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Method for detecting miRNA and kit

A technology for reagents and DNA molecules, applied in biochemical equipment and methods, and the determination/inspection of microorganisms. Effect

Pending Publication Date: 2018-12-28
FUZHOU UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

However, this invention needs to design a double hairpin structure, which is prone to misalignment and pairing in the process of rolling circle amplification, which reduces the sensitivity of detection

Method used

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  • Method for detecting miRNA and kit
  • Method for detecting miRNA and kit
  • Method for detecting miRNA and kit

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0084] A method for detecting liver cancer miRNA,

[0085] Step 1: Synthesize the DNA molecular machine and configure it into a solution with a concentration of 20nM,

[0086] Step 2: Pulverize mouse liver cancer tissue at room temperature, filter and extract the juice, and prepare a sample solution with a concentration of 2 μM,

[0087]Step 3: Take 4 μL of sample solution and 2 μL of DNA molecular machine solution, mix evenly by micro-oscillation to obtain a mixed solution,

[0088] Step 4: Add 10 μL buffer, 6 μL polymerase, 6 μL nickase, 6 μL Thioflavin T, 6 μL dNTP, 6 μL Mg 2+ Add to the mixed solution obtained in step 3 to obtain a reaction solution.

[0089] Wherein, the above dNTP concentration is 0.12mM; the polymerase concentration is 0.112U / uL; the nickase concentration is 0.01U / uL; the concentration of Thioflavin T is 0.1uM; the buffer solution includes Tris with a concentration of 30mM and pH8.3 HCL, tween20 at a concentration of 0.1%, KCl at a concentration of 1...

Embodiment 2

[0107] Step 1: Synthesize the DNA molecular machine and configure it into a solution with a concentration of 60nM,

[0108] Step 2: Grinding the mouse liver cancer tissue at room temperature, filtering and extracting the juice, and preparing a sample solution with a concentration of 8 μM,

[0109] Step 3: Take 8 μL of sample solution and 4 μL of DNA molecular machine solution, mix evenly by micro-oscillation to obtain a mixed solution,

[0110] Step 4: Add 40 μL buffer, 10 μL polymerase, 10 μL nickase, 10 μL Thioflavin T, 10 μL dNTP, 10 μL Mg 2+ Add to the mixed solution obtained in step 3 to obtain a reaction solution.

[0111] Wherein, the above-mentioned dNTP concentration is 0.50mM; the polymerase concentration is 0.236U / uL; the nickase concentration is 0.06U / uL; the buffer solution includes Tris HCL with a concentration of 70mM, pH9.0, and tween20 with a concentration of 0.6%. KCl at a concentration of 160mM, (NH4) at a concentration of 60mM 2 SO 4 , NaCl at a concent...

Embodiment 3

[0121] Step 1: Synthesize the DNA molecular machine and configure it into a solution with a concentration of 100nM,

[0122] Step 2: Pulverize mouse liver cancer tissue at room temperature, filter and extract the juice, and prepare a sample solution with a concentration of 2 μM,

[0123] Step 3: Take 4 μL of the sample solution and 4 μL of the DNA molecular machine solution, and mix evenly by micro-oscillation to obtain a mixed solution.

[0124] Step 4: Add 20 μL buffer, 6 μL polymerase, 7 μL nickase, 10 μL Thioflavin T, 3 μL dNTP, 36 μL Mg 2+ Add to the mixed solution obtained in step 3 to obtain a reaction solution.

[0125] Wherein, the above-mentioned dNTP concentration is 0.3mM; the polymerase concentration is 0.212U / uL; the nickase concentration is 0.03U / uL; the concentration of Thioflavin T is 0.1uM; HCL, tween20 at a concentration of 0.4%, KCl at a concentration of 145mM, (NH4) at a concentration of 45mM 2 SO 4 , a concentration of 46mM NaCl; Mg 2+ The concentrat...

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Abstract

The invention belongs to the biotechnological field and particularly relates to a method for detecting miRNA. The method comprises the steps that S1, a DNA molecular machine for specific recognition of miRNA is provided, the DNA molecular machine contains a nucleotide sequence of complementary sequences of a G-four-stranded-body sequence; S2, the DNA molecular machine and a sample to be detected are mutually recognized; S3, DNA polymerase and nickase are introduced to act on the DNA molecular machinery; S4, thioflavin T is introduced to induce production of the G-four-stranded-body structure and combination with a G-four-stranded-body; S5, the fluorescence radiation situation is recorded. The DNA molecule generating machine is a nucleotide sequence consisting of zones arranged in sequencefrom 3' to 5', namely a miRNA sequence recognition zone, a stable zone, nickase recognition zone and a G-four-stranded-body synthesis template. The method does not need fluorescent probe labeling, hashigher sensitivity and is simpler in operation.

Description

technical field [0001] The invention belongs to the field of biotechnology, and in particular relates to a method and a kit for detecting miRNA. Background technique [0002] MicroRNAs (miRNAs) are a class of endogenous non-coding RNAs 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 through complementary base pairing, and according to the degree of complementarity Differently direct the silencing complex to degrade the target mRNA or to repress the translation of the target mRNA. Recent studies have found that miRNA expression is associated with a variety of cancers, and about 50% of annotated miRNAs are located in fragile sites associated with tumors on the genome. This shows that miRNAs play a crucial role in the process of tumorigenesis. The r...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12Q1/6825C12Q1/682
CPCC12Q1/682C12Q1/6825C12Q2563/107
Inventor 张芳梁淑英陈雨浓陈雪雲张晓婷高飞
Owner FUZHOU UNIV
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