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Method for detecting DNA (deoxyribonucleic acid) binding protein

A technology of binding protein and detection method, which is applied in the field of detection of DNA binding protein, and can solve the problem of low sensitivity

Active Publication Date: 2013-12-25
深圳柏垠生物科技有限公司
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

However, the sensitivity of traditional DNA-binding protein detection methods is low

Method used

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  • Method for detecting DNA (deoxyribonucleic acid) binding protein
  • Method for detecting DNA (deoxyribonucleic acid) binding protein
  • Method for detecting DNA (deoxyribonucleic acid) binding protein

Examples

Experimental program
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Embodiment 1

[0059] Preparation of the gold nanoparticle solution: the gold nanoparticle solution was prepared by reducing chloroauric acid with sodium citrate. 3.7mL of HAuCl with a mass concentration of 1% 4 Add the aqueous solution to 90mL water and heat to boiling, then quickly add 9mL sodium citrate aqueous solution with a mass concentration of 1%, and continue boiling for 15 minutes.

[0060] Preparation of nano-gold detection probes: two different sulfhydryl-modified oligonucleotides were used to modify gold nanoparticles. 5.2 nanomoles of probe 1 (sequence shown in SEQ ID No.3) and 5.2 nanomoles of probe 2 (sequence shown in SEQ ID No.4) were added together in 2.5 ml of nano-gold solution After standing at room temperature for 16 hours, add 0.1 mole per liter of phosphate buffer (pH7.0) dropwise to adjust to a final concentration of 10 mmol per liter, and simultaneously add NaCl to adjust to a concentration of 0.1 mole per liter, Stand at room temperature for 40 hours. Then at 4...

Embodiment 2

[0068] 1. Characteristics of gold nanoparticles and DNA-modified gold nanoparticles

[0069] In order to obtain uniformly sized gold nanoparticles, the particle size of the synthesized gold nanoparticles observed by a scanning transmission electron microscope is about 14 nanometers, almost all round, with good dispersion, and the results are as follows image 3 shown.

[0070] 2. Feasibility experiment of colorimetric detection of NF-κB p50 based on amplification of gold nanoparticles

[0071] To determine whether amplification-based gold nanocolorimetry can detect DNA-binding proteins, we chose the NF-κB p50 transcription factor as a model for detection. NF-κB p50 binds to DNA, and after DNA exonuclease III cleavage and isothermal amplification at 40°C, it is first verified by 14% non-denaturing polyacrylamide gel electrophoresis whether there is a 24nt oligonucleotide sequence. The result is as Figure 4 It shows that 24nt oligonucleotide sequence can be produced when NF-κB...

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Abstract

The invention discloses a method for detecting DNA (deoxyribonucleic acid) binding protein. The method comprises the following steps: providing a sample solution of the DNA binding protein to be detected, and carrying out digestion reaction under the condition that Exo III exists, thus obtaining a digestive juice after complete reaction; adding the digestive juice in an amplification system, carrying out isothermal amplification reaction under the condition that a polymerase, an incision enzyme and an amplification template exist to obtain an amplification solution; mixing a gold nanoparticle solution with a solution containing a first detection probe and a second detection probe and then carrying out incubation to obtain a nano gold detection probe solution; mixing the amplification solution with the nano gold detection probe solution and then detecting a mixed solution to complete the detection for the DNA binding protein. The first detection probe and the second detection probe are combined with a target DNA in an amplification product so that gold nanoparticles of the detection probes gather to cause that the color of the mixed solution changes, thus the method is visual in phenomenon and high in sensitivity.

Description

technical field [0001] The invention relates to the field of gene detection, in particular to a detection method of DNA binding protein. Background technique [0002] Simple and sensitive detection of transcription factors in human cells is of great significance for clinical diagnosis and drug screening. The current detection methods are all based on electrophoretic migration, immunochemical or fluorescence detection methods, which are directly detected by non-amplification, so the sensitivity is low. Moreover, these methods have radioactive contamination or require specific antibodies and fluorescently labeled probes, which greatly increase the cost and complexity of the experiment. [0003] DNA-binding proteins play crucial roles in genome replication, gene transcription, cell division and DNA repair processes. Most of the DNA-binding proteins play the role of transcription factors, regulating cell development, differentiation and proliferation, so transcription factors ...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): G01N33/68G01N33/52
Inventor 张春阳张艳
Owner 深圳柏垠生物科技有限公司
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