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Method for detecting miRNA (micro ribonucleic acid) based on graphene/nucleic acid dye platform

A nucleic acid dye and graphene technology, which is applied in the field of nucleic acid fluorescence detection, can solve the problems of cumbersome operation, low sensitivity, and time-consuming, and achieve the effects of simple operation, common detection devices, and low cost

Inactive Publication Date: 2012-10-03
SOUTH CHINA NORMAL UNIVERSITY
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0003] At present, the conventional miRNA detection methods mainly include Northern hybridization (Northern blotting) method, but it is time-consuming and the sensitivity is not high
Other methods based on capillary electrophoresis, nanoparticle signal amplification, and surface plasmon resonance have high detection sensitivity, but the required operations are cumbersome and require expensive instrument support

Method used

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  • Method for detecting miRNA (micro ribonucleic acid) based on graphene/nucleic acid dye platform
  • Method for detecting miRNA (micro ribonucleic acid) based on graphene/nucleic acid dye platform
  • Method for detecting miRNA (micro ribonucleic acid) based on graphene/nucleic acid dye platform

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Embodiment 1

[0052] The construction of embodiment 1 graphene oxide / SYBR Green I detection platform

[0053] (1) Design a DNA hairpin probe for miR-21 as the target molecule, and the binding subsequence of the DNA hairpin probe is TT with both 5' and 3' ends;

[0054] (2) Take 1 mL of graphene oxide with a diameter of 1 μm (Pioneer Nanomaterials Co., Ltd.), wash it twice with double distilled water at room temperature, and centrifuge it for later use;

[0055] (3) Add the designed DNA hairpin probe, graphene oxide, SYBR Green I, different concentrations of miR-21 target molecules and detection buffer to a 96-well plate for hybridization reaction; reaction specific parameters For: 50nM DNA hairpin probe, 8μg / mL graphene oxide, 2μM SYBR Green I nucleic acid dye (invitrogen company), Tris-HCl 10mM and MgCl in 50μL buffer (pH 8.2) reaction system 2 5mM, hybridization at 37°C for 30 minutes; the concentrations of miR-21 target molecules in the five samples were 0nM, 2nM, 10nM, 50nM, and 250nM;...

Embodiment 2

[0057] Example 2 Strand Displacement Isothermal Amplification Reaction

[0058] (1) Prepare strand displacement constant temperature amplification reaction system: 20 μL reaction system contains 200 nM DNA hairpin probe, 400 nM primers, 2U Klenow fragment exo-polymerase (MBI company), Klenow fragment exo-polymerase buffer, dNTPs (each 0.1mM), volume ratio of 3% DMSO and 50fmol target miR-21;

[0059] (2) The reaction system in step (1) is reacted at 37°C for 30-60 minutes;

[0060] The result of the strand displacement isothermal amplification reaction is as follows: image 3 Shown: 1. DNA hairpin probe, 2. DNA hairpin probe and primers, 3. DNA hairpin probe, primers and strand displacement constant temperature amplification enzyme, 4. DNA hairpin probe, target miR-21 and primers, 5 .DNA hairpin probe, target miR-21, primers and strand displacement constant temperature amplification enzyme (react at 37°C for 30 minutes), 6. Same as 5 (react at 37°C for 60 minutes); In the p...

Embodiment 3

[0061] Example 3 Detection of miR-21 using graphene oxide / SYBR Green I detection platform combined with strand displacement constant temperature amplification technology

[0062] (1) Design DNA hairpin probes and primers according to the sequence for detecting miR-21 target molecules;

[0063] (2) DNA hairpin probes, primers, different amounts of miR-21 target molecules, DMSO, enzymes with strand displacement amplification properties and necessary reactants (Klenow fragment exo-polymerase from MBI company and its standard Buffer, dNTPs) were mixed for strand displacement constant temperature amplification reaction; the specific parameters of the reaction were: 20μL reaction system containing 200nM hairpin probe, 400nM primers, 2U Klenow fragment exo-polymerase, dNTPs (each 0.1mM), volume The ratio of 3% DMSO and the amount of miR-21 target molecules are 0.4fmol, 0.8fmol, 4fmol, 20fmol, 40fmol, 60fmol, 100fmol respectively, and react at 37°C for 60 minutes;

[0064] (3) Add 60...

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Abstract

The invention discloses a method for detecting a miRNA (micro ribonucleic acid) based on a graphene oxide / nucleic acid dye detection platform and a nucleic acid constant-temperature amplification technology. The method comprises the following steps of: first, designing a specific DNA (deoxyribonucleic acid) hairpin probe and an amplification primer according to the sequence of a target miRNA, mixing the DNA hairpin probe, the primer, the target miRNA, DMSO (dimethylsulfoxide), an enzyme with a strand displacement amplification property and necessary reactants to carry out a constant-temperature amplification reaction, and then, adding the graphene oxide / nucleic acid dye detection platform to incubate, and carrying out fluorescence detection. When the target miRNA exists, a detection system has an obvious fluorescent signal enhancement phenomenon, and in a condition that the target miRNA does not exist, a fluorescent signal of the detection system is quite weak. By using the method, the purpose of highly sensitively, specifically, simply and quickly detecting a target molecule by using a non-modifying probe is realized, and the method is quite suitable to popularize in the application of actual detection.

Description

technical field [0001] The invention belongs to the technical field of nucleic acid fluorescence detection, in particular to a method for detecting microRNA based on a graphene oxide / nucleic acid dye detection platform combined with nucleic acid constant temperature amplification technology. Background technique [0002] microRNA (miRNA) is a class of endogenous non-coding RNA with regulatory functions found in eukaryotes, with a size of about 20-25 nucleotides. Mature miRNAs are produced by a series of nuclease cleavage and processing of longer primary transcripts, and then assembled into RNA-induced silencing complexes, which recognize target mRNAs by base complementary pairing, and Differently direct the silencing complex to degrade the target mRNA or to repress the translation of the target mRNA. In the past 10 years, research on miRNA has made great progress. According to reports, in mammalian genes, about 30% of the protein-coding genes are regulated by miRNA. Recen...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12Q1/68G01N21/64
Inventor 邢达朱啸
Owner SOUTH CHINA NORMAL UNIVERSITY
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