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Fluorescence detection method for detecting desoxyribonucleic acid binding protein

A deoxyribonucleic acid and protein-binding technology, which is applied in the fields of fluorescence/phosphorescence, biological testing, and material inspection products, can solve the problems of complicated experiments, contamination of radioactive substances, and low analysis efficiency, and achieve high selectivity and sensitivity, shorten The effect of detection time and high analysis efficiency

Inactive Publication Date: 2012-03-21
NANJING UNIV OF POSTS & TELECOMM
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0003] There are many methods currently used to study the interaction between DNA and proteins, such as gel mobility analysis, blotting, etc. Although these conventional methods have played an important role in the study of DNA-binding proteins, these techniques The workload is heavy, time-consuming and labor-intensive, and it is easy to cause radioactive material contamination during the operation
In recent years, scientists have successively reported some detection methods based on fluorescent dyes, such as molecular beacons (Boon, E.M.; Salas, J.E.; Barton, J.K., Nat.Biotech.2002, 20(3):282-286.), Fluorescence resonance energy transfer (Xu, X.; Zhao, Z.; Qin, L.; Wei, W.; Levine, J.E.; Mirkin C.A., Anal.Chem.2008, 80(14):5616-5621.), etc.; And based on the colorimetric detection of gold nanoparticles (Ou, L.J.; Jin, P.Y.; Chu, X.; Jiang, J.H.; Yu, R.Q., Anal.Chem.2010, 82, (14), 6015-24.) etc., these The method effectively avoids the contamination of radioactive substances, but because these fluorescent probes need to be double-labeled, the experiment is complicated and the analysis efficiency is low.

Method used

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  • Fluorescence detection method for detecting desoxyribonucleic acid binding protein
  • Fluorescence detection method for detecting desoxyribonucleic acid binding protein
  • Fluorescence detection method for detecting desoxyribonucleic acid binding protein

Examples

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Effect test

preparation example Construction

[0040] a) Preparation of fluorescein-labeled nucleic acid probes

[0041] NF-κB nucleic acid probe:

[0042] A: 5′-AGTTGA GGGGACTTTCC CAGGCTTTTT-3′

[0043] B: 3′-TCAACTC CCCTGAAAGG GTCCG-FAM-5′

[0044] Where ______ is the binding site of NF-κB, and FAM is fluorescein.

[0045] When preparing NF-κB nucleic acid probes, mix equimolar solutions of two oligonucleotide chains A and B in hybridization buffer (10mM Tris-HCl, 100mM NaCl, pH 8.0), incubate at 95°C for 10 minutes, and cool slowly to room temperature. Then the probe solution was diluted with hybridization solution to a concentration of 10 μM, and stored at 4° C. for future use.

[0046] b) The labeled nucleic acid probe binds to nuclear factor NF-κB

[0047] Add nuclear factor NF-κB protein to deoxyribose nucleic acid binding buffer, incubate at 37°C for 10 minutes, then add 10 μM nucleic acid probe, and incubate at 37°C for 20-30 minutes.

[0048] c) Exonuclease III digestion

[0049] The 10× digestion reac...

Embodiment 1

[0052] Example 1 Detection of nuclear factor NF-κB protein.

[0053] Detection 1: Add nuclear factor NF-κB protein to deoxyribonucleic acid binding buffer, incubate at 37°C for 10 minutes, then add 10 μM double-stranded nucleic acid probe, and incubate at 37°C for 20-30 minutes. Add 10× enzyme digestion reaction solution and 20U / μL exonuclease III to the reaction system, and incubate at 37°C for 5-10 minutes. After the enzyme cleavage reaction, add 0.4M ethylenediaminetetraacetic acid solution to terminate the enzyme cleavage reaction of exonuclease III, and then add it to the detection buffer containing water-soluble conjugated polymer, the detection excitation wavelength is 404nm, and the emission The wavelength is 410-650nm. Since the labeled nucleic acid probe will undergo a sequence-specific binding reaction with the nuclear factor protein NF-κB, the formed "nucleic acid probe / deoxyribonucleic acid binding protein" complex provides space protection for the nucleic acid p...

Embodiment 2

[0060] Example 2 detects different concentrations of nuclear factor protein NF-κB.

[0061] Different concentrations of nuclear factor NF-κB protein were detected, and other conditions were the same as detection 1 in Example 1.

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Abstract

Provided is a fluorescence detection method for detecting desoxyribonucleic acid binding protein. The method utilizes double-strand nucleic acid containing protein binding locus and marked by fluorescein as a probe, the probe is bound with target protein to form a nucleic acid probe / desoxyribonucleic acid binding protein composite which provides protection space for the nucleic acid probe, so that the nucleic acid probe can be prevented from being hydrolyzed by nuclease, the nucleic acid probe can be bound with cationic water-soluble conjugated polymer through a static reaction, the distance between the fluorescein and the polymer is small, and high-efficient fluorescence resonance energy transfer can be generated. Based on the water-soluble conjugated polymer and exonuclease III, the method includes the following steps: a, preparing the double-strand desoxyribonucleic acid fluorescence probe capable of binding protein with specificity; b, binding the double-strand desoxyribonucleic acid fluorescence probe with the desoxyribonucleic acid binding protein; c, enzyme restriction can be conducted on the composite through exonuclease III; and d, adding the water-soluble conjugated polymer into the reaction system for fluorescent detection.

Description

technical field [0001] The invention belongs to the field of biological analysis and sensing, in particular to a fluorescence detection method for detecting deoxyribonucleic acid (DNA) binding protein based on water-soluble conjugated polymer and exonuclease III. Background technique [0002] Deoxyribonucleic acid binding protein is a kind of protein in the biological proteome that can bind to sequence-specific deoxyribonucleic acid and produce important functions. This type of protein plays an important role in cellular processes such as gene regulation, gene recombination, and gene repair. , has become an important object of functional genomics and proteomics research; many diseases are associated with abnormalities in intracellular deoxyribonucleic acid binding proteins. The detection and analysis of deoxyribonucleic acid binding protein is the main means to study its function. Therefore, research on detection and analysis of DNA-binding proteins has always been highly v...

Claims

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Application Information

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IPC IPC(8): G01N33/68G01N21/64
Inventor 刘兴奋黄维欧阳斓黄艳琴
Owner NANJING UNIV OF POSTS & TELECOMM
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