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Recombinant protein structural domain and coding DNA thereof, enhanced TET enzyme and whole genome DNA methylation detection method

A recombinant protein and detection method technology, applied in the biological field, can solve the problems of large DNA damage, low accuracy and high background noise

Active Publication Date: 2021-11-12
YEASEN BIOTECHNOLOGY (SHANGHAI) CO LTD
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

This method suffers from large DNA damage, high background noise and low accuracy

Method used

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  • Recombinant protein structural domain and coding DNA thereof, enhanced TET enzyme and whole genome DNA methylation detection method
  • Recombinant protein structural domain and coding DNA thereof, enhanced TET enzyme and whole genome DNA methylation detection method
  • Recombinant protein structural domain and coding DNA thereof, enhanced TET enzyme and whole genome DNA methylation detection method

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0041] Example 1: Test of the specific activity of each recombinant protease of MBD-TET.

[0042] Epigentek's Epigenase 5mC-hydroxylase TET activity / inhibition assay kit (fluorescence method) was used to measure the enzyme specific activity of each MBD-TET recombinant protein according to the procedure in the manual.

[0043] The schematic diagram of the modified enzyme activity is shown in figure 1 , see the result figure 2 , TET enzymes fused with MBD can significantly enhance the activity of each TET enzyme.

Embodiment 2

[0044] Example 2: Determination of the oxidation ability of each recombinant protease of MBD-TET to 5mC oligo.

[0045] In this embodiment, the ratio of the oxidation product of MBD-TET enzyme to 5mC is determined, and the specific implementation method is as follows:

[0046] Table 1

[0047] components Dosage 5mC oligo 1-100ng 10×TET Enzyme Reaction Buffer 3 μL 10 μM each TET enzyme 2-10 μL Add ddH 2 O to

30μL

[0048] 10×TET enzyme reaction buffer contains 1-100 mM 3-(N-morpholino)propanesulfonic acid sodium salt, 1-100 mM bis(2-hydroxyethyl)amino-tris(hydroxymethyl)methane, 1 -100 mM hydroxyethylpiperazine ethanesulfonic acid, 1-300 mM sodium chloride, 0.1-10 mM ascorbic acid, 0.1-10 mM citric acid, 0.1-20 mM α-ketoglutarate, 0.1-20 mM 1,3- Acetone dicarboxylic acid, 0.1-20 mM adenosine triphosphate, 0.1-10 mM tetrafluoro-p-benzoquinone, 0.1-10 mM tetrachloro-p-benzoquinone, 0.1-10 mM tetrabromo-p-benzoquinone, 0.1-10 mM te...

Embodiment 3

[0054] Example 3: A simple high-throughput sequencing method based on MBD1-NgTET1.

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PUM

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Abstract

The invention provides a recombinant protein structural domain for enhancing TET enzyme activity. The recombinant protein structural domain is a DNA methylation binding structural domain (MBD), and the amino acid sequence of MBD1-4 is as shown in SEQ ID 1-4. The recombinant protein MBD-TET formed by fusing the MBD and the TET enzyme can be used for remarkably enhancing the oxidation activity of the TET enzymes, including NgTET1, mTET1CD, mTET2CDT, hTET1CD and hTET2CDT, on 5mC. In addition, the invention further provides a simple, convenient and rapid high-throughput DNA methylation detection process, and the sensitivity and accuracy of a TET enzymatic oxidation reaction-based DNA methylation detection technology are improved.

Description

technical field [0001] The patent of the invention relates to the recombinant protein domain and its coding DNA, enhanced TET enzyme and whole genome DNA methylation detection method, belonging to the field of biotechnology. Background technique [0002] DNA cytosine methylation (5mC) is the most common base modification in DNA, accounting for about 1%-8% of all cytosines, and is called the "fifth base". DNA methylation is significantly correlated with chromatin state and gene transcription activity, and is an effective basis for predicting gene expression levels. Therefore, the detection of DNA methylation level is an effective means for clinical disease diagnosis. The existing DNA methylation detection technology mainly relies on the bisulfite conversion method of reverse screening. The principle is to use bisulfite to convert unmethylated cytosine into uracil, and then amplify it by PCR. into thymine. This method suffers from large DNA damage, high background noise, an...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C07K14/00C12N15/11C12Q1/6886C07K19/00C12N9/10
CPCC07K14/00C12N9/1014C12Q1/6886C12Q2600/154C07K2319/00Y02P10/20Y02P20/55
Inventor 侯策韦磊江翱陈晶晶黄开喻滕以刚曹振宋东亮
Owner YEASEN BIOTECHNOLOGY (SHANGHAI) CO LTD
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