Triple-amplification-technology-based homogeneous-phase label-free method for detecting activity of telomerase

A technology of telomerase and amplification, applied in the field of telomerase activity, can solve problems such as high cost, cumbersome steps, and complicated operation

Active Publication Date: 2015-12-16
SHAANXI NORMAL UNIV
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  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0007] In order to overcome the defects of high cost, complicated operation, and cumbersome steps in the prior art, the present invention provides a non-labeled, non-enzyme-assisted signal amplification, and can be amplified without polymerase chain reaction, and has high sensitivity. Homogeneous non-standard detection method for telomerase activity based on triple amplification technology

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  • Triple-amplification-technology-based homogeneous-phase label-free method for detecting activity of telomerase
  • Triple-amplification-technology-based homogeneous-phase label-free method for detecting activity of telomerase
  • Triple-amplification-technology-based homogeneous-phase label-free method for detecting activity of telomerase

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Embodiment 1

[0029] Taking the detection of telomerase in HeLa (HeLa) cells as an example, combining figure 1 It can be seen that the detection method of the present embodiment is realized by the following steps:

[0030] (1) According to the conventional method, lyse the raised HeLa cells to extract telomerase, add 0.4 μL of 10 μmol / L telomerase substrate TS (sequence: : AATCCGTCGAGCAGAGTT) and the telomerase extract corresponding to 500 HeLa cells were incubated at 37°C for 1 hour to obtain the test solution system.

[0031] (2) Use a concentration of 50mmol / L, pH=7.4 and contain 5mmol / LMgCl 2 Tris-hydrochloric acid buffer solution will hairpin DNA probe H1 (sequence 5'-GGGATGGGTTAGGGCGGGAATCAGAGGGCGGGATGGGGATTCCCGCCCTAACCCTAACTC-3'), hairpin DNA probe H2 (sequence 5'-GGGTTGGGCGGGATGGGGATTAGGGTTAGGGCGGGAATCCCCATCCCGCCCTCTGA-3') and single-stranded A -DNA (5'-AACCCTAACCCTAACCCTAACTCTGCTC-3') and single strand (T-DNA5'-GAGTTAGGGTTAGGGCGGGAATC) were diluted to a concentration of 20 μmol / L...

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=7.4 2 ) with hairpin DNA probe H1 (sequence 5'-GGGATGGGTTAGGGCGGGAATCAGAGGGCGGGATGGGGATTCCCGCCCTAACCCTAACTC-3'), hairpin DNA probe H2 (sequence 5'-GGGTTGGGCGGGATGGGGATTAGGGTTAGGGCGGGAATCCCCATCCCGCCCTCTGA-3')...

Embodiment 3

[0047]The step (1) of the above-mentioned embodiment 1 or 2 is to lyse and extract telomerase from the cultured cells to be tested according to the conventional method, and add 0.4 μL of telomerase with a concentration of 10 μmol / L to 20 μL of the telomerase repeat amplification buffer solution. The telomerase substrate TS and the telomerase extract corresponding to 500 cells to be tested were incubated at 37° C. for 0.5 hour to obtain the test solution system. The H1 solution and H2 solution prepared in step (2) were placed in 1.5mL centrifuge tubes, heated to 95°C in a water bath for 5 minutes, naturally cooled to room temperature, and stored at 0°C for later use. Step (3) is to mix the A-DNA solution prepared in step (2) with the T-DNA solution in equal volumes to obtain the AT-DNA solution, which is placed in a 1.5mL centrifuge tube, heated to 95°C in a water bath for 5 minutes, Naturally cool to room temperature and store below 0°C for later use. Step (4) is to add 84 μ ...

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Abstract

The invention relates to a triple-amplification-technology-based homogeneous-phase label-free method for detecting the activity of telomerase. The method is characterized in that a telomerase extension product is combined with accessory DNA to release triggering DNA, and the triggering DNA can trigger a strand displacement reaction to form a hairpin H1:H2 compound; G-quadruplexes can be formed at the two tail ends of the compound respectively, fluorescence signals of NMM are weak and are remarkably enhanced after the G-quadruplexes are embedded and inserted, and the activity of the telomerase can be sensitively detected by detecting changes of the fluorescence signals of the NMM. According to the method, the strand displacement reaction is sufficiently adopted, and signal amplification can be achieved without assisting of enzymes; meanwhile, formation of the G-quadruplexes is ingeniously adopted, the method in which the activity of the telomerase can be sensitively detected without labeling is constructed, a new concept is provided for diagnosis and treatment of tumor diseases and researching of disease mechanisms, and the method is of great significance in the telomerase-based cancer treatment and diagnosis aspect.

Description

technical field [0001] The invention belongs to the technical field of tumor activity detection, and in particular relates to a simple, fast and low-cost method for detecting telomerase activity in cancer cells based on a strand substitution-assisted triple amplification technology. Background technique [0002] Telomere is a "cap-shaped" structure contained at the end of chromosomes in eukaryotic cells, which can prevent chromosome degradation, end-to-end fusion, recombination and chromosome loss, thereby protecting the stability of chromosome ends. Under normal circumstances, due to the end replication problem of chromosomes, telomeres lose 50 to 150 base pairs every time a cell divides. As the number of cell divisions increases, the ends of telomere DNA shorten. When shortened to a certain extent, the cells enter a dangerous period and eventually lead to cell death. Telomerase is a ribonucleoprotein reverse transcriptase, mainly composed of RNA template (hTR) catalytic s...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12Q1/68C12Q1/48
CPCC12Q1/485C12Q1/6844G01N2333/9128C12Q2525/151C12Q2525/301C12Q2537/1373C12Q2563/107
Inventor 金燕高艳芳
Owner SHAANXI NORMAL UNIV
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