Detection probe, kit and direct detection method for telomerase activity

A technology for detecting probes and telomerase, which can be used in biochemical equipment and methods, measuring devices, and microbial determination/inspection, etc., and can solve the problems of nanopore chip damage, single use of nanopores, and low enzyme modification efficiency. Achieve high signal-to-noise ratio and enhanced rigidity

Pending Publication Date: 2022-05-06
NANJING UNIV OF POSTS & TELECOMM
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

In view of the fact that in traditional nanopore sensing, the specific recognition of enzymes is mostly achieved by means of molecular modification in the nanopore channel. This method has certain advantages, but the modification process will inevitably cause damage to the nanopore chip, and the modification efficiency of the enzyme is low. And the modified nanopore has a single purpose

Method used

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

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0041] Embodiment 1: the design of detection probe

[0042] The invention provides a detection probe for high specificity and high sensitivity detection of telomerase activity. The detection probe comprises: using two gold nanoparticles (AuNPs) as carriers, respectively modifying two polymeric diblock DNA 1 and diblock DNA 2 , telomerase primer sequence TP DNA 3 ; Among them, the diameter of gold nanoparticles is 5nm, which is easy to assemble for single-molecule DNA labeling, has the best assembly efficiency, and has high signal-to-noise ratio and detection throughput in nanopores; among them, the telomerase primer sequence is AATCCGTCGAGCAGAGTT; double-embedded Segment DNA 1 and diblock DNA 2 The anchor chains are all polyA 40 sequence, DNA 1 The front part of the connecting strand is complementary to the sequence of the telomerase primer, and the back part is complementary to the DNA 2 The connecting strands are partially complementary to form a stable assembly struc...

Embodiment 2

[0047] Embodiment 2: Preparation of detection probe

[0048] Step (1): Take the AuNPs with a diameter of 5nm required for the experiment and prepare an AuNPs solution with a total volume of 400ul and a concentration of 200nM. Segment DNA 1 and diblock DNA 2 Take 20ul each to incubate the reaction at a molar ratio of 1:100, and mix and culture at 25°C for 24 hours. Specifically, the DNA in this example 1 and DNA 2 The sequences are respectively shown in SEQ ID NO.1 and SEQ ID NO.2 in Table 1;

[0049] Step (2): Add PBS buffer solution to the product obtained in step (1) every 30 minutes until the final concentration is 0.1M to obtain the initial sample;

[0050] Step (3): Salt out the initial sample obtained in step (2) with 0.1M PBS. This process is also completed by incubating at 25°C for 24 hours to obtain two AuNPs-DNA complexes;

[0051] Step (4): The product obtained in step (3) was centrifuged at 12,500 rpm for 30 minutes at 25°C, washed 3 times repeatedly to remove...

Embodiment 3

[0053] Example 3: Detection of Telomerase Activity

[0054] The detection probe prepared in Example 2 was used to detect telomerase activity, and a silicon nitride nanopore was used as a sensing reactor. Specifically, a solid-state nanopore sensor with a diameter of 24 nm is selected for signal detection.

[0055] Telomerase enzymatic reaction: Add 20ul of telomerase reverse transcription buffer (20mM Tris-HCl, pH 8.3, 1mM EDTA, 0.05% Tween 20, 63mM KCl, 0.1 mg / mL BSA, 1.5mM MgCl 2 ), 100mM dNTP solution 2ul and 18ul ultrapure water, mix evenly, then add 10uL telomerase extract, mix evenly, place on a shaker for reaction, the reaction conditions are 37°C, 500rpm, and incubate together for 1h.

[0056] Add 10ul of 1% SDS solution by mass ratio to the product solution after the telomerase enzymatic reaction; place the prepared telomerase extension reaction products at 4°C for about half an hour, centrifuge at 8000rpm to remove the precipitate, and use a freezer Centrifuge at ...

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Abstract

The invention discloses a detection probe, a kit and a direct detection method of telomerase activity. The detection probe comprises gold nanoparticles, and double-block DNA1 and DNA2 coating the surfaces of the gold nanoparticles. The double-block DNA1 can be hybridized and complemented with a telomerase primer sequence, and meanwhile, the DNA1 and the DNA2 have complementary sequence hybridization to trigger the gold nanoparticles to be assembled to form a dimer. After telomerase is added, the primer is taken as a template, telomeres are extended, and melting displacement of a DNA2 chain is initiated, so that nano-particle dimer dissociation is carried out to form a monomer; as the volume of the nanoparticles is in direct proportion to a blocking current signal, the detection probe generates an electric signal with more obvious difference when passing through the nanopore before and after the telomerase is added, so that a higher signal-to-noise ratio is achieved, and high-sensitivity telomerase activity detection is realized.

Description

technical field [0001] The invention belongs to the field of telomerase activity detection, in particular to a detection probe, a kit and a direct detection method for telomerase activity. Background technique [0002] Telomeres exist at the ends of chromosomes and are a structure like a "cap". It will gradually become shorter with the mitosis process of normal cells, and is closely related to cell aging and apoptosis. Telomerase is a reverse transcriptase that can maintain the length of chromosomes. It will continuously add (TTAGGG) n base sequences at the ends of chromosomes to maintain the length of telomeres during mitosis. Telomerase is ubiquitously present in human cells, but telomerase is inactive in most cells except hematopoietic cells, germ cells and stem cells. Once the telomerase activity in somatic cells is abnormal, it is usually related to the occurrence of cancer and tumor diseases, so telomerase has become an important target molecule for diseases such as c...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12Q1/6825C12Q1/48C12N15/11
CPCC12Q1/6825C12Q1/48G01N2333/9128C12Q2525/173C12Q2533/101C12Q2537/1373C12Q2563/137C12Q2521/113C12Q2565/631C12Q2565/607
Inventor 武灵芝龚靖哲王宇朋翁丽星胡岚
Owner NANJING UNIV OF POSTS & TELECOMM
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