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High-throughput single-cell genome 5-hydroxymethylpyrimidine single-molecule visual analysis method

A technology of hydroxymethylpyrimidine single and hydroxymethylcytosine, which is applied in the field of high-throughput single-cell genome 5-hydroxymethylpyrimidine single-molecule visualization analysis, achieving the effect of fast reaction speed, simple reaction conditions and high yield

Inactive Publication Date: 2020-04-10
XI AN JIAOTONG UNIV
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Problems solved by technology

According to these characteristics, there are various microfluidic droplet systems for capturing, culturing, sorting and detecting single cells, but currently no microfluidic method has been used to specifically detect single-cell 5-hmU and 5-hmC. related reports

Method used

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  • High-throughput single-cell genome 5-hydroxymethylpyrimidine single-molecule visual analysis method
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  • High-throughput single-cell genome 5-hydroxymethylpyrimidine single-molecule visual analysis method

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Embodiment 1

[0032] MCF-7 adherent cells were digested and a suspension of a certain cell density was prepared; a large number of agarose gel microspheres wrapped with single cells were obtained through a microfluidic chip, and after the single cells were lysed, each agarose coagulated Single-cell genomes are encapsulated in the gel microspheres. Then, primers were labeled with 5-hydroxymethylcytosine (5-hmC) and 5-hydroxymethyluracil (5-hmU) of the single-cell genome through specific chemical reactions, and then rolled circle amplification (RCA) And hybridize fluorescent probes, use SYBR Green I to stain the genome in the gel microspheres, and finally use fluorescent confocal microscopy for imaging analysis.

[0033] The number of 5-hmU and 5-hmC sites in a single cell can be assessed by the number of spots in each gel microsphere, enabling single-molecule visual analysis of the modified base 5-hmC and 5-hmU in a single cell .

[0034] The result is as figure 2 with image 3As shown,...

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Abstract

The invention discloses a high-throughput single-cell genome 5-hydroxymethylpyrimidine single-molecule visual analysis method, belonging to the technical field of analytical chemistry. According to the invention, a microfluidic chip technology is utilized to obtain agarose gel microspheres wrapping a single-cell genome; and after single cells in the agarose gel microspheres are subjected to cracking treatment, each agarose gel microsphere wraps a single cell genome. Primers are marked on 5-hmC and 5-hmU of the single-cell genome through a specific chemical reaction; rolling circle amplification is then performed; a fluorescent probe is hybridized; and single-molecule visual analysis of modified bases 5-hmC and 5-hmU is achieved. According to the invention, a large amount of liquid drops can be formed in a short time through a microfluidic liquid drop method; the high-throughput agarose gel microspheres wrapping the single-cell genome are efficiently obtained; thus, analysis of a high-throughput single-cell target object is realized; the difference caused by cell heterogeneity is avoided; accurate detection is realized; and the reaction has the advantages of specificity, high yield,simple reaction conditions, high reaction speed and the like.

Description

technical field [0001] The invention belongs to the field of analytical chemistry and relates to a high-throughput single-cell genome 5-hydroxymethylpyrimidine single-molecule visual analysis method. Background technique [0002] In addition to the four classic bases of A, T, C, and G, the human genome also contains chemically modified DNA bases. Usually these chemically modified bases are produced by endogenous enzymes or exogenous factors. At present, researchers have discovered a variety of chemically modified bases in mammalian genomes, the most famous of which is 5-methylcytosine (5-methylcytosine, 5-mC) and its oxidized derivative 5-hydroxymethylcytosine Pyrimidine (5-hmC), 5-formylcytosine (5-formylcytosine, 5-fC) and 5-carboxycytosine (5-carboxylcytosine, 5-caC). These chemically modified bases have the potential to profoundly affect genome function and cellular processes, and these epigenetic marks have now been shown to play important roles in regulating gene exp...

Claims

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Application Information

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IPC IPC(8): C12Q1/6844
CPCC12Q1/6844C12Q2565/629C12Q2563/159C12Q2525/117C12Q2531/125C12Q2563/107
Inventor 赵永席陈锋薛静张进
Owner XI AN JIAOTONG UNIV
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