Transcription factor detection method based on DNA-Ag nanoclusters molecular beacons and exonuclease III cycle signal amplification

An exonuclease and transcription factor technology, used in measurement devices, analytical materials, biological tests, etc., can solve the problems of high cost, cumbersome steps, low sensitivity, etc., and achieve low detection limit, increased sensitivity, and high selectivity. Effect

Inactive Publication Date: 2017-07-21
NANJING MEDICAL UNIV
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  • Abstract
  • Description
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Problems solved by technology

[0004] The object of the present invention is to provide a kind of DNA-silver nanocluster molecular beacon (DNA-Ag nanoclustersmolecular beacons, AgMBs) as fluorescent probe, and by exonuclease III (Exonuclease III, ExoIII) assisted circulation signal amplified transcription A new method for factor detection, so as to achieve the purpose of fast, sensitive and high selectivity, and overcome the disadvantages of low sensitivity, time-consuming detection, high cost and cumbersome steps of existing transcription factor detection methods

Method used

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  • Transcription factor detection method based on DNA-Ag nanoclusters molecular beacons and exonuclease III cycle signal amplification
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  • Transcription factor detection method based on DNA-Ag nanoclusters molecular beacons and exonuclease III cycle signal amplification

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Embodiment 1

[0031] A method for detecting a transcription factor, comprising the following steps:

[0032] 1) Synthesis of AgMBs: AgMBs were synthesized according to the method provided in the literature. Specifically: Dissolve 1OD template DNA in 975μL buffer (10mM Na 2 HPO 4 / NaH 2 PO 4 ,100mMCH 3 COONa, 5mM Mg(CH3COO) 2 ,pH7.5), heated to 95°C, maintained for 10min, then slowly cooled to 37°C, and the duration of cooling was 1h. Then add 12 μL of freshly prepared 10 mM silver nitrate solution, vortex and mix, and react in the dark at 4°C for 30 min. Next, 12 μL of freshly prepared 10 mM sodium borohydride solution was added quickly, shaken and mixed for 1 min, and reacted overnight at 4°C in the dark.

[0033] 2) Preparation of dsNF-κB p50probe: Dissolve the lyophilized oligonucleotide with buffer (10mM Tris-Ac, 0.1mM EDTA, 100mM NaAc) to obtain 100μM reporter and s-reporter, and then vortex the two with equal volume Rotate, heat to 95°C, maintain for 10 minutes, then slowly co...

Embodiment 2

[0038] Selective experiment of detection method of the present invention

[0039] Repeat steps 1)-4) in Example 1, replace NF-κB p50 in step 3) with interfering substances such as bovine serum albumin, human insulin, human serum albumin, NF-κB p65, and keep other conditions unchanged , detect the fluorescence, and obtain the selectivity result graph of this method for detecting NF-κBp50, see image 3 .

Embodiment 3

[0041] The recovery rate experiment of detection method of the present invention

[0042] Repeat the steps 1)-4) of 1) in the embodiment, in step 4), carry out sample addition detection, add the NF-κB p50 of 10, 50, 200, 500, 1000, 1500pM respectively, detect, obtain this method The recovery rate in the actual sample detection, the results are shown in Table 2.

[0043] Table 2 The recovery rate of NF-κB p50 detected by this method

[0044]

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Abstract

The invention discloses a transcription factor detection method based on DNA-Ag nanoclusters molecular beacons and exonuclease III cycle signal amplification. The novel nanomaterial DNA-Ag nanoclusters molecular beacon is applied as a fluorescent probe to transcription factor detection for the first time. Also, based on the characteristic that fluorescence adjustment of DNA-Ag nanoclusters molecular beacons is carried out by an enzyme cleavable DNA fragment, exonuclease III is added to participate in signal transformation and also participate in enhancement of the detection signal. The transcription factor detection method provided by the invention has the advantages of no label, high selectivity, low cost and high sensitivity, and can realize high throughput detection of transcription factors in biological samples.

Description

technical field [0001] The invention belongs to the technical field of analysis and detection, in particular to a transcription factor detection method based on DNA-silver nanocluster molecular beacon and exonuclease III cycle amplification. 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. Studies suggest that transcription factors determine the on, off and expression levels of regulated genes, and thus play a vital role in gene expression. At the same time, a large number of studies have pointed out that the abnormal expression and activation of transcription factors widely exist in the pathological process of the human body. These pathological processes include cancer metastasis, viral infection, autoimmune diseases, etc. Therefore, transcription factors It can be used not only as a p...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): G01N33/68
CPCG01N33/6872
Inventor 周学敏李昺之徐磊陈月朱婉莹沈心洪俊丽
Owner NANJING MEDICAL UNIV
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