Silver nanocluster fluorescent probe based T4 polynucleotide kinase activity detecting method

A polynucleotide and detection method technology, which is applied in the field of T4 polynucleotide kinase activity detection based on silver nanocluster fluorescent probes, and achieves the effects of simple operation and significant clinical application value.

Inactive Publication Date: 2018-11-16
THE SECOND HOSPITAL OF NANJING
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Benefits of technology

This new technology allows us to detect even small amounts (<0.1 ml or less), making it possible to study how certain chemicals act on cells more effectively than before they were introduced into our body.

Problems solved by technology

Technologies described in this patents involve developing improved ways to measure specific proteins called p66 protein fragments involved in regulating gene expression during various physio logical functions like transport, biosensitive assays, chemistry, immunology, and drug development. These improvements aim to enhance the accuracy and efficiency of current methods while reducing their limitations associated therewith.

Method used

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  • Silver nanocluster fluorescent probe based T4 polynucleotide kinase activity detecting method
  • Silver nanocluster fluorescent probe based T4 polynucleotide kinase activity detecting method
  • Silver nanocluster fluorescent probe based T4 polynucleotide kinase activity detecting method

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Embodiment 1D

[0043] The synthesis of embodiment 1 DNA sequence

[0044] In this example, two DNA sequences were synthesized by Shanghai Sangon Biotechnology Co., Ltd., and their base sequences are respectively:

[0045] DNA S1: 5′-CCCTTAATCCCCTATAATAAATTTTAAATATTATTTATTAAT-3′,

[0046] DNAS2: 5'-ATTAATAAATAATATTTAAAATTTATTATAGGGTGGGGTGGGGTGGGG-3';

Embodiment 2

[0047] Example 2 Preparation of Silver Nanocluster Templated DNA S1

[0048] Add 10 μM DNA S1 in Example 1 to the pH7 phosphate buffer, then heat at 90°C for 5min and cool to room temperature, add AgNO to the solution 3 , where Ag + The molar ratio to DNA S1 is 6.67:1, and incubated at room temperature for 30min to form DNA-Ag + Complex, the prepared DNA-Ag + Complex with NaBH 4 processing, where NaBH 4 and Ag + The molar ratio of the silver nanoclusters templated DNAS1 (DNA-AgNCs) solution is 1:1, and then stored in the dark for 12 hours to prepare the silver nanocluster-templated DNAS1 (DNA-AgNCs) solution.

Embodiment 3

[0049] Example 3 Activity detection of T4 PNK

[0050] Mix 6 μM DNA S2 dissolved in TE buffer in Example 1 and the DNA-AgNCs (DNA S1) solution prepared in Example 2 at a concentration of 1× in the DNA hybridization solution, and heat to 95°C for 50s, then slowly cool down to room temperature and incubate for 20min to hybridize the two DNA strands to obtain hybridized DNA, add the hybridized DNA to Tris-HCl buffer for DNA phosphorylation reaction to obtain a reaction mixture, and incubate the reaction mixture at 37°C After 30 minutes, heat to 75°C for 10 minutes to inactivate the activity of λexo to obtain the final reaction solution.

[0051] The hybridization buffer described therein is 10mM Tris-HCl, 1.0mM EDTA and 1.0M sodium chloride, pH 7.4;

[0052] Wherein, the Tris-HCl buffer is 10mM MgCl 2 , 5 mM DTT, 0.5 mM ATP, 10 units λexo and T4 PNK.

[0053] The T4 PNK concentration gradient in this embodiment is 0, 0.01, 0.1, 0.5, 1, 5, 10 and 12.5 U / mL;

[0054] The result...

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Abstract

The invention discloses a silver nanocluster fluorescent probe based T4 polynucleotide kinase activity detecting method. The silver nanocluster fluorescent probe based T4 polynucleotide kinase activity detecting method is characterized in that a marker-free 'turn-off' type fluorescent probe is manufactured for detecting activity of T4 PNK. A DNA chain for preparing silver nanocluster template is S1(DNA S1), and a DNA chain containing [3'-G4(TG4)2TG3] sequence is S2(DNA S2), and the two chains are hybridized; under action of a G-rich sequence, a silver nanocluster can generate strong red fluorescence, and 5-hydroxyl terminal of the double chains DNA is phosphorylated through T4 PNK, so that a 5-phosphoryl group terminal is generated; the 5-phosphoryl group terminal can be quickly distinguished and degraded by gamma exonuclease, so that two DNA chains are separated, the fluorescence signal is quickly weakened to generate a signal which is in inverse proportion with T4 PNK concentration.Therefore, the silver nanocluster fluorescent probe based T4 polynucleotide kinase activity detecting method can be used for precisely detecting activity of T4 PNK; and the detecting sensitivity of the method can be 0.01 U/mL according to the strength of the DNA-AgNCs fluorescence signal.

Description

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Claims

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Application Information

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Owner THE SECOND HOSPITAL OF NANJING
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