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Method for detecting structure-specific nuclease FEN1 by using biosensor combining DNA ligation reaction and rolling circle amplification

A biosensor and rolling circle amplification technology, which is applied in the field of biosensor detection of structure-specific nuclease FEN1, can solve the problems that conventional detection methods cannot meet clinical needs and the development of analysis technology is weak, so as to avoid instability and improve sensitivity , the effect of effective research tools

Active Publication Date: 2021-03-30
NANJING NORMAL UNIVERSITY
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, at present, the development of analytical technology for detecting FEN1 is still weak, and many conventional detection methods including Western blot and ELISA cannot meet clinical needs. Therefore, the invention of new detection technologies that can improve performance and reduce costs is very much needed

Method used

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  • Method for detecting structure-specific nuclease FEN1 by using biosensor combining DNA ligation reaction and rolling circle amplification
  • Method for detecting structure-specific nuclease FEN1 by using biosensor combining DNA ligation reaction and rolling circle amplification
  • Method for detecting structure-specific nuclease FEN1 by using biosensor combining DNA ligation reaction and rolling circle amplification

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Experimental program
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Effect test

Embodiment 1

[0031] Condition optimization experiment of detection method of the present invention

[0032] The specific method is as follows:

[0033] 1) Mix 10 μL of the designed dumbbell-shaped DNA probe at a concentration of 10 nM with a 5’ end sequence fragment, 10 μL of the test sample and 30 μL of reaction buffer, and incubate at 35°C-40°C for 20-40min. Then, 1 μL, 5 U / μL T4 DNA ligase and 1× reaction buffer were added to the solution, and incubated at 22° C. for 40 min. The reaction buffer is: 15mMKCl, 30mM Tris-HCl, 15mM (NH 4 ) 2 SO 4 , 3mM MgSO 4 , pH 8.8. 10 μL of the test sample is a solution that may contain the target substance, and the solvent is water or buffer.

[0034] 2) Drop primers and 5 μL of RCA mixture (2U phi29 DNA polymerase, 1 mM dNTPs, 1×phi29 reaction buffer and 1×SGI) into the sample obtained in 1) to start the rolling circle amplification RCA reaction. The primer sequences and template sequences in 1) are shown in the table below:

[0035] Table 1 DNA...

Embodiment 2

[0041] The test sample in step 1) in Example 1 was replaced with different concentrations of FEN1, and other conditions remained unchanged, and steps 1)-3) in Example 1 were repeated to detect fluorescence. Among them, the specific preparation method of different concentrations of FEN1 is: dilute the FEN1 standard with reaction buffer to obtain FEN1 solutions with concentrations of 0, 20, 100, 500, 1000, 2000, 3000, 5000, and 8000 fM.

[0042]Draw the fluorescence spectrum of the sensing system corresponding to different concentrations of FEN1, such as image 3 As shown in A. Plot the linear relationship between FEN1 concentration and fluorescence signal as image 3 Shown in B. According to linear fitting, the obtained linear range is 20-8000 fM, and according to the calculation rule of signal-to-noise ratio=3, the detection limit is 15 fM.

Embodiment 3

[0044] Selective experiment of detection method of the present invention

[0045] Replace the test sample in step 1) in Example 1 with corresponding concentrations of T3 DNA ligase, Exo III, Bst DNA pol, BSA, HAS, histone and IFN-γ, and repeat the steps in Example 1 with other conditions unchanged 1)-3), detect fluorescence, and obtain the selective result of detecting target FEN1 by the method of the present invention, such as image 3 C shown. from image 3 In C, it can be seen that the method of the present invention has good selectivity to the target FEN1.

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Abstract

The invention discloses a method for detecting structure-specific nuclease FEN1 by using a biosensor combining DNA ligation reaction and rolling circle amplification. The method comprises the following steps: mixing and incubating a dumbbell-shaped DNA probe with a 5 '-terminal sequence fragment, a test sample and a reaction buffer solution, then adding T4DNA ligase and the reaction buffer solution into a solution, incubating, dropwise adding a primer and an RCA mixture into the obtained sample to start an RCA reaction, and measuring the fluorescence of the obtained sample, and finally, detecting the fluorescence values of the test samples with different concentrations, drawing a relational graph between the concentrations and the fluorescence intensities, detecting the fluorescence valuesof the samples to be detected, and substituting the fluorescence values into the relational graph to calculate the concentration of FEN1. The FEN1 detection method has the advantages of high sensitivity, strong selectivity and low cost, and can realize efficient detection of FEN1 in tumors.

Description

technical field [0001] The invention relates to a method for detecting a certain target by a biosensor, in particular to a method for detecting structure-specific nuclease FEN1 by a biosensor combined with DNA ligation reaction and rolling circle amplification. Background technique [0002] The pivotal role of biomarkers in diagnosis and prognosis has been generally recognized, and the advantages of their accurate detection are also of great significance in the development of precision medicine. FEN1 (Flap endonuclease 1) plays an important role in the process of DNA replication and repair. At the same time, due to the vigorous vitality of cancer cells, they often replicate rapidly in the human body, resulting in the overexpression of DNA polymerase, which leads to FEN1 overexpression. Therefore, the specific detection of FEN1 can accurately locate cancer cells and carry out subsequent treatment, which is of great significance. However, at present, the development of analy...

Claims

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Application Information

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IPC IPC(8): C12Q1/6844
CPCC12Q1/6844C12Q2521/501C12Q2521/327C12Q2525/30C12Q2531/125C12Q2563/107C12Q2565/607
Inventor 李昺之张幸夏安祺吉峙润谢思盈锁缇莹罗毅轩
Owner NANJING NORMAL UNIVERSITY
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