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Method for detecting polynucleotide kinase in high-salt high-protein biological sample

A nucleotide kinase and high protein technology, which is applied in the field of detecting polynucleotide kinases in high-salt and high-protein biological samples, can solve the problems of complex operation and inability to detect complex samples, and achieves simple experimental operation and flexible design. , good stability

Pending Publication Date: 2022-02-08
TIANJIN NORMAL UNIVERSITY
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

Despite their good performance, some of these methods are operationally complex and do not allow detection of complex samples

Method used

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  • Method for detecting polynucleotide kinase in high-salt high-protein biological sample
  • Method for detecting polynucleotide kinase in high-salt high-protein biological sample

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Embodiment 1

[0044] The present invention will be described in detail below in conjunction with the accompanying drawings and specific implementation examples.

[0045] An oligonucleotide sequence-stabilized silver nanocluster (ssDNA / AgNCs) for high-salt, high-protein complex biological matrix samples T 4 polynucleotide kinase (T 4 PNK) activity detection method, characterized in that: after 1 mMZn 2+ , Ce 4+ , Pb 2+ Simple processing, high-salt, high-protein complex biological matrix samples can be directly used for sample detection without multiple filtration and dilution operations; ssDNA / AgNCs can still produce stable fluorescence output in high-salt, high-protein complex biological matrix samples, so that ssDNA / AgNCs has strong anti-interference ability and wider application range. The specific operation is as follows:

[0046] (1) with single chain T 2 -DNA as the starting point; first, T 4 In the presence of PNK, catalyzes the transfer and exchange of the phosphate group (Pi...

Embodiment 2

[0050] Quantitative detection of T in Hela cell extracts 4 PNK activity

[0051] 1. The characteristics of the preparation method of AgNCs rich in C sequence ssDNA stability include the following steps:

[0052] (a) Prepare 0.02 mM 100 mL pH7.0 PBS solution: weigh 0.7 g Na 2 HPO 4 and 1.7 g NaNO 3 , dissolved in deionized water, and diluted to 100 mL. Weigh 0.3 g NaH 2 PO 4 2H 2 O and 1.7 g NaNO 3 , dissolved in deionized water, and diluted to 100 mL. The first two solutions were mixed in any proportion to obtain a PBS solution with a final pH of 7.0;

[0053] (b) Prepare 0.3 mM 4 mL AgNO 3 Solution: weigh 40 mg AgNO 3 , dissolved in 8 mL deionized water, diluted 100 times to obtain 0.3 mM 4 mL AgNO 3 solution;

[0054] (c) Prepare 0.6 M 4 mL NaBH 4 Solution: weigh 18 mg NaBH 4 , dissolved in 8 mL deionized water, diluted 100 times to obtain 0.6 mM 4 mL NaBH 4 solution;

[0055] (d) 75 µL 0.02 mM PBS, 10 µL 50 µM ssDNA, and 10 µL 0.3 mM fresh AgNO 3 solution...

Embodiment 3

[0062] Quantitative detection of T in human serum 4 PNK activity

[0063] 1. The characteristics of the preparation method of AgNCs rich in C sequence ssDNA stability include the following steps:

[0064] (a) Prepare 0.02 mM 100 mL pH7.0 PBS solution: weigh 0.7 g Na 2 HPO 4 and 1.7 g NaNO 3 , dissolved in deionized water, and diluted to 100 mL. Weigh 0.3 g NaH 2 PO 4 2H 2 O and 1.7 g NaNO 3 , dissolved in deionized water, and diluted to 100 mL. The first two solutions were mixed in any proportion to obtain a PBS solution with a final pH of 7.0;

[0065] (b) Prepare 0.3 mM 4 mL AgNO 3 Solution: weigh 40 mg AgNO 3 , dissolved in 8 mL deionized water, diluted 100 times to obtain 0.3 mM 4 mL AgNO 3 solution;

[0066] (c) Prepare 0.6 M 4 mL NaBH 4 Solution: weigh 18 mg NaBH 4 , dissolved in 8 mL deionized water, diluted 100 times to obtain 0.6 mM 4 mL NaBH 4 solution;

[0067] (d) 75 µL 0.02 mM PBS, 10 µL 50 µM ssDNA, and 10 µL 0.3 mM fresh AgNO 3 solution, vorte...

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Abstract

The invention discloses a novel method for detecting the activity of T4 polynucleotide kinase (T4 PNK) in complex biological matrix samples with high salt, high protein and the like by using silver nanoclusters (ssDNA / AgNCs) with stable oligonucleotide sequences. A large amount of salt and protein exist in a complex biological matrix sample, and the complex biological matrix sample needs to be subjected to high-multiple dilution, otherwise, fluorescence detection of T4 PNK is difficult to realize. Researches find that when enzymatic reactions such as T4 PNK and the like are completed, firstly, a quantitative AgNO3 solution is added, secondly, NaIO4 and Zn (NO3)2 solutions are added to oxidize protein, finally, dark-state ssDNA / AgNCs is added into the solution, bright-state ssDNA / AgNCs can be obtained after incubation, and fluorescence detection of the activity of T4 PNK is carried out. The method has the advantages of high detection sensitivity and high anti-interference capability, and can be suitable for high-salt and high-protein complex biological matrix samples without dilution.

Description

technical field [0001] The invention relates to genetic engineering tool enzymes and fluorescent nanomaterials ssDNA / AgNCs in the field of molecular biology, and discloses a ssDNA / AgNCs used in high-salt and high-protein complex biological matrix samples T 4 polynucleotide kinase (T 4 PNK) activity detection method, expanding the application of ssDNA / AgNCs detection in complex samples. Background technique [0002] Genetic engineering tool enzyme is a general term for various enzymes used in genetic engineering, including enzymes required in procedures such as nucleic acid sequence analysis, labeled probe preparation, vector construction, target gene selection, and recombinant DNA preparation. The tool enzymes commonly used in genetic engineering are mainly nucleases, polymerases, ligases, DNAzymes and other modification enzymes. This experiment focuses on four genetic engineering tool enzymes, which are T 4 PNK enzyme, T 4 DNA Ligase, Vent ® (exo-)DNase and Nt.BstN...

Claims

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Application Information

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IPC IPC(8): G01N21/64
CPCG01N21/6428Y02A50/30
Inventor 刘越姚美荣李琰
Owner TIANJIN NORMAL UNIVERSITY
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