Single-molecule analysis method for detecting methylated DNA

An analytical method and methylation technology, applied in the direction of electrochemical variables of materials, etc., can solve the problems of complexity, high technical requirements, poor reproducibility of results, etc., and achieve the effect of small sample volume, high sensitivity, and simple and easy operation.

Active Publication Date: 2016-03-02
NORTHWEST UNIV
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  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

All of the above methods are very complicated, the results ar

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  • Single-molecule analysis method for detecting methylated DNA
  • Single-molecule analysis method for detecting methylated DNA
  • Single-molecule analysis method for detecting methylated DNA

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Embodiment 1

[0046] Specifically, the detection of methylated hairpin DNA by the present invention comprises the following steps:

[0047] (1) Install the pre-punched Telfon membrane in the center of the electrolytic cell, divide the electrolytic cell into two parts, and drop a mixture of pentane and hexadecane containing 1% hexadecane around the hole with a capillary tube. Add 4M tetramethylammonium chloride, 10mM Tris-HCl, pH8.5 buffer solution into the tank, add a small amount of phospholipid dropwise above the liquid surface, add buffer solution to make the liquid surface cross the small hole, and form a stable phospholipid bilayer. The electrolytic cell is cis-added with wild-type α-hemolysin, which is inserted into the phospholipid bilayer to form a nano single channel.

[0048] (2) Add the hairpin DNA containing 0 / 1 / 2 methylated cytosine in the stem region in cis to the electrolytic cell, stir evenly, and detect the DNA current signal under positive voltage (+40mV~+120mV). The tail...

Embodiment 2

[0053] Potassium chloride is a traditional nanopore single-molecule detection electrolyte, similar to Example 1, under the condition that other experimental conditions are kept constant, the detection results of methylated hairpin DNA in potassium chloride electrolyte are as follows figure 2 as shown, figure 2 -a, b, c are schematic diagrams of DNA detection principle (left), single-channel current signal diagram (right). figure 2 -a is hairpin DNA that does not contain methylated cytosine; figure 2 -b is hairpin DNA containing 1 methylated cytosine; figure 2 -c is hairpin DNA containing 2 methylated cytosines; figure 2 -d is the distribution statistical graph of the residence time of the three kinds of hairpin DNA.

[0054] First of all, in the KCl electrolyte, the difference in the retention time of the three hairpin DNAs is only ~10ms, which is much smaller than the ~2600ms in the tetramethylammonium chloride electrolyte; and, the retention time of the hairpin DNA ...

Embodiment 3

[0056] Detection of methylated single-stranded DNA includes the following steps:

[0057] (1) Hairpin probe DNA and target DNA were mixed at a concentration ratio of 1:1, heated at 95°C for 5 minutes, and cooled naturally.

[0058] (2) Install the pre-punched Telfon membrane in the center of the electrolytic cell, divide the electrolytic cell into two parts, and drop a mixture of pentane and hexadecane containing 1% hexadecane around the hole with a capillary tube. Add 4M Tetramethylammonium Chloride, 10mM Tris-HCl, pH8.5 buffer solution into the tank, add a small amount of phospholipid dropwise above the liquid surface, add buffer solution to make the liquid surface submerge the small holes, and form a stable phospholipid bilayer. The electrolytic cell is cis-added with wild-type α-hemolysin, which is inserted into the phospholipid bilayer to form a nano single channel.

[0059] (3) Add the mixture of hairpin probe DNA and target DNA containing 0 / 1 / 2 methylated cytosine in c...

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Abstract

The invention relates to a single-molecule analysis method for detecting methylated DNA. According to the method, in an electrolytic tank containing a tetramethyl ammonium salt electrolyte or a tetraethyl ammonium salt electrolyte or a tetrapropyl ammonium salt electrolyte or a tetrabutyl ammonium salt electrolyte, a nanopore sensor distinguishes and detects DNA of different methylation levels through current electrochemical signals, wherein DNA of different methylation levels includes un-methylated DNA, mono-methylated DNA and multi-methylated DNA, and the structural forms of DNA include a single-stranded structural form, a double-stranded structural form and a hairpin structural form. According to the single-molecule analysis method for detecting methylated DNA, the quantity of needed samples is small, sensitivity is high, and single-base resolution is achieved; nanopore and DNA modification, DNA amplification and marking are not needed, operation is easy, and high applicability is achieved.

Description

technical field [0001] The invention relates to a single-molecule analysis method for detecting methylated DNA, belonging to the technical field of single-molecule analysis. Background technique [0002] Nanopore sensors can realize single-molecule detection at the micro / nano scale, and have unique value and significant advantages in the fields of single-molecule analysis and single-molecule chemical reaction research. Traditional nanopore sensors use potassium chloride or sodium chloride as electrolytes. When electrophoresis drives analyte molecules to pass through the nanopore channel, it will cause an instantaneous blocking effect on the ion flow in the channel. According to the amplitude, residence time and frequency of the current blockage, information such as the concentration and chemical structure of the measured sample can be obtained. Nanopore technology has made great progress as a low-cost and high-efficiency DNA detection technology. At present, nanopore senso...

Claims

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Application Information

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IPC IPC(8): G01N27/26
Inventor 亢晓峰王莹
Owner NORTHWEST UNIV
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