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A method for detecting miRNA without enzyme-labeled dna logic system

A logic system, enzyme labeling technology, applied in measurement devices, material excitation analysis, fluorescence/phosphorescence, etc., can solve the problems of expensive equipment, high experimental cost, poor sensitivity, etc., and achieve low detection cost, high sensitivity and specificity. Good results

Active Publication Date: 2020-04-17
UNIV OF JINAN
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

These methods have been improved many times, but there are still many problems, such as complicated process, expensive equipment and high experimental cost, etc., and their sensitivity is poor, so they are limited to a certain extent in clinical application.

Method used

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  • A method for detecting miRNA without enzyme-labeled dna logic system
  • A method for detecting miRNA without enzyme-labeled dna logic system
  • A method for detecting miRNA without enzyme-labeled dna logic system

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0027] A method for detecting miRNA by an enzyme-free labeling DNA logic system is characterized in that it comprises the following steps:

[0028] (1) Preparation of the main solution

[0029] Tris-EDTA-KCl buffer: Take 0.0605 g tris, 0.0146 g ethylenediaminetetraacetic acid, 5.84 g sodium chloride, 0.375 g potassium chloride, dissolve in sterilized water, and dilute to 50 mL Store in a bottle at 4°C for later use, and adjust the pH to 8.0 when used.

[0030] 6 mM protoporphyrin solution: take 0.1879 g of protoporphyrin, dissolve it in sterilized water, dilute it to a 50 mL volumetric flask, and store it at 4 ℃ in the dark for future use. The whole process is carried out in the dark.

[0031] 10% ammonium persulfate: Take 0.2 g of ammonium persulfate, add 2 mL of sterilized water, mix well, and store at 4 °C for later use.

[0032] (2) Probe design

[0033] Design the corresponding G1, G2 and I chains according to the target.

[0034] (3) Construct DNA logic system

[00...

Embodiment 2

[0040] The detection steps were the same as in Example 1, except that the initial concentrations of the I solution were 4 μM, 2 μM, 0.4 μM, 0.3 μM, 0.2 μM, 0.1 μM and 0.05 μM.

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Abstract

The invention discloses a method for detecting miRNAs by using an enzyme-label-free DNA logic system, belonging to the field of fluorescence detection of miRNAs. According to the invention, specific recognition of a target object is realized through designing of complementary probes without addition of corresponding labels and specific enzymes; the purpose of detecting the target object is achieved by characterizing simplification of optical signals in virtue of the characteristic that fluorescence is enhanced after conjugation of protoporphyrin and G-quadruplex; and the complementary probes capable of spontaneously carrying out a strand displacement reaction destroy the above conjugation to realize NOT-gate output. The method provided by the invention is low in cost and high in sensitivity.

Description

technical field [0001] miRNA widely exists in various animals and plants, and participates in many important life activities. As of June 2014, the latest miRNA database Release 21.0 of the Sanger Institute has included 28,645 miRNA precursors and 35,828 mature miRNAs from 223 species. At present, more than 2,000 miRNAs have been discovered in the human body, accounting for 1% of the human genome, and they regulate the expression of 30% of genes. Its regulatory scope is not limited to normal physiological processes in the human body (such as cell proliferation, differentiation, development, apoptosis, etc.), but is also closely related to the occurrence and development of heart disease and tumors. Background technique [0002] miRNA widely exists in various animals and plants, and participates in many important life activities. As of June 2014, the latest miRNA database Release 21.0 of the Sanger Institute has included 28,645 miRNA precursors and 35,828 mature miRNAs from 2...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): G01N21/64
CPCG01N21/6486
Inventor 刘海云祝琳岳森何涛于京华
Owner UNIV OF JINAN