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Nanosensor for detecting DNA methyltransferase based on single quantum dot

A nano-sensor, methyltransferase technology, applied in the field of biological analysis, to avoid signal amplification steps, improve simplicity, and reduce sample consumption.

Inactive Publication Date: 2017-07-28
SHANDONG NORMAL UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

However, so far, there are few reports on the detection of DNA methyltransferases by integrating single-molecule fluorescence detection and quantum dot technology.

Method used

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  • Nanosensor for detecting DNA methyltransferase based on single quantum dot
  • Nanosensor for detecting DNA methyltransferase based on single quantum dot
  • Nanosensor for detecting DNA methyltransferase based on single quantum dot

Examples

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Embodiment 1

[0062] Detection of DNA methyltransferases and formation of quantum dot / probe / cyanine 5 / black hole quencher nanostructures: The detection of DNA methyltransferases involves three sequential steps: first, all oligonucleotides are Stock solutions were prepared by dilution in 10× Tris-ethylenediaminetetraacetic acid (Tris-EDTA) buffer. 1 micromole per liter of hairpin probe at 95°C in an annealing buffer solution (50 mmol per liter of NaCl, 5 mmol per liter of pH 8.0 tris-hydrochloric acid (Tris-HCl) ) for 4 minutes, then slowly lowered to room temperature, and the formed hairpin probe substrate was stored at 4°C for use. Second, the methylation and cleavage reactions of hairpin probes were carried out in a 50-microliter reaction system, including 96 nanomoles per liter of hairpin probes, different concentrations of DNA methyltransferase, 1× methyltransferase Enzyme reaction buffer solution (50 mmol / L Tris-HCl, 10 mmol / L EDTA, 5 mmol / L 2-mercaptoethanol, pH 7.5 ), 160 μmol per ...

Embodiment 2

[0068] 2.1 Experimental verification of the principle

[0069] The principle of the technical solution of the present invention: the technical solution of the present invention first designs a biotinylated hairpin detection probe, the 5' end of which is labeled with the fluorophore cyanine 5 (Cy5), and the 3' end is labeled with the quenching group black hole quencher (BHQ2), the stem region contains the recognition sequence 5'-GATC-3' for the target DNA methyltransferase. The probes were attached to the surface of streptavidin-modified quantum dots through streptavidin-biotin interactions to form quantum dot / probe / cyanine 5 / black hole quencher complexes. In this complex, due to the negligible emission crosstalk between quantum dots and cyanine 5 and the significant spectral overlap between the emission of quantum dots and the excitation of cyanine 5, the quantum dots are used as donors for fluorescence resonance energy transfer, and the flower Cyanine 5 was used as an accept...

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Abstract

The invention discloses a nanosensor for detecting DNA methyltransferase based on a single quantum dot. The nanosensor only comprises a single detection probe thereby preventing complex detection design and cumbersome signal amplification steps. The nanosensor has high selectivity and high sensitivity to DNA methyltransferase. The nanosensor comprises a tail end 3'-biotinylated hairpin detection probe. The tail end 5' of the detection probe is labeled with a fluorophore cyanine 5(Cy5). The tail end 3' is labeled with a quenching group black hole quenching agent (BHQ2). The stem region contains a recognition sequence 5'-GATC-3' of a target DNA methyltransferase. The surface is coated with a streptavidin quantum dot. Through interaction of streptavidin and biotin, the probe is connected to the streptavidin-modified quantum dot surface so that a quantum dot / probe / cyanine 5 / black hole quenching agent complex is formed. The nanosensor also comprises a methylation-specific endonuclease (DpnI).

Description

technical field [0001] The invention belongs to the technical field of biological analysis, in particular to a single quantum dot-based nanometer sensor for detecting DNA methyltransferase. Background technique [0002] DNA methylation is a major epigenetic modification in cells and plays a key role in various important cellular processes such as gene regulation, maintenance of chromatin structure and cell development. DNA methylation is catalyzed by a family of DNA methyltransferases (MTases). DNA methyltransferases can establish and maintain certain genomic methylation patterns by transferring methyl groups from S-adenosylmethionine (SAM) to adenine or cytosine residues in specific DNA sequences. Abnormal DNA methyltransferase activity may interfere with normal methylation patterns, leading to the development of many diseases including cancer. For example, overexpression of DNA methyltransferases (1, 3a and 3b) is strongly associated with retinoblastoma tumorigenesis. T...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): G01N21/64
CPCG01N21/6486
Inventor 张春阳马飞刘文静
Owner SHANDONG NORMAL UNIV
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