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Method for determining transcriptional control complex

A technology of transcriptional regulation and complex, which is applied to the measurement/testing of microorganisms, biochemical equipment and methods, etc., can solve the problems of simultaneous detection of protein-nucleic acid interaction, inability to fully respond to information, incompatibility, etc., to achieve Alleviation of incompatibility, improvement of measurement efficiency, and large library capacity

Active Publication Date: 2015-07-15
HUAZHONG AGRI UNIV
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Problems solved by technology

[0008] Various existing technologies for detecting interaction have their unique advantages and application ranges, but for protein interaction and nucleic acid protein interaction, the detection systems are independent and incompatible, that is, they cannot be combined in one Simultaneously detect protein-protein interaction and protein-nucleic acid interaction in the same system
Existing research methods have certain limitations when constructing multi-component interactome networks such as transcriptional regulatory networks, which combine protein interactions (transcription factors forming complexes) with nucleic acid-protein interactions (transcription factors and DNA binding sites ) to separate the research, the information obtained cannot fully reflect the real situation, so it is necessary to establish a new analysis method

Method used

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  • Method for determining transcriptional control complex
  • Method for determining transcriptional control complex
  • Method for determining transcriptional control complex

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Embodiment 1

[0071] Example 1: Solid Phase Screening

[0072] An embodiment of the method for determining the transcriptional regulatory complex of the present invention, the method for determining the transcriptional regulatory complex described in this embodiment comprises the following steps: 1. Preparation of a bait DNA sequence; 2. Preparation of a DNA sequence encoding a barcode protein; 3. . Construction of barcoded protein library; 4. Solid-phase screening of transcriptional regulatory complexes; 5. In situ proximity ligation capture of transcriptional regulatory complexes; 6. Identification of transcriptional regulatory complexes. Specific steps are as follows:

[0073] 1. Preparation of bait DNA sequence

[0074] Firstly, the double-strand DNA was obtained by overlapping extension PCR. Add 1 μL MDF, 1 μL MDR, 23 μL H 2O, 25 μL premix (Takara), annealing temperature of 60 ° C, 8 cycles, 2% agarose gel detection, PCR products were purified using a High pure PCR product purificat...

Embodiment 2

[0125] Example 2 Liquid Phase Screening

[0126]An embodiment of the method for determining the transcriptional regulatory complex of the present invention, the method for determining the transcriptional regulatory complex described in this embodiment comprises the following steps: 1. Preparation of a bait DNA sequence; 2. Preparation of a DNA sequence encoding a barcode protein; 3. . Construction of barcoded protein library; 4. Liquid-phase screening of transcriptional regulatory complexes; 5. Proximity junction capture of transcriptional regulatory complexes; 6. Identification of transcriptional regulatory complexes. Wherein, steps 1, 2, 3, and 6 are respectively the same as steps 1, 2, 3, and 6 in Example 1 (solid-phase screening), and there are differences in steps 4, 5, and steps 4, 5 are as follows:

[0127] 4. Liquid Phase Screening of Transcription Regulatory Complexes

[0128] 1) Millproe 3K ultrafiltration tube replacement buffer: add 9.6mL pre-cooled transcription ...

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Abstract

The invention relates to a method for determining a transcriptional control complex, and belongs to the field of molecular biology. The method for determining the transcriptional control complex includes the following steps: preparation of a bait DNA sequence, preparation of a DNA sequence for encoding a bar code protein, construction of a bar code protein library, screening of the transcriptional control complex, and in situ adjacent connection capture of the transcriptional control complex, and the identification of the transcriptional control complex. The invention combines cDNA display technology and the adjacent connection technology to establish a novel method for determining the transcriptional control complex; the determination of transcriptional control complex involves simultaneous determination of a variety of interaction effects of protein-protein and protein-DNA; and the method can realize simultaneous determination of a variety of interaction effects of protein-protein and protein-DNA in a same experimental system, and effectively solve the incompatibility of the determination methods for the protein-protein interaction and protein-DNA interaction in the same experimental system in the prior art.

Description

technical field [0001] The invention relates to a method for measuring the transcription regulation complex, in particular to a method for simultaneously measuring the transcription regulation complex in the same experimental system. Background technique [0002] A cell is an ordered basic unit of life activities formed by the interactions between different biomolecules, and the occurrence and regulation of major life activities in cells are completed through the interactions between molecules. With the advent of the post-genome era, interactomics, which focuses on the dynamic interrelationships between the components of life endome, has become one of the hotspots in systems biology research today. However, simple pairwise interaction research does not conform to the dynamic characteristics of life. The interactome network should be studied systematically from an overall perspective. Therefore, in-depth research on the interactions between molecules, especially the use of hi...

Claims

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Application Information

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IPC IPC(8): C12Q1/68
CPCC12Q1/6853C12Q2531/113C12Q2549/119
Inventor 张红雨康寿凯
Owner HUAZHONG AGRI UNIV
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