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A method to study the sequence preference of tet2

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

Active Publication Date: 2022-07-12
SUN YAT SEN UNIV
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  • Abstract
  • Description
  • 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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  • A method to study the sequence preference of tet2
  • A method to study the sequence preference of tet2
  • A method to study the sequence preference of tet2

Examples

Experimental program
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Effect test

Embodiment 1

[0053] Analysis of sequence binding preference of Tet2

[0054] The binding preference of Tet2 for the four most commonly methylated DNA sequences was investigated. Based on the different preferences of Tet2 for the flanking sites of the action site, different sequences bind different amounts of proteins, resulting in different DNA polymerase extension rates and different fluorescence amplification curves during the sequence amplification process.

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

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

[0057] 10mM HEPE solution, 200nM Fe solution were added to the reaction tube. 2 SO 4 solution, 4mM ascorbic acid, 2mM α-ketoglutarate, 150mM NaCl, 1mM ATP, 168nM Tet2 protein, continue to add 1×Cutsmart buffer (20mM Tris-acetate, 50mM potassium acetate, 10mM mag...

Embodiment 2

[0063] Oxidation preference of Tet2 for different sequences

[0064] The DNA sequence after Tet2 oxidation was modified by glycosylation, and 3-carboxyphenylboronic acid (3-CPBA) was further used to condense the cis-diol on the sugar ring to obtain the modified site CPBA-5gmC, which contained 3CPBA. - The sequence of the gmC site and the unmodified DNA sequence were amplified respectively, 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. The amplification reaction conditions were optimized first.

[0066] (a) Template dosage

[0067] Prepare template solutions of different concentrations to optimize the optimal amount of template. The reaction was prepared in two parts, Part A and Part B. Part A incl...

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Abstract

The invention discloses a method for studying the sequence preference of Tet2, which is characterized in that: the sequence preference of Tet2 is studied by the difference of amplification signals. Based on the isothermal amplification technology, the invention amplifies the signal of the slight change of the flanking position sequence of the Tet2 action site, and detects the difference in the content of the main oxidation product, so as to realize the research on the sequence preference behavior of Tet2 in the process of active demethylation. It has the following characteristics: high-throughput detection, which can realize the simultaneous detection of multiple samples; low detection cost and does not depend on expensive and complex large-scale instruments; good biocompatibility and will not cause damage to protein or DNA samples; Convert differences in protein behavior into differences in amplification signals.

Description

technical field [0001] The invention relates to the field of biotechnology, in particular to a method for researching Tet2 sequence preference. Background technique [0002] Dioxygenated protease 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 demethylation of DNA can regulate the overall level of 5mC in the genome or specific gene segments, revealing that DNA methylation is a reversible and dynamically changing modification state. [0003] It is reported in the literature that Tet2 catalyzes almost only the oxidation of 5mC at the CpG site on the DNA sequence, which means that a base downstream of the methylation site has a significant impact on the recognition and catalysis of Tet2. Ther...

Claims

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Application Information

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Patent Type & Authority Patents(China)
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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