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Thio probe for detecting nuclease with 3'-5' exo activity

A nuclease and probe technology, applied in the field of sulfur-modified oligonucleotide fluorescent probes, can solve the problems of increasing design cost and workload, losing the detection advantages of fluorescent nucleic acid probes, etc., and achieves good fluorescence recovery effect, The effect of improved fluorescence quenching efficiency and high sensitivity

Active Publication Date: 2015-01-21
PEKING UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

In this case, it is often necessary to analyze the product of the enzyme reaction, which not only increases the design cost and workload, but also loses the detection advantages of fluorescent nucleic acid probes

Method used

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  • Thio probe for detecting nuclease with 3'-5' exo activity
  • Thio probe for detecting nuclease with 3'-5' exo activity
  • Thio probe for detecting nuclease with 3'-5' exo activity

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0036] Embodiment 1

[0037] In this example, thio-modified oligonucleotide fluorescent probe 1 was used to detect the activity of the non-restrictive endonuclease DNase I and compared with the signals generated by other enzymes.

[0038] Specific steps are as follows:

[0039] 1. The fluorescent signal of the thio-modified oligonucleotide fluorescent probe 1 is in a quenched state at the beginning, and it is mixed with different concentrations of DNase I and placed in a suitable solution condition to form a reaction system. The phosphodiester bond of the non-sulfonylated part of the probe is hydrolyzed into small fragments under the catalysis of DNase I, resulting in the separation of the fluorescent group and the quencher group, and the release of the fluorescent signal, which is detected by a real-time fluorescent PCR instrument. As the reaction progresses, the number of probes hydrolyzed increases, and the fluorescence signal increases rapidly until the reaction balances...

Embodiment 2

[0052] Embodiment 2

[0053] In this example, the activity of the non-restrictive endonuclease Exonuclease III was detected using the thio-modified oligonucleotide fluorescent probe 2 and compared with the signals generated by other enzymes.

[0054] Specific steps are as follows:

[0055] 1. The fluorescent signal of the thio-modified oligonucleotide fluorescent probe 2 is in a quenched state at the beginning, and it is mixed with different concentrations of Exonuclease III and placed in a suitable solution condition to form a reaction system. The phosphodiester bond of the probe 3' connected to the fluorescent group is hydrolyzed under the catalysis of Exonuclease III, causing the fluorescent group to leave the probe, separate from the quencher group, and the fluorescent signal is released, which is detected by a real-time fluorescent PCR instrument. As the reaction progresses, the number of probes hydrolyzed increases, and the fluorescence signal increases rapidly until th...

Embodiment 3

[0069] Example 3

[0070] In this example, the activity of the apurinic / apyrimidinic site endonuclease Endonuclease IV was detected using the fluorescent probe 3 of the thio-modified oligonucleotide and compared with the signals generated by other enzymes.

[0071] Specific steps are as follows:

[0072] 1. The fluorescent signal of the thio-modified oligonucleotide fluorescent probe 3 is in a quenched state at the beginning, and it is mixed with different concentrations of Endonuclease IV and placed in a suitable solution condition to form a reaction system. The phosphodiester bond at the 5' of the AP site of the probe is hydrolyzed under catalysis, the small fragment oligonucleotide with the fluorescent group leaves the probe, the fluorescent group is separated from the quencher group, and the fluorescent signal is released. Real-time fluorescent PCR instrument for detection. As the reaction progresses, the number of probes hydrolyzed increases, and the fluorescence signa...

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Abstract

The invention discloses a thio probe for detecting nuclease with 3'-5' exo activity. The thio modified oligonucleotide fluorescent probe has a stem-loop structure, wherein the loop part is a single-chain structure which consists of 4-8 nucleotide residues and the stem part consists of 8-15 pairs of nucleotide residues; the phosphodiester bond between every two basic groups of the probe undergoes sulfur acylation; the phosphate group connected between the basic group at the tail end of 3' and the fluorescent group marked on the basic group does not undergo sulfur acylation; the quenching group is marked on the fourth basic group from 3' to 5'. The thio probe can be used for detecting the exo activity of the nuclease in real time, is strong in selectivity, high in sensitivity, rapid in speed, low in cost, correct and reliable, and is capable of overcoming the limits in the prior art.

Description

[0001] This application is a divisional application of the invention patent application "thio-modified oligonucleotide fluorescent probe and its application in nuclease detection" with the filing date of June 17, 2013 and the application number of 201310239614.0. technical field [0002] The present invention relates to the detection of nucleases, more specifically, to thio (sulfonylated) modified oligonucleotide fluorescent probes, and the use of the fluorescent probes to have 3'-5' excision activity in actual biological systems nuclease detection. Background technique [0003] DNA is the carrier of the genetic information of living organisms, and nuclease ensures the accuracy of gene replication and the stability of inheritance. Nucleases are widely found in biological systems. Interactions between nucleic acids and nucleases are important processes in the transfer of genetic information, including replication, recombination, and repair. Abnormal enzyme activities usuall...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12Q1/68C12Q1/44C12N15/11
CPCC12Q1/44
Inventor 赵美萍苏昕张晨柳杨肖先金
Owner PEKING UNIV
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