Method for detecting Dam methyltransferase activity based on base excision repair induction

A methyltransferase and detection method technology, applied in the field of biological detection, can solve the problems of reducing the sensitivity of the analysis method, cumbersome operation steps, long analysis time, etc., and achieve the effects of improving the detection sensitivity, good selectivity, and highly sensitive detection

Active Publication Date: 2019-01-22
LANZHOU UNIVERSITY
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Problems solved by technology

Although SDA-based colorimetric assays have been successfully used for the detection of methyltransferase activity, their sensitivity is relatively low
Compared with the SDA method, the sensitivity of the RCA method is relatively high, but the operation steps are cumbersome and the analysis time is long
Although the isothermal exponential amplification method based on restriction enzymes has high amplification efficiency, non-specific amplification is inevitable, resulting in increased background and reduced sensitivity of the analytical method

Method used

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  • Method for detecting Dam methyltransferase activity based on base excision repair induction
  • Method for detecting Dam methyltransferase activity based on base excision repair induction
  • Method for detecting Dam methyltransferase activity based on base excision repair induction

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Embodiment 1

[0033] Example 1 A method for detecting Dam methyltransferase activity based on base excision repair-induced strand displacement and isothermal exponential amplification fluorescence

[0034] Dam methyltransferase activity assay:

[0035] All nucleic acids were diluted to 100 μM with EDTA buffer before use, then the EXPAR template and signal probe were diluted to 1 μM and 10 μM with water, respectively, and the hairpin substrate and hairpin template were annealed in a 95°C water bath for 5 min, and then naturally cooled to room temperature so that the final concentrations were 5 μM and 1 μM, respectively.

[0036] Add 0.5 μM hairpin substrate, 1×dam buffer, Buffer, 160μM SAM, 10U Dpn I and different concentrations of Dam were reacted at 37°C for 2h and then inactivated at 80°C for 20min. Then, in a 20 μL reaction system, add 4 μL of the above methylated cleavage product, 50 nM hairpin template, 100 nM EXPAR template, 350 nM signal probe, 2.8U Bst DNA polymerase, 1U UDG, 5U ...

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Abstract

The present invention discloses a method for detecting Dam methyltransferase activity based on base excision repair-induced strand displacement amplification and exponential isothermal amplification reaction fluorescence. The method has simple operation steps, good selectivity and high sensitivity. Experimental results show that the linear range of determination of Dam methyltransferase is 0.02-10U/mL, and the detection limit is 0.014 U/mL. The method can also be used for detection of endogenous Dam methyltransferase in E. coli cells. The detection limit of Dam methyltransferase in E. coli is0.61*10<-6> mg/mL.

Description

technical field [0001] The invention belongs to the technical field of biological detection, and in particular relates to a method for detecting Dam methyltransferase activity based on base excision repair-induced strand displacement and isothermal exponential amplification fluorescence. Background technique [0002] DNA methylation is an important epigenetic phenomenon, which plays an important role in the process of cell proliferation, aging and gene expression. It regulates the function of cells by changing the expression of genes. Abnormal DNA methylation can lead to many cancers, such as breast cancer, ovarian cancer and lung cancer. The activity of methyltransferase determines the level of DNA methylation, and the abnormality of methyltransferase activity appears earlier than other cancer signs, so the activity of methyltransferase can be used as a biomarker for early diagnosis of cancer. Methyltransferase can recognize a specific DNA sequence and transfer the active ...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12Q1/48C12Q1/6844
CPCC12Q1/48C12Q1/6844C12Q2531/119C12Q2563/107
Inventor 张会鸽王莉莉陈宏丽
Owner LANZHOU UNIVERSITY
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