Probe and method for simultaneously detecting plurality of microRNAs under constant-temperature conditions based on DNA assembly

A probe and probe set technology, applied in DNA/RNA fragments, recombinant DNA technology, biochemical equipment and methods, etc., can solve problems such as complex systems, achieve simple amplification system, good accuracy, and expand the scope of application Effect

Active Publication Date: 2018-12-21
SUN YAT SEN UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Solve the complex problem of the conventional constant temperature amplification method syst

Method used

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  • Probe and method for simultaneously detecting plurality of microRNAs under constant-temperature conditions based on DNA assembly
  • Probe and method for simultaneously detecting plurality of microRNAs under constant-temperature conditions based on DNA assembly
  • Probe and method for simultaneously detecting plurality of microRNAs under constant-temperature conditions based on DNA assembly

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0058] Example 1 A probe set for detecting microRNA

[0059] The probe set for detecting microRNA includes Y-1n, Y-2n and fluorescent probe LP; probes Y-1n, Y-2n, LP and target microRNA can self-assemble to form a stable "I"-shaped structure;

[0060] Part of the bases in the middle of the probe Y-1n are reverse-complementary paired with the middle part of the bases in Y-2n to form a stem sequence in the middle of the "I" shape; the length of the stem sequence is 7 to 9 bp; the The stem sequence contains the recognition site for the nicking endonuclease.

[0061] The other part of the base sequence of the probes Y-1n and Y-2n is reverse-complementary paired with the two ends of the target microRNA respectively, forming a "one"-shaped head at one end of the "I" shape;

[0062] There are also base sequences in the probes Y-1n and Y-2n that can undergo reverse complementary pairing with the sequences at both ends of the fluorescent probe LP, forming a "one"-shaped head at the ot...

Embodiment 2

[0067] Embodiment 2 A method for detecting microRNA

[0068] method:

[0069] A target miRNA (miR-373) was selected and a probe was designed by NUPACK software, and DNA probes Y-11, Y-21 and fluorescent probe LP partially complementary to the sequence were synthesized. The specific sequences of each probe are shown in Table 1 shown.

[0070] Mix Y-11, Y-21, LP, Nt.BstNBI, Nt.BstNBI buffer and target miRNA, and react at 45°C for 30 minutes; simultaneously detect the change of fluorescence intensity.

[0071] At the same time, three groups of control experiments were set up:

[0072] Y-11+Y-21+LP1 group: when only Y-11, Y-21 and LP exist in the reaction system of the control group, the nicking endonuclease Nt.BstNBI and target miRNA (miR-373) are not included, other The conditions were the same as the above experimental group.

[0073] Y-11+Y-21+LP1+NEase group: the reaction system of the control group does not contain the target miRNA (miR-373), and other conditions are the...

Embodiment 3

[0081] The investigation of embodiment 3 reaction temperature

[0082] method:

[0083] According to the physical and chemical properties of the restriction endonuclease Nt.BstNBI, its catalytic activity is higher at about 55°C, and its activity will be significantly reduced if the reaction temperature is too high or too low. In addition, the melting temperature of the "I"-shaped bridge structure in the system also has a great impact on the reaction. When the reaction temperature is too high, it will be difficult to form this structure, thus affecting the enzymatic digestion cycle amplification process. In this system, keeping other conditions constant, 35, 40, 45, 50, 55 and 60°C were selected as reaction temperatures to investigate the effect of temperature on the fluorescence intensity of the reaction system. The specific operation method is: mix Y-11, Y-21, LP, Nt.BstNBI, Nt.BstNBI buffer and target miRNA (miR-373), respectively, at 35, 40, 45, 50, 55 and 60 ° C The reac...

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Abstract

The invention discloses a probe and a method for simultaneously detecting a plurality of microRNAs under constant-temperature conditions based on DNA assembly. The probe comprises three probe sets; the three probe sets and target microRNAs can be self-assembled into a stable 'I'-shaped structure; according to the high specificity of restriction endonucleases and the cutting property of the same tosingle-stranded DNAs, DNA modules with non-sequence-dependent nuclease functions are realized, and are applied to simultaneous fluorescence detection of the miRNAs under the constant-temperature conditions. The probe and the method have the advantages that the system complication of conventional constant-temperature amplification methods is solved, and a new method is provided for simultaneous sensitive detection of the miRNAs; only the restriction endonucleases and the simply-designed probe are needed, and the constant-temperature signal amplification detection of nucleic acids can be realized without addition of dNTPs; the probe has high precision, repeatability and stability, and is suitably developed into a kit and popularized to the market.

Description

technical field [0001] The invention belongs to the technical field of biological detection, and in particular relates to a method for simultaneous fluorescence detection of multiple microRNAs under constant temperature conditions based on DNA assembly. Background technique [0002] MicroRNA (miRNA) is a class of non-coding small molecule RNA that regulates gene expression and plays an important role in the growth, development, differentiation and reproduction of animals and plants. About 30% of human genes are regulated by miRNA, and the expression level of miRNA is closely related to major human diseases. Quantitative detection of miRNA is helpful for in-depth understanding of its mechanism of action, which is of great significance to the diagnosis and treatment of diseases and the development of related gene medicines. Studies have shown that the expression level of one miRNA is often associated with multiple diseases. For example, miR-373 expression is increased in bre...

Claims

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Application Information

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IPC IPC(8): C12Q1/6883C12N15/11C12Q1/6816
CPCC12Q1/6816C12Q1/6883C12Q2600/178C12Q2563/107
Inventor 戴宗陈俊吕品田邹小勇
Owner SUN YAT SEN UNIV
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