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A kind of detection method of telomerase activity

A detection method and telomerase technology, applied in the biological field, can solve the problems of complicated methods, undetectable telomerase activity, and difficulty in handling pollutants, and achieve the effect of avoiding false positive results.

Active Publication Date: 2011-11-30
CHANGZHOU INST OF ENERGY STORAGE MATERIALS &DEVICES
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Moreover, the primer dimer can also form an artificial product (artifact) through the elongation mechanism of the above-mentioned staggered annealing with CX, and the artificial product also appears as a ladder with an interval of 6 bp after PCR amplification, which is indistinguishable from the real amplified product. Distinguish, causing false positive results
[0008] Thirdly, when using the TRAP method to detect the activity of telomerase with poor continuous synthesis ability, its short end is not conducive to PCR amplification, so the telomerase activity cannot be detected, which limits the practical application effect of TRAP reaction
[0009] In addition, the detection of telomerase activity by TRAP requires electrophoresis, silver staining or radioactive isotope labeling. The method is very complicated and time-consuming, and the use of radioactive isotopes is harmful to a certain extent, and the treatment of pollutants is difficult, which limits the application of TRAP.

Method used

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  • A kind of detection method of telomerase activity
  • A kind of detection method of telomerase activity
  • A kind of detection method of telomerase activity

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0053] 2×10 6 293T cells plus CHAPS lysate (10mmol / L Tris-HCl, pH 7.5, 1mmol / L MgCl 2 , 1mmol / L EGTA, 0.1mmol / L PMSF, 5mmol / L mercaptoethanol, 0.5%w / v CHAPS (3-[(3-cholamidopropyl)-diethylammonium]-propanesulfonic acid), 10%w / v glycerol) 200 μL, lyse in an ice bath for 30 min, during which the pipette was repeatedly sucked 3 times to ensure full lysis. Centrifuge at 14000rpm for 20min, absorb the supernatant and store it at -80°C for later use.

[0054] Extension reaction system: 50 μL system, including 10×TRAP buffer (200mmol / L Tris-HCl, pH 8.3, 15mmol / L MgCl 2 , 1% Tween-20, 630mmol / L KCl, 10mmol / L EGTA) 5μL, dATP, dTTP and dGTP each 50μmol / L, TS primer 150nmol / L, add sterile DEPC water to 46μL, add 2μL telomerase The extract was used as a positive extension, and redistilled water was added as a negative extension, and incubated at 25°C for 20 minutes, and the telomerase extension product was synthesized by reverse transcription.

[0055] Pipette 1 μL of the extension pro...

Embodiment 2

[0062] 2×10 6 Hella cells plus CHAPS lysate (10mmol / L Tris-HCl, pH 7.5, 1mmol / L MgCl 2 , 1mmol / L EGTA, 0.1mmol / L PMSF, 5mmol / L mercaptoethanol, 0.5%w / v CHAPS (3-[(3-cholamidopropyl)-diethylammonium]-propanesulfonic acid), 10%w / v glycerol) 200 μL, lyse in an ice bath for 30 min, during which the pipette was repeatedly sucked 3 times to ensure full lysis. Centrifuge at 14000rpm for 20min, absorb the supernatant and store it at -80°C for later use.

[0063] Extension reaction system: 50 μL system, including 10×TRAP buffer (200mmol / L Tris-HCl, pH 8.3, 15mmol / L MgCl 2 , 1% Tween-20, 630mmol / L KCl, 10mmol / L EGTA) 5μL, dATP, dTTP and dGTP each 50μmol / L, TS primer 150nmol / L, add sterile DEPC water to 46μL, add 2μL telomerase The extract was used as a positive extension, and redistilled water was added as a negative extension, and incubated at 25°C for 20 minutes, and the telomerase extension product was synthesized by reverse transcription.

[0064] Pipette 1 μL of the extension ...

Embodiment 3

[0070] 2×10 6 293T cells plus CHAPS lysate (10mmol / L Tris-HCl, pH 7.5, 1mmol / L MgCl 2 , 1mmol / L EGTA, 0.1mmol / L PMSF, 5mmol / L mercaptoethanol, 0.5%w / v CHAPS (3-[(3-cholamidopropyl)-diethylammonium]-propanesulfonic acid), 10%w / v glycerol) 200 μL, lyse in an ice bath for 30 min, during which the pipette was repeatedly sucked 3 times to ensure full lysis. Centrifuge at 14000rpm for 20min, absorb the supernatant and store it at -80°C for later use.

[0071] Extension reaction system: 50 μL system, including 10×TRAP buffer (200mmol / L Tris-HCl, pH 8.3, 15mmol / L MgCl 2 , 1% Tween-20, 630mmol / L KCl, 10mmol / L EGTA) 5μL, dATP, dTTP and dGTP each 50μmol / L, TS primer 150nmol / L, add sterile DEPC water to 46μL, add 2μL telomerase The extract was used as a positive extension, and redistilled water was added as a negative extension, and incubated at 25°C for 20 minutes, and the telomerase extension product was synthesized by reverse transcription.

[0072] Pipette 1 μL of the extension p...

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Abstract

The invention relates to the field of biotechnology, specifically to a telomerase activity detection method. The detection method comprises the following steps of: acquiring active telomerase from a sample to be measured, extending TS primers to obtain a telomerase extension product, using the telomerase extension product as a template, carrying out gap-ligase chain reaction amplification in the presence of four primers to obtain an amplification product, mixing the amplification product and a molecular beacon to react, and carrying out a fluorescence detection to obtain the telomerase activity. The detection method provided by the invention has high sensitivity and good specificity, and can be used to effectively detect the activity of telomerase in the sample to be measured.

Description

technical field [0001] The invention relates to the field of biotechnology, in particular to a detection method for telomerase activity. Background technique [0002] Telomerase is a ribonucleoprotein, which contains protein and its own RNA template complementary to telomere DNA, and catalyzes the synthesis and maintenance of a certain sequence of telomeres. Telomeres play an important role in maintaining chromosome stability and cell activity in cells of different species. Telomerase can extend shortened telomeres (shortened telomeres have limited cell replication ability), thereby enhancing the proliferation ability of cells in vitro. It plays an important role in maintaining telomere stability, genome integrity, long-term cell activity and potential continued proliferation. [0003] However, in normal human cells, the activity of telomerase is quite tightly regulated, and active telomerase can only be detected in hematopoietic cells, stem cells and germ cells, which must...

Claims

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Application Information

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IPC IPC(8): C12Q1/68C12Q1/25
Inventor 于聪张玉静焦虎平王斌廖冬丽王方远杨越李文英张青峰陈健刘丹
Owner CHANGZHOU INST OF ENERGY STORAGE MATERIALS &DEVICES
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