Detection probe, kit and direct detection method of telomerase activity

A technology for detecting probes and telomerase, which is used in biochemical equipment and methods, microbial determination/inspection, DNA/RNA fragments, etc., to achieve real-time detection, unique optoelectronic characteristics, and easy real-time detection.

Pending Publication Date: 2021-05-14
NANJING UNIV OF POSTS & TELECOMM
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  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

At the same time, it is difficult to provide a real-time detection process that can characterize the kinetic process of the telomerase extension s...

Method used

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  • Detection probe, kit and direct detection method of telomerase activity
  • Detection probe, kit and direct detection method of telomerase activity
  • Detection probe, kit and direct detection method of telomerase activity

Examples

Experimental program
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Effect test

Embodiment 1

[0044] Embodiment 1: the preparation of detection probe

[0045] Step (1): Take 200uL 50nM gold nanoparticle solution with a diameter of 5nm, add 50uL 5×TBE solution and 2uL 100uM sulfhydryl-modified telomerase primer sequence (SH-ttttttaatccgtcgagcagagtt), use an oscillator to shake evenly, put React on a small shaker at 37°C and 500rpm for 10h.

[0046] Step (2): Add salt to the product obtained in step (1) for aging, use 3M NaCl solution, add the sample in 5 times, add 10uL each time, the final concentration of NaCl in the sample is 500mM, and continue to place it on a small shaker at 37°C , 500rpm reaction 10h.

[0047] Step (3): Use a balance to balance the sample obtained in step (2), and use a refrigerated centrifuge to centrifuge at 15,000 rpm for 30 minutes at 25°C, remove the supernatant and retain the precipitate, redissolve with 50-100uL ultrapure water, and repeat three times to obtain the detection Store the probe in a 4°C refrigerator.

[0048] Among them, th...

Embodiment 2

[0050] Example 2: Preparation of DNA-AuNPs complex

[0051] Use telomerase extracted from Hela cells to activate and extend the telomeres enriched on the surface of gold nanoparticles, and the operation steps are as follows:

[0052] Step (1): Use mild CHAPS lysate (aladdin) to lyse the cultured Hela cells and extract the telomerase therein. According to the operating instructions of the kit, in the cell sediment obtained by centrifugation, about 10 6 Add 200uL cell lysate to each cell, oscillate, lyse, suck it out and put it into a 1.5mL centrifuge tube; place the centrifuge tube in an ice bath, place it in a 4°C refrigerator for 10min, use a refrigerated centrifuge at 4°C, centrifuge at 15000rpm for 30min, and take out the supernatant. Pack and store in -80°C refrigerator.

[0053] Step (2): Take 50uL of the detection probe solution (about 20nM) prepared in Example 1, add 10uL TRAP solution, 2uL100mM dNTP solution and 28uL ultrapure water, mix well, add the telomerase extra...

Embodiment 3

[0056] Example 3: Detection of extension products of DNA-AuNPs complex by nanopore sensor

[0057] According to the method of Example 2, through the regulation of SDS denaturant, prepare the telomerase extension telomeric DNA sequence at different times to produce DNA-golden sphere complexes with different chain lengths, and use the resulting product using a 20nm silicon nitride nanopore The sensor uses a bias voltage of 600mV to detect DNA-AuNps at different time gradients. After the telomeres on the surface of the AuNps are extended by telomerase, the surface of the DNA-AuNps will be induced. The charge carried and the hydrated particle size change. These changes in properties can be detected by the change of the highly sensitive nanopore ion current signal and the generated current blocking signal to distinguish products under different time gradients. Enzyme activity and monitoring of the kinetic process of telomeric sequence extension on the surface of gold spheres.

[0...

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Abstract

The invention discloses a detection probe, a kit and a direct detection method for telomerase activity. The detection probe comprises gold nanoparticles and a probe sequence connected with the gold nanoparticles, wherein the probe sequence comprises a connecting section and a primer section for telomerase recognition; the sequence length of the connecting segment is 5-8 nt; the length of the primer section is 15 to 20 nt. The probe sequence marked on the surfaces of the gold nanoparticles is extended by telomerase; after telomeres extend, the gold nanospheres with the surfaces covered with DNA products of different lengths are recognized through the solid nanopores, and real-time high-sensitivity monitoring of the solid nanopores on the telomerase activity is achieved. According to the invention, telomere DNA chains are fixed on the surfaces of the gold nanospheres, enzyme capture is facilitated, and the activity reaction of telomerase is improved; meanwhile, the DNA-gold nanosphere composite structure plays a signal amplification function in nanopore detection, and the telomerase activity can be directly monitored in real time.

Description

technical field [0001] The invention belongs to the detection field of solid-state nanopore sensors, in particular to a detection probe, a kit and a direct detection method for telomerase activity. Background technique [0002] A telomere is a DNA sequence with a repeating sequence (TTAGGG) that usually exists at the end of a cell's chromosomes. During cell division, the duplication of chromosomes leads to the continuous shortening of telomeres. As a reverse transcriptase widely present in the body, telomerase plays a key role in maintaining telomere length and in the process of cell growth and differentiation. Telomerase achieves the purpose of maintaining telomere length and cell activity by continuously replicating the TTAGGG repeat sequence at the 3' end of the telomere. In 2009, ELIZABETN and others won the Nobel Prize in Medicine for their discovery of telomerase and the protective mechanism of telomeres on cell activity. Since then, many studies have shown that exc...

Claims

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Application Information

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IPC IPC(8): C12N15/11C12Q1/6825C12Q1/48
CPCC12Q1/6825C12Q1/48C12Q2563/137C12Q2521/113C12Q2565/607C12Q2565/631
Inventor 武灵芝叶媛翁丽星鲍碧清
Owner NANJING UNIV OF POSTS & TELECOMM
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