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A kit for detecting methylation at the 5' end of gp5 gene and its detection method

A technology of terminal methylation and kit, which is applied in the field of kits for detecting methylation at the 5' end of GP5 gene, can solve the problems of false positives, expensive instruments, complicated operations, etc., to prevent competition inhibition, high sensitivity, and result analysis. easy effect

Active Publication Date: 2020-07-24
天津康博尔生物基因技术有限公司
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Among them, MSP is the most common method, but it is prone to false positives
Both Methylight and Heavymethyl are based on fluorescent PCR, requiring expensive instruments and complex operations and analysis processes

Method used

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  • A kit for detecting methylation at the 5' end of gp5 gene and its detection method
  • A kit for detecting methylation at the 5' end of gp5 gene and its detection method
  • A kit for detecting methylation at the 5' end of gp5 gene and its detection method

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0029] Example 1: Preparation of Lateral Flow Nucleic Acid Detection Test Paper

[0030] See attached Figure 1A , wherein, SP: sample pad CP: binding pad NM: nitrocellulose membrane detection area AP: absorbent pad.

[0031] (1) Preparation of nitrocellulose membrane detection area:

[0032] The nitrocellulose detection membrane was cut into long strips, placed on the platform of the dot-spraying apparatus, and digoxinumab, FITC monoclonal antibody and biotin-BSA were sprayed on the detection membrane respectively to form T1, T2 and C lines. Dry at 42°C for 30 minutes or dry naturally at room temperature.

[0033] (2) Preparation of binding pad:

[0034] Cut the glass wool into long strips, place it on the platform of the dot sprayer, take the nanoparticle markers, add a certain volume of pretreatment solution, spray dots on the glass wool, and dry at 50°C for 30 minutes.

[0035] (3) Preparation of sample pad:

[0036] Cut glass wool into long strips, soak with a certain...

Embodiment 2

[0039] Embodiment 2: sulfite solution treatment DNA

[0040] (1) Use a commercial DNA extraction kit or other appropriate methods to extract human genomic DNA;

[0041](2) Take 10 μL of DNA solution (100ng-2μg) and 90 μL of sulfite solution, mix well, centrifuge briefly, and place in a PCR instrument for reaction;

[0042] (3) The reaction conditions are as follows:

[0043] The first stage: 95°C for 5 minutes, 1 cycle;

[0044] The second stage: 95°C 30S, 70°C 10min, 15 cycles;

[0045] (4) Purify the DNA using a commercial DNA purification kit or other suitable methods.

Embodiment 3

[0046] Example 3: Methylation-dependent restriction endonuclease treatment of DNA

[0047] (1) Use a commercial DNA extraction kit or other appropriate methods to extract human genomic DNA;

[0048] (2) Take 10 μL DNA solution (100ng-2μg), 5 μL 10x reaction buffer, 2U methylation-dependent restriction endonuclease and 35 μL deionized water, mix well, centrifuge briefly, and place in a PCR instrument for reaction;

[0049] (3) The reaction conditions are as follows:

[0050] The first stage: 30 ℃ 1hr, 1 cycle,;

[0051] The second stage: 15min at 70°C, 1 cycle.

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Abstract

The invention discloses a kit for detecting GP5 gene 5' end methylation and a detecting method of the kit. The kit comprises GP5 gene 5' end methylation specific amplification reaction liquid and lateral flow nucleic acid test paper. The GP5 gene 5' end methylation is identified and amplified on the basis of a PCR method, the amplification product is marked, and the GP5 gene 5' end methylation is detected through the lateral flow technology. The kit and the detecting method have the advantages that the simple and efficient DNA methylation technology and PCR amplification technology are used, nano colored latex microspheres are used as the visual signals of nucleic acid test to replace optical signals such as fluorescent probes, complex operation and expensive instruments are not needed, better detection sensitivity and specificity are achieved as compared with similar technology or products, the GP5 gene 5' end methylation can be detected fast, conveniently, sensitively and economically, and the kit is suitable for popularization, application and industrialization.

Description

technical field [0001] The invention relates to the field of biological detection, in particular to a kit for detecting methylation at the 5' end of GP5 gene and a detection method thereof. Background technique [0002] Breast cancer (BC) is the most common malignancy in women, causing more than 500,000 deaths per year. Currently, with the advancement of medicine, especially in stage I and stage II breast cancer, the 5-year survival rate is quite impressive. But because many breast cancer patients are relatively young. The high recurrence and metastasis of breast cancer are still the main obstacles to the cure of patients. [0003] In recent years, many studies have shown that the DNA methylation of certain genes is closely related to the development of breast cancer. Aberrant DNA methylation in the genome may contribute to malignant transformation by silencing multiple tumor suppressor genes. Such epigenetic changes are thought to occur early in tumor development, possi...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12Q1/6886C12Q1/6858C12Q1/6804
CPCC12Q1/6804C12Q1/6858C12Q1/6886C12Q2600/154C12Q2531/113C12Q2565/625C12Q2523/125C12Q2521/301C12Q2563/155C12Q2525/301C12Q2563/131
Inventor 杨光张亮
Owner 天津康博尔生物基因技术有限公司
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