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Method for detecting telomerase activity with colorimetric method based on strand displacement reaction

A technology of telomerase and colorimetry, which is applied in the field of detecting telomerase activity in cancer cells, can solve the problems of low practicability, complicated operation, complicated steps, etc., achieve reliable visual detection, and realize visual detection. Effect

Inactive Publication Date: 2016-11-23
SHAANXI NORMAL UNIV
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  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

[0007] In order to overcome the defects of high cost, complex operation, cumbersome steps, and poor practicability in the prior art, the present invention provides a method for detecting telomerase activity by a colorimetric method based on strand substitution reaction, which does not require polymerase chain The reaction can be visualized and sensitively detects telomerase activity, and the operation is simple and fast

Method used

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  • Method for detecting telomerase activity with colorimetric method based on strand displacement reaction
  • Method for detecting telomerase activity with colorimetric method based on strand displacement reaction
  • Method for detecting telomerase activity with colorimetric method based on strand displacement reaction

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Embodiment 1

[0027] Taking the detection of telomerase in HeLa (HeLa) cells as an example, the detection method of this embodiment is realized by the following steps:

[0028] (1) Lyse the raised HeLa cells according to the conventional method to extract telomerase, add 0.4 μL of 10 μmol / L telomerase substrate TS (sequence: AAT CCG TCG AGCAGA GTT) and the telomerase extract corresponding to 500 HeLa cells were incubated at 37°C for 1 hour to obtain the test solution system.

[0029] (2) Tris-hydrochloric acid buffer solution (containing 5mmol / LMgCl) with a concentration of 50mmol / L and pH=7.0 2 ) hairpin DNA probe H1 (sequence is 5'-GGGATGGGTTAGGGCGGGAATCAGAGGGCGGGATGGGGATTCCCGCCCTAACCCTAACTC-3'), hairpin DNA probe H2 (sequence is 5'-GGGTTGGGCGGGATGGGGATTAGGGTTAGGGCGGGAATCCCCATCCCGCCCTCTGA-3') and single-stranded A-DNA (5'-AGCCCTAACCTTAACCCTA '), single-stranded T-DNA (sequence 5'-GAGTTAGGGTTAGGGCGGGAATC) were diluted to a concentration of 20 μmol / L to prepare H1 solution, H2 solution, A-...

Embodiment 2

[0039] Taking the detection of telomerase in human acute lymphoblastic leukemia T lymphocytes (CCRF-CEM) as an example, the detection method of this embodiment is realized by the following steps:

[0040] (1) Lyse and extract telomerase from raised human acute lymphoblastic leukemia T lymphocytes (CCRF-CEM) according to conventional methods, and add 0.4 μL to 20 μL of telomerase repeat amplification buffer solution at a concentration of 10 μmol / L The telomerase substrate TS and the active telomerase extract corresponding to 500 human acute lymphoblastic leukemia T lymphocytes (CCRF-CEM) were incubated at 30° C. for 2 hours to obtain the test solution system.

[0041] (2) Tris-hydrochloric acid buffer solution (containing 5mmol / LMgCl) with a concentration of 50mmol / L and pH=7.0 2 ) hairpin DNA probe H1 (sequence is 5'-GGGATGGGTTAGGGCGGGAATCAGAGGGCGGGATGGGGATTCCCGCCCTAACCCTAACTC-3'), hairpin DNA probe H2 (sequence is 5'-GGGTTGGGCGGGATGGGGATTAGGGTTAGGGCGGGAATCCCCATCCCGCCCTCTGA-3'...

Embodiment 3

[0049] Taking the detection of telomerase in lung cancer cells (SCLC) as an example, the detection method of this embodiment is realized by the following steps:

[0050] (1) Lyse and extract telomerase from raised lung cancer cells (SCLC) according to conventional methods, add 0.4 μL of 10 μmol / L telomerase substrate TS and The active telomerase extract corresponding to 500 lung cancer cells (SCLC) was incubated at 35° C. for 0.5 hour to obtain the test solution system.

[0051] (2) Tris-hydrochloric acid buffer solution (containing 5mmol / LMgCl) with a concentration of 50mmol / L and pH=7.0 2 ) hairpin DNA probe H1 (sequence is 5'-GGGATGGGTTAGGGCGGGAATCAGAGGGCGGGATGGGGATTCCCGCCCTAACCCTAACTC-3'), hairpin DNA probe H2 (sequence is 5'-GGGTTGGGCGGGATGGGGATTAGGGTTAGGGCGGGAATCCCCATCCCGCCCTCTGA-3') and single-stranded A-DNA (sequence is 5'-AA CCCTAACCCTAACCCTAACTCTGCTC-3'), single-stranded T-DNA (sequence is 5'-GAGTTAGGGTTAGGGCGGGAATC) were diluted to a concentration of 20 μmol / L, res...

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Abstract

The invention relates to a method for detecting telomerase activity with a colorimetric method based on strand displacement reaction. According to the method, in the presence of active telomerase, hybridization is performed on a prolonging product of a substrate with assistant DNA so as to release a plurality of triggering DNAs, the triggering DNAs can trigger hairpin DNA probes H1 and H2 to have self-assembled strand displacement reaction, then G-4 chains are respectively formed at two tail ends of H1:H2, hemin reacts with the G-4 chains, thus G-4 chain DNA enzyme with catalytic activity can be formed, a TMB-H2O2 system is catalyzed to have reaction, the color of a solution is changed into blue from colorless, and the solution can be oxidized into yellow if acid is added; due to addition of active telomerase, the color of the solution can be changed, visible detection on telomerase activity can be achieved, meanwhile an ultraviolet absorption spectrum of the solution can be tested, and the telomerase activity can be more accurately and sensitively tested.

Description

technical field [0001] The invention belongs to the technical field of tumor activity detection, and in particular relates to a simple and rapid colorimetric method for detecting telomerase activity in cancer cells based on chain substitution reaction enhanced catalytic enzyme action. Background technique [0002] Telomere is a special structure consisting of telomere DNA and telomere binding protein that exists at the end of linear chromosomes in eukaryotic cells. Telomere DNA mainly includes a G-rich double-stranded part composed of several thousand bases and a G-rich The 3'overhang end, which sits like a tall hat on the chromosome head, is called the clock of life. Every time a normal cell replicates, the telomere will be shortened by 50-200bp bases. When the telomere is shortened to a certain extent, the chromosome will be unstable and the cell will tend to age. Telomerase is a ribonucleoproteinase mainly composed of RNA template (hTR) catalytic subunit (hTERT) and telo...

Claims

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Application Information

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IPC IPC(8): C12Q1/68C12Q1/48
CPCC12Q1/6853C12Q1/48C12Q2565/519
Inventor 金燕高艳芳
Owner SHAANXI NORMAL UNIV
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