Vulcanizing agent in gene methylation detection and application thereof

A vulcanizing agent and methylation technology, applied in the biological field, can solve the problems of long vulcanization time, weak vulcanization strength, and inconvenient preparation, and achieve the effects of simple and time-saving preparation, complete vulcanization, and time-saving and cheap reagent preparation

Inactive Publication Date: 2017-05-31
JIANGSU MICRODIAG BIOMEDICINE TECH CO LTD
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

High-concentration sodium bisulfite is extremely difficult to dissolve, it is inconvenient to prepare and wastes time, and its vulcanization strength is weak, and it takes a long time for vulcanization, usually 2-3 hours or even overnight

Method used

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  • Vulcanizing agent in gene methylation detection and application thereof
  • Vulcanizing agent in gene methylation detection and application thereof
  • Vulcanizing agent in gene methylation detection and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0027] Materials: Plasma samples to be tested, 0.1ng / ul methylation-positive DNA samples, 1ng / ul methylation-negative DNA samples, both methylation-positive DNA samples and methylation-negative DNA samples were derived from cells.

[0028] Instruments: Lightcycler 480, water bath, sterilizer.

[0029] Reagents: ammonium bisulfite (WAKO Japan), anhydrous sodium sulfite (Aladdin), tetrahydrofuran (Aladdin), VE (Aldrich)

[0030] The DNA is derived from plasma DNA and cell DNA. Plasma DNA is extracted by the plasma free DNA extraction reagent of Suzhou Industrial Park Weizhen Biomedical Co., Ltd.; cell DNA is extracted by blood tissue cell DNA extraction reagent (Tiangen Biochemical Technology Co., Ltd., article number: DP304) for extraction, the total amount used for the reaction is generally 0.1ng ~ 2ng.

[0031] The system and reaction conditions of the vulcanization reaction: the composition of the vulcanization reaction system is: 100ul DNA with a total amount of 0.5ng+190u...

Embodiment 2

[0037] Material: 1ng / ul methylation-positive DNA sample, the methylation-positive DNA sample comes from cells.

[0038] Instruments: Lightcycler 480, water bath, sterilizer.

[0039] Reagents: ammonium bisulfite (WAKO Japan), anhydrous sodium sulfite (Aladdin), tetrahydrofuran (Aladdin), VE (Aldrich)

[0040] Cellular DNA is extracted with blood tissue cell DNA extraction reagent (Tiangen Biochemical Technology Co., Ltd., product number: DP304), and the total amount used for the reaction is generally 0.1ng-2ng.

[0041] The composition of the vulcanization reaction system is: 100ul DNA with a total amount of 1.0ng+190ul vulcanization reagent+30ul DNA protection solution.

[0042] The vulcanization reagent is a mixture of 55% (v / v) ammonium bisulfite and 0.15 g / mL anhydrous sodium sulfite. The DNA protection solution is a mixture of 0.125g / mL VE and tetrahydrofuran.

[0043] The specific conditions of the vulcanization conversion reaction are: react at 80° C. for 1 hour. Af...

Embodiment 3

[0045] Material: 1ng / ul methylation-positive DNA sample, the methylation-positive DNA sample comes from cells.

[0046] Instruments: Lightcycler 480, water bath, sterilizer.

[0047] Reagents: ammonium bisulfite (WAKO Japan), anhydrous sodium sulfite (Aladdin), tetrahydrofuran (Aladdin), VE (Aldrich)

[0048] Cellular DNA is extracted with blood tissue cell DNA extraction reagent (Tiangen Biochemical Technology Co., Ltd., product number: DP304), and the total amount used for the reaction is generally 0.1ng-2ng.

[0049] The composition of the vulcanization reaction system is: 100ul DNA with a total amount of 1.0ng+190ul vulcanization reagent+30ul DNA protection solution.

[0050] The vulcanization reagent is a mixture of 95% (v / v) ammonium bisulfite and 0.2 g / mL anhydrous sodium sulfite. The DNA protection solution is a mixture of 0.125g / mL VE and tetrahydrofuran.

[0051] The specific conditions of the vulcanization conversion reaction are: react at 80° C. for 1 hour. Aft...

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Abstract

The invention discloses a vulcanizing agent used in gene methylation detection, a matched kit and a detection method thereof. According to the vulcanizing agent, a mixed solution of ammonium hydrogen sulfite and anhydrous sodium sulfite is used as a DNA-methylation vulcanizing conversion reagent. Compared with the DNA-methylation vulcanizing conversion and modification method widely applied on the market, the method disclosed by the invention has the advantages that (1) anhydrous sodium sulfite is extremely easy to dissolve in ammonium hydrogen sulfite, the preparation of the vulcanizing reagent is simple and has a time-saving effect; (2) the whole process of the method only needs 1 hour, and high-temperature unwinding at 98 DEG C is not needed, so that the experimental operation is simple and fast; (3) the vulcanization is complete, and methylation of target genes can be still detected under the background of existence of (90%) non-methylated DNA; (4) the vulcanizing efficiency is high, and all 0.1ng positive DNA can be still detected by the vulcanizing conversion and modification method provided by the invention; and (5) the whole system does not contain toxic and harmful substances and has no hazard for testing personnel and environment, so that the whole system is safe.

Description

technical field [0001] The invention belongs to the field of biology, in particular to a vulcanizing agent used in gene methylation detection. Background technique [0002] DNA methylation refers to the process in which organisms transfer methyl groups to specific bases under the catalysis of DNA methyltransferase (DNMT), with S-adenosylmethionine as the methyl donor. In mammals, DNA methylation mainly occurs on 5'-CpG-3', in which 80% of CpG dinucleotides are methyl-modified, and the remaining 20% ​​are unmethylated CpG dinucleotides The acid is highly aggregated and widely distributed in the promoter region and the first exon region of the coding gene, named as CpG island. Abnormal methylation of CpG islands is an important factor in the occurrence and development of tumors. Since the local hypermethylation of CpG islands is earlier than the malignant proliferation of cells, the detection of its methylation can be used for tumor prediction. Methylation can be used as a b...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12Q1/68
CPCC12Q1/6858C12Q2523/125
Inventor 王弢李雪虹李虹侠
Owner JIANGSU MICRODIAG BIOMEDICINE TECH CO LTD
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