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A methylation, glioma technology, applied in biochemical equipment and methods, microbial determination/inspection, etc., can solve the problems of dependence, high cost, low cost, etc.
Active Publication Date: 2020-07-10
INST OF CHEM CHINESE ACAD OF SCI +1
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[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
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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] Put all the solution obtained in the previous step into the DNease adsorption column, add 500 μl AW1 buffer solution, 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 DNea...
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%.
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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) treating a DNA standard with a restriction enzyme that specifically recognizes and cleaves unmethylated sites; (2) amplifying the digested product by PCR; (3) combining the PCR product with a cationic conjugated polymer to enable the PCR product to undergo fluorescence energy resonance transfer, and respectively detecting fluorescence intensity of the cationic conjugated polymer and fluorescein; (4) calculating E value of gene methylation level in glioma according to the fluorescence intensity; (5) establishing a methylation degree quantitative standardization curve, determining an optimal detection condition, and detecting a glioma DNA sample to be detected under the optimal detection condition; and (6) judging a methylation degree of the glioma DNA sample, and performing combined analysis to evaluate CpG island methylation phenotype of the glioma. The method can realize purposes of detecting the methylation degree of the glioma gene 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...
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