A visualized miRNA detecting method by utilizing an exonuclease reaction to generate primers and dendritic rolling circle amplification

Abstract

A visualized miRNA detecting method by utilizing an exonuclease reaction to generate primers and dendritic rolling circle amplification is disclosed. The method includes three steps. The first step is a step of primer generation, wherein after a hairpin-structured probe I is opened by a target miRNA, a single-chain part is degraded by an exonuclease I, and then RNA in a DNA:RNA hybrid chain is degraded by ribonuclease H to leave a short-chain DNA segment. The second step is a step of dendritic rolling circle amplification, wherein the single-chain DNA generated in the former step is connected to a long single-chain DNA probe to form a ring adopted as a template, first-stage rolling circle amplification is performed by adopting the single-chain DNA as a primer, and a hairpin-structured stem-loop structure II introduced into a rolling circle amplification system is opened by a product of the first-stage rolling circle amplification, thus performing multi-stage dendritic rolling circle amplification. The third step is a step of performing detection based on a G-quadruplex in a product of the rolling circle amplification. The method is a sensitive, economical, convenient and in-situ visualized miRNA detecting method.

Description

technical field [0001] The invention belongs to the fields of molecular biology and nucleic acid chemistry, and relates to a method for visually detecting miRNA by using exonuclease reaction to generate primers combined with dendritic rolling circle amplification. Background technique [0002] MicroRNA (miRNA) is a kind of endogenous, non-coding small RNA with a length of about 20-24 nucleotides, which has a variety of important regulatory functions in cells. Mature miRNAs have a phosphate group at the 5' end and a hydroxyl group at the 3' end. miRNA genes are usually transcribed by nuclear RNA polymerase II, and the initial product is a large pri-miRNA with a cap structure and a polyA tail, which is then processed into a 60-70 base hairpin by Drosha in the nucleus The pre-RNA is transported into the cytoplasm by the transport protein Exprotin-5 complex, and then recognized and cleaved by Dicer enzyme to form mature miRNA. It is assumed that miRNAs regulate one-third of hu...

AI Technical Summary

Problems solved by technology

Some current methods for miRNA detection, such as the traditional northern blot method, require large sample volumes, long detection cycles, and poor ability to distinguish homologous sequences, which limits further applications

Some emerging methods based on nanoparticles and electrochemical detection methods have solved the problem of detection limit very well, but for the production of nanoparticles and electrochemical substrates, skilled professionals are required, and it is not easy to popularize and apply this method. RT -PCR method successfully solves the above problems, with high sensitivity, can detect RNA as low as 1-10ng, and the detection range is as high as 7 orders of magnitude, can detect low or high concentration miRNA, and can distinguish homologs very well sequence

However, complex primer design and expensive real-time fluorescence quantitative PCR instruments limit the popularization and application of RT-PCR to a certain extent.

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