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MGMT promoter methylation detection primer and detection method thereof

A technology for detection of primers and detection methods, applied in biochemical equipment and methods, measurement/testing of microorganisms, DNA/RNA fragments, etc., can solve problems such as uncomfortable clinical diagnosis, long detection cycle, cumbersome operation, etc., and achieve accurate and reliable treatment effect, the effect of high repeat stability

Inactive Publication Date: 2018-09-25
上海润达榕嘉生物科技有限公司
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

The above methods often have the disadvantages of cumbersome operation, long detection cycle, high false positive, etc., and are not suitable for clinical diagnosis.

Method used

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  • MGMT promoter methylation detection primer and detection method thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0053] A method for detecting MGMT gene methylation in glioma patient tissue:

[0054] 1. Sample collection and storage

[0055] During the operation, the tissue sampling was completed within 30 minutes after the tissue was isolated, and the sampling action was as fast as possible. The tissue was then cut into small pieces, placed in sterilized cryopreservation tubes, and frozen in liquid nitrogen.

[0056] 2. Genomic DNA extraction (select QIAGEN, Cat No. / ID: 69504)

[0057] Cut out no more than 30 mg of tumor tissue material from a glioma patient, put it into a centrifuge tube containing 200 ul of lysate, and vortex for 15 seconds. Add 20ul Proteinase K (20mg / ml) solution, vortex to mix, and briefly centrifuge to remove the water droplets on the inner wall of the tube cap. Place at 56°C until the tissue is completely dissolved, centrifuge briefly to remove water droplets on the inner wall of the tube cap, and proceed to the next step.

[0058] Add 200ul buffer GB, mix th...

Embodiment 2

[0107] A kit for the diagnosis of glioma MGMT gene methylation, including:

[0108] (1) Genomic DNA extraction reagents from tissue samples, specifically including: Proteinase K, buffer GB, absolute ethanol, buffer GD, rinse solution PW, eluent TE;

[0109](2) Genomic DNA bisulfite conversion and separation and purification reagents, specifically including Bisulfite Mix, RNasefree water, DNA Protect Buffer, BL buffer, BW buffer, BD buffer, EB eluate, carrierDNA;

[0110] (3) Reagents for amplifying the target region of the MGMT gene promoter: 2×Master Mix, 10×Concentrate, 3uM forward and reverse amplification primers (as shown in SEQ NO1, SEQ NO 2) and sterilized water;

[0111] (4) Reagents for purifying and separating single-stranded DNA: SSHP beads (Streptavidin Sepharose HighPerformance beads), binding buffer, sequencing primer (as shown in SEQ NO3), Annealing Buffer, 70% (V / V) ethanol solution;

[0112] (5) Reagents for detecting the methylation of the MGMT gene promoter...

Embodiment 3

[0116] Evaluation of sample amplification efficiency of this kit

[0117] Method: Use the kit amplification primers forward and reverse amplification primers (as shown in SEQ NO1, SEQ NO 2) to amplify the sample after bisulfite treatment, and the amplification result is detected by electrophoresis.

[0118] The length of the MGMT promoter region amplified by this kit meets the expected 394bp, the band is clear, the amplification efficiency is ideal, and it fully meets the DNA content required for subsequent methylation detection.

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Abstract

The invention belongs to the technical field of gene detection, and provides a primer for detecting the methylation level of a promoter area of an MGMT gene and a detection method of the primer. Sequencing is conducted by adopting a pyrosequencing method, the ratio of methylation and non-methylation of a CpG island in a target area is efficiently detected, and the methylation level of the MGMT promoter area is evaluated, so that the purposes of predicating the prognosis of a patient with glioma and making individualized chemotherapy and medication plan are achieved.

Description

technical field [0001] The invention relates to the technical field of gene detection, in particular to a glioma MGMT gene methylation detection primer and a detection method thereof. . technical background [0002] Glioma, a tumor originating from glial cells, is the most common primary intracranial tumor. Malignant glioma accounts for about 70% of primary malignant brain tumors, and the annual incidence is about 5-7 / 100,000 people. [0003] The full name of MGMT is O6-methylguanine-DNA methyltransferase (O6-methylguanine-DNAmethyltransferase), which is the only protein that can remove the O6 guanine complex from DNA, which can protect chromosomes from the mutagenesis of alkylating agents, Carcinogenic and cytotoxic damage. MGMT mainly protects cells from the damage of alkylating agents by irreversibly transferring the alkylating group from O6-mG to the cysteine ​​residue at position 145 of MGMT protein. In this process, MGMT acts as both a methyltransferase and a methy...

Claims

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Application Information

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IPC IPC(8): C12Q1/6886C12N15/11
CPCC12Q1/6886C12Q2600/106C12Q2600/118C12Q2600/154
Inventor 钱学庆黄欣秦炜
Owner 上海润达榕嘉生物科技有限公司
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