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DNA methylation real-time detection method and application thereof

A real-time detection and methylation technology, applied in the field of electrochemical detection, can solve problems such as simultaneous amplification of difficult signals, and achieve the effect of reducing the cost of measurement, shortening the analysis time, and realizing immediacy.

Active Publication Date: 2017-04-05
SUN YAT SEN UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

(2) It is difficult to achieve synchronous amplification of signals

Method used

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  • DNA methylation real-time detection method and application thereof
  • DNA methylation real-time detection method and application thereof
  • DNA methylation real-time detection method and application thereof

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Experimental program
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Embodiment 1

[0039]After the probe molecule containing the methylation site is labeled with mercaptoferrocene, it is co-modified with mercaptohexanol on the surface of the gold electrode, and hybridizes with complementary DNA to form a long-range electron transfer pathway. Under the catalysis of DNMT3a, after the probe DNA is methylated, a bromine derivatization reaction occurs immediately, and a bromine atom is bonded to the C4 position of methylcytosine. Under the joint action of the bromine atom and the methyl group, the The long-range electron transfer ability of double-stranded DNA is obviously reduced. Gaussian calculations confirmed that the reduction of the long-range electron transport ability comes from the influence of bromine on the distribution of π electron cloud in the region.

[0040] 1. Preparation of electrochemical biosensors

[0041] (1) Gold electrode pretreatment

[0042] After soaking the gold electrode with a hydrogen peroxide / sulfuric acid mixture with a volume r...

Embodiment 2

[0060] The technical scheme of the present invention will be further described below by taking the methylation site 5'-CCGG-3' of DNA methyltransferase DNMT3a as an example.

[0061] After the probe molecule containing a methylation site (5'-CCGG-3') is labeled with mercaptoferrocene, it is co-modified with mercaptohexanol on the surface of the gold electrode, and hybridizes with complementary DNA to form a long-range electron transfer pathway. Under the catalysis of DNMT3a, after the probe DNA is methylated, a bromine derivatization reaction occurs immediately, which significantly reduces the long-range electron transfer ability of the double-stranded DNA. Gaussian calculations confirmed that the reduction of the long-range electron transport ability comes from the influence of bromine on the distribution of π electron cloud in the region.

[0062] 1. Fabrication of Electrochemical Sensors

[0063] (1) Gold electrode pretreatment

[0064] The gold electrode (Φ=3mm) was imme...

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Abstract

The invention discloses a DNA methylation real-time detection method and an application thereof. Based on DNA double-stranded long-range electron transfer characteristic, a specific chemical derivative reaction is combined, a rapid and simple DNA methylation dynamic process real-time electrochemical detection method and an electrochemical biological sensor are established, which can be used for real-time detection of a DNA methylation process, enzyme inhibitor screening and enzyme sequence preference research.

Description

technical field [0001] The invention belongs to the technical field of electrochemical detection, in particular to a DNA methylation real-time detection method based on the bromine atom dilution DNA long-range electron transfer principle and its application. Background technique [0002] DNA methylation refers to the transfer of a methyl group to the C5 position of cytosine by catalyzing the substrate S-adenosylmethionine (SAM) under the action of methyltransferases (MTases) to generate 5-methylcytosine Pyrimidine (5-mC) process. As one of the important epigenetic modification methods, DNA methylation is closely related to embryonic development, chromosome structure, and gene imprinting. DNA methylation is a dynamic process, abnormal expression of MTases usually causes abnormal methylation modification, and is associated with various human diseases. Therefore, an in-depth understanding of the DNA methylation process and the function of MTases is of great significance in di...

Claims

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Application Information

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IPC IPC(8): G01N27/26G01N27/327
CPCG01N27/26G01N27/3278
Inventor 戴宗张立邹小勇
Owner SUN YAT SEN UNIV
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