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MicroRNA detection method

A detection method, a technology of RNA recombination, which can be applied to DNA/RNA fragments, recombinant DNA technology, biochemical equipment and methods, etc. The effect of improving reaction sensitivity, reducing cost, and simple synthesis

Inactive Publication Date: 2015-09-23
HUAZHONG UNIV OF SCI & TECH
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

However, all the probes in the above experiments were modified with fluorescent groups and quenching groups, and the double-labeled modification increases the difficulty of probe design and synthesis, which leads to the possibility of increasing the occurrence of false signals, so the probes The simplification of the structure is one of the ways to solve the above limitations in reality

Method used

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Examples

Experimental program
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Effect test

Embodiment 1

[0035] According to the sequence of miR-21 (SEQ NO:2), the probe molecule with DNA sequence such as SEQ NO:1 is designed, and the alkynyl group is modified at the 5' end, and 2-(4-(benzyl azide)phenyl- 1,1,2-Triphenylethylene reacts under the catalysis of cuprous bromide and sodium ascorbate, and the synthetic route is as follows:

[0036]

[0037] In the reaction, compound of formula II: compound of formula I: cuprous bromide: sodium ascorbate=1:10:11.5:23 (molar ratio); reaction condition: anaerobic environment, temperature is 25 ℃~30 ℃, stirs 24 hours or so. The reaction product was purified by reverse-phase high-performance liquid chromatography, and finally characterized by mass spectrometry to obtain the probe molecule with the molecular structure shown in formula III.

Embodiment 2

[0039] Step 1: Using the synthesized miR-21 as the test substance, prepare a 100nM solution. Add 23 μL RNA-free water, 5 μL NaCl solution (500 mM), 5 μL probe molecule (100 μM), 0.5 μL RNA recombinase inhibitor (40 U / μL), 5 μL miR-21 (100 nM), 5 μL into a 1.5 mL centrifuge tube, respectively. Graphene solution, 5 μL 10×DNA exonuclease III buffer, react in 37°C water bath for 30 minutes;

[0040] Step 2: Measure the fluorescence value immediately after adding 1.5 μL of DNA exonuclease III (200 U / μL); then measure the fluorescence value every 3 minutes at a temperature of 37°C, and obtain the curve of fluorescence intensity changing with time as shown in figure 2 , the measurement conditions of the fluorescence spectrum are: the excitation slit and the emission slit are both 5nm, the voltage is 600v, the excitation wavelength is 350nm, the emission scanning range is 400-600nm, and a 50μL quartz cuvette is used for measurement.

Embodiment 3

[0042] Repeat Example 1 with the same steps as described, the difference is that the concentration of miR-21 in Step 1 is 100aM (10 -16 mol / L); the reaction temperature in step 2 was plus 4°C, and the fluorescence intensity was measured after 3 days.

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Abstract

The invention discloses a MicroRNA detection method. The method includes that through combination of a DNA (deoxyribonucleic acid) probe molecule modified with aggregation induced luminescence compounds (AIE molecules) and to-be-detected MicroRNA and under the action of DNA excision enzyme III, hydrophilic desoxyribonucleic acid in a probe can be hydrolyzed, remained hydrophobe AIE substances aggregate to give out light, the targeted MicroRNA is released to be combined with the probe, the probe is hydrolyzed once more, fluorescent light is enhanced after circulation for multiple times, and the MicroRNA is detected. The invention further discloses the probe molecule and a reagent box used for detecting miR-21 with the method. The method used for detection of the MicroRNA is high in speed and low in detection limit, and the probe is simple in process and low in cost.

Description

technical field [0001] The invention belongs to the field of biological detection, and more specifically relates to a method for detecting MicroRNA. Background technique [0002] MicroRNA is a type of single-stranded non-coding RNA with a length of 17-25 nt, with an average length of 22 nt. It exists widely in plants and animals and has a wide range of regulatory functions. It is an important regulator of the growth and development of higher plants and animals. It is highly evolved keep. In 1993, Lee and his colleagues first confirmed the first MicroRNA (Lin-4) in the body of Caenorhabditis elegans. Since then, with the development of high-throughput sequencing, bioinformatics and other technologies, a large number of MicroRNAs have been discovered one after another. MicroRNAs are ubiquitous in various organisms. By the beginning of 2009, a total of about 8,000 MicroRNAs had been discovered in various species, including nearly 700 MicroRNAs in humans. MicroRNA can cause th...

Claims

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Application Information

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IPC IPC(8): C12Q1/68C12N15/11
CPCC12Q1/6816C12Q2525/207C12Q2521/319C12Q2527/127
Inventor 夏帆闵雪红娄筱叮
Owner HUAZHONG UNIV OF SCI & TECH
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