Double-stranded nucleic acid and application thereof, and ribonuclease detection method

A ribonuclease, double-stranded nucleic acid technology, which is applied in biochemical equipment and methods, microbial determination/inspection, DNA/RNA fragments, etc. Use value, wide applicability, simple operation effect

Inactive Publication Date: 2013-04-03
SUZHOU RIBO LIFE SCIENCE CO LTD
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

[0021] In summary, although the above existing detection methods have played a certain role in improving the sensitivity and accuracy of ribonuclease activity detection, they all have certain defects and are not suitable for use as a general method in samples. Highly sensitive detection of ribonuclease activity, there is still a lot of room for improvement in related technical solutions

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  • Double-stranded nucleic acid and application thereof, and ribonuclease detection method
  • Double-stranded nucleic acid and application thereof, and ribonuclease detection method
  • Double-stranded nucleic acid and application thereof, and ribonuclease detection method

Examples

Experimental program
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Effect test

Embodiment 1

[0078] This example is used to illustrate the sequence structure of the oligoribonucleotide used in the present invention and the optical parameters of the fluorescent group. Entrust Guangzhou Ruibo Biotechnology Co., Ltd. to synthesize a series of oligoribonucleotides. A fluorophore is labeled at the 5' end of the oligoribonucleotide. Complementary oligoribonucleotides can be annealed to form double-stranded ribonucleic acid according to the method in "Molecular Cloning: A Laboratory Manual". Table 1 shows the sequences of the synthesized oligoribonucleotides. Wherein, in the oligoribonucleotide represented by SEQ ID NO: 11, the pentose sugar of the guanylic acid residue at position 3 is modified with 2'-oxymethyl (2'-OME). In the oligoribonucleotide represented by SEQ ID NO: 12, the pentose sugar of the uridine acid residue at position 14 is modified by 2'-fluoro substitution (2'-Fluro substitution). In the oligoribonucleotide represented by SEQ ID NO: 13, the pentose sug...

Embodiment 2

[0084] This example is used to illustrate the identification of the fluorescence resonance energy transfer phenomenon of the present invention. In the technical scheme for detecting ribonuclease levels by using fluorescence resonance energy transfer method provided by the present invention, a key condition is that labeling can occur between the fluorescent donor group and the fluorescent acceptor group at both ends of the double-stranded nucleic acid substrate. Fluorescence resonance energy transfer.

[0085] In order to verify this, at first the oligoribonucleotide (5'-FAM-AUGAGCCUGAUUU) of SEQ ID NO:1 and its complementary SEQ ID NO:2 oligoribonucleotide (UACUCGGACUAAA-TAMRA-5' ) anneal to form double-stranded ribonucleic acid substrate DS1. Take 4 μl of DS1 and add it to 2ml fluorescence resonance energy transfer buffer (0.01M Tris-HCl, pH 7.4, 0.002M MgCl 2 ), the final concentration of DS1 was 10nM. Subsequently, the reaction system is added into a quartz detection cup...

Embodiment 3

[0088]This example is used to illustrate the method for detecting ribonuclease by using the fluorescence resonance energy transfer method of the present invention. In order to utilize the fluorescence resonance energy transfer method to detect the activity and content of ribonuclease in the sample, take 4 μ l of double-stranded RNA substrate DS1 and join in 2 ml fluorescence resonance energy transfer buffer (0.01MTris-HCl, pH 7.4, 0.002M MgCl 2 ), the final concentration of DS1 was 10nM. Subsequently, the reaction system is added into a quartz detection cup of a fluorescence spectrometer, and a micro-magnetic rotor is placed in the quartz cup. After adding 20 μl of RNase A at a certain concentration to the reaction system, the reaction system was continuously excited at 480 nm and detected at 515 nm at the same time, and the fluorescence intensity values ​​at intervals of seconds were obtained. Using these values ​​to plot the curve of time versus fluorescence intensity, the ...

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Abstract

The present invention provides a double-stranded nucleic acid, a ribonuclease detection method, and applications of the double-stranded nucleic acid and the ribonuclease detection method in ribonuclease detection. Specifically the double-stranded nucleic acid substrate contains at least one ribonuclease-sensitive site, wherein double-stranded nucleic acid substrate degradation by the ribonuclease is analyzed to detect activity and content of the ribonuclease in a sample. The present invention further provides applications of the double-stranded nucleic acid substrate and the detection method in sample ribonuclease detection.

Description

technical field [0001] The invention relates to a double-stranded nucleic acid, a ribonuclease detection method and a detection kit using the double-stranded nucleic acid. Specifically, the present invention relates to a double-stranded nucleic acid substrate, a method for detecting ribonuclease by using the double-stranded nucleic acid substrate by fluorescence resonance transfer method, and the method is used in the detection of ribonuclease activity and application in content. Background technique [0002] Ribonuclease (RNase) or RNase is a large class of enzymes including dozens of species that can hydrolyze RNA substrates into small molecular nucleic acids. According to the position of the cleavage site, these enzymes can be divided into two types: endonuclease and exonuclease, belonging to multiple subclassifications in EC 2.7 (phosphorylase) and EC 3.1 (hydrolase). Ribonucleases from different sources have different specificities and modes of action. For example, t...

Claims

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Application Information

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IPC IPC(8): C12N15/11C12Q1/68C12Q1/34
Inventor 不公告发明人
Owner SUZHOU RIBO LIFE SCIENCE CO LTD
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