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, failure, and DNA degradation, and achieve the effect of real-time monitoring efficiency

Active Publication Date: 2019-04-26
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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  • Kit for bisulfite conversion of free DNA
  • Kit for bisulfite conversion of free DNA
  • Kit for bisulfite conversion of free DNA

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0037] Embodiment 1 is used for the kit of free DNA bisulfite conversion

[0038] The kit includes the following components: bisulfite solution, protection 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, each component is by weight percentage: 50% ammonium bisulfite, 4% sodium bisulfite, 2% ammonium sulfite monohydrate, 0.8% TritionX-100, 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, each component is calculated by weight percentage: tetrahydrofurfuryl alcohol 83%, D-alpha-tocopheryl acid polyethylene glycol 1000 succinic acid (TPGS) 12%, tetrahydroglycol acetate 0.25% %, 0.08% of ethoxylated yellow chil, and the balance is water.

[0041] The lotion A is composed of the following components, each component is calculated by weight percentage: 22% of guanid...

Embodiment 2

[0098] Embodiment 2 is used for the kit of free DNA bisulfite conversion

[0099] The kit includes the following components: bisulfite solution, protection 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, each component is calculated by weight percentage: 50% ammonium bisulfite, 2% sodium bisulfite, 3% ammonium sulfite monohydrate, 0.5% TritionX-100, 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 by weight percentage: tetrahydrofurfuryl alcohol 80%, D-alpha-tocopheryl acid polyethylene glycol 1000 succinic acid (TPGS) 15%, tetrahydroglycol acetate 0.2% %, 0.1% of ethoxylated yellow chil, and the balance is water.

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

Embodiment 3

[0107] Embodiment 3 is used for the test kit of free DNA bisulfite conversion

[0108] The kit includes the following components: bisulfite solution, protection 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, each component is by weight percentage: 50% ammonium bisulfite, 6% sodium bisulfite, 1% ammonium sulfite monohydrate, 1% TritionX-100, The balance is water, and the pH value is between 5.3 and 5.6.

[0110] The protection solution is composed of the following components, each component is calculated by weight percentage: tetrahydrofurfuryl alcohol 85%, D-alpha-tocopheryl acid polyethylene glycol 1000 succinic acid (TPGS) 10%, tetrahydroglycol acetate 0.3% %, 0.05% of ethoxylated yellow chil, and the balance is water.

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

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Abstract

The invention discloses a kit for bisulfite conversion of free DNA. The kit comprises a bisulfite solution, a protective solution, washing liquor A, a magnetic bead suspension, washing liquor B, washing liquor C and eluate, wherein the protective solution is prepared from the following ingredients: 80% to 85% of tetrahydrofuran furfuryl alcohol, 10% to 15% of D-alpha tocopherol cetomacrogol 1000 succinate, 0.2% to 0.3% of tetrahydroglycosyl acetate, 0.05% to 0.1% of ethoxychrysoidine and the balance of water. The invention further provides a method for bisulfite conversion and DNA purificationof the free DNA by using the kit. According to the kit, a sulfite conversion method is subjected to great technological improvement, so that ctDNA conversion can be completed rapidly (40 to 60 minutes) under mild conditions (70 DEG C to 80 DEG C), pH of a system can be stabilized, ctDNA degradation is prevented, high-conversion-ratio, high-quality and high-purity free DNA can be rapidly obtained,and full automated operation of methylation researches is facilitated.

Description

technical field [0001] The invention relates to a kit for free DNA bisulfite conversion, which belongs to the technical field of gene detection. Background technique [0002] The field of epigenetics research is the fastest-growing discipline among many biological research directions in recent years. It is a supplement and further development of classical genetics. It is the most fully studied epigenetics in human cells. The phenomenon is DNA methylation. DNA methylation refers to the transfer of a methyl group to a base in DNA by DNA methyltransferase, the common one in vertebrates is cytosine, namely 5-methylcytosine. 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, abnormal methylation is a sign of malignant tumors and plays a key rol...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N15/10
CPCC12N15/1013C12Q2523/125
Inventor 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
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