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Fluorescent probe based on oxidative damage bases, and test kit and method for directly detecting DNA methylation

A fluorescent probe and oxidative damage technology, applied in the field of biological analysis, to achieve the effect of simplifying experimental procedures, high sensitivity, and improving detection sensitivity

Active Publication Date: 2020-10-16
SHANDONG NORMAL UNIV
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Problems solved by technology

At present, most of the methods are suitable for the detection of multiple methylation sites of CpG islands, but the inventors found that there is very little method that can directly analyze the methylation of a specific single site on genomic DNA

Method used

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  • Fluorescent probe based on oxidative damage bases, and test kit and method for directly detecting DNA methylation
  • Fluorescent probe based on oxidative damage bases, and test kit and method for directly detecting DNA methylation
  • Fluorescent probe based on oxidative damage bases, and test kit and method for directly detecting DNA methylation

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Embodiment 1

[0056] Cell culture and preparation of genomic DNA samples: Human colon cancer cells (SW480 cells) were cultured in Dulbecco's modified Eagle's medium DMEM containing 10% fetal bovine serum (FBS) and 1% penicillin-streptomycin at 37 ℃ with 5% CO 2 cultivated in a humid environment. Genomic DNA was extracted according to the instructions of the QIAamp DNA mini kit (QIAGEN). First, cells were thoroughly lysed with lysis buffer containing proteinase K, incubated at 56°C for 10 min, and then an equal volume of 100% ethanol was added. Genomic DNA is bound to the QIAamp column, washed twice and then eluted to collect pure genomic DNA. Genomic DNA concentration was measured with a NanoDrop 2000 spectrophotometer. According to the instructions of the EZ-DNA methylation-gold kit kit, 500pg-2μg of genomic DNA was treated with bisulfite.

[0057] Bisulfite treatment of DNA: The bisulfite treatment of DNA was carried out according to the instructions of the EZ-DNA methylation-gold kit...

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Abstract

The invention belongs to the field of biological analysis, and relates to a fluorescent probe based on oxidative damage bases, and a test kit and a method for directly detecting DNA methylation. The fluorescent probe based on oxidative damage bases is developed for the first time, and DNA methylation of a single site is accurately detected through cyclic cutting signal amplification. hOGG1 enzymecan specifically cut an 8-oxoG base group in an 8-oxoG / 5-mC base pair, but has no activity on the 8-oxoG base group in an 8-oxoG / U base pair. The presence of the target methylated DNA can induce cyclic cleavage of the fluorescent probe with the aid of the hOGG1 enzyme resulting in an enhanced fluorescent signal. The method is high in sensitivity and high in specificity, a single methylation site can be detected, and the lower detection limit reaches 3.458 * 10 <-15> M. And the method can distinguish 0.01% of methylation level in a complex DNA mixed system, and can also detect the DNA methylation level in the genome.

Description

technical field [0001] The invention belongs to the field of biological analysis, in particular to a fluorescent probe based on oxidative damage bases for direct detection of DNA methylation. Background technique [0002] The information disclosed in this background section is only intended to increase the understanding of the general background of the present invention, and is not necessarily taken as an acknowledgment or any form of suggestion that the information constitutes the prior art already known to those skilled in the art. [0003] Epigenetic modification of DNA produces heritable changes in gene expression without altering the coding sequence of the gene. DNA methylation is one of the most important epigenetic events regulating gene transcription, cell differentiation, and pathogenicity in a variety of diseases including cancer. DNA methylation in mammalian cells occurs almost exclusively at the C-5 position of cytosine in CpG islands. In normal cells, most of ...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12Q1/6827C12Q1/682C12Q1/6886C12N15/11
CPCC12Q1/6827C12Q1/682C12Q1/6886C12Q2600/154C12Q2521/531C12Q2523/125C12Q2525/117C12Q2563/107
Inventor 张春阳张艳李琛琛
Owner SHANDONG NORMAL UNIV
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