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Method for detecting target nucleic acid based on cationic conjugated polymer and nuclease-assisted cyclic amplification

A nuclease and nucleic acid technology, applied in the field of biomedicine, can solve problems such as low content, difficulty in detecting sequence similarity of miRNA family members, and difficult miRNA

Active Publication Date: 2020-10-30
SHENZHEN GRADUATE SCHOOL TSINGHUA UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

However, the unique characteristics of miRNAs, such as short sequences, low abundance in tissues and cells, and sequence similarity of miRNA family members pose many difficulties in detection
In addition, the occurrence of a cancer is usually closely related to changes in the expression levels of multiple miRNAs, but the detection of multiple miRNAs in the same system has always been a major problem in the field of miRNA detection

Method used

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  • Method for detecting target nucleic acid based on cationic conjugated polymer and nuclease-assisted cyclic amplification
  • Method for detecting target nucleic acid based on cationic conjugated polymer and nuclease-assisted cyclic amplification
  • Method for detecting target nucleic acid based on cationic conjugated polymer and nuclease-assisted cyclic amplification

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0072] Embodiment 1, the establishment of the method for detecting miRNA or DNA using azo-PPE(+)

[0073] 1. Establishment of a method for detecting target DNA using azo-PPE(+) and DNA exonuclease III (Exo III)

[0074]The inventors of the present invention have established a method for detecting target DNA using azo-PPE(+) and Exo III through a large number of experiments. Digestion by Exo III into oligonucleotides or oligonucleotide fragments (Exo III can remove a single nucleotide from the 3' blunt end or cohesive end of dsDNA, respectively, followed by release of ssDNA, which does not require a specific recognition site ), as a result, the electrostatic interaction becomes weaker, resulting in the fluorescence of the probe being unable to be quenched, thereby forming a "turn on" phenomenon of the fluorescent signal; when the target DNA does not exist, the probe cannot form a double-stranded structure because it cannot Cut into oligonucleotide fragments by Exo III, the str...

Embodiment 2

[0096] The feasibility analysis of the method that embodiment 2, embodiment 1 establish

[0097] In this example, P1 was selected as the model probe, and the feasibility of detecting the target DNA T1 and the target miRNA-21 by the biosensor was explored according to the method established in Example 1.

[0098] 1. Obtain the fluorescence emission spectrum of the DNA detection system under different conditions according to the method established in step 1 of Example 1

[0099] Condition 1: Reaction system 1 is 100 uL, consisting of reaction buffer 1.

[0100] Condition 2: Reaction system 1 is 100 uL, consisting of probe P1 and reaction buffer 1; in reaction system 1, the concentration of probe P1 is 100 nM. In system 1, the concentration of azo-PPE(+) was 3.6 μM.

[0101] Condition 3: Reaction system 1 is 100 uL, consisting of probe P1, reaction buffer 1 and target DNA T1; in reaction system 1, the concentrations of probe P1 and target DNA T1 are both 100 nM. In system 1, t...

Embodiment 3

[0113] Embodiment 3, sensitivity analysis

[0114] 1. Sensitivity analysis of DNA detection

[0115] According to the method established in step 1 of Example 1, the fluorescence emission spectra of the DNA detection system were obtained under different reaction systems.

[0116] Reaction system 1-1: Reaction system 1-1 is 100 uL, consisting of probe P1, reaction buffer 1 and Exo III (about 5 U); in reaction system 1-1, the concentration of probe P1 is 100 nM.

[0117] Reaction system 1-2: Reaction system 1-2 is 100uL, consisting of probe P1, reaction buffer 1, Exo III (about 5U) and target DNA T1; in reaction system 1-2, the concentration of probe P1 is 100nM , the concentration of target DNA T1 was 40 nM.

[0118] Reaction system 1-3: Reaction system 1-3 is 100uL, composed of probe P1, reaction buffer 1, Exo III (about 5U) and target DNA T1; in reaction system 1-3, the concentration of probe P1 is 100nM , the concentration of target DNA T1 was 50 nM.

[0119] Reaction sys...

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Abstract

The invention discloses a method for detecting target nucleic acid based on cationic conjugated polymer and nuclease-assisted cyclic amplification. The method comprises the following steps: taking a reaction system containing nucleic acid, a reaction buffer solution, a DNA probe and nuclease, and incubating; adding azo-PPE (+), and standing for 5 min or longer; detecting fluorescence intensity; judging whether the nucleic acid contains target nucleic acid or not or the content of the target nucleic acid according to the fluorescence intensity; wherein the nucleotide sequences of the DNA probeand the target nucleic acid are the same or reversely complementary; the tail end of the DNA probe is subjected to fluorescence labeling; the nuclease is used for digesting one nucleic acid strand ofa double-stranded nucleic acid molecule formed by the DNA probe and the target nucleic acid so as to release the other nucleic acid strand. Experiments prove that the method provided by the inventioncan be used for simultaneously detecting various target nucleic acids, and is high in accuracy, high in sensitivity and good in specificity. The invention has important application value.

Description

technical field [0001] The invention belongs to the field of biomedicine, and in particular relates to a method for detecting target nucleic acid based on cationic conjugated polymer and nuclease-assisted cycle amplification. Background technique [0002] Cancer survival rates are often poor, likely due to late diagnosis and lack of treatment options. Early and accurate diagnosis of cancer is undoubtedly crucial for the clinical diagnosis of cancer, the judgment of cancer staging and the evaluation of the efficacy of cancer treatment. The detection of cancer biomarkers has brought great hope to the early diagnosis of cancer and has become a research hotspot. Cancer biomarkers exist in tumor tissue or serum and encompass a variety of molecules, including DNA, miRNA, enzymes, metabolites, transcription factors, and cell surface receptors, among others. [0003] Tumor cells exist in the human circulatory system, so the DNA in the cells will also exist in the circulatory syste...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12Q1/6858C12Q1/6851C12Q1/6876
CPCC12Q1/6858C12Q1/6851C12Q1/6876C12Q2600/178C12Q2600/16C12Q2600/158C12Q2600/156C12Q2521/319C12Q2537/143C12Q2563/107
Inventor 谭英陈俊粤谭春燕金天
Owner SHENZHEN GRADUATE SCHOOL TSINGHUA UNIV
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