Double electrical signal and DNA circulating amplification technique-based Dam methyltransferase activity detection method

A methyltransferase and cyclic amplification technology, applied in the field of electrochemical sensing, can solve problems such as insufficient, and achieve the effects of good selectivity, high sensitivity and low detection limit

Inactive Publication Date: 2015-08-12
NANCHANG UNIV
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  • Application Information

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Problems solved by technology

The method of detecting methyltransferase activity and screening methyltransferase inhibitors based on

Method used

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  • Double electrical signal and DNA circulating amplification technique-based Dam methyltransferase activity detection method
  • Double electrical signal and DNA circulating amplification technique-based Dam methyltransferase activity detection method
  • Double electrical signal and DNA circulating amplification technique-based Dam methyltransferase activity detection method

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Embodiment 1

[0022] (1) Electrode pretreatment: the gold electrode is freshly prepared with H at a volume ratio of 7:3 2 SO 4 :H 2 o 2 Soak in the solution for 20 minutes, rinse the electrode with ultrapure water, and then polish the suede with 0.3 μm and 0.05 μm alumina powder until the surface of the gold electrode is mirror-like, and place the electrodes in HNO with a volume ratio of 1:1. 3 :H 2 O, ethanol, and ultrapure water were sonicated for 1 minute, and the cleaned electrode was placed in 0.1 M H 2 SO 4 Perform cyclic voltammetry scans in the solution in the potential range from -0.2 V to +1.55 V until a stable cyclic voltammetry peak is obtained;

[0023] (2) Preparation of ferrocene-DNA modified gold electrode: activate thiolated DNA with 100 μL of 1 mM dithiothreitol solution for 3 hours and pass through the column; soak the gold electrode treated in step (1) in 50 μL of 1 μM In the activated thiolated DNA solution, react at room temperature for 16 hours, wash the electro...

Embodiment 2

[0028] Detection of Dam methyltransferase activity

[0029] (1) Optimization of SAM concentration, Dpn I concentration, methylation reaction time, and Exo III reaction time

[0030] image 3 A is the effect of different SAM concentrations on the detection of Dam. It can be seen from the figure that in the presence of Dam methyltransferase, the ratio of the current intensity of ferrocene to G-quadruplex gradually decreases with the increase of SAM concentration, and tends to be stable when the SAM concentration reaches 80 μM. Therefore, the optimal concentration of SAM is 80 μM. image 3 B is the effect of Dpn I concentration on Dam methyltransferase. It can be seen from the figure that in the presence of Dam methyltransferase, as the concentration of Dpn I increases, the ratio of the current intensity of ferrocene to G-quadruplex gradually decreases, and when the concentration of Dpn I reaches 80 U mL -1 stabilized afterwards. Therefore, the optimal concentration of Dpn I...

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Abstract

The invention discloses a double electrical signal and DNA circulating amplification technique-based Dam methyltransferase activity detection method, and belongs to the technical field of electrochemical sensing. After methylation of hairpin-shaped DNA containing methylation sites under the effect of Dam methyltransferase and cutting by DpnI endonuclease, double-stranded DNA is formed by hybridization of released single stranded DNA and protruding parts of dicyclopentadienyl iron-DNA on the electrode surface, and then is cut into DNA fragments by endonuclease III, so that dicyclopentadienyl iron leaves away from the electrode surface so as to reduce the current, current of G-four chain body-heme complex formed by the the single stranded DNA on the electrode surface and heme is enhanced, and Dam methyltransferase activity sensitive detection can be realized by proportion of current intensity of the two.

Description

technical field [0001] The invention relates to a method for detecting Dam methyltransferase activity based on dual electric signal and DNA cycle amplification technology, belonging to the technical field of electrochemical sensing. Background technique [0002] DNA methylation plays an important role in human gene expression. In prokaryotes, DNA methylation has important links with DNA repair, gene expression regulation, and cell cycle regulation. In eukaryotes, DNA methylation affects a series of biological and physiological processes such as cell differentiation, gene expression, and chromosome structure formation. The methylation process of DNA is realized under the action of DNA methyltransferase, which catalyzes the methyl donor S-adenosylmethionine (SAM) to transfer the methyl group from SAM to adenosine or pyrimidine. Aberrant DNA methylation can lead to disruption of normal gene expression and cellular function. Studies have shown that the methylation of promoter...

Claims

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Application Information

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IPC IPC(8): G01N27/26G01N27/327
Inventor 邱建丁李志坚梁汝萍
Owner NANCHANG UNIV
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