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Method for separating DNA (deoxyribonucleic acid) binding protein and accurately positioning DNA binding site

A technology for binding proteins and binding sites, which is applied in the field of DNA binding proteins, can solve the problems of complex operation, low efficiency, and unnecessary use, and achieve the effect of simple method and short cycle

Inactive Publication Date: 2015-06-24
CAPITAL UNIVERSITY OF MEDICAL SCIENCES
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Problems solved by technology

[0003] The intracellular method is suitable for high-throughput screening to find unknown genes and proteins because it is an intracellular screening, which is in line with the physiological state. However, it can only screen for proteins that can specifically bind to the promoter DNA, but cannot detect the precise protein binding site. point, and its specificity needs to be further verified
Extracellular methods can detect precise protein binding sites, but are inefficient and complicated to operate, and are generally not used to find unknown genes and proteins

Method used

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  • Method for separating DNA (deoxyribonucleic acid) binding protein and accurately positioning DNA binding site
  • Method for separating DNA (deoxyribonucleic acid) binding protein and accurately positioning DNA binding site
  • Method for separating DNA (deoxyribonucleic acid) binding protein and accurately positioning DNA binding site

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Embodiment 1

[0042] Example 1 Isolation of hCLP46 gene promoter CpG island binding protein and localization of binding site

[0043] 1. Amplification of hCLP46 promoter region

[0044] Apply Primer Premier 5.0 to design primers (Table 1), 100 μL reaction system contains 10 μL of 10×PCR Buffer, 8 μL of 2.5mM dNTP mix, 4.0 μL of 10uM upstream and downstream primers, 2.0 μL of U937 DNA, 1.0 μL of 5U / μL Taq (TakaRa ) and 71.0 μL ddH2O. After the mixture was mixed and centrifuged, the amplification reaction was performed on a PCR machine (95°C for 3 min; 95°C for 30 s, 59°C for 30 s, 72°C for 1 min, 45 cycles; 72°C for 5 min; 4°C forever).

[0045] Table 1 hCLP46 promoter amplification primers

[0046]

[0047]

[0048] 2. U937 cell cytoplasmic protein and nuclear protein extraction

[0049] U937 cells were cultured according to the above method, and nuclear and cytoplasmic proteins were extracted using a nuclear and cytoplasmic protein extraction kit (Nanjing Kaiji). The simple steps ...

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Abstract

The invention provides a method for separating DNA (deoxyribonucleic acid) binding protein and accurately positioning DNA binding site, which comprises the following steps: DNA binding protein separation: carrying out PCR (polymerase chain reaction) amplification on a DNA segment to be researched by using biotin-labeled primers, sequentially adding nucleoprotein or cytoplast protein and avidin-coated magnetic beads, co-incubating, washing off nonspecific binding protein, and adding SDS (sodium dodecylsulfate) to denature and release the protein; and protein DNA binding site positioning: by using the DNA segment to be researched as a template, carrying out PCR reaction by using different primers to obtain overlapping DNA segments, and positioning the DNA binding site of the isolated protein according to the affinity difference of the overlapping DNA segments for the isolated protein.

Description

technical field [0001] The present invention relates to DNA-binding proteins, in particular, to a method for isolating DNA-binding proteins and precisely locating their DNA-binding sites. Background technique [0002] The current research methods on promoter DNA-binding proteins can be divided into two categories. The first type is the intracellular method, which uses the known promoter DNA sequence to screen the protein-coding gene that binds to it, and determines the protein through bioinformatics analysis, such as yeast one hybrid system (yeast one hybrid system, Y1H), phage Display technology (phage display, PD), etc. The second type is the extracellular method, that is, the recombinant known protein is combined with the promoter DNA in vitro, such as gel retardation experiment, DNase I footprint experiment and so on. [0003] The intracellular method is suitable for high-throughput screening to find unknown genes and proteins because it is an intracellular screening, ...

Claims

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Application Information

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IPC IPC(8): C07K1/14C12Q1/68
Inventor 王友信王嵬
Owner CAPITAL UNIVERSITY OF MEDICAL SCIENCES
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