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A gene for in situ detection of microrna-34 in living cells and its preparation method and application

A technology for in situ detection and living cells, applied in the field of genetic engineering, to achieve enhanced clinical significance, high sensitivity, and high specificity

Inactive Publication Date: 2020-07-28
THE FIRST AFFILIATED HOSPITAL OF ARMY MEDICAL UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

However, at present, most of the intracellular detection of nucleic acid molecules needs to be supplemented by various nanomaterials as carriers, and problems such as cytotoxicity and metabolic efficiency in vivo are unavoidable.

Method used

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  • A gene for in situ detection of microrna-34 in living cells and its preparation method and application
  • A gene for in situ detection of microrna-34 in living cells and its preparation method and application
  • A gene for in situ detection of microrna-34 in living cells and its preparation method and application

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preparation example Construction

[0027] The present invention also provides a preparation method of the gene, comprising the following steps: after mixing equal volumes of P1, P2, P3, P4, P5 and P6, denaturing at 95° C. for 5 minutes, and cooling to obtain the gene.

[0028] In the present invention, the concentrations of P1, P2, P3, P4, P5 and P6 are all preferably 10 μM. The present invention has no special limitation on the instrument for said denaturation, preferably a PCR instrument. The temperature reduction in the present invention is preferably natural cooling to room temperature (18-25° C.).

[0029] The present invention also provides a living cell detection kit for microRNA-34 in situ, which includes the gene or the gene prepared by the preparation method.

[0030] The present invention also provides the application of the kit in the in situ detection of microRNA-34 in living cells.

[0031]The in situ detection of live cells in the present invention is preferably for non-diagnostic purposes. In...

Embodiment 1

[0034] The assembled strands of the three-dimensional DNA tweezers were mixed in equal proportions at a concentration of 10 μM, denatured at 95°C for 5 minutes, and then slowly cooled to room temperature to form a three-dimensional DNA tweezers structure. The sequence of the three-dimensional DNA tweezers assembly chain is shown in Table 1.

[0035] Table 1 The sequence of assembled strands of three-dimensional DNA tweezers

[0036]

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Abstract

The invention provides a gene for detecting microRNA-34 of living cells in situ and relates to the technical field of genetic engineering. The gene realizes rapid detection of microRNAs in situ on thebasis of a DNA stereoscopic tweezer structure, detection in the living cells can be realized rapidly, and a series of operation such as microRNA extraction, reverse transcription and PCR is avoided.Meanwhile, in order to deal with the current situation that the specificity of microRNAs for disease diagnosis is poor, in-situ detection can well realize the detection of cells with abnormal microRNAexpression, so that the sources of cells and tissue with diseases are determined, and the clinical significance of microRNA detection can be greatly enhanced. In the embodiment, the to-be-detected microRNA-34 which is subjected to gradient dilution is detected, the detection limit reaches 1.499 nm, and the sensitivity is high. Meanwhile, compared with other microRNAs with similar sequences, the microRNA-34 shows high specificity.

Description

technical field [0001] The invention belongs to the technical field of genetic engineering, and in particular relates to a gene for in situ detection of microRNA-34 in living cells, a preparation method and application thereof. Background technique [0002] As a class of endogenous, non-coding RNA molecules, MicroRNA plays an important regulatory role in many processes of life activities, especially closely related to the occurrence and development of many diseases including cancer. The close relationship between the abnormal expression of MicroRNA and diseases such as tumors makes it a biomarker for diagnosis and prediction of cancer. However, due to the characteristics of low content, easy degradation, small size, similar sequence, and widespread abnormal expression in tumors, microRNA itself is difficult to detect directly by simple methods, so it is urgent to develop high-sensitivity, high-specificity in situ detection methods. . Currently commonly used microRNA detect...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12Q1/6841C12N15/11
CPCC12Q1/6841C12Q2563/107C12Q2543/10C12Q2525/207
Inventor 周琳陈鸣唱凯
Owner THE FIRST AFFILIATED HOSPITAL OF ARMY MEDICAL UNIV