Quantitative analysis method aiming at DNA methylation monitoring

A quantitative analysis and methylation technology, applied in the direction of material electrochemical variables, etc., can solve the problems of large consumption of monomer samples and complicated detection methods, and achieve low cost, reduced time-consuming experiments, and low consumption Effect

Inactive Publication Date: 2014-09-24
FUZHOU UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0004] Aiming at the existing problems of complicated DNA methylation detection methods and large consumption of monomer samples, the present invention proposes a sensitive, portable, easy-to-operate electrochemical quantitative monitoring method based on a modification-free ITO microchip.

Method used

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  • Quantitative analysis method aiming at DNA methylation monitoring
  • Quantitative analysis method aiming at DNA methylation monitoring
  • Quantitative analysis method aiming at DNA methylation monitoring

Examples

Experimental program
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Embodiment 1

[0027] Example 1 Fabrication of ITO Microelectrode Chips

[0028] Use CorelDraw12 software to draw the two-dimensional structure of the chip and make a mask. Use soft lithography technology to transfer the pattern to the ITO glass plate, develop and remove the exposure layer, and then place the chip in the acid etching solution (HCl:H2O:NHO3=50:50:3) for wet etching for 45 minutes, namely The ITO working electrode structure of the chip can be obtained, and the remaining photoresist is removed. Photoetching the reference electrode and counter electrode pattern on the ITO glass plate again, after developing and removing the photoresist of the exposure layer, metal sputtering and sputtering metal platinum layer, the platinum reference electrode and counter electrode structure of the chip can be obtained. And remove the remaining photoresist. Rinse the microchip with secondary water and blow dry with nitrogen. Soak the prepared ITO microchip in Alconox cleaning solution (10 ...

Embodiment 2

[0029] Example 2 Monitoring of DNA methylation process

[0030] with 10 mM Tris-HCl buffer (containing 10 mM MgCl 2 , 50 mM NaCl, and pH 7.5) as a solvent mix 1 μM hairpin DNA, 160 μM SAM (methylated adenosine), 1 mM DTT (dithiothreitol), 50 U / mL Dam MTase (methyltransferase) and 20 U / mL of Dpn I enzyme. React at a constant temperature of 37°C, take out 2μL samples every 10 minutes and drop them on the working unit of the ITO microelectrode chip, use the electrochemical workstation to collect the DPV differential pulse electrical signal, the level of the DPV electrical signal indicates DNA methyl The level of DNA methylation can be monitored in real time. image 3 It is a schematic diagram of the principle of the mixed reaction, that is, the hairpin DNA will first co-react with SAM and Dam MTase to convert into methylated hairpin DNA, and the generated DNA methylation site will be cleaved by Dpn I enzyme , so that the fragmented nucleotide chain with methylene blue is close...

Embodiment 3

[0031] Example 3 Screening of DNA Methylation Inhibiting Drugs

[0032] with 10 mM Tris-HCl buffer (containing 10 mM MgCl 2, 50 mM NaCl, and pH 7.5) as a solvent mix 1 μM hairpin DNA, 160 μM SAM (methylated adenosine), 1 mM DTT (dithiothreitol), 50 U / mL Dam MTase (methyltransferase) and drug candidates. React in a water bath at 37°C for 2 hours to complete DNA methylation. Then add (20 U / mL) Dpn I enzyme into the sample and react for another 2 hours (37°C). Finally, 2 μL of sample was taken out and dropped onto the working unit of the ITO microelectrode chip, and the electrochemical workstation was used to collect the DPV differential pulse electrical signal. If the DPV electrical signal is strong, it means that the inhibitory effect of the drug on DNA methylation is not obvious; otherwise, better DNA methylation inhibitory drugs can be screened. According to the designed protocol, DNA probes (sequence: 5'-AG GATC CCGC TTCT TTTG AAGC GGGA TCCTC-3') were used to detect amox...

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Abstract

The invention discloses a quantitative analysis method aiming at DNA methylation monitoring. The quantitative analysis method comprises the following steps: (1) designing a hairpin type DNA secondary structure according to a DNA methylation palindromic sequence, and modifying a methylene blue electroactive material on the structure; (2) reacting hairpin type DNA with transmethylase and methylation adenosine to synthesize a methylated hairpin type DNA; (3) performing specific enzymolysis on a DNA methylation site through DpnI enzyme to release DNA fragments connected with methylene blue; (4) performing sample monitoring through an ITO micro-electrode chip with negative electricity, and acquiring electrochemical signals through a multi-pass electric signal reading device; (5) recording a monitoring result; and (6) adding methylation suppression chemicals into a DNA methylation process to screen the DNA methylation suppression chemicals. The quantitative analysis method has the advantages of simplicity in operation, portability, low cost and the like, and an electrode does not need complicated modification.

Description

technical field [0001] The invention relates to an electrochemical quantitative analysis method for monitoring DNA methylation level, belonging to the technical field of electrochemical quantitative analysis methods. Background technique [0002] DNA methylation refers to the transfer of methyl groups from adenosylmethionine to adenine or cytosine nucleotides with special sequence DNA under the catalysis of methyltransferase, which is commonly found in gene 5'-CpG- 3', 5'-G-A-T-C-3' sequences, etc., form 5'-mCpG-3' and 5'-G-mA-T-C-3' respectively after methylation. In epigenetics research, DNA methylation is one of the earliest gene modification pathways discovered. DNA methylation plays a protective role in prokaryotic / eukaryotic organisms, and affects mammalian gene transcription and replication, as well as embryo formation and development. For humans, DNA methylation aberrations can inhibit DNA transcription, making DNA expression out of control, and causing a series of...

Claims

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Application Information

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IPC IPC(8): G01N27/26G01N27/30
Inventor 林振宇魏晓峰马小明郭隆华邱彬陈国南
Owner FUZHOU UNIV
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