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Enzyme DNA repair cascade drive fluorophore coding/de-coding-based biosensor and application thereof in telomerase detection

A biosensor and telomerase technology, applied in the field of fluorescence detection, can solve the problems of false positive signals of contamination, complex surface modification process required for sequences, etc., and achieve the effect of high sensitivity, high signal-to-noise ratio and reducing background signal.

Pending Publication Date: 2022-05-17
SHANDONG NORMAL UNIV
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Problems solved by technology

These methods improve the deficiencies of previous methods and have good sensitivity and specificity, but these methods rely on strict sequence requirements, mild reaction conditions and complex surface modification processes
In addition, it is worth noting that polymerase chain reaction (PCR), rolling circle amplification (RCA), and exponential isothermal amplification reaction (EXPAR) are template-dependent amplifications, while exonuclease-assisted signal amplification (EASA), Catalyzed hairpin assembly (CHA) and hybridization chain reaction (HCR) rely on DNA probe sequences, and their amplification processes also exhibit template dependence, which leads to inevitable cross-contamination of amplicons generated by non-specific amplification false positive signal

Method used

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  • Enzyme DNA repair cascade drive fluorophore coding/de-coding-based biosensor and application thereof in telomerase detection
  • Enzyme DNA repair cascade drive fluorophore coding/de-coding-based biosensor and application thereof in telomerase detection
  • Enzyme DNA repair cascade drive fluorophore coding/de-coding-based biosensor and application thereof in telomerase detection

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Embodiment

[0086] Experimental method and steps

[0087] 1. Cell culture and preparation of telomerase extract: cervical cancer cell line HeLa, lung adenocarcinoma cell line A549 and human breast cancer cell line MCF-7 cells in Dulbecco's modified Eagle medium (DMEM) For growth, 1% penicillin-streptomycin and 10% fetal bovine serum were added to DMEM, and the culture medium was placed in an incubator containing 5% carbon dioxide at 37°C until the cells matured. The human fetal lung fibroblast cell line MRC-5 was grown in minimal essential medium (MEM), supplemented with 1% penicillin-streptomycin and 10% fetal bovine serum, and the medium was placed at 37°C with 5% Cells were grown in a carbon dioxide incubator until they matured. Cells were harvested by trypsinization during the exponential growth phase of cells, washed twice with ice-cold PBS, and centrifuged at 2000 rpm for 10 min at 4 °C. Then, about 1 million cells were evenly dispersed in 200 μL of ice-cold 1×CHAPS lysis buffer, ...

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Abstract

The invention provides a biosensor based on enzyme DNA repair cascade drive fluorophore coding / de-coding and application of the biosensor in telomerase detection, and belongs to the technical field of fluorescence detection. The biosensor at least comprises an AP probe, a TS primer, a purine-free / pyrimidine-free endonuclease (APE1), dATP modified by a fluorophore, terminal deoxynucleotidyl transferase (TdT) and a magnetic bead coated by streptavidin. When the biosensor is used for telomerase detection, non-specific expansion can be effectively prevented, background signals are reduced, quantification is performed by using total internal reflection fluorescence (TIRF)-based single molecule detection, and sensitive detection of telomerase at a single cell level can be realized, so that the biosensor has great potential in the aspects of early clinical diagnosis, anti-cancer drug development and the like; therefore, the method has good practical application value.

Description

technical field [0001] The invention belongs to the technical field of fluorescence detection, and in particular relates to a biosensor based on enzymatic DNA repair cascade driving fluorophore encoding / decoding and its application in telomerase detection. Background technique [0002] The information disclosed in the Background of the Invention is only intended to increase the understanding of the general background of the invention, and is not necessarily to be taken as an acknowledgment or any form of suggestion that the information constitutes the prior art that is already known to those skilled in the art. [0003] Telomeres are special non-coding tandem DNA repeat (TTAGGG)n structures located at the ends of chromosomes, which gradually shorten as cells divide, leading to genome instability and cellular senescence. Human telomerase is an important ribonucleoprotein that maintains telomere length and cell division potential by adding the telomeric repeat sequence 5-(TTAG...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12Q1/48C12Q1/682C12Q1/6825
CPCC12Q1/485C12Q1/682C12Q1/6825G01N2333/9128C12Q2533/101C12Q2521/131C12Q2563/107C12Q2563/131C12Q2563/143C12Q2563/149C12Q2565/607
Inventor 张春阳刘明昊于宛彤
Owner SHANDONG NORMAL UNIV
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