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Method for detecting tumor biomarker by using palindrome lock-type probe

A technology for detection of biomarkers and probes, applied in biochemical equipment and methods, measurement/inspection of microorganisms, etc., can solve problems such as insufficient sensitivity and high temperature control, and achieve high sensitivity and good specificity

Inactive Publication Date: 2018-10-12
FUZHOU UNIV
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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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  • Method for detecting tumor biomarker by using palindrome lock-type probe
  • Method for detecting tumor biomarker by using palindrome lock-type probe
  • Method for detecting tumor biomarker by using palindrome lock-type probe

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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 invention provides a method for detecting a tumor biomarker by using a palindrome lock-type probe. A palindrome-sequence non-fluorescent mark lock-type RCA probe is designed. The probe is composedof a target identification part and a locus cutting part. The target identification part and target miRNA can completely complement each other and is located at a 3' end and a 5' end of the lock-typeprobe; a semi-identification locus of restriction enzyme Nt.AlWI is fused with a palindrome alkali segment. The probe is combined with nicking enzyme, an N-RCA concept is provided, a double-directionchain replacement reaction (D-SDA) is conducted for combination, and the probe is used for detecting the tumor marker miRNA. By means of the method, let-7a miRNA can be efficiently detected through amplification, and the probe is economical and practical and is expected to be applied to 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 Applications(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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