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Transcription factor multi-channel detection method combining DNA nanotechnology and liquid chromatography technology

A technology of transcription factor and liquid chromatography, applied in the field of multi-channel detection of transcription factor, can solve the problems of inability to realize multi-target detection, cumbersome detection process, immature application, etc., achieve good application prospects, improve sensitivity, and improve clinical detection efficiency Effect

Active Publication Date: 2020-05-29
NANJING MEDICAL UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, the traditional detection techniques for TFs are usually immunoblotting, DNA footprinting, nuclear chromatin immunoprecipitation and other methods. These methods have the technical problems of cumbersome detection process, low efficiency, narrow application range, and inability to achieve multi-target detection. , leading to immature clinical application of TFs as diagnostic indicators

Method used

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  • Transcription factor multi-channel detection method combining DNA nanotechnology and liquid chromatography technology
  • Transcription factor multi-channel detection method combining DNA nanotechnology and liquid chromatography technology
  • Transcription factor multi-channel detection method combining DNA nanotechnology and liquid chromatography technology

Examples

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Effect test

Embodiment 1

[0028] The detection method of the present invention is used to detect the transcription factor p50 to be detected, and the detection method comprises the following steps:

[0029] (1) Masking strand signal transduction: Dissolve the probe (p50-capture probe) capturing the transcription factor to be tested to 5 μM in citrate buffer, heat to 95°C, and cool to room temperature after 10 minutes (at least 1h), incubate with magnetic beads at 37°C for 30 minutes to obtain p50-capture probe modified magnetic beads; use p50-capture probe modified magnetic beads to capture p50, and add 10uL, 45nM p50-masking chain, in Disperse culture at 37°C for 30 minutes, incubate and mix well, magnetically separate the suspension, and collect the supernatant;

[0030] (2) Isothermal amplification: mix the above supernatant with p50-template (100nm), 5u / μL Nt.BstNBI endonuclease, 0.8u / μL Bst 2.0 dna polymerase, 100μM dNTPs mixture and 1× Mix the constant temperature amplification buffer, react at ...

Embodiment 2

[0034] Embodiment 2 High selectivity experiment of detection method of the present invention to detect transcription factor

[0035] Bovine serum albumin (BSA), human serum albumin (HSA), thrombin, γ-interferon (IFN-γ), p53, AP-1, MITF, c-Myc were used as comparison samples, and the same concentration of The buffer solution is used as a blank sample, and the transcription factor p50 to be tested is detected by the detection method of the present invention, and the detection method comprises the following steps:

[0036] (1) Masking strand signal transduction: Dissolve the probe (p50-capture probe) capturing the transcription factor to be tested to 5 μM in citrate buffer, heat to 95°C, and cool to room temperature after 10 minutes (at least 1h), incubate with magnetic beads at 37°C for 30 minutes to obtain p50-capture probe-modified magnetic beads; use p50-capture probe-modified magnetic beads to capture p50, bovine serum albumin (BSA), human serum albumin (HSA ), thrombin (th...

Embodiment 3

[0041] Example 3 The detection method of the present invention is used to detect multiple transcription factors to be tested

[0042] Detection of comparative sample 1: comparative sample 1 is p53; the transcription factor p50 to be tested in Example 1 is replaced by p53, the p50-capture probe is replaced by p53-capture probe, and the p50-masked strand is replaced by p53-masked strand , the p50-template was replaced with a p53-template, and the rest of the steps and conditions were the same as in Example 1;

[0043] Detection of comparative sample 2: comparative sample 1 is p53; the transcription factor p50 to be tested in Example 1 is replaced by p53, the p50-capture probe is replaced by p53-capture probe, and the p50-masked strand is replaced by p53-masked strand , the p50-template was replaced with a p53-template, and the rest of the steps and conditions were the same as in Example 1;

[0044] Detection of comparative sample 3: comparative sample 1 is MITF; the transcripti...

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Abstract

The invention discloses a transcription factor multi-channel detection method combining a DNA nanotechnology and a liquid chromatography technology. The method comprises the following steps of firstly, capturing a transcription factor to be detected by using a magnetic bead modified by a probe with a captured to-be-detected transcription factor, combining the captured transcription factor to be detected with a corresponding masking chain sequence, and converting a signal of the transcription factor to be detected into release of a nucleic acid signal; then triggering multi-channel isothermal amplification by the nucleic acid signal under the action of a corresponding amplification template, Bst 2.0 DNA polymerase and Nt. BstNBI endonuclease to generate oligonucleotides with a plurality ofcodes as signal DNA; and finally, detecting the signal DNA by adopting high performance liquid chromatography to obtain the concentration of the transcription factor to be detected. The detection method disclosed by the invention is simple in process, can simultaneously detect a plurality of transcription factors to be detected at one time, realizes multi-target detection, obtains a good detectionresult in multi-channel detection, conveniently and reliably performs quantitative and qualitative analysis on DNA reports with different retention behaviors, and improves the detection efficiency.

Description

technical field [0001] The invention relates to the field of analytical technology detection, and more specifically, relates to a multi-path detection method for transcription factors combined with DNA nanotechnology and liquid chromatography technology. Background technique [0002] Transcription factors (Transcription factors, TFs) are a kind of regulatory proteins in cells, which initiate and regulate the transcription process of genes by combining with DNA cis-acting elements in genes. At the same time, a large number of studies have pointed out that the abnormal expression and activation of transcription factors are widely present in the pathological processes of the human body, including the occurrence and metastasis of cancer, viral infection, autoimmune diseases, etc. Therefore, transcription factors can be used not only as a potential diagnostic marker, but also as a target for clinical treatment. However, the traditional detection techniques for TFs are usually im...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12Q1/6844
CPCC12Q1/6844C12Q2545/114C12Q2537/143C12Q2565/137
Inventor 周学敏李昺之陈月严孝强朱婉莹王晶卢巧云洪俊丽
Owner NANJING MEDICAL UNIV
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