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A highly specific microRNA fluorescence detection method based on short-chain nucleic acid probes and double-strand-specific endonucleases

A technology of endonuclease and nucleic acid probe, which is applied in the direction of biochemical equipment and methods, microbial determination/inspection, etc. It can solve the problems of reducing the overall speed of hybridization-enzyme digestion reaction, slow miRNA hybridization speed, and difficulty in distinguishing single bases Mismatch and other problems, to achieve the effect of improved sensitivity, low cost, high specificity constant temperature detection

Active Publication Date: 2021-02-26
SUN YAT SEN UNIV
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  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

[0005] Although the constant temperature amplification method based on DSN enzyme has been combined with electrochemistry, fluorescence spectroscopy and other technologies to realize the sensitive detection of miRNA, but this kind of method still faces the following bottleneck problems: (1) The amplification efficiency is low
Conventional DSN enzyme-based methods use long-chain DNA probes, which hybridize slowly with the miRNA to be tested, which reduces the overall speed of the hybridization-enzyme digestion reaction and affects the amplification efficiency
(2) The specificity is not high
This method relies only on Watson-Crick interactions between bases to provide hybridization specificity; for long DNA probes, it is difficult to distinguish single base mismatches, especially when distinguishing miRNA families with high homology (such as the let-7miRNA family) When the performance is unsatisfactory, there is a high

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  • A highly specific microRNA fluorescence detection method based on short-chain nucleic acid probes and double-strand-specific endonucleases
  • A highly specific microRNA fluorescence detection method based on short-chain nucleic acid probes and double-strand-specific endonucleases
  • A highly specific microRNA fluorescence detection method based on short-chain nucleic acid probes and double-strand-specific endonucleases

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Embodiment 1

[0065] Embodiment 1 A kind of microRNA (hereinafter referred to as miRNA) fluorescent detection method based on short-chain nucleic acid probe and double-strand specific endonuclease

[0066] Design a short-chain DNA probe, which is labeled with a fluorescent group and a quencher group at both ends. Add the solution containing DNA probe and DSN enzyme to the solution to be tested. When there is no target miRNA in the solution to be tested, the fluorescent group labeled on the DNA probe is quenched and there is no fluorescence emission; when the solution contains target miRNA, The DNA probe hybridizes with the target miRNA, and the DSN enzyme cleaves the DNA probe in the DNA / miRNA duplex to release a fluorescent signal. The hybridization-enzyme digestion reaction occurs in a continuous cycle, and the fluorescent signal continues to increase. By optimizing the reaction conditions, a calibration curve for the detection of the target miRNA is obtained to achieve quantitative dete...

Embodiment 2

[0091] Embodiment 2 A kind of fluorescence detection method based on the microRNA let-7 family of short-chain nucleic acid probe and double-strand specific endonuclease

[0092] 1) Divide the target microRNA sequence into two sections, the 3' end section and the 5' end section, the base number of both sections is not less than 8bp, and the probe P 1 Make it possible to detect the 3' end segment or the 5' end segment;

[0093] Probe P 1 The number of bases is not less than 8bp, and does not exceed the base number of the 3' end segment or the 5' end segment; probe P 1 With fluorescent groups and quenching groups.

[0094] 2) with probe P 1 The microRNA sample to be tested is subjected to double-strand specific endonuclease-mediated microRNA fluorescence detection, and the fluorescence value F 1 , if with probe P 1 Complementary paired sequences only exist in the target microRNA, according to the fluorescence value F obtained by detection 1 Calculate the microRNA content, w...

Embodiment 3

[0099] Example 3 A microRNA let-7a fluorescence detection method based on short-chain nucleic acid probe and double-strand specific endonuclease

[0100] This embodiment takes the detection of microRNA let-7a: 5'-UGA GGU AGU AGGUUG UAU AGUU-3' (SEQ ID NO: 4) in the microRNA let-7 family as an example to further illustrate the method of the present invention.

[0101] (1) Divide the target microRNA let-7a sequence into two segments, the 3' end segment and the 5' end segment, and detect the probe 7a-P of the 3' end segment according to the design 1 ; Simultaneously design a probe 7a–P that is completely complementary to the entire sequence of microRNA let-7a 0 As a control, all probes are labeled with fluorescent groups and quenchers, and the specific sequences are as follows:

[0102] Probe 7a-P 1 : FAM-5'-CTATACAACC-3'-BHQ-1 (SEQ ID NO: 1),

[0103] Probe 7a–P 0 : FAM-5'-AACTATACAACCTACTACCTCA-3'-BHQ-1 (SEQ ID NO: 3).

[0104] (2) Probe 7a-P 1 ,7a–P 0 Optimization of De...

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Abstract

The invention discloses a highly specific microRNA fluorescence detection method based on a short-chain nucleic acid probe and a double-strand specific endonuclease. The present invention uses short-chain DNA probes combined with DSN enzymes to accelerate the probe / miRNA hybridization rate and the hybridization-enzyme digestion cycle rate while improving the sensitivity of the probes to base mismatches in hybridization reactions and achieving human body temperature High-sensitivity and high-specificity constant temperature detection of miRNAs under high temperature; it can sensitively distinguish homologous similar miRNAs with single base mismatches. This method can obtain high-efficiency amplification reaction at 37°C. Compared with other amplification reactions based on DSN enzyme (the temperature needs to be 55-60°C), it is more suitable for in situ or living detection of cells. The amplification reaction is a constant temperature reaction, without precise temperature control or temperature cycle device, simple operation, low cost, and more suitable for market promotion.

Description

technical field [0001] The technical field of biological detection of the present invention specifically relates to a highly specific microRNA fluorescence detection method based on short-chain nucleic acid probes and double-strand specific endonucleases. Background technique [0002] MicroRNAs (miRNAs) are a class of non-coding RNAs with a length of about 18-24 bases. By specifically binding to the 3'-untranslated region of its target messenger RNA (mRNA), miRNAs can regulate gene expression at the post-transcriptional level, widely participate in the regulation of all levels of life activities, and are closely related to the occurrence and development of major diseases such as malignant tumors . The sensitive detection of miRNAs has important theoretical significance and practical value in in-depth study of the relationship between miRNA and disease occurrence and development, early diagnosis of diseases, etc. [0003] Due to the short miRNA sequence, extremely low expre...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12Q1/6816
CPCC12Q1/686C12Q2563/107C12Q2545/114C12Q2521/301
Inventor 戴宗马颖君邹小勇
Owner SUN YAT SEN UNIV