阅读说明：本技术 一种基于DNA网状结构灵敏检测miRNA的方法 (Method for sensitively detecting miRNA based on DNA network structure ) 是由 刘海云 高奕 何涛 颜梅 于京华 于 2020-04-21 设计创作，主要内容包括：本专利公开了一种基于DNA网状结构灵敏检测miRNA的方法,涉及生物传感领域。通过设计结构可切换的哑铃型探针,提高了目标识别的特异性,同时又使得滚环扩增产物具有串联重复茎环结构的特点；滚环扩增产物与修饰DNA-AuNPs杂交形成DNA网状结构,使得AuNPs发生聚集,利用动态光散射技术对AuNPs尺寸进行测量,从而实现miRNA的检测。本发明灵敏度高、特异性好。(The patent discloses a method for sensitively detecting miRNA based on a DNA mesh structure, and relates to the field of biosensing. By designing the dumbbell-shaped probe with switchable structure, the specificity of target recognition is improved, and meanwhile, the rolling circle amplification product has the characteristic of a tandem repeat stem-loop structure; the rolling circle amplification product and the modified DNA-AuNPs are hybridized to form a DNA net structure, so that the AuNPs are aggregated, and the size of the AuNPs is measured by using a dynamic light scattering technology, thereby realizing the detection of miRNA. The invention has high sensitivity and good specificity.)

1. A method for sensitively detecting miRNA based on a DNA reticular structure is characterized by comprising the following steps:

(1) probe design

Designing corresponding dumbbell-shaped padlock probes PP and DNA sequences for modifying AuNPs according to the target chain sequence; wherein, 5 end of the dumbbell-shaped padlock probe PP sequence is modified with phosphate group, 3 end of the DNA sequence of AuNPs is modified with sulfhydryl (C)₆-SH）；

(2) Connection of dumbbell padlock probe PP

The ligation of the dumbbell padlock probe PP was performed in a 10. mu.L volume of solution containing 10. mu.M padlock probe PP, 2. mu.L 10 XT 4 DNA ligase reaction buffer, 1. mu.M let-7a, 20U/. mu. L T4 DNA ligase and 5. mu.L DEPC treated water, and the reaction mixture was heated at 55 ℃ for 5 minutes before adding T4 DNA ligase, then annealed at 39 ℃ for 30 minutes, cooled to room temperature, then added with T4 DNA ligase and subjected to ligation at 16 ℃ for 2 hours;

(3) rolling circle amplification reaction

Adding 2.5. mu.L of 10 XPhi 29 DNA polymerase reaction buffer, 0.5. mu.LBSA, 3. mu.L of dNTPs with the concentration of 25 mM, 1. mu.L of phi29 DNA polymerase and 8. mu.L of DEPC treated water to the solution obtained in step (2); the resulting mixture was reacted at 30 ℃ for 6 hours and then incubated at 65 ℃ for 10 minutes to terminate the reaction;

(4) dynamic light scattering technology detection

Adding 10 mu L of DNA-AuNPs into the solution obtained in the step (3), and incubating the mixed solution at 37 ℃ for 30 minutes; dynamic light scattering measurements were then performed, with the following experimental parameters: 25 ℃, equilibration stabilization time 10 seconds and measurement angle 90 °.

2. The method for sensitively detecting miRNA based on the DNA network structure of claim 1, wherein the rolling circle amplification product with the tandem stem-loop structure is hybridized with DNA-AuNPs to form the DNA network structure, so that the AuNPs are dispersed to be aggregated and have sizes that are changed from small to large.

3. The method for sensitively detecting miRNA based on the DNA network structure of claim 1, wherein the dumbbell-shaped padlock probe is connected into the dumbbell-shaped sealing probe by taking the padlock probe as a template, and the sealing probe can be activated from the dumbbell shape to a circular shape through a strand displacement reaction only in the presence of the target miRNA, thereby starting the rolling circle amplification reaction.

4. The method for sensitively detecting miRNA based on DNA network structure of claim 1, wherein the DNA sequences of the dumbbell-shaped padlock probe PP and the modified AuNPs in the step (A) have the nucleotide sequences shown in the sequence list.

Technical Field

The invention relates to the field of detection of a dynamic light scattering technology, in particular to a method for detecting miRNA (micro ribonucleic acid) by constructing a DNA (deoxyribonucleic acid) network structure based on a nucleic acid rolling circle amplification technology, inducing nanoparticles to be in an aggregation state from a monodisperse state and detecting the particle size of the nanoparticles by using the dynamic light scattering technology.

Background

MicroRNA (miRNA) is a very active research field at present, and has great significance for deeply discussing the nature of life phenomenon, explaining cell behavior and disease generating mechanism, and developing disease diagnosis and gene therapy medicines. mirnas are widely present in various animals and plants, and participate in a variety of important life activities. It is an important regulatory factor for gene regulation, plays an important role in many biological processes, but its regulation range is not limited to normal physiological activities, and has close relation with the occurrence of many tumors and cancers.

MiRNA is considered as a novel biomarker and potential therapeutic target for various diseases. Its wide existence and conservation in evolution suggest that it has essential regulation in life activities, and they are involved in various processes such as growth and development of animals and plants, cell differentiation, cell proliferation and apoptosis, hormone secretion, tumor formation, etc. Mirnas, both quantitatively and functionally, may far exceed current findings, and therefore detection of mirnas is a hot spot and frontier of current research. However, due to the inherent characteristics of easy degradation and low abundance of mirnas, it is difficult to detect mirnas with high sensitivity.

In order to solve the problem that miRNA can be detected even at low concentration, identification and signal amplification detection strategies for miRNA become especially important. However, currently common miRNA detection methods, such as optical detection methods of fluorescence colorimetry, fluorescence method, chemiluminescence method, and the like, have low sensitivity and are susceptible to photostability and complex biological samples. Therefore, the development of a stable and high-sensitivity miRNA detection method is of great significance. The experiment tries to develop a sensitive and stable method, a dumbbell-shaped probe with a switchable structure is designed, a rolling circle amplification technology is utilized to construct a DNA mesh structure, and a stable dynamic light scattering technology is utilized to realize the high-sensitive detection of miRNA.

Disclosure of Invention

The invention aims to solve the technical problem of constructing a DNA reticular structure by a rolling circle amplification technology to realize high-sensitivity detection of miRNA.

A method for sensitively detecting miRNA based on a DNA reticular structure is characterized by comprising the following steps:

(1) probe design

Designing corresponding DNA sequences of a padlock probe PP and modified gold nanoparticles (AuNPs) according to a target chain sequence; wherein is dumbThe 5 end of the bell-shaped padlock probe PP sequence is modified with phosphate group, the 3 end of the DNA sequence of AuNPs is modified with sulfhydryl (C)₆-SH）；

(2) Connection of dumbbell padlock probe PP

The ligation reaction of the dumbbell padlock probe PP was performed in a 10. mu.L solution volume containing 10. mu.M padlock probe PP, 2. mu.L of 10 XT 4 DNA ligase reaction buffer, 1. mu.M target miRNA, 20U/. mu. L T4 DNA ligase and 5. mu.L of DEPC treated water, and before adding T4 DNA ligase, the reaction mixture was heated at 55 ℃ for 5 minutes, then annealed at 39 ℃ for 30 minutes, cooled to room temperature, then T4 DNA ligase was added, and ligation reaction was performed at 16 ℃ for 2 hours;

(3) rolling circle amplification reaction

Adding 2.5. mu.L of 10 XPhi 29 DNA polymerase reaction buffer, 0.5. mu.LBSA, 3. mu.L of dNTPs with the concentration of 25 mM, 1. mu.L of phi29 DNA polymerase and 8. mu.L of DEPC treated water to the solution obtained in step (2); the resulting mixture was reacted at 30 ℃ for 6 hours and then incubated at 65 ℃ for 10 minutes to terminate the reaction;

(4) dynamic light scattering technology detection

Adding 10 mu L of DNA-AuNPs into the solution obtained in the step (3), and incubating the mixed solution at 37 ℃ for 30 minutes; dynamic light scattering measurements were then performed, with the following experimental parameters: 25 ℃, equilibration stabilization time 10 seconds and measurement angle 90 °.

The invention has the advantages of

(1) The dumbbell-shaped structure of the probe enables the rolling circle amplification product to have a tandem repeat stem-loop sequence, and the rolling circle amplification product is hybridized with DNA-AuNPs to form a DNA net structure, so that the AuNPs are aggregated and the size of the AuNPs is increased;

(2) the dumbbell type probe with a switchable structure is used for specifically recognizing miRNA, a rolling circle amplification reaction is started, and amplification of signals is realized;

(3) the method of the invention has high sensitivity and good specificity.

Drawings

FIG. one is an experimental schematic of the method described herein.

Detailed Description

For a better understanding of the invention, the following further illustrates the invention with reference to examples and drawings, but the invention is not limited to the following embodiments.