Method for initiating rolling circle amplification and FRET to detect miRNA based on Toehold-mediated strand displacement reaction

A chain displacement reaction and detection method technology, applied in the field of molecular detection, can solve the problem of low signal output and achieve good selectivity

Active Publication Date: 2019-07-12
SHANDONG NORMAL UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

However, the inventors of the present disclosure found that TMSD has a defect of low signal output, which is not conducive to the application of TMSD to the detection of miRNA

Method used

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  • Method for initiating rolling circle amplification and FRET to detect miRNA based on Toehold-mediated strand displacement reaction
  • Method for initiating rolling circle amplification and FRET to detect miRNA based on Toehold-mediated strand displacement reaction
  • Method for initiating rolling circle amplification and FRET to detect miRNA based on Toehold-mediated strand displacement reaction

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Experimental program
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Effect test

Embodiment

[0052] The principle of this embodiment is as figure 1 As shown, the experimental procedure is as follows.

[0053] Preparation of dumbbell-shaped DNA probes

[0054] Prepare 20 μL reaction system for each DNA probe of T6, T7, T8, T9 and T10, including 1 μL DNA probe (100 μM), 2 μL 10×T4 DNA ligase reaction buffer (500 mM Tris-HCl, 100 mM MgCl 2 , 100 mMDTT, 10 mM ATP, pH 7.5, 25°C), 1 μL T4 DNA ligase (400 U / μL), 16 μL DEPC water. The system was reacted at 23°C for 2 hours, and then heated at 65°C for 10 minutes to terminate the reaction. Add 3 μL NEB buffer Ⅰ (100mM BisTris Propane-HCl, 100mM MgCl 2 , 10mM DTT, pH 7.5, 25℃), 1μL exonuclease Ⅰ (20U / μL), 1μL exonuclease Ⅲ (100U / μL), incubate at 37℃ for 30 minutes, then heat at 80℃ for 20 minutes Enzyme inactivation. The prepared dumbbell-shaped DNA probes were stored at -20°C.

[0055] Real-time fluorescence detection of dumbbell-shaped DNA probe using TMSD reaction to initiate RCA reaction (hereinafter referred to as TI...

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Abstract

The invention provides a method for initiating rolling circle amplification and FRET to detect miRNA based on a Toehold-mediated strand displacement reaction. The method comprises the steps that a rolling circle amplification reaction of the to-be-detected miRNA is initiated by utilizing the toehold-mediated strand displacement reaction, and the detection of the fluorescence resonance energy transfer is achieved by utilizing a DNA single strand, a DNA single-strand probe containing donor fluorophores and a DNA single-strand probe containing receptor fluorophores of the rolling circle amplification reaction. According to the method, by adopting the DNA single strand generated by combining the TMSD and the rolling circle amplification, a large quantity of fluorophores can be gathered, so that the gradual increasing tendency is generated instead of weakening the fluorescence intensity of the donor fluorophores, and therefore the effect of analyzing the content of the to-be-detected targetmiRNA is improved.

Description

technical field [0001] The disclosure belongs to the field of molecular detection, and relates to a method for detecting miRNA based on Toehold-mediated strand displacement reaction triggering rolling circle amplification and FRET. Background technique [0002] The statements herein merely provide background information related to the present invention and may not necessarily constitute prior art. [0003] MicroRNAs (miRNAs) are a class of non-coding single-stranded RNA molecules encoded by endogenous genes with a length of about 22 nucleotides, which have the function of regulating gene expression at the translational or post-transcriptional level. Abnormal expression of miRNAs is associated with various diseases, such as cancer and metabolic diseases. Therefore, miRNAs have become important markers for early clinical diagnosis. As far as the inventors of the present disclosure know, at present, the method for qualitative detection of RNA content is mainly polymerase chai...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12Q1/6844C12N15/11C12Q1/6886C12Q1/6883
CPCC12Q1/6844C12Q1/6886C12Q1/6883C12Q2531/125C12Q2531/119C12Q2521/501
Inventor 唐波刘炬王慧李平梁凯丽
Owner SHANDONG NORMAL UNIV
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