Looking for breakthrough ideas for innovation challenges? Try Patsnap Eureka!

Method for detecting gene methylation in glioma

A technology of glioma and methylation, applied in the direction of biochemical equipment and methods, microbial measurement/testing, etc., can solve the problems of insufficient sensitivity, low cost, high cost, etc.

Active Publication Date: 2021-08-20
INST OF CHEM CHINESE ACAD OF SCI +1
View PDF5 Cites 0 Cited by
  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0003] The purpose of the present invention is to provide a method for detecting gene methylation in glioma in order to overcome the problems of insufficient sensitivity, high cost, and dependence on professional equipment and personnel in the detection of glioma gene methylation in the prior art , this method uses cationic conjugated polymer (CCP)-based fluorescence energy resonance transfer (FERT) technology to establish quantitative standard curves for the methylation levels of glioma key genes MGMT, CDKN2A / P16, and TERT promoters to achieve high sensitivity , Low-cost detection of the degree of methylation of glioma genes

Method used

the structure of the environmentally friendly knitted fabric provided by the present invention; figure 2 Flow chart of the yarn wrapping machine for environmentally friendly knitted fabrics and storage devices; image 3 Is the parameter map of the yarn covering machine
View more

Image

Smart Image Click on the blue labels to locate them in the text.
Viewing Examples
Smart Image
  • Method for detecting gene methylation in glioma
  • Method for detecting gene methylation in glioma
  • Method for detecting gene methylation in glioma

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0059] 1. Collection of specimens, DNA extraction and preparation

[0060] ①Sample collection and storage: After the glioma sample was removed, it was immediately stored in pieces in a -80°C refrigerator for later use.

[0061] ② DNA extraction

[0062] Take 25 mg of specimen tissue, cut it into pieces and process it into a cell suspension, put it into a 1.5 ml centrifuge tube; add 180 μl of ATL buffer, shake until completely suspended.

[0063] Add 20 μl proteinase K solution with a concentration of 20mg / μl, mix well, incubate at 56°C until the tissue is completely dissolved, add 4 μl RNase A, and mix well.

[0064] Add 200 μl of AL buffer and alcohol (about 98%), and mix well.

[0065] Add all the solution obtained in the previous step to the DNease adsorption column, add 500 μl AW1 buffer, centrifuge at 12000 rpm for 1 min, discard the waste liquid, and put the adsorption column back into the collection tube.

[0066] Add 500 μl AW2 buffer solution to the DNease adsorpti...

Embodiment 2

[0106] According to the method of Example 1, the difference is that the weight ratio of the PCR product to the cationic conjugated polymer in step (3) is 1:7.68. And record the lowest DNA concentration and the lowest methylation level that it can detect.

[0107] The results are shown in Table 7.

[0108] The result of table 7 embodiment 1

[0109]

[0110]

[0111] *The TERT methylation level of the sample cannot be detected within the detection range of this method, so it is expressed as 0%.

the structure of the environmentally friendly knitted fabric provided by the present invention; figure 2 Flow chart of the yarn wrapping machine for environmentally friendly knitted fabrics and storage devices; image 3 Is the parameter map of the yarn covering machine
Login to View More

PUM

No PUM Login to View More

Abstract

The invention relates to the field of gene detection, and discloses a method for detecting gene methylation in glioma. The method comprises the following steps: (1) using a restriction enzyme that specifically recognizes and cuts off unmethylated sites processing the DNA standard; (2) amplifying the enzyme cleavage product by PCR; (3) combining the PCR product with a cationic conjugated polymer to cause fluorescence energy resonance transfer of the PCR product, and detecting the cationic conjugated polymer separately and fluorescence intensity of fluorescein; (4) calculate the E value of gene methylation level in glioma according to the fluorescence intensity; (5) establish a quantitative standardization curve of methylation degree, determine the optimal detection conditions, and determine the optimal detection conditions. The glioma DNA sample to be detected is detected under the detection conditions; (6) the methylation degree of the glioma DNA sample is determined, and a combined analysis is performed to evaluate the methylation phenotype of the glioma CpG island. This method can achieve the purpose of detecting the methylation degree of glioma genes with high sensitivity and low cost.

Description

technical field [0001] The invention relates to the field of gene detection, in particular to a method for detecting gene methylation in glioma. Background technique [0002] Promoter methylation is the most important and common mechanism for transcriptional silencing of tumor suppressor genes, and is a key factor in tumor formation and progression. Integrating the methylation levels of multiple key glioma genes MGMT, CDKN2A / P16, and TERT promoter can assist in the diagnosis of glioma and the prognosis of patients. At present, the methods for detecting glioma promoter methylation mainly include methylation-specific PCR (MSP), MethyLight, high-resolution melting curve (HRM), combined with sodium bisulfite treatment and enzymatic analysis (COBRA). , Pyrosequencing, etc., most of them require bisulfite treatment, the process is cumbersome, the sensitivity is low, and the cost is high. Methylation-specific PCR (MSP) is a qualitative detection method that can only determine the...

Claims

the structure of the environmentally friendly knitted fabric provided by the present invention; figure 2 Flow chart of the yarn wrapping machine for environmentally friendly knitted fabrics and storage devices; image 3 Is the parameter map of the yarn covering machine
Login to View More

Application Information

Patent Timeline
no application Login to View More
Patent Type & Authority Patents(China)
IPC IPC(8): C12Q1/6858
CPCC12Q1/6858C12Q2521/301C12Q2531/113C12Q2565/101C12Q2563/107C12Q2545/113
Inventor 王树马立新刘礼兵于春江张宏伟
Owner INST OF CHEM CHINESE ACAD OF SCI
Who we serve
  • R&D Engineer
  • R&D Manager
  • IP Professional
Why Patsnap Eureka
  • Industry Leading Data Capabilities
  • Powerful AI technology
  • Patent DNA Extraction
Social media
Patsnap Eureka Blog
Learn More
PatSnap group products

Browse by: Latest US Patents, China's latest patents, Technical Efficacy Thesaurus, Application Domain, Technology Topic, Popular Technical Reports.

© 2024 PatSnap. All rights reserved.Legal|Privacy policy|Modern Slavery Act Transparency Statement|Sitemap|About US| Contact US: help@patsnap.com