Method for identification of gene promoter region related transcription factor gene

A technology for gene promoter regions and transcription factors, which is applied in the field of identifying transcription factor genes related to gene promoter regions, can solve problems such as multiple labor costs, costs of reagents, consumables, complicated operations, etc., and achieves enhanced accuracy and convenience. Results with credible effects

Inactive Publication Date: 2017-07-07
GUIZHOU UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

At present, methods for identifying the interaction between promoters and transcription factors include yeast one-hybridization, chromatin immunoprecipitation, electrophoretic mobility shift analysis, etc.; however, the above methods are relatively complicated to operate and require more labor and reagents and consumables.

Method used

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  • Method for identification of gene promoter region related transcription factor gene
  • Method for identification of gene promoter region related transcription factor gene
  • Method for identification of gene promoter region related transcription factor gene

Examples

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Embodiment 1

[0020] Embodiment 1 of the present invention: The method for identifying transcription factor genes related to gene promoter regions is specifically as follows:

[0021] 1. Preparation of strains and reagents

[0022] Strain: Escherichia coli competent cell DH5α was purchased from Dalian Takara Company;

[0023] Reagent source: high-fidelity DNA polymerase, liposome 2000 was purchased from Invitrogen, USA, dNTP Mix was purchased from Takara, Dalian, T 4 DNA Ligase was purchased from Dalian Takara Company, DNA Marker was purchased from Dalian Takara Company; fetal bovine serum, DMEM high glucose medium, OPTI-MEM, pancreatin were purchased from gibco Company, and C2C12 cells were purchased from Shanghai Cell Bank of Chinese Academy of Sciences.

[0024] The RNA extraction kit was purchased from Omega, the column-type plasmid DNA small-amount extraction kit, the column-type DNA gel recovery kit was purchased from (Axygen), and the endotoxin-free plasmid extraction kit (QIAGEN).

[0025] Ot...

Embodiment 2

[0126] Example 2 of the present invention: The method, experimental method and procedure for identifying the transcription factor genes related to the gene promoter region are the same as those in the example, verifying the MyoD1 gene promoter and the transcription factor SP1 gene, and constructing and obtaining the pEGFP-N3-MyoD1-SP1 recombinant expression vector , Screened out positive clones, verified by sequencing, and transfected C2C12 cells with the correct vector, and no green fluorescence signal was observed by fluorescence microscope. The experimental results proved that the SP1 gene is not a transcription factor that binds to the MyoD1 gene promoter.

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Abstract

The invention discloses a method for identification of a gene promoter region related transcription factor gene. According to the invention, amplified transcription factor gene fragment and vector-promoter fragment amplified with high-fidelity DNA polymerase are subjected to blunt-end ligation so as to construct recombinant plasmid. The method can be adopted to simply and accurately obtain a recombinant vector and will not be limited by any restriction enzyme cutting sites. By the method, any related transcription factor gene fragments which need research and verification can be accurately connected. Accuracy and convenience of the experimental result are enhanced, and the experimental result is more credible.

Description

Technical field [0001] The present invention relates to the field of biotechnology, in particular to a method for identifying transcription factor genes related to gene promoter regions. Background technique [0002] In addition to RNA polymerase, DNA transcription also requires a variety of cofactors. These cofactors are called transcription factors, which can recognize and bind to the cis-acting site of DNA, thereby regulating the expression of the target gene at a specific time and space with a specific intensity. Therefore, studying the interaction between transcription factors and promoters is of great significance for understanding the function of promoters. The current methods for identifying the combination of promoters and transcription factors include yeast one-hybrid, chromatin immunoprecipitation, electrophoretic mobility change analysis, etc.; however, the above methods are relatively complicated to operate and require more manpower and reagents and consumables. . ...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12Q1/68C12N15/85C12N15/65
CPCC12N15/65C12N15/85C12Q1/6897
Inventor 许厚强周迪陈琨陈伟赵佳福段志强王圆圆
Owner GUIZHOU UNIV
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