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Fluorescent biosensor for detecting activity of DNA transmethylase and preparation method of fluorescent biosensor

A biosensor and methyltransferase technology, applied in the field of biosensors, can solve the problems of long detection period, low specificity and sensitivity, and achieve the effects of fast detection speed, simple operation and high specificity

Pending Publication Date: 2019-03-01
UNIV OF JINAN
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0004] In order to solve the problems of relatively low specificity and sensitivity and long detection period of the method for detecting DNA methyltransferase activity in the above prior art, the present invention provides a catalytic hairpin-based method with high specificity and sensitivity and fast detection speed. Assembly of amplified fluorescent biosensor for detection of DNA methyltransferase activity

Method used

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  • Fluorescent biosensor for detecting activity of DNA transmethylase and preparation method of fluorescent biosensor
  • Fluorescent biosensor for detecting activity of DNA transmethylase and preparation method of fluorescent biosensor
  • Fluorescent biosensor for detecting activity of DNA transmethylase and preparation method of fluorescent biosensor

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0051] Embodiment 1 Fluorescence intensity changes with endonuclease IV concentration

[0052] The preparation method of described fluorescent biosensor comprises the following steps:

[0053] a. Add sterilized water, 1× Dam buffer (buffer), 0.5 μM MTase chain, 160 μM SAM, and 10 U of Dam to the pre-prepared sterilized EP tube. Incubate at 37°C for 60 min.

[0054] b. Add the target substance (5 μL) that has been reacted in a to the EP tube, add 1 μL Dpnl (20 U / μL), 1×cutsmart reaction buffer 2 μL, H1 H2 H3 1 μM each, Signal probe 5 μM, 1 μL nucleic acid Dicer IV (concentrations are 0.1U / μL, 0.2U / μL, 0.3U / μL, 0.5U / μL, 0.8U / μL, 1.0 U / μL), incubate at 37°C for 90min;

[0055] c. Dilute the solution (10 μL) after the reaction in step b to 70 μL, and detect the fluorescence at 518 nm with a fluorometer.

[0056] The excitation wavelength of the fluorescence instrument is set to 486nm, the emission wavelength is 518nm, the detection range is 450nm-530nm, and the change of the fl...

Embodiment 2

[0061] Example 2 Variation of Fluorescence Intensity with Target Concentration

[0062] A preparation method of the fluorescent biosensor of the present invention:

[0063] The preparation method of described fluorescent biosensor comprises the following steps:

[0064] a. Add sterilized water, 1× Dam buffer (buffer), 0.5μM MTase chain and 160μM SAM, Dam (0.005to 40 U / mL) into the pre-prepared sterilized EP tube. Incubate at 37°C for 60 min.

[0065] b. Add the target substance (5 μL) that has been reacted in a to the EP tube, add 1 μL Pnl (20 U / μL), 1×cutsmart reaction buffer 2 μL, H1 H2 H3 each 1 μM, Signal probe 5 μM, 0.5 U / μL Incubate with endonuclease IV in an incubator at 37°C for 90 minutes;

[0066] c. Dilute the solution (10 μL) after the reaction in step b to 70 μL, and detect the fluorescence at 518 nm with a fluorometer.

[0067] The excitation wavelength of the fluorescence instrument is set to 486nm, the emission wavelength is 518nm, the detection range is 45...

Embodiment 3

[0072] Example 3 Fluorescence Intensity Changes with H1 H2 H3 Chain Concentration

[0073] The preparation method of described fluorescent biosensor comprises the following steps:

[0074] a. Add sterilized water, 1× Dam buffer (buffer), 0.5 μM MTase chain, 160 μM SAM, and 10 U of Dam to the pre-prepared sterilized EP tube. Incubate at 37°C for 60 min.

[0075] b. Add the target substance (5 μL) after the reaction in a to the EP tube, add 1 μL Pnl (20 U / μL), 2 μL of 1×cutsmart reaction buffer, H1 H2 H3 (concentrations are 10 μM, 1 μM, 0.5 μM, 0.1 μM, 0.01μM), Signalprobe take 5μM, 0.5U / μL acid endonuclease IV, incubate at 37℃ for 90min;

[0076] c. Dilute the solution (10 μL) after the reaction in step b to 70 μL, and detect the fluorescence at 518 nm with a fluorometer.

[0077] The excitation wavelength of the fluorescence instrument is set to 486nm, the emission wavelength is 518nm, the detection range is 450nm-530nm, and the change of the fluorescence signal is read to ...

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Abstract

The invention relates to the technical field of biosensors, in particular to a fluorescent biosensor for detecting the activity of DNA transmethylase based on the catalytic hairpin self-assembling. Based on the specific identification of a methylated target object and nucleic acid probe, a three-way circuit structure is formed continuously by utilizing the catalytic hairpin self-assembling, a trigger is circularly amplified, the tail end of the three-way circuit is combined with a signal probe comprising a fluorescent group by virtue of complementary pairing of basic groups, finally a cleavagesite (AP site) of endonuclease IV is used for implementing the positioning cut, and the fluorescent group is released to generate a fluorescent signal of a given intensity. The sensor has the advantages of high detection speed, low detection limit, high specificity and the like, overcomes the defects for detecting the methylation of the DNA at present, and realizes the rapid and accurate quantitative detection for the methylation.

Description

technical field [0001] The invention relates to the technical field of biosensors, in particular to a fluorescent biosensor for detecting DNA methyltransferase activity based on catalyzed hairpin self-assembly, and to a preparation method thereof. Background technique [0002] DNA methylation plays an important role in cell proliferation, aging and gene transcription. Both excessive methylation (hypermethylation) and lack of methylation (hypomethylation) have been identified in various types of cancer, such as breast, ovarian, and lung cancers. In many human cases, abnormal DNA methylation patterns have been associated with abnormal DNA MTase activity, and thus, DNA methylation has been recognized as a new generation of cancer biomarkers and pharmacological targets for new antitumor therapies. [0003] A variety of methods have been developed for the determination of DNA MTase, such as radioactive assays, high-performance liquid chromatography (HPLC), immunochemical methods...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): G01N21/64
CPCG01N21/6402G01N21/6486
Inventor 黄加栋赵一菡刘素王玉瞿晓南张儒峰李莎莎王业茹孙文玉张曼茹江龙赵崇政
Owner UNIV OF JINAN
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