Method for detecting methylation of brain glioma MGMT promoter gene

A technology for glioma and detection methods, applied in biochemical equipment and methods, microbe measurement/inspection, etc., can solve problems such as inconvenient operation and poor detection accuracy, and achieve convenient operation, good sensitivity, and high detection accuracy Effect

Inactive Publication Date: 2017-09-22
安徽安龙基因科技有限公司
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  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

[0006] The technical problem to be solved by the present invention is to provide a method for detecting the methylation of the MGMT promoter gene in glioma in order to overcome the defects of poor detection accuracy and inconvenient operation in the prior art

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  • Method for detecting methylation of brain glioma MGMT promoter gene
  • Method for detecting methylation of brain glioma MGMT promoter gene
  • Method for detecting methylation of brain glioma MGMT promoter gene

Examples

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Embodiment 1

[0043] MGMT promoter methylation detection in patients with glioma A1

[0044] (1) DNA was extracted with a tissue DNA extraction kit from A1 glioma tissue to ensure that the DNA concentration was 100 ng / ul, 260 / 280=1.82.

[0045] (2), take 20ul of DNA and use the methylation modification kit to sulfurize the extracted DNA;

[0046] (3) Use the methylated primers of SEQ NO 1 and SEQ NO 2 to perform drop-down PCR on the sulfur-modified DNA template. The reaction conditions are as follows: pre-denaturation at 95°C for 5 minutes, followed by cycling, denaturation at 95°C for 30s, 57°C Anneal from ℃ to 42℃ for 30s for 2 cycles per degree, finally anneal at 42℃ for 30s for 10 cycles, extend at 72℃ for 45s, a total of 42 cycles, after that, continue to extend at 72℃ for 5min, store at 4℃, the reaction system is 25uL system, It includes: template (sulfurized product): 2uL, SEQ NO 1: 1uL, SEQ NO 2: 1uL, 10×MSP buffer: 2.5uL, 20mM dNTP: 2ul, Hot start taq: 0.5ul, deionized water: 16uL; ...

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Abstract

The invention discloses a method for detecting the methylation of a brain glioma MGMT promoter gene. The specific steps comprise: extracting the DNA from the brain glioma tissue of a patient by using a tissue DNA extraction kit; carrying out sulfurization treatment on the extracted DNA by using a methylation modification kit; carrying out touchdown PCR on the DNA site template being subjected to the sulfurization modification by using methylation primers represented by SEQ ID NO 1 and SEQ ID 2; diluting the site template 1000 times by using the product obtained through PCR amplification with the methylation primers represented by SEQ ID NO 1 and SEQ ID 2, and carrying out common PCR amplification with primers represented by SEQ ID NO 3 and SEQ ID 4; carrying out 1% agarose gel electrophoresis on the product obtained through the common PCR amplification with the primers represented by SEQ ID NO 3 and SEQ ID 4; recovering the target strip from the 1% agarose gel electrophoresis strips by using an agarose gel recovery kit; carrying out linking transformation on the PCR product and a T carrier, and carrying out blue-white screening; screening recon through bacterial colony PCR; sequencing the positive clones; and analyzing the sequencing results by using biq-analyzer software. The method of the present invention has advantages of high detection accuracy, convenient use, and the like.

Description

technical field [0001] The invention relates to the field of biological detection, in particular to a method for detecting the methylation of MGMT promoter gene of brain glioma. Background technique [0002] DNA methylation is a major form of genomic DNA epigenetic modification and an important means of genome function regulation. There are many ways to modify DNA methylation. The base to be modified can be the N-6 position of adenine, the N-4 position of cytosine, or the N-7 position of guanine or the C-position of cytosine. 5 digits. DNA methylation specifically refers to the covalent bonding of a methyl group at the 5' carbon position of cytosine of a genomic CpG dinucleotide under the action of DNA methyltransferase. Recent studies have shown that DNA methylation plays an important role in gene mutation, expression regulation, cell proliferation, differentiation, and development, and is closely related to the occurrence and development of tumors. [0003] Improving th...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12Q1/68
CPCC12Q1/6869C12Q1/6886C12Q2600/106C12Q2600/154C12Q2531/113C12Q2565/125
Inventor 韦玉军李航崔俊生苏军吴远航
Owner 安徽安龙基因科技有限公司
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