Fluorescent biosensor and method for detecting transcription factor and application of fluorescent biosensor

A technology of biosensors and transcription factors, applied in the field of detection and analysis, can solve the problems of high-concentration protein and probe consumption, sensitivity needs to be improved, and long time consumption for temperature control, so as to reduce cost and operation complexity, improve Resource utilization and the effect of reducing experimental costs

Pending Publication Date: 2020-11-24
SHANDONG NORMAL UNIV
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Problems solved by technology

[0004] In the current experiments for the detection of transcription factor activity, traditional gel electrophoresis and autoradiography detection methods mostly require complex nucleic acid labeling and gel electrophoresis procedures, which are low in sensitivity and time-consuming.
Later, new technologies for the detection of transcription factor activity were developed, including the use of dual fluorescent dye-labeled signal probes and the design of double-stem-loop molecular beacon-assisted detection, as well as various amplification strategies that have recently emerged, such as real-t

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  • Fluorescent biosensor and method for detecting transcription factor and application of fluorescent biosensor
  • Fluorescent biosensor and method for detecting transcription factor and application of fluorescent biosensor
  • Fluorescent biosensor and method for detecting transcription factor and application of fluorescent biosensor

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Embodiment

[0081] Protein-DNA interaction and exonuclease digestion: Dissolve 10 μM p50-s and 10 μM p50-antis in annealing buffer containing 100 mM NaCl and 10 mM Tris-HCl (pH 7.5) at 95 Incubate for 5 minutes at °C, then cool slowly to room temperature. Add 1 μl of purified recombinant NF-κB p50 and 500 nanomolar TF binding probe at various concentrations to 10 μl of binding buffer (10 mmol Tris-HCl (pH 7.5), 100 mmol KCl, 2 mM Molar MgCl 2 , 0.1 mmol EDTA, 0.1 mg / ml yeast tRNA, 10% glycerol, 0.25 mmol DTT). Exo III was then inactivated by adding 10 units of ExoIII and 1 μl of 10× NEB buffer 1 and incubating for another 10 min at 37°C, followed by heating at 80°C for 10 min.

[0082] Amplification reaction and fluorescence measurement: Add Exo III digested product to 50 microliters of reaction solution containing 500 nanomolar dNTP, 2.5 units of Klenow fragment polymerase, 5 units of Nt.BstI, 300 nanomolar signal probe , 5 units of Endo IV, 5 microliters of 10×NEBuffer 2 and 5 microl...

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Abstract

The invention provides a fluorescent biosensor and method for detecting a transcription factor and an application of the fluorescent biosensor and belongs to the technical field of detection analysis.The invention provides a two-way EXPAR and endonuclease IV-assisted circulatory digestion signal probe-based super-sensitive fluorescence strategy for detecting the transcription factor. Compared with conventional EXPAR, two-way EXPAR can initiate extension reaction in two directions to generate abundant trigger signals, so as to improve the amplification efficiency. The method is simple in operation, economic, high in sensitivity, low in background and good in specificity, and super-sensitive detection for the activity of the transcription factor in cells can be achieved by using the method,therefore, the method has good practical application value.

Description

technical field [0001] The invention belongs to the technical field of detection and analysis, and in particular relates to a fluorescent biosensor for detecting transcription factors and a detection method and application thereof. Background technique [0002] The information disclosed in this background section is only intended to increase the understanding of the general background of the present invention, and is not necessarily taken as an acknowledgment or any form of suggestion that the information constitutes the prior art already known to those skilled in the art. [0003] Transcription factors can regulate gene expression by specifically binding to promoters / enhancers in DNA duplexes. Under the precise regulation of transcription factors, many normal biological processes, such as cell cycle progression, intracellular maintenance of metabolic and physiological balance, cell differentiation and development, and other processes can be carried out in an orderly manner....

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Application Information

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IPC IPC(8): C12Q1/6825C12Q1/6844C12Q1/6883
CPCC12Q1/6825C12Q1/6844C12Q1/6883C12Q2600/136C12Q2525/113C12Q2563/107C12Q2525/131C12Q2525/151C12Q2521/101C12Q2521/301C12Q2521/319
Inventor 张春阳张艳李庆楠
Owner SHANDONG NORMAL UNIV
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