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A method for detecting tumor biomarkers using palindromic lock probes

A technology of biomarkers and padlock probes, which is applied in the field of detection of tumor biomarkers using palindromic padlock probes, can solve the problems of high temperature control and insufficient sensitivity, and achieve high sensitivity and good specificity

Inactive Publication Date: 2021-11-30
FUZHOU UNIV
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  • Claims
  • Application Information

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Problems solved by technology

Although these methods have their advantages, at the same time, they have many limitations, such as insufficient sensitivity, strict temperature control, and high experimental costs

Method used

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  • A method for detecting tumor biomarkers using palindromic lock probes
  • A method for detecting tumor biomarkers using palindromic lock probes
  • A method for detecting tumor biomarkers using palindromic lock probes

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Embodiment 1

[0023] In the verification process of this invention, 20 microliters is used as the reaction system, and 200 microliters is used as an example for the spectroscopic measurement. The sequence used is as follows:

[0024] 2. Feasibility study of palindromic lock probes for miRNA detection:

[0025] In order to prove the feasibility of this invention, we set up the experimental group (with target miRNA) and control group (without target miRNA) respectively. First, the palindromic lock probe (final concentration 1 μM) and miRNA (final concentration 1 μM) were added to T4 ligase buffer at a ratio of 1:1, and ligated at 350 U / μL T4 DNA Under the action of the enzyme, incubate at 16°C for 2 hours to allow the padlock probe to undergo a ligation reaction.

[0026] Next, add 10 mM 2 μL dNTPs, 3 U phi29 polymerase, 5 UNt.AlWI cutting enzyme, 0.5 μL 100×BSA, 2 μL 10×phi29 buffer solution and 4.7 μL secondary water to 10 μL of the above mixture, Incubate the reaction at 37°C for 2 ho...

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Abstract

The present invention provides a method for detecting tumor biomarkers using palindromic lock probes. A palindromic sequence non-fluorescence labeled lock RCA probe is designed. 3' end and 5' end of the formula probe; (2) Cutting site part: the half-recognition site of the restriction endonuclease Nt.AlWI, which is fused with a palindromic base fragment. It is combined with the nicking enzyme to propose the concept of N-RCA, and combined with the bidirectional strand displacement reaction (D-SDA), it is used for the detection of tumor marker miRNA. The invention can realize high-efficiency amplified detection of let-7a miRNA, is economical and is expected to be used in the development of clinical diagnostic kits.

Description

technical field [0001] The invention belongs to the field of biotechnology, and in particular relates to a method for detecting tumor biomarkers by using a palindromic lock probe. Background technique [0002] Currently, many traditional amplification techniques, including blotting, real-time quantitative PCR, and microarrays, are widely used for miRNA detection. Although these methods have their advantages, at the same time, they have many limitations, such as insufficient sensitivity, strict temperature control, and high experimental expense. In order to overcome the above difficulties, we designed a palindromic sequence non-fluorescence-labeled locked RCA probe, combined with nicking cutting enzymes, proposed a new concept of N-RCA, and used it in conjunction with the double-way strand displacement reaction (D-SDA) for tumor Detection of marker miRNA. Contents of the invention [0003] The purpose of the present invention is to provide a method for detecting tumor bio...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12Q1/682
CPCC12Q1/682C12Q2531/119C12Q2531/125C12Q2525/207C12Q2521/301C12Q2521/501C12Q2563/107
Inventor 吴再生许钬孙萌泽
Owner FUZHOU UNIV
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