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A kit for bisulfite conversion of free DNA

A bisulfite and kit technology, which is applied in the field of free DNA bisulfite conversion kits, can solve the problems of DNA methylation information loss, short fragments, and inability to avoid DNA degradation and real-time monitoring efficiency , the effect of shortening the operation time

Active Publication Date: 2022-06-24
银丰基因科技有限公司 +1
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0003] 5-Methylcytosine cannot be directly detected by conventional molecular biology methods, because the PCR or cloning system does not contain methyltransferases, and DNA methylation information is lost during amplification
However, long-term high temperature and low pH treatment will lead to serious degradation of DNA
For circulating tumor DNA with extremely low content (not higher than 1%), extremely short fragments (average 160bp) and easy degradation (average half-life in blood: 2h), the degradation will be more serious, making subsequent related operations impossible. Therefore, there are currently few kits for bisulfite conversion of free DNA

Method used

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  • A kit for bisulfite conversion of free DNA
  • A kit for bisulfite conversion of free DNA
  • A kit for bisulfite conversion of free DNA

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0037] Example 1 Kit for Free DNA Bisulfite Conversion

[0038] The kit includes the following components: bisulfite solution, protective solution, washing solution A, magnetic bead suspension, washing solution B, washing solution C and eluent.

[0039] The bisulfite solution is composed of the following components, and each component is calculated in percentage by weight: ammonium bisulfite 50%, sodium bisulfite 4%, ammonium sulfite monohydrate 2%, TritionX-100 0.8%, The balance is water, and the pH value is between 5.3 and 5.6.

[0040] The protective solution is composed of the following components, and each component is calculated in percentage by weight: tetrahydrofuran furfuryl alcohol 83%, D-α-tocopheryl acid polyethylene glycol 1000 succinic acid (TPGS) 12%, tetrahydrosugar acetate 0.25% %, ethoxylated aspartame 0.08%, and the balance is water.

[0041] The lotion A is composed of the following components, each component is calculated in percentage by weight: guanidi...

Embodiment 2

[0098] Example 2 Kit for Free DNA Bisulfite Conversion

[0099] The kit includes the following components: bisulfite solution, protective solution, washing solution A, magnetic bead suspension, washing solution B, washing solution C and eluent.

[0100] The bisulfite solution is composed of the following components, and each component is calculated in percentage by weight: ammonium bisulfite 50%, sodium bisulfite 2%, ammonium sulfite monohydrate 3%, TritionX-100 0.5%, The balance is water, and the pH value is between 5.3 and 5.6.

[0101] The protective solution is composed of the following components, each component is calculated in percentage by weight: tetrahydrofuran furfuryl alcohol 80%, D-α-tocopheryl acid polyethylene glycol 1000 succinic acid (TPGS) 15%, tetrahydrosugar acetate 0.2% %, 0.1% of ethoxylated aspartame, and the balance is water.

[0102] The lotion A is composed of the following components, each component is calculated in percentage by weight: guanidine ...

Embodiment 3

[0107] Example 3 Kit for Free DNA Bisulfite Conversion

[0108] The kit includes the following components: bisulfite solution, protective solution, washing solution A, magnetic bead suspension, washing solution B, washing solution C and eluent.

[0109] The bisulfite solution is composed of the following components, and each component is calculated in percentage by weight: ammonium bisulfite 50%, sodium bisulfite 6%, ammonium sulfite monohydrate 1%, TritionX-100 1%, The balance is water, and the pH value is between 5.3 and 5.6.

[0110] The protective solution is composed of the following components, and each component is calculated in percentage by weight: tetrahydrofuran furfuryl alcohol 85%, D-α-tocopheryl acid polyethylene glycol 1000 succinic acid (TPGS) 10%, tetrahydrosugar acetate 0.3% %, ethoxylated aspartame 0.05%, and the balance is water.

[0111] The lotion A is composed of the following components, each component is calculated in percentage by weight: guanidine ...

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Abstract

The invention discloses a kit for bisulfite conversion of free DNA, comprising a bisulfite solution, a protection solution, a washing solution A, a magnetic bead suspension, a washing solution B, a washing solution C and an eluent, The protective solution is composed of the following components: tetrahydrofurfuryl alcohol 80% to 85%, D-α-tocopheryl acid polyethylene glycol 1000 succinic acid 10% to 15%, tetrahydroglycol acetate 0.2% to 0.3%, ethoxy Huang Chi Jing 0.05% ~ 0.1%, the balance is water. The invention also provides a method for using the kit to convert free DNA bisulfite and purify DNA. The present invention greatly improves the sulfite conversion method, so that the ctDNA conversion can be completed quickly (40-60 minutes) under mild conditions (70-80° C.), can stabilize the pH of the system and prevent ctDNA from degrading, and can quickly Obtain high conversion rate, high quality, high purity free DNA, which is conducive to the fully automated operation of methylation research.

Description

technical field [0001] The invention relates to a kit for free DNA bisulfite conversion, belonging to the technical field of gene detection. Background technique [0002] The field of epigenetics research is the most rapidly developing subject among many biological research directions in recent years. It is the supplement and further development of classical genetics. The phenomenon is DNA methylation. DNA methylation refers to the transfer of methyl groups to bases in DNA by DNA methyltransferases, commonly cytosine, or 5-methylcytosine, in vertebrates. DNA methylation is an essential DNA modification in mammals. Almost all methylated cytosines occur at CpG sites. DNA methylation of cytosines in CpG islands plays an important role in biological processes such as cell differentiation and development. play an important role. In addition, aberrant methylation is a hallmark of malignant tumors and plays a key role in the process of carcinogenesis. Some DNA methylation biomar...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12N15/10
CPCC12N15/1013C12Q2523/125
Inventor 洪轲常晴云杨海星徐明刘长胜张茜茜冯孟秋许晓丹绳红丹
Owner 银丰基因科技有限公司