and/or the gene library constructed by the construction method in the methylation process of the target gene based on high-throughput sequencing.
In order to achieve the above object, the present invention is realized by the following means:
A primer composition for detecting the methylation degree of a target gene based on high-throughput sequencing, which comprises an upstream primer, a downstream primer, an extension primer 1, an extension primer 2 and a universal primer;
The upstream primer sequentially comprises a read 1 primer binding sequence and a specific primer sequence 1 from a 5 'end to a 3' end; the downstream primer sequentially comprises a read 2 primer binding sequence and a specific primer sequence 2 from a 5 'end to a 3' end;
The length of the read 1 primer binding sequence is 21-33 bp, and the length of the read 2 primer binding sequence is 21-34 bp; the specific primer sequence 1 and the specific primer sequence 2 respectively target the upstream region and the downstream region of the target gene and can specifically amplify the target gene;
the extension primer 1 sequentially comprises a joint sequence 1, an index sequence 1 and an identification sequence 1 from the 5 'end to the 3' end; the linker sequence 1 is a P5 linker sequence or a P7 linker sequence, and the recognition sequence 1 is 15-33 bp before the 5 'end of the read 1 primer binding sequence or 15-34 bp before the 5' end of the read 2 primer binding sequence;
The extension primer 2 sequentially comprises a connector sequence 2, an index sequence 2 and a recognition sequence 2 from the 5' end to the 3' end, wherein a blocking group is marked at the 3' end of the extension primer 2; the linker sequence 2 is a P7 linker sequence or a P5 linker sequence, and the recognition sequence 2 is 15-33 bp before the 5 'end of the read 1 primer binding sequence or 15-34 bp before the 5' end of the read 2 primer binding sequence;
The linker sequence 1 of the extension primer 1 is a P5 linker sequence, and the linker sequence 2 of the extension primer 2 is a P7 linker sequence; or the linker sequence 1 of the extension primer 1 is a P7 linker sequence, and the linker sequence 2 of the extension primer 2 is a P5 linker sequence;
The identification sequence 1 of the extension primer 1 is 15-33 bp before the 5 'end of the primer binding sequence of the read 1, and the identification sequence 2 of the extension primer 2 is 15-34 bp before the 5' end of the primer binding sequence of the read 2; or the identification sequence 1 of the extension primer 1 is 15-34 bp before the 5 'end of the primer binding sequence of the read 2, and the identification sequence 2 of the extension primer 2 is 15-33 bp before the 5' end of the primer binding sequence of the read 1;
the general primer is 15-29 bp before the 5' end of the connector sequence 1.
Preferably, the upstream primer and the downstream primer are used for specifically amplifying a target gene.
Preferably, the target gene is MGMT gene, and the primer composition contains a nucleotide sequence shown in SEQ ID NO:1, and the nucleotide sequence of the upstream primer is shown as SEQ ID NO:2, and the nucleotide sequence of the downstream primer is shown as SEQ ID NO:3, the nucleotide sequence of the extension primer 1 is shown as SEQ ID NO:4 and the nucleotide sequence of the extension primer 2 is shown as SEQ ID NO: 5;
Or the primer composition contains a nucleotide sequence shown as SEQ ID NO:1, and the nucleotide sequence of the upstream primer is shown as SEQ ID NO:2, and the nucleotide sequence of the downstream primer is shown as SEQ ID NO:6, the nucleotide sequence of the extension primer 1 is shown as SEQ ID NO:7 and the nucleotide sequence of the extension primer 2 is shown as SEQ ID NO: 8.
Preferably, the target gene is a TWIST1 gene, and the primer composition comprises a nucleotide sequence shown in SEQ ID NO:9, and the nucleotide sequence of the upstream primer is shown as SEQ ID NO:10, and the nucleotide sequence of the downstream primer is shown as SEQ ID NO:3, the nucleotide sequence of the extension primer 1 is shown as SEQ ID NO:4 and the nucleotide sequence of the extension primer 2 is shown as SEQ ID NO: 5;
or the primer composition contains a nucleotide sequence shown as SEQ ID NO:9, and the nucleotide sequence of the upstream primer is shown as SEQ ID NO:10, and the nucleotide sequence of the downstream primer is shown as SEQ ID NO:6, the nucleotide sequence of the extension primer 1 is shown as SEQ ID NO:7 and the nucleotide sequence of the extension primer 2 is shown as SEQ ID NO: 8.
The invention also discloses a detection system for detecting the methylation degree of the target gene based on high-throughput sequencing, which comprises the primer composition.
Preferably, the detection system further comprises TaqDNA polymerase and a buffer.
Preferably, the concentration of the upstream primer in the primer composition in the detection system is 200-750 nM; the concentration of the downstream primer in the primer composition in the detection system is 200-750 nM.
More preferably, the concentration of the upstream primer in the primer composition in the detection system is 50nM; the concentration of the downstream primer in the primer composition in the detection system was 50nM.
Preferably, the concentration of the extension primer 1 in the primer composition in the detection system is 250 to 500nM.
More preferably, the concentration of extension primer 1 in the primer composition in the detection system is 500nM.
Preferably, the concentration of the extension primer 2 in the primer composition in the detection system is 50-100 nM.
More preferably, the concentration of extension primer 2 in the primer composition in the detection system is 100nM.
Further preferably, the 3' -end-labeled blocking group of the extension primer 2 is a Spacer C3, spacer C6, dSpacer, INVERTED DA/G/C/T, ddC, pi, or NH 2C6.
Further preferably, the 3' -end-labeled blocking group of the extension primer 2 is NH 2C6.
Preferably, the concentration of the universal primer in the primer composition in the detection system is 200-750 nM.
More preferably, the concentration of the universal primer in the primer composition in the detection system is 500nM.
The invention also claims a construction method of a gene library for detecting the methylation degree of a target gene based on high-throughput sequencing, which comprises the following steps:
S1, extracting DNA of a sample to be detected, and performing bisulfite conversion to serve as an amplification template;
S2, carrying out PCR amplification on the amplification template obtained in the step S1 by using any detection system, and collecting an amplification product.
Preferably, the PCR amplification in step S2 is performed as follows: 95 ℃ and 3mins;95 ℃,15s,60 ℃,1min,45 cycles; stored at 60℃with 2mins at 4 ℃.
The invention also claims the application of the primer composition, the detection system and/or the gene library constructed by the construction method in the methylation process of the detection target gene based on high-throughput sequencing.
Compared with the prior art, the invention has the following beneficial effects:
The invention provides a primer composition for detecting the methylation degree of a target gene based on high-throughput sequencing, a detection system is constructed based on the primer composition, and a DNA template of a sample to be detected can be amplified by using the detection system by a one-step method, so that the library building time is shortened; and the gene library of the target gene region of the sample to be detected obtained by the one-step amplification has excellent specificity, and the target product is single. When the detection system is used for detecting the methylation degree of MGMT or TWIST1 of a sample to be detected, other bands can not appear except the MGMT gene band or TWIST1 gene band in gel electrophoresis detection of the amplified gene library, and meanwhile, when the methylation degree analysis is carried out by combining the amplified gene library with targeted second-generation sequencing and bioinformatics, the detection accuracy reaches 100%.
Drawings
FIG. 1 is an amplification schematic of example 1;
FIG. 2 is an amplification schematic of example 2;
FIG. 3 is an amplification schematic of comparative example 6;
FIG. 4 is a graph showing the results of gel electrophoresis detection of gene libraries 1 to 9 in test example 1;
FIG. 5 is a graph showing the results of electrophoresis detection of sequencing libraries constructed in the experimental group of test example 2;
FIG. 6 is a library strip size distribution plot of the sequencing library of sample 1 in the experimental set of test example 2;
FIG. 7 is a graph showing the results of electrophoresis detection of a sequencing library constructed in the control group of test example 2;
FIG. 8 is a library strip size distribution plot of the sequencing library of sample 1 in the control group of test example 2.
Detailed Description
The invention will be further described in detail with reference to the drawings and specific examples, which are given solely for the purpose of illustration and are not intended to limit the scope of the invention. The test methods used in the following examples are conventional methods unless otherwise specified; the materials, reagents and the like used, unless otherwise specified, are those commercially available.
The experimental procedure for MGMT methylation analysis of sequencing off-machine data in the test examples refers to the prior art:
(1)Singer B D.A Practical Guide to the Measurement and Analysis of DNA Methylation[J].American Thoracic Society,2019(4).DOI:10.1165/RCMB.2019-0150TR.;
(2)Rauluseviciute I,Drabls F,Rye M B.DNA methylation data by sequencing:experimental approaches and recommendations for tools and pipelines for data analysis[J].Clinical Epigenetics,2019,11.DOI:10.1186/s13148-019-0795-x..
Example 1A Gene library for quantitative determination of the methylation level of MGMT
Extracting genomic DNA (gDNA) of a fresh tumor tissue sample of a cancer patient by using a DNA extraction kit, and carrying out bisulphite conversion modification on 500ng of gDNA by using a bisulphite conversion kit (Novozan biotechnology Co., ltd., EM 102-01) to obtain a DNA sample after conversion modification; and taking a 25ng transformation modified DNA sample as a DNA template to construct a gene library.
The synthetic nucleotide sequence is shown as SEQ ID NO:1 (upstream primer of specific amplification MGMT gene), and the nucleotide sequence is shown in SEQ ID NO:2 (downstream primer of specific amplified MGMT gene), and nucleotide sequence is shown in SEQ ID NO:3 (extension primer 1), and the nucleotide sequence is shown as SEQ ID NO:4 (extension primer 2) and the nucleotide sequence of which is shown in SEQ ID NO:5, a universal primer R; specific sequence information is shown below.
MGMT specific primer F (SEQ ID NO: 1):
MGMT specific primer R (SEQ ID NO: 2):
p5 sequence (SEQ ID NO: 3):
P7-Blocker(SEQ ID NO：4)：
universal primer R (SEQ ID NO: 5): 5'-CAAGCAGAAGACGGCATACGA-3'.
In the sequence shownIs a Read primer binding sequence; "" is a specific sequence targeting MGMT; is a linker primer; Is an Index sequence; "_" is a truncated Read primer binding sequence;
wherein the nucleotide sequence is shown in SEQ ID NO: the MGMT specific primer F shown in 1 is a read 1 primer binding sequence and a specific primer sequence 1 of 33bp from a 5 'end to a 3' end in sequence; the nucleotide sequence is shown in SEQ ID NO:2, the MGMT specific primer R is a read 2 primer binding sequence and a specific primer sequence 2 which are 34bp in sequence from a 5 'end to a 3' end; the specific primer sequence 1 and the specific primer sequence 2 respectively target the upstream region and the downstream region of the MGMT gene, so that the MGMT gene can be specifically amplified;
The nucleotide sequence is shown in SEQ ID NO:3 from the 5' end to the 3' end, the P5 linker sequence, index sequence 1 and the front 21bp of the 5' end of the read 1 primer binding sequence are sequentially shown;
The nucleotide sequence is shown in SEQ ID NO:4, the P7-Blocker is sequentially a P7 joint sequence, an index sequence 2 and a read 2 primer binding sequence from the 5' end to the 3' end, wherein the front 21bp of the 5' end is shown in the formula; the 3' end of the P7-Blocker is marked with a blocking group (NH 2C6);
the nucleotide sequence is shown in SEQ ID NO:5, wherein the general primer R is 21bp before the 5' -end of the P7 joint sequence.
PCR amplification was performed according to the PCR amplification system 1 shown in Table 1, and the amplified product was collected to obtain a gene library 1 for quantitative detection of the methylation level of MGMT.
Wherein the amplification was performed according to the PCR amplification system 1 shown in Table 1, and the schematic amplification scheme is shown in FIG. 1.
TABLE 1PCR amplification System 1
| Composition of the components | Volume (mu L) |
| DNA Taq polymerase Mix (RR 390Q) | 10 |
| MGMT-specific primer F (SEQ ID NO: 1) at 1. Mu.M | 1 |
| MGMT-specific primer R (SEQ ID NO: 2) at 1. Mu.M | 1 |
| 10 Mu M P7-Blocker (SEQ ID NO: 4) | 0.2 |
| 10 Mu M universal primer R (SEQ ID NO: 5) | 1 |
| 10 Mu M P5 sequence (SEQ ID NO: 3) | 1 |
| DNA template (10 ng/. Mu.L) | 3 |
| H2O | To 20 |
The PCR amplification procedure was: 95 ℃ and 3mins;95 ℃,15s,60 ℃,1min,45 cycles; stored at 60℃with 2mins at 4 ℃.
Example 2A Gene library for quantitative determination of the methylation level of MGMT
Extracting genomic DNA (gDNA) of a fresh tumor tissue sample of a cancer patient by using a DNA extraction kit, and carrying out bisulphite conversion modification on 500ng of gDNA by using a bisulphite conversion kit (Novozan biotechnology Co., ltd., EM 102-01) to obtain a DNA sample after conversion modification; and taking a 25ng transformation modified DNA sample as a DNA template to construct a gene library.
The synthetic nucleotide sequence is shown as SEQ ID NO:1 (upstream primer of specific amplification MGMT gene), and the nucleotide sequence is shown in SEQ ID NO:2 (downstream primer of specificity amplification MGTM gene), and the nucleotide sequence of the MGMT specific primer R is shown as SEQ ID NO:6 (extension primer 1'), and the nucleotide sequence is shown as SEQ ID NO:7 (extension primer 2') and the nucleotide sequence of which is shown in SEQ ID NO:8, a universal primer F; specific sequence information is shown below.
P7 sequence (SEQ ID NO: 6):
P5-Blocker(SEQ ID NO：7)：
universal primer F (SEQ ID NO: 8): 5'-AATGATACGGCGACCACCGA-3'.
In the sequence shownIs a linker primer; Is an Index sequence; "_" is a truncated Read primer binding sequence;
The nucleotide sequence is shown in SEQ ID NO: the P7 sequence shown in 6 is a P7 joint sequence, an index sequence 2 and 21bp before the 5' end of a read 2 primer binding sequence from the 5' end to the 3' end in sequence;
The nucleotide sequence is shown in SEQ ID NO:7, the P5-Blocker is sequentially a P5 joint sequence, an index sequence 1 and a read 1 primer binding sequence from the 5' end to the 3' end, wherein the front 21bp of the 5' end of the primer binding sequence is shown in the formula I; the 3' end of the P5-Blocker is marked with a blocking group (NH 2C6);
The nucleotide sequence is shown in SEQ ID NO:8 is the front 21bp of the 5' end of the P5 linker sequence.
PCR amplification was performed according to the PCR amplification system 2 shown in Table 2, and the amplified product was collected to obtain a gene library 2 for quantitative detection of the methylation level of MGMT.
Wherein the amplification was performed according to the PCR amplification system 2 shown in Table 2, and the schematic amplification scheme is shown in FIG. 2.
TABLE 2 PCR amplification System 2
| Composition of the components | Volume (mu L) |
| DNA Taq polymerase Mix (RR 390Q) | 10 |
| MGMT-specific primer F (SEQ ID NO: 1) at 1. Mu.M | 1 |
| MGMT-specific primer R (SEQ ID NO: 2) at 1. Mu.M | 1 |
| 10 Mu M P5-Blocker (SEQ ID NO: 7) | 0.2 |
| 10 Mu M universal primer F (SEQ ID NO: 8) | 1 |
| 10 Mu M P7 sequence (SEQ ID NO: 6) | 1 |
| DNA template (10 ng/. Mu.L) | 3 |
| H2O | To 20 |
The PCR amplification procedure was: 95 ℃ and 3mins;95 ℃,15s,60 ℃,1min,45 cycles; stored at 60℃with 2mins at 4 ℃.
Example 3A Gene library for quantitative determination of the methylation level of MGMT
Extracting genomic DNA (gDNA) of a fresh tumor tissue sample of a cancer patient by using a DNA extraction kit, and carrying out bisulphite conversion modification on 500ng of gDNA by using a bisulphite conversion kit (Novozan biotechnology Co., ltd., EM 102-01) to obtain a DNA sample after conversion modification; and taking a 25ng transformation modified DNA sample as a DNA template to construct a gene library.
Using the nucleotide sequence shown in SEQ ID NO:1, wherein the nucleotide sequence of the MGMT specific primer F is shown as SEQ ID NO:2, wherein the nucleotide sequence of the MGMT specific primer R is shown as SEQ ID NO:3, and the nucleotide sequence of the P5 sequence is shown as SEQ ID NO:4 and the nucleotide sequence of the P7-Blocker is shown as SEQ ID NO:5, performing PCR amplification according to a PCR amplification system 3 shown in Table 3, and collecting amplification products to obtain a gene library 3 for quantitatively detecting the methylation level of MGMT.
TABLE 3 PCR amplification System 3
| Composition of the components | Volume (mu L) |
| DNA Taq polymerase Mix (RR 390Q) | 10 |
| MGMT-specific primer F (SEQ ID NO: 1) at 1. Mu.M | 1 |
| MGMT-specific primer R (SEQ ID NO: 2) at 1. Mu.M | 1 |
| 10 Mu M P7-Blocker (SEQ ID NO: 4) | 0.2 |
| 10 Mu M universal primer R (SEQ ID NO: 5) | 1 |
| 10 Mu M P5 sequence (SEQ ID NO: 3) | 0.5 |
| DNA template (10 ng/. Mu.L) | 3 |
| H2O | To 20 |
The PCR amplification procedure was: 95 ℃ and 3mins;95 ℃,15s,60 ℃,1min,45 cycles; stored at 60℃with 2mins at 4 ℃.
Comparative example 1A Gene library for quantitative determination of the methylation level of MGMT
Extracting genomic DNA (gDNA) of a fresh tumor tissue sample of a cancer patient by using a DNA extraction kit, and carrying out bisulphite conversion modification on 500ng of gDNA by using a bisulphite conversion kit (Novozan biotechnology Co., ltd., EM 102-01) to obtain a DNA sample after conversion modification; and taking a 25ng transformation modified DNA sample as a DNA template to construct a gene library.
Using the nucleotide sequence shown in SEQ ID NO:1, wherein the nucleotide sequence of the MGMT specific primer F is shown as SEQ ID NO:2, wherein the nucleotide sequence of the MGMT specific primer R is shown as SEQ ID NO:3, and the nucleotide sequence of the P5 sequence is shown as SEQ ID NO:4 and the nucleotide sequence of the P7-Blocker is shown as SEQ ID NO:5, performing PCR amplification according to a PCR amplification system 4 shown in Table 4, and collecting amplification products to obtain a gene library 4 for quantitatively detecting the methylation level of MGMT.
TABLE 4 PCR amplification System 4
| Composition of the components | Volume (mu L) |
| DNA Taq polymerase Mix (RR 390Q) | 10 |
| MGMT-specific primer F (SEQ ID NO: 1) at 1. Mu.M | 1 |
| MGMT-specific primer R (SEQ ID NO: 2) at 1. Mu.M | 1 |
| 10 Mu M P7-Blocker (SEQ ID NO: 4) | 0.6 |
| 10 Mu M universal primer R (SEQ ID NO: 5) | 1 |
| 10 Mu M P5 sequence (SEQ ID NO: 3) | 0.5 |
| DNA template (10 ng/. Mu.L) | 3 |
| H2O | To 20 |
The PCR amplification procedure was: 95 ℃ and 3mins;95 ℃,15s,60 ℃,1min,45 cycles; stored at 60℃with 2mins at 4 ℃.
Comparative example 2A Gene library for quantitative determination of the methylation level of MGMT
Extracting genomic DNA (gDNA) of a fresh tumor tissue sample of a cancer patient by using a DNA extraction kit, and carrying out bisulphite conversion modification on 500ng of gDNA by using a bisulphite conversion kit (Novozan biotechnology Co., ltd., EM 102-01) to obtain a DNA sample after conversion modification; and taking a 25ng transformation modified DNA sample as a DNA template to construct a gene library.
Using the nucleotide sequence shown in SEQ ID NO:1, wherein the nucleotide sequence of the MGMT specific primer F is shown as SEQ ID NO:2, wherein the nucleotide sequence of the MGMT specific primer R is shown as SEQ ID NO:3, and the nucleotide sequence of the P5 sequence is shown as SEQ ID NO:4 and the nucleotide sequence of the P7-Blocker is shown as SEQ ID NO:5, performing PCR amplification according to a PCR amplification system 5 shown in Table 5, and collecting amplification products to obtain a gene library 5 for quantitatively detecting the methylation level of MGMT.
TABLE 5 PCR amplification System 5
The PCR amplification procedure was: 95 ℃ and 3mins;95 ℃,15s,60 ℃,1min,45 cycles; stored at 60℃with 2mins at 4 ℃.
Comparative example 3A Gene library for quantitative determination of the methylation level of MGMT
Extracting genomic DNA (gDNA) of a fresh tumor tissue sample of a cancer patient by using a DNA extraction kit, and carrying out bisulphite conversion modification on 500ng of gDNA by using a bisulphite conversion kit (Novozan biotechnology Co., ltd., EM 102-01) to obtain a DNA sample after conversion modification; and taking a 25ng transformation modified DNA sample as a DNA template to construct a gene library.
Using the nucleotide sequence shown in SEQ ID NO:1, wherein the nucleotide sequence of the MGMT specific primer F is shown as SEQ ID NO:2, wherein the nucleotide sequence of the MGMT specific primer R is shown as SEQ ID NO:3, and the nucleotide sequence of the P5 sequence is shown as SEQ ID NO:4 and the nucleotide sequence of the P7-Blocker is shown as SEQ ID NO:5, performing PCR amplification according to a PCR amplification system 6 shown in Table 6, and collecting amplification products to obtain a gene library 6 for quantitatively detecting the methylation level of MGMT.
TABLE 6 PCR amplification System 6
| Composition of the components | Volume (mu L) |
| DNA Taq polymerase Mix (RR 390Q) | 10 |
| MGMT-specific primer F (SEQ ID NO: 1) at 1. Mu.M | 1 |
| MGMT-specific primer R (SEQ ID NO: 2) at 1. Mu.M | 1 |
| 10 Mu M P7-Blocker (SEQ ID NO: 4) | 1 |
| 10 Mu M universal primer R (SEQ ID NO: 5) | 1 |
| 10 Mu M P5 sequence (SEQ ID NO: 3) | 0.5 |
| DNA template (10 ng/. Mu.L) | 3 |
| H2O | To 20 |
The PCR amplification procedure was: 95 ℃ and 3mins;95 ℃,15s,60 ℃,1min,45 cycles; stored at 60℃with 2mins at 4 ℃.
Comparative example 4A Gene library for quantitative determination of the methylation level of MGMT
Extracting genomic DNA (gDNA) of a fresh tumor tissue sample of a cancer patient by using a DNA extraction kit, and carrying out bisulphite conversion modification on 500ng of gDNA by using a bisulphite conversion kit (Novozan biotechnology Co., ltd., EM 102-01) to obtain a DNA sample after conversion modification; and taking a 25ng transformation modified DNA sample as a DNA template to construct a gene library.
Using the nucleotide sequence shown in SEQ ID NO:1, wherein the nucleotide sequence of the MGMT specific primer F is shown as SEQ ID NO:2, wherein the nucleotide sequence of the MGMT specific primer R is shown as SEQ ID NO:3 and the nucleotide sequence of the P5 is shown as SEQ ID NO:4, performing PCR amplification according to a PCR amplification system 7 shown in Table 7, and collecting amplification products to obtain a gene library 7 for quantitatively detecting the methylation level of MGMT.
TABLE 7 PCR amplification System 7
| Composition of the components | Volume (mu L) |
| DNA Taq polymerase Mix (RR 390Q) | 10 |
| MGMT-specific primer F (SEQ ID NO: 1) at 1. Mu.M | 1 |
| MGMT-specific primer R (SEQ ID NO: 2) at 1. Mu.M | 1 |
| 10 Mu M P7-Blocker (SEQ ID NO: 4) | 0.6 |
| 10 Mu M P5 sequence (SEQ ID NO: 3) | 0.5 |
| DNA template (10 ng/. Mu.L) | 3 |
| H2O | To 20 |
The PCR amplification procedure was: 95 ℃ and 3mins;95 ℃,15s,60 ℃,1min,45 cycles; stored at 60℃with 2mins at 4 ℃.
Comparative example 5A Gene library for quantitative determination of the methylation level of MGMT
Extracting genomic DNA (gDNA) of a fresh tumor tissue sample of a cancer patient by using a DNA extraction kit, and carrying out bisulphite conversion modification on 500ng of gDNA by using a bisulphite conversion kit (Novozan biotechnology Co., ltd., EM 102-01) to obtain a DNA sample after conversion modification; and taking a 25ng transformation modified DNA sample as a DNA template to construct a gene library.
Using the nucleotide sequence shown in SEQ ID NO:1, wherein the nucleotide sequence of the MGMT specific primer F is shown as SEQ ID NO:2, wherein the nucleotide sequence of the MGMT specific primer R is shown as SEQ ID NO:4, the nucleotide sequence of the P7-Blocker is shown as SEQ ID NO:5, the general primer R and the nucleotide sequence are shown as SEQ ID NO:7, and the nucleotide sequence of the P5-Blocker is shown as SEQ ID NO:8, performing PCR amplification according to a PCR amplification system 8 shown in Table 8, and collecting amplification products to obtain a gene library 8 for quantitatively detecting the methylation level of MGMT.
TABLE 8 PCR amplification System 8
| Composition of the components | Volume (mu L) |
| DNA Taq polymerase Mix (RR 390Q) | 10 |
| MGMT-specific primer F (SEQ ID NO: 1) at 1. Mu.M | 1 |
| MGMT-specific primer R (SEQ ID NO: 2) at 1. Mu.M | 1 |
| 10 Mu M P7-Blocker (SEQ ID NO: 4) | 0.2 |
| 10 Mu M P5-Blocker (SEQ ID NO: 7) | 0.2 |
| 10 Mu M universal primer R (SEQ ID NO: 5) | 1 |
| 10 Mu M universal primer F (SEQ ID NO: 8) | 1 |
| DNA template (10 ng/. Mu.L) | 3 |
| H2O | To 20 |
The PCR amplification procedure was: 95 ℃ and 3mins;95 ℃,15s,60 ℃,1min,45 cycles; stored at 60℃with 2mins at 4 ℃.
Comparative example 6A Gene library for quantitative determination of the methylation level of MGMT
Extracting genomic DNA (gDNA) of a fresh cancer tumor tissue sample by using a DNA extraction kit, and carrying out bisulphite conversion modification on 500ng of gDNA by using a bisulphite conversion kit (Novain Biotech Co., ltd., EM 102-01) to obtain a DNA sample after conversion modification; and taking a 25ng transformation modified DNA sample as a DNA template to construct a gene library.
Using the nucleotide sequence shown in SEQ ID NO:1, wherein the nucleotide sequence of the MGMT specific primer F is shown as SEQ ID NO:2, wherein the nucleotide sequence of the MGMT specific primer R is shown as SEQ ID NO:3 and the nucleotide sequence of the P5 is shown as SEQ ID NO:6, performing PCR amplification according to a PCR amplification system 9 shown in Table 9, and collecting amplification products to obtain a gene library 9 for quantitatively detecting the methylation level of MGMT.
When amplification was performed in accordance with the PCR amplification system 9 shown in Table 9, the amplification scheme is shown in FIG. 3.
TABLE 9 PCR amplification System 9
The PCR amplification procedure was: 95 ℃ and 3mins;95 ℃,15s,55 ℃,30s,72 ℃,30s,6 cycles; 95 ℃,15s,60 ℃,1min,31 cycles; stored at 72℃and 2mins at 4 ℃.
Test example 1 gel electrophoresis detection of Gene library for quantitative detection of MGMT methylation level
1. Experimental method
Gel electrophoresis was performed on the gene libraries 1 to 3 constructed in examples 1 to 3 and the gene libraries 4 to 9 constructed in comparative examples 1 to 6, respectively, and the specific procedures were as follows:
Preparing 3% (w/w) agarose gel, mixing 10 mu L of gene library 1-9 and 1 mu L of 10× Loading Buffer after the agarose gel is solidified, spotting on the solidified agarose gel, running the gel for 50mins under 120V voltage, and observing electrophoresis bands under a gel imager after the running of the gel is finished.
2. Experimental results
The gel electrophoresis detection results of the gene libraries 1 to 9 are shown in FIG. 4, and the results show that: (1) The clear target bands appear in the gel electrophoresis detection results of the gene libraries 1-3 constructed in the examples 1-3, other impurity bands are not seen at the same time, and the target bands of the example 1 are clearer compared with the example 3, which shows that the amplified gene library contains more target sequences when the concentration of the P5 sequence in an amplification system is 500 nM; (2) The gel electrophoresis detection results of the gene libraries 4-7 constructed in comparative examples 1-4 show NO obvious target band, and obvious impurity bands are generated, which indicates that the nucleotide sequences are shown as SEQ ID NO:4 can influence the specificity of the finally amplified gene library, wherein the concentration of the P7-Blocker in the amplification system is 200nM, 300nM and 400nM, and obvious impurity bands are generated in the amplified gene library; (3) The gel electrophoresis detection result of the gene library 8 constructed in the comparative example 5 shows that a distinct band appears at a position of 100bp, and a target band region does not appear, which indicates that the target gene region cannot be amplified by extension only using the P5-Blocker and the P7-Blocker.
Therefore, the PCR amplification system shown in examples 1 to 3 can specifically amplify the MGMT gene region, and the constructed gene library only contains the MGMT gene, so that the library construction time is shortened, and the sequencing data volume in the follow-up quantitative detection is reduced.
Test example 2 quantitative determination of the degree of methylation of MGMT Using Gene library
1. Experimental method
(1) Acquisition of DNA templates for construction of Gene libraries
Taking tumor tissues of 12 brain glioma patients with known MGMT methylation degrees as detection samples (namely, samples 1 to 12, wherein the MGMT methylation degrees are all obtained through pyrosequencing detection), respectively extracting genome DNA (gDNA) of each detection sample by using a DNA extraction kit, and carrying out bisulfite conversion modification on 500ng of gDNA by using a bisulfite conversion kit (Novone Biotechnology Co., ltd., EM 102-01) to obtain a DNA sample after conversion modification; taking 25ng of the DNA sample after transformation modification as a DNA template of each detection sample to construct a gene library;
the 12 brain glioma patients are all from Shenzhen Heplosis medical test laboratory, and the patients are authorized before MGMT methylation detection by using tumor tissues from the 12 brain glioma patients.
(2) Gene library construction and electrophoresis detection of experimental group and control group
The experimental group was set as follows: according to the construction method of the gene library shown in example 1, the gene libraries of samples 1 to 12 were constructed, respectively, while the non-methylated standard (Zymo Research, D5014) and the methylated standard (Zymo Research, D5014) were used as templates, and the gene library of the non-methylated standard and the gene library of the methylated standard were constructed, respectively.
10. Mu.L of the gene library of samples 1 to 12 constructed by the construction method of the gene library shown in example 1, the gene library of the unmethylated standard and the gene library of the methylated standard were purified by the magnetic bead method, respectively, to obtain a sequencing library of samples 1 to 12, a sequencing library of the unmethylated standard and a sequencing library of the methylated standard of the experimental group.
The sequencing library of samples 1 to 12 of the experimental group, the sequencing library of the unmethylated standard, and the sequencing library of the methylated standard were electrophoresed using an align 4200 bioanalyzer, and the sequencing library of sample 1 of the experimental group was library-band analyzed using an align 4200 bioanalyzer.
The control group was set as follows: according to the construction method of the gene library shown in comparative example 6, the gene libraries of samples 1 to 12 were respectively constructed, and simultaneously, the gene library of the unmethylated standard and the gene library of the methylated standard were respectively constructed by using the unmethylated standard (Zymo Research, D5014) and the methylated standard (Zymo Research, D5014) as templates;
The 10. Mu.L gene library of sample 1 to sample 12, the gene library of the unmethylated standard and the gene library of the methylated standard constructed by the construction method of the gene library shown in comparative example 6 were purified by a magnetic bead method, respectively, to obtain a sequencing library of sample 1 to sample 12, a sequencing library of the unmethylated standard and a sequencing library of the methylated standard of a control group.
The sequencing library of samples 1 to 12 of the control group, the sequencing library of the unmethylated standard, and the sequencing library of the methylated standard were electrophoresed using an align 4200 bioanalyzer, and the sequencing library of sample 1 of the control group was subjected to library banding analysis using an align 4200 bioanalyzer.
(3) Quantitative determination of the degree of methylation of MGMT in the experimental group
The MGMT methylation quantitative detection was performed using the sequencing library of samples 1 to 12 of the experimental group, the sequencing library of the unmethylated standard, and the sequencing library of the methylated standard, and specifically the steps are as follows:
the sequencing library of samples 1 to 12 of the experimental group, the sequencing library of the non-methylated standard and the sequencing library of the methylated standard were subjected to MGMT gene methylation sequencing using Novaseq sequencer (Illumina company) and MGMT methylation analysis based on sequencing lower machine data, respectively.
2. Experimental results
The electrophoresis detection result diagram of the sequencing library constructed in the experimental group is shown in FIG. 5, and the library strip size diagram distribution of the sequencing library of the sample 1 in the experimental group is shown in FIG. 6; the result of electrophoresis detection of the sequencing library constructed in the control group is shown in FIG. 7, and the library band size distribution diagram of the sequencing library of sample 1 in the control group is shown in FIG. 8.
Compared with the electrophoresis detection results of each sample sequencing library in the experimental group, the electrophoresis detection results of each sample sequencing library in the control group can detect target bands, but a plurality of bands with similar sizes exist; the library band size distribution of the sequencing library of sample 1 in the control group shows (FIG. 8) that there are many closely sized bands (187 bp, 232bp, and 344 bp) around the target library band (276 bp); the library band size distribution of the sequencing library of sample 1 in the experimental group showed (FIG. 6), the library product was more concentrated and no other bands were found around the target library band (281 bp).
The quantitative detection results of MGMT methylation of samples 1 to 12 in the experimental group are shown in table 10.
TABLE 10 quantitative determination of MGMT methylation
With 8% as the cut-off value, the agreement between the quantitative detection results of MGMT of samples 1 to 12 in the experimental group and the known degree of methylation of MGMT (pyrosequencing result) of each sample was 100%.
The results illustrate: according to the construction method of the gene library shown in example 1, the gene library is constructed on the sample to detect the methylation degree of MGMT, and the constructed gene library does not contain amplification products of non-target regions, and can accurately detect the methylation degree of MGMT of the sample.
Example 4A Gene library for quantitative detection of TWIST1 methylation level
Extracting genomic DNA (gDNA) of a fresh tumor tissue sample of a cancer patient by using a DNA extraction kit, and carrying out bisulphite conversion modification on 500ng of gDNA by using a bisulphite conversion kit (Novozan biotechnology Co., ltd., EM 102-01) to obtain a DNA sample after conversion modification; and taking a 25ng transformation modified DNA sample as a DNA template to construct a gene library.
The synthetic nucleotide sequence is shown as SEQ ID NO:9 (upstream primer for specifically amplifying TWIST1 gene) and a nucleotide sequence shown in SEQ ID NO: TWIST 1-specific primer R shown in FIG. 10 (specific downstream primers for sexually amplified TWIST1 gene).
TWIST1 specific primer F (SEQ ID NO: 9):
TWIST1 specific primer R (SEQ ID NO: 10):
In the sequence shown Is a Read primer binding sequence; "_" is a specific sequence targeting TWIST 1.
Wherein the nucleotide sequence is shown in SEQ ID NO: the TWIST1 specific primer F shown in 9 is a read 1 primer binding sequence and a specific primer sequence 3 of 33bp from the 5 'end to the 3' end in sequence; the nucleotide sequence is shown in SEQ ID NO:2, the TWIST1 specific primer R is a read 2 primer binding sequence and a specific primer sequence 4 which are 34bp in sequence from a 5 'end to a 3' end; the specific primer sequence 3 and the specific primer sequence 4 target the upstream region and the downstream region of the TWIST1 gene respectively, and can specifically amplify the TWIST1 gene.
Using the nucleotide sequence shown in SEQ ID NO:9, the TWIST1 specific primer F and the nucleotide sequence are shown as SEQ ID NO:10, wherein the nucleotide sequence of the TWIST1 specific primer R is shown as SEQ ID NO:3, and the nucleotide sequence of the P5 sequence is shown as SEQ ID NO:4 and the nucleotide sequence of the P7-Blocker is shown as SEQ ID NO:5, performing PCR amplification according to a PCR amplification system 11 shown in Table 11, and collecting amplification products to obtain a gene library 11 for quantitatively detecting the methylation level of TWIST 1.
TABLE 11 PCR amplification System 11
| Composition of the components | Volume (mu L) |
| DNA Taq polymerase Mix (RR 390Q) | 10 |
| TWIST 1-specific primer F (SEQ ID NO: 9) at 1. Mu.M | 1 |
| TWIST 1-specific primer R (SEQ ID NO: 10) at 1. Mu.M | 1 |
| 10 Mu M P7-Blocker (SEQ ID NO: 4) | 0.2 |
| 10 Mu M universal primer R (SEQ ID NO: 5) | 1 |
| 10 Mu M P5 sequence (SEQ ID NO: 3) | 1 |
| DNA template (10 ng/. Mu.L) | 3 |
| H2O | To 20 |
The PCR amplification procedure was: 95 ℃ and 3mins;95 ℃,15s,60 ℃,1min,45 cycles; stored at 60℃with 2mins at 4 ℃.
Example 5A Gene library for quantitative detection of TWIST1 methylation level
Extracting genomic DNA (gDNA) of a fresh tumor tissue sample of a cancer patient by using a DNA extraction kit, and carrying out bisulphite conversion modification on 500ng of gDNA by using a bisulphite conversion kit (Novozan biotechnology Co., ltd., EM 102-01) to obtain a DNA sample after conversion modification; and taking a 25ng transformation modified DNA sample as a DNA template to construct a gene library.
Using the nucleotide sequence shown in SEQ ID NO:9, the TWIST1 specific primer F and the nucleotide sequence are shown as SEQ ID NO:10, wherein the nucleotide sequence of the TWIST1 specific primer R is shown as SEQ ID NO:6, and the nucleotide sequence of the P7 sequence is shown as SEQ ID NO:7 and the nucleotide sequence of the P5-Blocker is shown as SEQ ID NO:8, and carrying out PCR amplification according to a PCR amplification system 12 shown in Table 12, and collecting amplification products to obtain a gene library 12 for quantitatively detecting the methylation level of TWIST 1.
TABLE 12 PCR amplification System 12
The PCR amplification procedure was: 95 ℃ and 3mins;95 ℃,15s,60 ℃,1min,45 cycles; stored at 60℃with 2mins at 4 ℃.
Test example 3 gel electrophoresis detection of Gene library for quantitative detection of TWIST1 methylation level
1. Experimental method
The gene library 11 constructed in example 4 and the gene library 12 constructed in example 5 were subjected to gel electrophoresis detection, respectively, as shown in step 1 of test example 1.
2. Experimental results
The gene library 11 constructed in example 4 and the gene library 12 constructed in example 5 both showed clear bands of interest (TWIST 1 gene bands) in the gel electrophoresis test results, and no other bands were seen; it was demonstrated that the PCR amplification systems shown in examples 4 and 5 can specifically amplify the TWIST1 gene region, and the constructed gene library only contains the TWIST1 gene, thereby shortening the library construction time and reducing the sequencing data amount in the subsequent quantitative detection.
Finally, it should be noted that the above embodiments are merely for illustrating the technical solution of the present invention and not for limiting the scope of the present invention, and that other various changes and modifications can be made by one skilled in the art based on the above description and the idea, and it is not necessary or exhaustive of all the embodiments. Any modification, equivalent replacement, improvement, etc. which come within the spirit and principles of the invention are desired to be protected by the following claims.
Claims (5)
1. The detection system for detecting the methylation degree of the target gene based on high-throughput sequencing is characterized by comprising an upstream primer, a downstream primer, an extension primer 1, an extension primer 2 and a universal primer;
The target gene is MGMT gene, and the primer composition contains nucleotide sequences shown in SEQ ID NO:1, and the nucleotide sequence of the upstream primer is shown as SEQ ID NO:2, and the nucleotide sequence of the downstream primer is shown as SEQ ID NO:3, the nucleotide sequence of the extension primer 1 is shown as SEQ ID NO:4 and the nucleotide sequence of the extension primer 2 is shown as SEQ ID NO: 5;
or the primer composition contains a nucleotide sequence shown as SEQ ID NO:1, and the nucleotide sequence of the upstream primer is shown as SEQ ID NO:2, and the nucleotide sequence of the downstream primer is shown as SEQ ID NO:6, the nucleotide sequence of the extension primer 1 is shown as SEQ ID NO:7 and the nucleotide sequence of the extension primer 2 is shown as SEQ ID NO: 8;
The concentration of the extension primer 1 in the detection system is 500 nM, the concentration of the extension primer 2 in the detection system is 100 nM, and the concentration of the universal primer in the detection system is 500 nM.
2. The detection system of claim 1, wherein the concentration of the upstream primer in the primer composition in the detection system is 200 nM to 750nM; the concentration of the downstream primer in the primer composition in the detection system is 200-750 nM.
3. The detection system for detecting the methylation degree of the target gene based on high-throughput sequencing is characterized by comprising an upstream primer, a downstream primer, an extension primer 1, an extension primer 2 and a universal primer;
The target gene is TWIST1 gene, and the primer composition contains a nucleotide sequence shown in SEQ ID NO:9, and the nucleotide sequence of the upstream primer is shown as SEQ ID NO:10, and the nucleotide sequence of the downstream primer is shown as SEQ ID NO:3, the nucleotide sequence of the extension primer 1 is shown as SEQ ID NO:4 and the nucleotide sequence of the extension primer 2 is shown as SEQ ID NO: 5;
Or the primer composition contains a nucleotide sequence shown as SEQ ID NO:9, and the nucleotide sequence of the upstream primer is shown as SEQ ID NO:10, and the nucleotide sequence of the downstream primer is shown as SEQ ID NO:6, the nucleotide sequence of the extension primer 1 is shown as SEQ ID NO:7 and the nucleotide sequence of the extension primer 2 is shown as SEQ ID NO: 8;
The concentration of the extension primer 1 in the detection system is 500 nM, the concentration of the extension primer 2 in the detection system is 100 nM, and the concentration of the universal primer in the detection system is 500 nM.
4. The detection system of claim 3, wherein the concentration of the upstream primer in the primer composition in the detection system is 200 nM to 750nM; the concentration of the downstream primer in the primer composition in the detection system is 200-750 nM.
5. The construction method of the gene library for detecting the methylation degree of the target gene based on high-throughput sequencing is characterized by comprising the following steps:
s1, extracting DNA of a sample to be detected, and performing bisulfite conversion to obtain a DNA template;
s2, carrying out PCR amplification on the DNA template obtained in the step S1 by using the detection system shown in any one of claim 1 and/or claim 3, and collecting an amplification product.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN202311862466.8A CN117778578B (en) | 2023-12-29 | 2023-12-29 | A primer combination for detecting the methylation degree of a target gene based on high-throughput sequencing and its application |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN202311862466.8A CN117778578B (en) | 2023-12-29 | 2023-12-29 | A primer combination for detecting the methylation degree of a target gene based on high-throughput sequencing and its application |
Publications (2)
| Publication Number | Publication Date |
|---|---|
| CN117778578A CN117778578A (en) | 2024-03-29 |
| CN117778578B true CN117778578B (en) | 2024-10-15 |
Family
ID=90400193
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN202311862466.8A Active CN117778578B (en) | 2023-12-29 | 2023-12-29 | A primer combination for detecting the methylation degree of a target gene based on high-throughput sequencing and its application |
Country Status (1)
| Country | Link |
|---|---|
| CN (1) | CN117778578B (en) |
Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN106497926A (en) * | 2016-11-03 | 2017-03-15 | 承启医学（深圳）科技有限公司 | A kind of amplicon primer and construction method for building microbial bacterial 16s rDNA variable regions sequencing library |
| CN107208019A (en) * | 2014-11-11 | 2017-09-26 | 伊鲁米纳剑桥有限公司 | Method and array for generation and the sequencing of nucleic acid monoclonal cluster |
Family Cites Families (5)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| WO2016181128A1 (en) * | 2015-05-11 | 2016-11-17 | Genefirst Ltd | Methods, compositions, and kits for preparing sequencing library |
| DK3408408T3 (en) * | 2016-01-27 | 2019-07-22 | Devyser Holding Ab | SELECTIVE AMPLIFICATION OF DESIRED NUCLEIC ACID AREAS IN A TARGET SEQUENCE |
| WO2019062614A1 (en) * | 2017-09-26 | 2019-04-04 | Phocus Technology ( Shanghai) Co. Ltd | A method of amplifying a target nucleic acid |
| WO2020232635A1 (en) * | 2019-05-21 | 2020-11-26 | 深圳华大智造科技有限公司 | Method and system for constructing sequencing library on the basis of methylated dna target region, and use thereof |
| CN111705135B (en) * | 2020-08-19 | 2020-11-24 | 至本医疗科技（上海）有限公司 | A method for detecting methylation of MGMT promoter region |
-
2023
- 2023-12-29 CN CN202311862466.8A patent/CN117778578B/en active Active
Patent Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN107208019A (en) * | 2014-11-11 | 2017-09-26 | 伊鲁米纳剑桥有限公司 | Method and array for generation and the sequencing of nucleic acid monoclonal cluster |
| CN106497926A (en) * | 2016-11-03 | 2017-03-15 | 承启医学（深圳）科技有限公司 | A kind of amplicon primer and construction method for building microbial bacterial 16s rDNA variable regions sequencing library |
Also Published As
| Publication number | Publication date |
|---|---|
| CN117778578A (en) | 2024-03-29 |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| EP3481403B1 (en) | Solid tumor methylation markers and uses thereof | |
| CN109952381B (en) | Method for multiplex detection of methylated DNA | |
| CN110982907B (en) | Thyroid nodule-related rDNA methylation marker and application thereof | |
| EP3481952A1 (en) | Leukemia methylation markers and uses thereof | |
| WO2023193357A1 (en) | Method for constructing free dna methylation sequencing library and use thereof | |
| CN108315424A (en) | PCR specific primers, detection kit and the detection method of Benign Thyroid Nodules tumor- associated gene | |
| EP2814987A1 (en) | A system and method of genomic profiling | |
| WO2024056008A1 (en) | Methylation marker for identifying cancer and use thereof | |
| CN117778578B (en) | A primer combination for detecting the methylation degree of a target gene based on high-throughput sequencing and its application | |
| KR101735075B1 (en) | Composition and method for prediction of swine fecundity using genomic differentially methylated region | |
| US20210155972A1 (en) | Targeted rare allele crispr enrichment | |
| CN108315425A (en) | PCR specific primers, kit and its application method of metastasis of thyroid carcinoma related gene detection | |
| CN113493835A (en) | Method and kit for screening large intestine tumor by detecting methylation state of BCAN gene region | |
| KR101683086B1 (en) | Prediction method for swine fecundity using gene expression level and methylation profile | |
| CN116676393A (en) | Methylation markers, primer pairs and methods for early screening of cervical cancer | |
| CN117778568A (en) | Marker for identifying gastric cancer and application thereof | |
| CN102533992B (en) | Method and kit for sequencing phenylalanine hydroxylase gene | |
| US20220127601A1 (en) | Method of determining the origin of nucleic acids in a mixed sample | |
| KR102799841B1 (en) | Methylation marker genes for ovarian cancer diagnosis and use thereof | |
| CN118531103A (en) | A method and detection kit for detecting SDC2 gene promoter methylation based on nanopore targeted sequencing technology | |
| CN116656823A (en) | Kit for detecting marker and identifying histological subtype of non-small cell lung cancer | |
| WO2025078657A1 (en) | Amplification-free target enrichment workflow for direct detection of nucleic acid modifications | |
| CN118531102A (en) | Method and detection kit for detecting septin9 gene promoter methylation based on nano Kong Ba-way sequencing technology | |
| CN118547068A (en) | Method and detection kit for detecting MLH1 gene promoter methylation based on nano Kong Ba-direction sequencing technology | |
| CN118389510A (en) | A method and detection kit for detecting SHOX2 gene promoter methylation based on nanopore targeted sequencing technology |
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 | ||
| GR01 | Patent grant | ||
| GR01 | Patent grant |