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Method for transforming, recovering and purifying total DNA sulfite in human feces

A technology of sulfite and feces, applied in the biological field, can solve the problems of the influence of human DNA methylation analysis, low CT conversion rate and recovery rate, and difficulty in automatic operation, so as to save experimental operation time and simplify equipment , The effect of high CT conversion efficiency

Inactive Publication Date: 2018-12-07
HANGZHOU HEYI GENE TECH
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

However, the main problems of this type of sulfite conversion kit are: 1) DNA is denatured by sodium hydroxide, and then a long-term conversion and variable temperature process is carried out, and sodium hydroxide solution is required for desulfonation
During this process, the DNA will be severely degraded and fragmented; 2) The spin column method is basically used for product purification, and some kits also require carrier RNA, resulting in high DNA loss, low recovery efficiency, and difficulty in automatic operation; 3) At present, there is no kit specifically for sulfite treatment of human fecal DNA samples on the market, and the existing commercial kits have relatively low CT conversion and recovery rates for sulfite treatment of human fecal DNA. Subsequent methylation analysis of DNA has a large impact

Method used

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  • Method for transforming, recovering and purifying total DNA sulfite in human feces
  • Method for transforming, recovering and purifying total DNA sulfite in human feces
  • Method for transforming, recovering and purifying total DNA sulfite in human feces

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Embodiment 1

[0047] Example 1, the methylation conversion and recovery of total human feces DNA, the method of the present invention was compared with the method of Z brand commercial sulfite conversion kit.

[0048] The acquisition of human feces total DNA: the human feces total DNA was extracted by a commercial human feces DNA extraction kit.

[0049] The specific implementation steps of the sulfite conversion and recovery and purification of human feces total DNA in the present invention:

[0050] (1) Add 1 μg of human feces total DNA (methylated DNA or unmethylated DNA) to a 2.0 mL centrifuge tube. If the concentration of human feces total DNA is 10 ng / μL, the dosage is 100 μL, set 3 Parallel samples (A, B, C);

[0051] (2) Add 300 μL of sulfite conversion solution and 50 μL of protection solution to the centrifuge tube in step (1), cover the centrifuge tube tightly, vortex to mix, and centrifuge instantaneously;

[0052] The sulfite conversion solution is an aqueous solution containin...

Embodiment 2

[0089] Embodiment 2, for methylated and unmethylated human genomic DNA standard substance, the method of the present invention is compared with the method of Z brand commercialized sulfite conversion kit;

[0090] Methylated and unmethylated human genomic DNA standards were obtained from Merck, Germany.

[0091] Sulphite conversion of the present invention and purification specific implementation steps:

[0092] (1) Add 1 μg of genomic DNA (methylated DNA or unmethylated DNA) to be processed into a 2.0 mL centrifuge tube. If the DNA concentration is 10 ng / μL, the dosage is 100 μL. Set up 3 parallel samples (A, B, C);

[0093] (2) Add 200 μL of sulfite conversion solution and 50 μL of protection solution to the centrifuge tube in step (1), cover the centrifuge tube tightly, vortex to mix, and centrifuge instantaneously;

[0094] The sulfite conversion solution is an aqueous solution containing 40% (m / v) sodium bisulfite and 10% (m / v) sodium sulfite, and the pH value is 5.4; ...

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Abstract

The invention provides a method for transforming and purifying total DNA sulfite in human feces. The method comprises the following steps: carrying out thermal denaturation at 95 DEG C for 10-20 minutes, then immediately placing in an iced environment for 5-10 minutes to prevent renaturation, then transforming DNA at 75 DEG C for 45-60 minutes, and adopting immunomagnetic beads and a binding solution containing PEG6000 / 8000 for purifying the transformed DNA to obtain methylated DNA with high transformation rate and high quality, thereby being conducive to standardized and fully automated operation of methylation detection, and being used for analyzing the level of methylation of genes in human DNA in subsequent samples of feces.

Description

technical field [0001] The invention belongs to the field of biotechnology, more specifically, it relates to a method for converting total DNA of human feces under the conditions of constant temperature, denaturing agent and DNA protection reagent, using sulfite solution to convert DNA, and using purification reagent to recover the conversion product method. Background technique [0002] Methylation refers to the process of catalytic transfer of methyl groups from reactive methyl compounds such as S-adenosylmethionine to other compounds. The most common methylation modifications are DNA methylation and histone methylation, and the present invention relates to DNA methylation. [0003] DNA methylation refers to the transfer of active methyl groups to specific bases in the DNA chain under the catalysis of DNA methyltransferase (DNMT) using S-adenosylmethionine as the methyl donor. chemical modification process. DNA methylation generally occurs at the CpG site (cytosine-phos...

Claims

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Application Information

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IPC IPC(8): C12N15/10C12Q1/6806
CPCC12N15/1013C12Q1/6806C12Q2523/125
Inventor 王军一肖雯王巍刘杰舒平尚慧捷高金龙
Owner HANGZHOU HEYI GENE TECH
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