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DNA sulfite conversion treatment method and kit

A sulfite conversion treatment technology, applied in the field of methods and kits, DNA sulfite conversion treatment, can solve the problems of decreased detection rate of methylation sites of long fragments, limited research and detection applications, etc., to achieve The effect of ensuring integrity

Inactive Publication Date: 2019-08-02
上海埃文生物科技有限公司
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  • Description
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  • Application Information

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Problems solved by technology

With the improvement of technology, the current commercial sulfite conversion kit can realize the rapid conversion of DNA, which effectively shortens the time of gene DNA sulfite conversion, but significantly improves the fragmentation and degradation level of DNA, making long fragments DNA becomes shorter, resulting in a decrease in the detection rate of methylation sites in long fragments, which limits the application of research and detection of gene methylation levels, so there is an urgent need for efficient and rapid DNA sulfite conversion and purification Methods

Method used

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  • DNA sulfite conversion treatment method and kit

Examples

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Embodiment 1

[0071] In this embodiment, the DNA to be treated is subjected to sulfite conversion and purification to obtain a DNA conversion solution, and then the conversion efficiency detection and methylation-specific PCR detection are performed on the obtained DNA conversion solution. In this example, the DNA of the cell lines HCT116 and Hela genome was extracted by a commercial kit as the DNA to be processed.

[0072] (Transformation and purification of cell genomic DNA)

[0073] (1) Use Quanshijin's commercial genomic DNA extraction kit to extract genomic DNA of cell line HCT116. The specific operation is carried out according to the manufacturer's instructions. After the extracted DNA is detected by the UV spectrophotometer for nucleic acid concentration, take 5μL of the DNA to be processed Add 200 μL to the centrifuge tube.

[0074] (2) Take 120μL of the conversion solution containing 2.5mol / L sodium metabisulfite, 1mol / L sodium bisulfite, 0.5mol / L ammonium sulfite monohydrate, pH 5.0, a...

Embodiment 2

[0122] In this example, compared with Example 1, the use contains 2.5mol / L sodium metabisulfite, 1mol / L sodium bisulfite, 0.5mol / L ammonium sulfite monohydrate and 0.3mol / L sodium hydroxide (pH The procedure for sulfite conversion treatment in the PCR machine for the conditioning agent) is: 60° C., 40 min, and then treated in the same manner as in Example 1 to obtain a converted and purified DNA solution. Then take 10 μL and perform methylation-specific PCR detection in the same manner as in Example 1. The detection results are shown in Table 10.

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Abstract

The invention provides a DNA sulfite conversion treatment method. The method comprises the following steps of (a) preparing a DNA solution to be treated; (b) adding a conversion agent and a protectionagent into the DNA solution, and mixing to obtain a DNA mixed solution, wherein the conversion agent is a water solution of at least one of materials selected from sodium metabisulfite, sodium hydrogen sulfite, amine hydrogen sulfite, magnesium bisulfite and ammonium sulfite; the protection agent is a water solution of at least one of ingredients of hydroquinone and water-soluble vitamin E and mycose or agarose; (c) converting the DNA mixed solution under the specified condition to obtain a DNA conversion solution. According to the disclosure, the method with the advantages that the DNA sulfite conversion can be efficiently and fast realized, and the DNA integrity can be effectively ensured can be provided.

Description

Technical field [0001] The present invention particularly relates to a method and kit for DNA sulfite conversion treatment. Background technique [0002] Methylation modification is a way to achieve epigenetic regulation of genes. It plays an important role in regulating gene expression models and genome stability without changing the DNA sequence. DNA methylation mainly occurs at the CpG site. The DNA methyltransferase catalyzes the conversion of cytosine to 5-methylcytosine. The degree of CpG methylation is inversely proportional to the transcription activity. The organism changes the CpG in the gene promoter region. The degree of methylation regulates the expression level of genes, which in turn affects the characteristics of cells and the entire organism. Therefore, the methylation level of genomic DNA is closely related to cell proliferation, differentiation and canceration. Current studies have found that abnormal methylation levels of many genes are related to the occurr...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N15/10
CPCC12N15/10C12N15/1013C12Q2523/125
Inventor 樊晓婷黄青红张笑人王伦善
Owner 上海埃文生物科技有限公司
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