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Telomere length detecting method based on biological nano channel of aerolysin

An Aeromonas lysin and biological nano-channel technology, applied in the field of life science, can solve the problems of complex detection process, inability to directly measure telomere length, inability to detect the actual length of telomere, etc. The effect of low consumption and low price

Inactive Publication Date: 2016-05-04
EAST CHINA UNIV OF SCI & TECH
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

However, in actual operation, none of the above methods can directly measure the length of telomeres, neither can detect the actual length of telomeres, but also need to carry out enzyme digestion, molecular hybridization, fluorescent labeling and other operations, the detection process is relatively complicated

Method used

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  • Telomere length detecting method based on biological nano channel of aerolysin
  • Telomere length detecting method based on biological nano channel of aerolysin
  • Telomere length detecting method based on biological nano channel of aerolysin

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Embodiment 1

[0042] A method for detecting telomere length based on an aerolysin biological nanochannel, comprising the following steps:

[0043] (1) Open the G-quadruplex structure of telomeric DNA

[0044] The determined human telomere DNA is telomere 1——5'-TTAGGG-3'.

[0045]Telomere 1 was added to 1 mL of electrolyte solution, which was 1 M magnesium chloride (MgCl 2 ) and 10mM tris(hydroxymethyl)aminomethane buffer (Tris), pH=7.4, were mixed with a constant temperature magnetic stirrer at 25°C for 20min.

[0046] Since the formation of the G-quadruplex structure is cation-selective, different configurations will be formed in different kinds of alkali metal salt solutions: in the most commonly used potassium chloride (KCl) The formation of the conjoined structure can promote the formation, but this structure is not conducive to the precise detection of DNA by the nanochannel, and will also hinder the passage of DNA through the nanochannel.

[0047] Since the electrolyte solution use...

Embodiment 2

[0068] A method for detecting telomere length based on an aerolysin biological nanochannel, comprising the following steps:

[0069] (1) Open the G-quadruplex structure of telomeric DNA

[0070] Basic content is the same as embodiment 1. The difference is that the measured human telomere DNA is telomere 2——5’-(TTAGGG) 2 -3.

[0071] (2) Preparation of Aerolysin Bionanochannels

[0072] Basic content is the same as embodiment 1. The difference is: the electrolyte solution containing the telomere 2 is added to the detection cell A (cis end).

[0073] (3) Detection of telomere length by aerolysin bionanochannel

[0074] Basic content is the same as embodiment 1. The difference is: Example 2 is measured by the detection data analysis, the blocking time of telomere 2 is 3.0ms, and the blocking degree is 92% (see image 3 ).

Embodiment 3

[0076] A method for detecting telomere length based on an aerolysin biological nanochannel, comprising the following steps:

[0077] (1) Open the G-quadruplex structure of telomeric DNA

[0078] Basic content is the same as embodiment 1. The difference is that the measured human telomere DNA is telomere 3——5’-(TTAGGG) 3 -3.

[0079] (2) Preparation of Aerolysin Bionanochannels

[0080] Basic content is the same as embodiment 1. The difference is: the electrolyte solution containing the telomere 3 is added to the detection cell A (cis end).

[0081] (3) Detection of telomere length by aerolysin bionanochannel

[0082] Basic content is the same as embodiment 1. The difference is that in Example 3, the blocking time of telomere 3 is 6.0 ms and the blocking degree is 95% (see Figure 4 ).

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Abstract

The invention discloses a telomere length detecting method based on a biological nano channel of aerolysin. The method comprises the following steps: (1) opening the G-quadruplet structure of telomere DNA of human; (2) preparing a biological nano channel of aerolysin: (a) activating aerolysin; (b) preparing a phospholipid n-decane solution; (c) preparing phospholipid bilayer; (d) forming a biological nano channel of aerolysin; (3) using the biological nano channel of aerolysin to detect the telomere length: (a) collecting monomolecular signal data; (2) analyzing the monomolecular signal data. Compared with the prior art, the provided method has the advantages that the operation is simple and efficient; enzyme digestion, molecular hybridization, or fluorescence labeling is not needed; the consumption of DNA substrate is little; the detection sensitivity and resolution are high; the price is cheap; the absolute length of telomere can be precisely and directly measured in real time; and the method has a positive meaning for bioscience research.

Description

Technical field [0001] The DNA single -molecule detection technology involved in the field of life sciences is specifically a method based on the test telometer length based on gas monocylocycin -soluble biological nano channels. Background technique [0002] Telomey is a small DNA-protein complex located at the end of the chromosome, which has the effect of stable chromosomal end structure and controlling cell division cycle.The telomere DNA is a single-chain multi-repeated non-transcription sequence 5 '-(TTAGGG) n -3 '.Each cell is split, each chromosomal telomere will be missing for a period. When it is missing to a certain degree, the cell will activate the apoptosis mechanism, that is, the apoptosis.Studies have found that the length of the telomere has a close connection with the life and disease. Therefore, the length of measurement of telomeres is of great significance to studying life science. [0003] Biological nano channels are a single molecular detection method for ...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): G01N27/00
CPCG01N27/00
Inventor 龙亿涛曹婵廖冬芳
Owner EAST CHINA UNIV OF SCI & TECH
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