Method for detecting transcription factors on basis of cooperative masking effect and binding protection effect mediation cascade signal amplification strategies

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

Active Publication Date: 2016-12-07
SHANDONG UNIV
View PDF2 Cites 8 Cited by
  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Exonuclease cleavage-assisted fluorescence amplification detection method is the most commonly used amplification detection method for transcription factors. It has high sensitivity, but it needs to use one or two exonucleases to cut excess recognition probes. Incomplete cleavage will lead to recognition Probe enters downstream signal output process, causing false positive signal

Method used

the structure of the environmentally friendly knitted fabric provided by the present invention; figure 2 Flow chart of the yarn wrapping machine for environmentally friendly knitted fabrics and storage devices; image 3 Is the parameter map of the yarn covering machine
View more

Image

Smart Image Click on the blue labels to locate them in the text.
Viewing Examples
Smart Image
  • Method for detecting transcription factors on basis of cooperative masking effect and binding protection effect mediation cascade signal amplification strategies
  • Method for detecting transcription factors on basis of cooperative masking effect and binding protection effect mediation cascade signal amplification strategies
  • Method for detecting transcription factors on basis of cooperative masking effect and binding protection effect mediation cascade signal amplification strategies

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0062] Example 1: Detection of transcription factors (NF-κB p50) based on a cascade signal amplification strategy mediated by cooperative masking effects and binding protective effects

[0063] The specific method is as follows:

[0064] (1) Binding of target and hairpin recognition probe

[0065] 1 μL Cutsmart, 40 nM hairpin recognition probe, different concentrations of NF-κBp50 or actual samples were included in a 10 μL reaction system, the volume of the reaction system was 10 μL, and placed in a 37°C incubator after slight shaking for 3 hours.

[0066] (2) masking reaction

[0067] The 11 μL reaction system contained 10 μL of the binding product, 1 μM DNA masking strand, placed in a 37 ° C incubator after slight shaking, and reacted for 0.5 h.

[0068] (3) Strand Displacement Amplification (SDA)

[0069] In a 20 μL reaction system, 11 μL masked reaction product, 2 μL Cutsmart, 1.5 mM dNTPs, 1UKlenow fragment, 2U Nt.BbvCI, placed in a 37°C incubator after slight shaking,...

Embodiment 2

[0075] Embodiment 2: Feasibility study of detection method of the present invention

[0076] In order to verify the feasibility of the detection method of the present invention, the present invention examines the feasibility of the novel detection strategy proposed herein by measuring the fluorescence intensity under different conditions, such as figure 2 shown. It can be seen that the dyes ThT (curve a) and negative (curve b) show relatively weak fluorescence, indicating that in the absence of the target NF-κB p50, the masking strand hybridizes with the recognition probe to form a stable double-stranded structure, with purple ends Enclosed in the double strand, it cannot hybridize with the template strand, triggering the subsequent cascade of signal amplification. When the target exists, the fluorescence intensity is significantly enhanced (curve c), indicating that the target binds to the recognition probe to form a complex, and the steric hindrance effect prevents the mas...

Embodiment 3

[0077] Embodiment 3: the sensitivity investigation of detection method of the present invention

[0078] 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 1.5×10 -13 M-1.0×10 -8 In the M range, ΔF has a linear relationship with the logarithm of the target concentration (R 2=0.993), the estimated LOD of this detection method is 1.0×10 -13 M, better than most reported sensitivities in the literature.

the structure of the environmentally friendly knitted fabric provided by the present invention; figure 2 Flow chart of the yarn wrapping machine for environmentally friendly knitted fabrics and storage devices; image 3 Is the parameter map of the yarn covering machine
Login to view more

PUM

No PUM Login to view more

Abstract

The invention discloses a method for detecting transcription factors on the basis of cooperative masking effect and binding protection effect mediation cascade signal amplification strategies. The method has the advantages that masking effects are applied to biosensing, DNA (deoxyribonucleic acid) masking single strands designed and are hybridized with excessive recognition probes to obtain stable double strands, and accordingly false positive signals due to incompletely removed excessive recognition probes can be eliminated; the method is bound with binding protection effects of targets, cascade signal amplification, including SDA (strand displacement amplification) and ERCA (exponent rolling circle amplification), can be triggered, and accordingly the transcription factors and practical samples can be detected in a high-sensitivity manner.

Description

technical field [0001] The invention relates to a method for detecting transcription factors based on a cascade signal amplification strategy mediated by a synergistic masking effect and a binding protective effect. Background technique [0002] Transcription factors (TFs) play a key role in human gene regulation. TFs transmit genetic information (expression from DNA to RNA) by recognizing a short double-stranded DNA sequence located in the gene regulatory region. The normal regulation of TFs is crucial to the process of cell development, cell differentiation and growth, and 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 immunity by binding to the κB site in the genome when it i...

Claims

the structure of the environmentally friendly knitted fabric provided by the present invention; figure 2 Flow chart of the yarn wrapping machine for environmentally friendly knitted fabrics and storage devices; image 3 Is the parameter map of the yarn covering machine
Login to view more

Application Information

Patent Timeline
no application Login to view more
Patent Type & Authority Applications(China)
IPC IPC(8): C12Q1/68C12N15/11
CPCC12Q1/682C12Q2525/301C12Q2531/119C12Q2531/125
Inventor 姜玮王磊朱德颂
Owner SHANDONG UNIV
Who we serve
  • R&D Engineer
  • R&D Manager
  • IP Professional
Why Eureka
  • Industry Leading Data Capabilities
  • Powerful AI technology
  • Patent DNA Extraction
Social media
Try Eureka
PatSnap group products

Browse by: Latest US Patents, China's latest patents, Technical Efficacy Thesaurus, Application Domain, Technology Topic.

© 2024 PatSnap. All rights reserved.Legal|Privacy policy|Modern Slavery Act Transparency Statement|Sitemap