Method for detecting gene promoter methylation

A detection method, a methylation technology, applied in the fields of life science-biochemistry and molecular biology, which can solve the problems of difficult repeatability of methylation detection, low amplification efficiency and MSP

Inactive Publication Date: 2008-02-27
冯景 +1
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Problems solved by technology

[0003] The present invention proposes a method for detecting methylation of gene promoters to solve the problems of repeated methylation detection, sodium bisulfi

Method used

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Embodiment Construction

[0010] (1). Sodium bisulfite modification

[0011] 1.1 Reagent preparation and modification process

[0012] 1.1.1 Reagent preparation Hydroquinone 55mg in 5ml ddH 2 In O, 5.2g of sodium bisulfite was dissolved in 10ml of water and about 1.4ml of 3M sodium hydroxide was used to accurately adjust the pH to 5.0 (prepared before use). The modified concentration is above 3.0mol / L to ensure thorough modification.

[0013] 1.1.2 Modification process Take 1 μg of DNA, add double distilled water to 50 μl, add 3M NaOH, 5.5 μl, and put it at 54°C for 20 minutes to denature. Add 30 μl of 10 mmol / L hydroquinone (prepared just before use) and 520 μl of 3.9M sodium bisulfite (pH 5.0) (prepared just before use) to the denatured DNA, and incubate at 54° C. for 16 hours. Then purify with a purification column, add 50 μl of pre-warmed 80°C double distilled water to dissolve the purified DNA, add 5.5 μl of 3M NaOH, let stand at 25 degrees for 15 minutes, add 56 μl of 10M NH4Ac to stand at roo...

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Abstract

The present invention relates to a detection method of gene promoter methylation. Said method includes the following steps: firstly, using freshly-prepared modification reagent to modify extracted DNA, after the modified DNA is passed through column, making purification and desulfurization reaction, then using absolute ethyl alcohol and salmon sperm DNA to enrich micro modified DNA, further selecting and using specific PCR GC Buffer to make multiple methylation specific PCR amplification, then adopting electrophoresis technique to analyze result.

Description

technical field [0001] The invention belongs to the field of life science-biochemistry and molecular biology, and relates to a method for detecting gene promoter methylation. Background technique [0002] DNA methylation is an enzyme-mediated chemical modification process in higher vertebrate organisms, which affects gene transcription and expression, and is closely related to tumorigenesis. Changes in DNA methylation are a key factor in tumorigenesis and one of the inactivation methods of tumor suppressor genes. It leads to abnormal expression of genes related to cell proliferation and differentiation through genetic and epigenetic mechanisms, resulting in cell loss. Normal gene regulation results in malignant transformation to form tumors. At present, there are not many methods for detecting DNA methylation status. Methylation-specific PCR (methylation-specific PCR, MSP) is a fast and sensitive method for gene methylation analysis[2]. It can detect tumor cells in which 1...

Claims

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Application Information

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IPC IPC(8): C12Q1/68
Inventor 冯景吕军王玉平张吉才
Owner 冯景
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