Method for detecting transcription factor through cascade signal amplification strategy on basis of colocalization recognition and activation

A technology for transcription factors and site recognition, applied in biochemical equipment and methods, microbiological determination/testing, DNA/RNA fragments, etc., can solve problems such as false positive signals, and achieve the effect of eliminating false positive signals

Active Publication Date: 2016-12-14
SHANDONG UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Exonuclease cleavage-assisted fluorescence amplification detection method has high sensitivity, but one or two exonucleases need to be used to cut excess recognition probes. Incomplete cleavage will cause the recognition probes to enter the subsequent signal output process, causing false positives Signal

Method used

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  • Method for detecting transcription factor through cascade signal amplification strategy on basis of colocalization recognition and activation
  • Method for detecting transcription factor through cascade signal amplification strategy on basis of colocalization recognition and activation
  • Method for detecting transcription factor through cascade signal amplification strategy on basis of colocalization recognition and activation

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Experimental program
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Effect test

Embodiment 1

[0066] Example 1: Detection of transcription factors (NF-κB p50) based on a cascade signal amplification strategy based on colocalization recognition activation

[0067] The specific method is as follows:

[0068] (1) Co-regional identification

[0069] 1 μL Cutsmart, 40 nM S1, S2, S3, different concentrations of NF-κB p50 or actual samples were included in a 10 μL reaction system, placed in a 37°C incubator after slight shaking, and reacted for 4 hours.

[0070] (2) Strand Displacement Amplification (SDA)

[0071] In a 20 μL reaction system, 10 μL colocalization recognition product, 2 μL Cutsmart, 1.5mM dNTPs, 1U Klenow fragment, 2U Nt.BbvCI, placed in a 37°C incubator after slight shaking, reacted for 0.5h, and then inactivated at 85°C 0.5h.

[0072] (3) Exponential Rolling Circle Amplification (ERCA)

[0073] ERCA includes ligation and amplification reactions. The ligation reaction was carried out in a 30 μL reaction system, including 20 μL of SDA product, 3 μL of T4 l...

Embodiment 2

[0077] Embodiment 2: Feasibility study of detection method of the present invention

[0078] In order to verify the feasibility of the detection method of the present invention, the present invention investigates the feasibility of the detection method of the present invention by measuring and comparing the fluorescence intensity under different conditions, such as figure 2shown. It can be seen that both the dyes ThT (a) and negative (b, close to coincide with a) show weak fluorescence, indicating that in the absence of the target NF-κB p50, the three split recognition components cannot self-hybridize into a double-stranded structure, triggering The subsequent cascade signal amplification; when the target NF-κB p50 exists, the fluorescence intensity increases sharply (c), indicating that the affinity induction of the target makes the split recognition components approach each other, and then hybridize into a double-stranded structure, triggering the subsequent cascade signal...

Embodiment 3

[0079] Embodiment 3: the sensitivity investigation of detection method of the present invention

[0080] The linear range and sensitivity of the method can be determined by measuring the fluorescence intensity corresponding to different concentrations of the target. Such as image 3 shown. It can be seen that the target concentration is 3.8×10 -13 M-1.5×10 -8 In the M range, ΔF has a linear relationship with the logarithm of the target concentration (R 2 =0.994), the estimated LOD of this detection method is 2.5×10 -13 M, better than most reported sensitivities in the literature.

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Abstract

The invention discloses a recognition probe, a regent and a method for detecting a transcription factor through a cascade signal amplification strategy on the basis of colocalization recognition and activation. By constructing new recognition mode and colocalization recognition of the transcription factor, subsequent cascade chain replacement and amplification are activated, high-sensitivity NF-kB p50 detection is achieved, and the detection sensitivity is 0.2 pM. By means of the detection method, detection of NF-kB p50 in actual samples is also achieved. Excessive fission recognition assemblies in the detection process cannot be autonomously hybridized into two-chain structures, the later cascade signal amplification process cannot be triggered, and the false positive influence caused by incomplete removal of excessive recognition probes is eliminated.

Description

technical field [0001] The invention relates to a method for detecting transcription factors based on a cascade signal amplification strategy of colocalization recognition activation. Background technique [0002] Transcription factors (TFs) are a class of sequence-specific DNA-binding proteins that play key roles in gene regulation. They regulate genetic information (expression from DNA to RNA) by recognizing a small stretch of double-stranded DNA sequence located in the regulatory region of the gene. The normal regulation of TFs is crucial to life processes such as cell development, cell differentiation and repair, while the abnormal regulation of TFs will lead to a series of diseases, such as developmental disorders, abnormal hormone responses, inflammation and cancer. Nuclear factor κB (NF-κB) is a type of transcription factor that widely exists in various cells, usually exists in the cytoplasm in the form of dimers, and plays an important role in inflammation and immun...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12Q1/68C12N15/11
CPCC12Q1/682C12Q2521/301C12Q2521/501C12Q2531/119C12Q2531/125
Inventor 姜玮王磊朱德颂
Owner SHANDONG UNIV
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