DNA protective agent and DNA sulfite conversion method

A technology of sulfite and protective agent, applied in the field of DNA protective agent and DNA sulfite conversion, can solve the problems that hinder the scientific research and clinical application of DNA methylation, the amount of DNA cannot meet the needs of PCR amplification and sequencing, and the ease of DNA. Fracture and degradation and other problems, to achieve the effect of shortening the high temperature denaturation time, the conversion method is simple and easy, and eliminating the problem of fracture degradation

Pending Publication Date: 2021-12-28
WUHAN YZY MEDICAL SCI & TECH
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

[0006] In the process of treating DNA with sulfite, DNA is easily broken and degraded. Currently, sulfite conversion kits on the market have obvious DNA degradation problems, and the amount of remaining DNA cannot meet the needs of downstream PCR amplification and sequencing.
DNA degradation has become the main bottleneck of sulfite conversion technology, which seriously hinders the scientific research and clinical application of DNA methylation. Once the DNA degradation problem in the process of sulfite conversion is solved, it will greatly promote the progress of DNA methylation. Basic research and promote the application of DNA methylation markers in disease diagnosis

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  • DNA protective agent and DNA sulfite conversion method
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  • DNA protective agent and DNA sulfite conversion method

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Embodiment 1

[0058] This embodiment provides a DNA protecting agent and a corresponding DNA sulfite conversion method.

[0059] The DNA protecting agent contains 125mmol / L hydroquinone, 5% dimethyl sulfoxide and 0.8% glucomannan, and the solvent is water.

[0060] The DNA sulfite conversion method is as follows:

[0061] In this example, genomic DNA of the cell line HCT116 was extracted as methylation-positive DNA with a commercially available DNA extraction kit, and plasmid DNA containing human gene fragments was used as methylation-negative DNA. The above-mentioned DNA to be transformed was subjected to sulfite conversion.

[0062] (1) After detecting the concentration of the DNA to be converted with an ultraviolet spectrophotometer, take 10ul (containing 50ngDNA) of the DNA to be converted and add it to a 200ul centrifuge tube.

[0063] (2) Get 100ul of conversion agent and protective agent mixed solution (pH is 5) containing 3.5mol / L sodium sulfite, 0.8% glucomannan, 125mmol / L hydroq...

Embodiment 2

[0068] This embodiment provides a DNA protecting agent and a corresponding DNA sulfite conversion method.

[0069] The DNA protection agent contains 100mmol / L hydroquinone, 8% dimethyl sulfoxide and 0.5% glucomannan (Shanghai Aladdin Biochemical Technology Co., Ltd.).

[0070] The DNA sulfite conversion method is as follows:

[0071] In this example, genomic DNA of the cell line HCT116 was extracted as methylation-positive DNA with a commercially available DNA extraction kit, and plasmid DNA containing human gene fragments was used as methylation-negative DNA. The above-mentioned DNA to be transformed was subjected to sulfite conversion.

[0072] (1) After detecting the concentration of the DNA to be transformed with an ultraviolet spectrophotometer, take 10ul (containing 30ngDNA) of the DNA to be transformed and add it to a 200ul centrifuge tube.

[0073] (2) Get 100ul of conversion agent and protective agent mixed solution (pH is 5) containing 5mol / L magnesium bisulfite, 0...

Embodiment 3

[0078] This embodiment provides a DNA protecting agent and a corresponding DNA sulfite conversion method.

[0079] The DNA protecting agent contains 75mmol / L hydroquinone, 10% dimethyl sulfoxide, 0.5% gum arabic and 0.3% glucomannan.

[0080] The DNA sulfite conversion method is as follows:

[0081] (1) After detecting the concentration of the DNA to be converted with a UV spectrophotometer, take 10ul (containing 80ngDNA) of the DNA to be converted and add it to a 200ul centrifuge tube.

[0082] (2) Get 100ul of conversion agent and protective agent mixed solution ( pH is 5) into the centrifuge tube in step (1), and mix well.

[0083] (3) After the conversion system in step (2) was heated on a 98°C metal bath for 5 minutes, it was quickly cooled in an ice bath for 5 minutes, and then converted on a 65°C metal bath for 4 hours.

[0084] (4) Purify the transformed DNA with a commercially available magnetic bead purification kit (AMPure XP kit from Beckman Coulter), and refer ...

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Abstract

The invention discloses a DNA protective agent and a DNA sulfite conversion method, and relates to the technical field of DNA methylation state analysis and detection. The DNA protective agent comprises hydroquinone, dimethyl sulfoxide and a DNA anti-degradation agent, and the DNA anti-degradation agent is glucomannan and / or arabic gum. The DNA protective agent can obviously reduce damage to DNA in the transformation process, avoid DNA degradation and maintain the integrity of the DNA. Besides, the high conversion rate of the non-methylated cytosine can be realized to a great extent, and the melting temperature of the DNA can be reduced to a great extent, so that the high-temperature denaturation time is indirectly shortened, and the problems of breakage and degradation caused by high-temperature damage of the DNA are solved.

Description

technical field [0001] The invention relates to the technical field of analysis and detection of DNA methylation status, in particular to a DNA protection agent and a DNA sulfite conversion method. Background technique [0002] Inheritance in the traditional sense means that the genetic information of the genome is encoded by the sequence of the four bases of AGCT. With the development of biological science and technology, biologists have more and more comprehensive understanding of genetic laws. Scientists have gradually discovered that the methylation of the base cytosine (C) and the acetylation of histones also affect the phenotype of individuals. These biochemical modifications are the main research content of epigenetics. Epigenetics refers to the regulation of gene expression through the modification of DNA and histones without changing the genome sequence, among which DNA methylation is the most common and plays an important role, thus constituting epigenetics. The ...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N15/10
CPCC12N15/10
Inventor 朱燕华王维旭吴亚邱宇邓魏鑫
Owner WUHAN YZY MEDICAL SCI & TECH
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