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Method for determining activity of T4 polynucleotide kinase based on magnetic nano material and biological signal amplification technology

A polynucleotide and magnetic nanotechnology, applied in the field of biochemical analysis, can solve the problems of application limitation, complicated operation process and high cost

Active Publication Date: 2021-04-27
YUNNAN MINZU UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Although this type of method has high sensitivity, the operation process is cumbersome and the cost is high, and its application is limited to a certain extent.
[0005] At present, there is a lack of a method for the determination of T4 polynucleotide kinase activity based on magnetic nanomaterials and biological signal amplification technology that achieves highly sensitive, rapid and quantitative determination of T4 PNK activity

Method used

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  • Method for determining activity of T4 polynucleotide kinase based on magnetic nano material and biological signal amplification technology
  • Method for determining activity of T4 polynucleotide kinase based on magnetic nano material and biological signal amplification technology

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Embodiment 1

[0039] The present invention is based on Fe 3 o 4 @TiO 2 The principle of the method for detecting T4PNK activity by dual signal amplification technology of magnetic core-shell nanoparticles and biological signals is as follows: figure 1 shown. First prepare the Fe 3 o 4 @TiO 2 In the presence of magnetic core-shell nanoparticles, ATP and T4 PNK, the 5′ end of the primer strand S1 in the rolling circle amplification reaction was phosphorylated, and the phosphorylated primer strand S1 passed through the reaction with TiO 2 Specific reaction, modified to Fe 3 o 4 @TiO 2 Magnetic nanoparticle surfaces. Join Ring Template S 2 , in the presence of T4 DNA ligase, S2 and Fe 3 o 4 @TiO 2 The S1 hybridization of the primer strand on the surface of the magnetic nanoparticle forms a circular mixture. In the presence of phi29 DNA polymerase and dNTPs, Fe 3 o 4 @TiO 2 RCA reaction occurs on the surface of magnetic nanoparticles. Ferrocene-labeled nucleic acid chain S3 (Fc...

Embodiment 2

[0041] Oligonucleotide sequence design

[0042] The oligonucleotide sequence designed in the present invention was synthesized by China Shanghai Sangon Bioengineering Co., Ltd., purified and tested by HPLC, and freeze-dried. The oligonucleotide sequence designed by the present invention is as follows:

[0043] S1: 5'-TTTTTTCACAGAGGATAGGACATGA-3';

[0044] S2: 5′-PO 4 - CTCAGCTGTGAACAACTAGAAGATAACTGTGAAGATCGCTTATCATGTCCTATC-3';

[0045] S3: 5′-NH 2 -TTTTTTTTTAAGATAACTGTGATTTTTTTT-NH 2 -3';

[0046] The oligonucleotides were dissolved in ultrapure sterile water and stored at -18°C for later use.

Embodiment 3

[0048] Fe 3 o 4 @TiO 2 Preparation of Magnetic Core-Shell Nanoparticles

[0049] Weigh 0.615g FeCl 3 ·6H 2 Dissolve O in 20 mL of ethylene glycol, add 1.8 g of NaAc and 0.5 g of polyethylene glycol, stir the solution, transfer it to a 100 mL Teflon-lined stainless steel autoclave, react at 200 °C for 8 hours, and cool to room temperature , washed several times with ethanol, separated by magnetic force, and air-dried at room temperature to obtain Fe 3 o 4 magnetic nanoparticles. Take 9.6mL glacial acetic acid and 6.8mL butyl titanate and dissolve in 60mL ethanol, add 0.4g Fe 3 o 4 Magnetic nanoparticles were ultrasonically dispersed for 15 min, 2.4 g of polyethylene glycol and 3.6 g of urea were added, stirred electrically for 1 h, and then the mixture was transferred to a reaction vessel for crystallization at 180° C. for 8 h. After cooling to room temperature, the precipitate was filtered and washed several times with ethanol, and dried at 80 °C for 12 h to give Fe ...

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Abstract

The invention discloses a method for determining the activity of T4 polynucleotide kinase based on a magnetic nano material and a biological signal amplification technology. Fe3O4@TiO2 magnetic core-shell nanoparticles are prepared by adopting a hydrothermal method; when ATP and T4 PNK exist, the 5'end of a rolling circle amplification reaction primer chain S1 is phosphorylated and modified to the surfaces of the Fe3O4@TiO2 magnetic nanoparticles; an annular template S2 is added, and a nucleic acid chain S2 is hybridized with the Fe3O4@TiO2 magnetic nanoparticle surface primer chain S1 to form an annular mixture; an RCA reaction is carried out on the surfaces of the Fe3O4@ TiO2 magnetic nanoparticles; a ferrocene-labeled nucleic acid chain S3 is added, the Fc-S3 is hybridized with an RCA reaction product, an obtained product is modified onto the surfaces of the magnetic nanoparticles, and an electrochemical response signal is enhanced; and through magnetic enrichment of the GME and the electrochemical response signal, high-sensitivity and quantitative determination of the activity of T4 PNK is realized.

Description

technical field [0001] The invention relates to the technical field of biochemical analysis, in particular to a method for measuring T4 polynucleotide kinase activity based on magnetic nanomaterials and biological signal amplification technology. Background technique [0002] T4 polynucleotide kinase (T4 PNK) is a protein encoded by the pseT gene of bacteriophage. It has 5' kinase activity and can catalyze the phosphorylation of the 5' hydroxyl terminal of nucleic acid, and DNA recombination, Normal cellular activities such as replication and damage repair are closely related. In addition, T4 PNK is also an important molecular biology tool, and the discovery and application of T4 PNK has promoted the development of molecular biology to a certain extent. At present, T4 PNK has become an indispensable tool enzyme in genetic engineering and bioanalysis research, and is further used in the research of nucleic acid damage repair and enzyme inhibitors. Therefore, the determinati...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): G01N27/327C12Q1/6844C12Q1/682
CPCG01N27/3278G01N27/3277C12Q1/682C12Q1/6844C12Q2531/125
Inventor 张艳丽刘在琼庞鹏飞王红斌
Owner YUNNAN MINZU UNIV
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