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Research method of Tet2 sequence preference

A preference and sequence technology, applied in the research field of Tet2 sequence preference, can solve problems such as the influence of antibody affinity and specificity, subtle changes cannot provide effective quantitative analysis data, etc., and achieve low detection cost, simple operation, and biological good compatibility

Active Publication Date: 2019-09-17
SUN YAT SEN UNIV
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  • Abstract
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  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Electrophoresis-based gel shift assay (EMSA) technology is mainly used to study the interaction between DNA-binding proteins and their related DNA-binding sequences. This method is simple and intuitive, but it is only suitable for qualitative or semi-quantitative use and requires radioactive isotopes. Markers, cannot provide effective quantitative analysis data for small changes that occur at the site of action ±1 or ±2
Chromosomal immunoprecipitation (ChIP) can obtain DNA-protein interaction information, but is easily affected by antibody affinity and specificity

Method used

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  • Research method of Tet2 sequence preference
  • Research method of Tet2 sequence preference
  • Research method of Tet2 sequence preference

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0053] Tet2 pair sequence binding preference analysis

[0054] The binding preference of Tet2 to the four most commonly methylated DNA sequences was investigated. Based on Tet2's different preference for the flanking position of the action site, different sequences bind different amounts of protein, which leads to different extension rates of DNA polymerases and differences in fluorescence amplification curves during the sequence amplification process.

[0055] The effect of Tet2 concentration on the sequence amplification reaction from low to high, respectively 27.9, 55.9, 112, 168 and 224nM was studied.

[0056] (1) Isothermal amplification reaction to detect the effect of Tet2 on different sequences

[0057] 10mM HEPE solution, 200nM Fe 2 SO 4 Solution, 4mM ascorbic acid, 2mMα-ketoglutarate, 150mMNaCl, 1mM ATP, 168nM Tet2 protein, continue to add 1×Cutsmart buffer (20mM Tris-acetate, 50mM potassium acetate, 10mM magnesium acetate, 0.1mg mL –1 BSA, pH 7.9), 125 μM dNTPs mi...

Embodiment 2

[0063] Study on the Oxidation Preference of Tet2 to Different Sequences

[0064] Carry out glycosylation modification on the DNA sequence after Tet2 oxidation, and further use 3-carboxyphenylboronic acid (3-CPBA) to condense with the cis-ortho-diol on the sugar ring to obtain the modified site CPBA-5gmC, which will contain 3CPBA The sequence of the -gmC site and the unmodified DNA sequence were amplified separately, and the amplification efficiency of the two was compared to determine the oxidation preference of Tet2.

[0065] The amplification reaction system includes two parts, A and B, wherein part A contains nickase buffer, dNTPs, primers and templates, and part B contains polymerase buffer, nucleic acid dye, polymerase and nickase. Optimize the amplification reaction conditions first.

[0066] (a) template usage

[0067] Prepare template solutions with different concentrations to optimize the optimal template dosage. The reaction is divided into two parts, Part A and P...

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Abstract

The invention discloses a study method of Tet2 sequence preference, and the study method is characterized in that the Tet2 sequence preference is studied by amplified signal differences. According to the invention, small changes of Tet2 action site flanking sequences are performed with signal amplification based on the isothermal amplification technique, differences of main oxidation product contents are detected, so as to realize the study on sequence preference behaviors of Tet2 in the active demethylation process. The study method has the following characteristics: high-throughput detection, capable of achieving simultaneous detection of multiple samples; low detection cost, independent of expensive and complex large-scale instruments; good biocompatibility, unable to cause damage to protein or DNA samples; and simple operation, capable of achieving the transformation of protein behavior differences into amplified signal differences.

Description

technical field [0001] The invention relates to the field of biotechnology, in particular to a research method for Tet2 sequence preference. Background technique [0002] Dioxyprotease Ten-eleven translocation (Tet) can oxidize 5mC in DNA to 5hmC under the action of α-ketoglutarate (α-KG), ferrous ions, oxygen, etc., and then continue to oxidize to 5fC and 5caC, Among them, 5fC and 5caC can be recognized and removed by TDG protein, and finally restored to cytosine through the base repair pathway. Tet-mediated active DNA demethylation can regulate the overall level of 5mC in the genome or specific gene fragments, revealing that DNA methylation is a reversible and dynamically changing modification state. [0003] It has been reported that Tet2 almost only catalyzes the oxidation of 5mC at the CpG site on the DNA sequence, which means that a base downstream of the methylation site has a major impact on Tet2 recognition and catalysis. Therefore, the bases at the ±1 or ±2 posit...

Claims

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Application Information

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IPC IPC(8): C12Q1/26
CPCC12Q1/26G01N2333/90245
Inventor 戴宗陈丹萍邹小勇
Owner SUN YAT SEN UNIV
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