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Homogeneous phase detection method for methylation state of epidermal growth factor receptor (EGFR) gene promoter based on fluorescence polarization

A methylation and promoter technology, applied in fluorescence/phosphorescence, microbial determination/inspection, biochemical equipment and methods, etc., to achieve the effects of easy standardization, simple result analysis, and simple operation

Inactive Publication Date: 2012-08-01
FOURTH MILITARY MEDICAL UNIVERSITY
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  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

[0003] At present, there have been some reports abroad on the determination and analysis of the methylation status of the EGFR gene promoter, such as Bisulfite Sequencing PCR (BSP), MSP (methylation Specific PCR) , COBRA assay, DHPLC, Methylight, microarray, pyrosequencing technology and other technical methods, these detection methods require a variety of reaction steps, and the operation is complicated. Most of the detection requires elution and purification of the molecules to be detected and electrophoresis detection. Multi-step post-processing is time-consuming and laborious , high cost, expensive instruments and equipment, can not only complete the reaction quickly and simply and objectively detect and analyze, the efficiency needs to be improved, so it is difficult to popularize and apply

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  • Homogeneous phase detection method for methylation state of epidermal growth factor receptor (EGFR) gene promoter based on fluorescence polarization
  • Homogeneous phase detection method for methylation state of epidermal growth factor receptor (EGFR) gene promoter based on fluorescence polarization
  • Homogeneous phase detection method for methylation state of epidermal growth factor receptor (EGFR) gene promoter based on fluorescence polarization

Examples

Experimental program
Comparison scheme
Effect test

Embodiment approach 1

[0034] Homogeneous detection of methylation status of EGFR gene promoter in peripheral blood of patients with lung cancer

[0035] 1), materials and methods

[0036] The DNA extracted from the peripheral blood of healthy volunteers and the DNA extracted from the peripheral blood of healthy volunteers treated with SssI methyltransferase (New England Biolabs, Boston, USA) were treated with sodium bisulfate and transformed, and the promoter of EGFR gene was used to target CpG Forward primer 5'-ggttttttgattttgtttagtattg-3' of the island region, reverse primer 5'-ccttacctttcttttcctcc-3' amplified, and the amplified product was cloned into

[0037] Negative and positive controls were constructed with pGEM-T-easy Vectors (Promega, USA). The methylation status of the control was verified by sequencing. The plasmid pGEM-T-methylated-EGFR containing DNA extracted from the peripheral blood of healthy volunteers treated with SssI methyltransferase (New England Biolabs, Boston, USA) ...

Embodiment approach 2

[0052] Homogeneous detection of EGFR gene promoter methylation status in tumor tissues of patients with cervical cancer

[0053] 1), materials and methods

[0054] The plasmid pGEM-T-methylated-EGFR containing 1-1000 copies / mL of EGFR gene promoter methylated DNA cloned in this laboratory is used as a positive control standard to contain 1-10 7 Copies / ml of EGFR gene promoter unmethylated DNA from the plasmid pGEM-T-Umethylated-EGFR was used as a negative control standard. Take 1-10 mg of tumor tissue from patients C and D respectively, and use TIANamp Genomic DNA Kit kits (Tiangen Biotech, Beijing) to extract DNA from the samples, and the extracted products should be converted to genomic DNA using bisulfite or methylation kits. For the methylation modification, the unmethylated cytosine is converted into uracil, the methylated cytosine remains unchanged, and the product is recovered. The extraction and methylation treatment of genomic DNA were performed according to the pro...

Embodiment approach 3

[0061] Epigenetics studies: homogeneous detection of EGFR gene promoter methylation status in normal humans

[0062] 1), materials and methods

[0063] Take 1 ml of peripheral blood from blood donor No. 1, and use TIANamp Genomic DNA Kit kit (Tiangen Biotechnology, Beijing) to extract DNA from peripheral blood samples. The fluorescently labeled probe of the target gene is 5' R110-agtcggagcgagtttttcggg-3', and other steps As in Example 1.

[0064] 2) The results are summarized in Table 2 below.

[0065] Table 3. The mean (means) and standard deviation (S.D.) of FP values ​​of samples, positive and negative control reaction final solutions

[0066]

[0067] *Data are expressed as FP value mean (means) ± standard deviation S.D.

[0068] These results show that the fluorescence polarization FP value of the present invention has a good correlation with the methylation status of the EGFR gene promoter. The distribution of polarization FP values ​​was significantly different, ...

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Abstract

The invention relates to a homogeneous phase detection method for a methylation state of an epidermal growth factor receptor (EGFR) gene promoter based on fluorescence polarization. The method solves the problems that the cost is high, the operation steps are fussy, pollution is easily produced and the accuracy of the detection result is affected in the prior art; the method is simple and easy to operate; and the method is finished by one step in a closed tube, and does not produce pollution. The method is low in cost, and does not require any special reagent and fluorescence quenching or micro groove bonding agent. Methylation of a target region is detected by using change of a fluorescence polarization value due to a single end-marked methylated specific probe, so that the detection result is objective and accurate. The method is simple in result analysis; and only digital comparison is required during result judgment, so the method easily realizes standardization and automation, has a wide application range and can be used for detecting clinical blood or tissue specimens.

Description

Technical field: [0001] The invention relates to the detection field of methylation state of EGFR gene promoter. In particular, it relates to a homogeneous detection method, which monitors the methylation state of the EGFR gene promoter present in the sample by changing the fluorescence polarization value. Background technique : [0002] The epidermal growth factor receptor (EGFR) gene is located in the p13-q22 region of chromosome 7, with a total length of about 200kb. It is a member of the HER / ERB-B transmembrane receptor kinase family. domain and an intracellular kinase active domain. The main function of the extracellular part of EGFR is to bind to ligands such as epidermal growth factor (EGF), activate the activity of tyrosine kinase in the intracellular part, initiate signal transduction pathways, and play a role in regulating cell proliferation and differentiation in tissues. The 5′ regulatory region of the EGFR gene includes a promoter rich in GC islands. Abnorm...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12Q1/68C12N15/11G01N21/64
Inventor 张菊姜英浩梁平王萌强少盈颜真
Owner FOURTH MILITARY MEDICAL UNIVERSITY
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