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High-specificity microRNA fluorescence detection method based on short chain nucleic acid probe and double-chain specific endonuclease

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

Active Publication Date: 2017-10-20
SUN YAT SEN UNIV
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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

Method used

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  • High-specificity microRNA fluorescence detection method based on short chain nucleic acid probe and double-chain specific endonuclease
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  • High-specificity microRNA fluorescence detection method based on short chain nucleic acid probe and double-chain specific endonuclease

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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] In this embodiment, the method of the present invention is further described by taking 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.

[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 Optimizatio...

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Abstract

The invention discloses a high-specificity microRNA fluorescence detection method based on a short chain nucleic acid probe and a double-chain specific endonuclease. According to the invention, the short chain DNA probe is used and the DSN enzyme is combined, so that probe / miRNA hybridization rate and hybridization-digestion circulating rate are increased, and meanwhile, the sensitivity of the probe for the hybridization reaction mismatch is promoted and the miRNA high-sensitivity high-specificity constant temperature detection under human body temperature is realized; the homologous similar miRNAs of single-base mismatch can be sensitively distinguished; according to the method, the efficient amplified reaction at 37 DEG C can be acquired; compared with other amplified reaction based on DSN enzyme (under the temperature at 55-60 DEG C), the efficient amplified reaction is more suitable for in situ or vivo detection of cells; the amplified reaction is thermostatic reaction and requires no accurate temperature control or temperature circulating device; and the operation is simple, the cost is low and the method is more suitable for marketing.

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 . For the sensitive detection of miRNAs, it has important theoretical significance and practical value in the aspects of in-depth study of the relationship between miRNA and disease occurrence and development, and early diagnosis of diseases. [0003] Due to the short miRNA sequenc...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12Q1/68
CPCC12Q1/686C12Q2563/107C12Q2545/114C12Q2521/301
Inventor 戴宗马颖君邹小勇
Owner SUN YAT SEN UNIV
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