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Isothermal detection method of RNA (Ribonucleic Acid)

A detection method, isothermal technology, applied in the field of thermally amplified RNA, can solve the problems of easy vibration, instability and weak fluorescence of the chemical structure, and achieve the effect of simple operation, convenient operation and high sensitivity

Inactive Publication Date: 2013-04-03
CHENGDU INST OF BIOLOGY CHINESE ACAD OF S
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, the strand displacement isothermal amplification technique finally produces a large amount of single-stranded DNA. The chemical structure of the triphenylmethane dye is easy to vibrate and unstable, and the fluorescence emitted when it exists alone is very weak.

Method used

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  • Isothermal detection method of RNA (Ribonucleic Acid)
  • Isothermal detection method of RNA (Ribonucleic Acid)
  • Isothermal detection method of RNA (Ribonucleic Acid)

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0043] Example 1: Strand displacement amplification experiment using RNA as primer

[0044] (1) Extension reaction after RNA is sheared by DNAzyme that shears RNA

[0045] In this experiment, a DNAzyme is firstly used, and the reaction solution 10μL contains 50nm Flag-RNA, 2μM DNAzyme, 10mM Tris-HCl (pH8.5), 100mM KCl, 10mM MgCl 2 , and 0.1mg / ml BSA, 0.5unit / μl PNK and 0.4unit / μl Bsm DNA polymerase, reacted at 37°C for 1h, analyzed with denaturing polyacrylamide gel (PAGE), no extension band appeared. The improved DNAzyme is used here, that is, the bases at the end of the cut RNA are complementary paired with the improved DNAzyme (fCat). After the same reaction conditions as above, extended bands appeared. The function of PNK is to hydrolyze the 2' and 3' cyclic phosphate groups at the cut end of RNA, so that the cut RNA can be used as a primer for extension reaction. as attached figure 2 shown.

[0046] Add a certain concentration of Flag-RNA, 0.5μM fCat, 0.25unit / μl PN...

Embodiment 2

[0067] Example 2: Malachite green combined with Hum21 (a DNAzyme with a G-tetramer structure that can be combined with a dye to generate fluorescence) is developed as a quantitative reporter system experiment

[0068] The quantitative probe Tb of the present invention includes a sequence complementary to trgger from 3' to 5', a restriction endonuclease recognition sequence, a sequence complementary to hum21 that can combine with malachite green to generate fluorescence, and a part of the catalytic center of fCat the same sequence. When the primer is combined with Tb, continuous displacement amplification occurs, thereby producing a large amount of Hum21. Using the property of fluorescence generated by the combination of G-tetramer and malachite green, real-time detection can be achieved with the accumulation of signal. Therefore, it shows that malachite green combined with Hum21 can be developed as a quantitative reporter system.

[0069] Reaction solution 20μL, including 10m...

Embodiment 3

[0072] Example 3 The experiment of using DNAzyme and quantitative probe to quantitatively detect RNA

[0073] For the quantitative detection of RNA, the present invention adopts a one-step method to implement in a reaction container, which further simplifies and speeds up the operation and reduces the risk of contamination at the same time. In the one-step reaction, the improved DNAzyme (fCat) first cuts the RNA, and the function of using PNK is to hydrolyze the 2' and 3' cyclic phosphate groups at the cut end of the RNA, and then the RNA is amplified by continuous displacement. The purpose of detecting RNA is achieved by the quantitative reporter system.

[0074] Reaction solution 20μL, 10mM Tris-HCl, pH7.0, 100mM KCl, 10mM MgCl 2 ,0.1mg / ml BSA, dNTPs (250μM), Nb.Bpu10I (0.17unit / μl), Bsm polymerase (0.17unit / μl), PNK (0.08unit / μl), malachite green (15μmol), 0.25μM fCat, 0.25μM Tb, a certain concentration of Flag-RNA.

[0075] 1 negative control tube: just don't add Flag-R...

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Abstract

The invention provides a qualitative and quantitive isothermal detection method of RNA (Ribonucleic Acid). The method comprises the steps of after cutting RNA at fixed points by dnazyme (DNAzyme), amplifying RNA through a strand displacement isothermal amplification technique (SDA); and carrying out qualitative or quantitive detection by inspecting a reporter group G-tetramer released from an SDA product. The method can measure trace RNA (comprising mRNA and miRNA) quickly, simply, conveniently, highly sensitively and specifically, and meanwhile, the risk of pollution is reduced.

Description

technical field [0001] The invention belongs to the technical field of molecular biology, and relates to the analysis and detection of RNA amplification. More specifically, the invention relates to a detection method for using strand displacement isothermal amplification of RNA and releasing a reporter system. Background technique [0002] In vitro amplification of nucleic acid molecules is an important means of biotechnology research. It can not only amplify and isolate target genes, but also has important uses in clinical diagnosis, gene mutation detection, and forensic identification. Polymerase chain reaction (PCR) is the most widely used nucleic acid amplification technology. It requires repeated thermal denaturation to untie the double strands of DNA. It relies on high-quality thermal cyclers to achieve denaturation, annealing, Three steps are extended, causing trouble in temperature control. With the development of science and different research purposes, since the e...

Claims

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Application Information

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IPC IPC(8): C12Q1/68
Inventor 唐卓赵永云
Owner CHENGDU INST OF BIOLOGY CHINESE ACAD OF S
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