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Phosphorthioate-modified oligonucleotide fluorescence probe and application thereof in detection of nuclease

A technology of oligonucleotides and fluorescent probes, which is applied in the field of sulfurated (sulfonylated) modified oligonucleotide fluorescent probes, which can solve the problem of increasing design costs and workload, and losing the advantages of fluorescent nucleic acid probe detection etc. to achieve good fluorescence recovery effect, improved fluorescence quenching efficiency, and high sensitivity

Active Publication Date: 2013-10-02
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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  • Phosphorthioate-modified oligonucleotide fluorescence probe and application thereof in detection of nuclease
  • Phosphorthioate-modified oligonucleotide fluorescence probe and application thereof in detection of nuclease
  • Phosphorthioate-modified oligonucleotide fluorescence probe and application thereof in detection of nuclease

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0035] Embodiment 1

[0036] 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.

[0037] Specific steps are as follows:

[0038] 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

[0051] Embodiment 2

[0052] 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.

[0053] Specific steps are as follows:

[0054] 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

[0068] Example 3

[0069] 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.

[0070] Specific steps are as follows:

[0071] 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 phosphorthioate-modified oligonucleotide fluorescence probe and an application thereof in the detection of nuclease. The phosphorthioate-modified oligonucleotide fluorescence probe is provided with a stem-and-loop structure, the ring part of the phosphorthioate-modified oligonucleotide fluorescence probe is of a single-stranded structure and comprises 4-8 nucleotide residues, the stem part of the phosphorthioate-modified oligonucleotide fluorescence probe comprises 8-15 pairs of nucleotide residues, O- in part of phosphate diester bond of a probe is replaced by S-, and the probe is divided into a non-restriction endonuclease probe, an exonuclease probes and a depurination / depyrimidination site lyase probes according to the activity function of the nuclease to be detected. The phosphorthioate-modified oligonucleotide fluorescence probe is an accurate and reliable analytic method which can be used for detecting the activity of the nuclease in real time, has strong selectivity and high sensitivity and is fast in speed and low in cost, so that lots of limitations in the prior art are overcome.

Description

technical field [0001] The present invention relates to the detection of multiple nucleases such as non-restrictive endonucleases, exonucleases, apurinic / apyrimidinic site (AP site) endonucleases, more specifically, three kinds of thio (sulfoylated ) to modify oligonucleotide fluorescent probes, and use these three fluorescent probes to detect nucleases in actual biological systems. Background technique [0002] 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 usually lead to diseases, and the more important significance of nuclease omics research lies in the diagnosis of diseases and the development of drugs. Therefore, developing ...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N15/11C12Q1/68C12Q1/34
CPCC12Q1/44
Inventor 赵美萍苏昕张晨柳杨肖先金
Owner PEKING UNIV
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