Method for detecting miRNA by ultra-branch rolling circle amplification triggered by connection reaction

Abstract

The invention discloses a method for detecting miRNA by ultra-branch rolling circle amplification triggered by a connection reaction, and belongs to the field of molecular biology and nucleic acid chemistry. The method is mainly composed of three parts, wherein the first part is to mutually supplement and pair two terminals of a lock type probe of 5'end phosphorylation and a target marker minRNA, and then connect them by a SplintR ligase to be a ring; the second part is an ultra-branch roll ring amplification; after the lock type probe is used as a template after forming to be a ring, the target market miRNA is used as a primer; under the function of phi 29 DNA polymerase, the linear roll ring amplification is generated; through introducing a second-grade primer, the linear roll ring amplification is expanded along a branch chain direction, so as to further amplify the signal; the third part is to add SYBR GREEN I dye in the roll ring amplification product to detect. The method is simple in operation, low in cost, single in system, high in sensitivity, free from the background fluorescence signal disturbance, and suitable for detecting miRNA in a complex biotic environment.

Description

technical field [0001] The invention belongs to the fields of molecular biology and nucleic acid chemistry, and in particular relates to a method for detecting miRNA by hyperbranched rolling circle amplification triggered by a ligation reaction. Background technique [0002] miRNA (microRNA) is a class of non-coding single-stranded ribonucleic acid molecules encoded by endogenous genes with a length of about 18-25 nucleotides, which participate in post-transcriptional gene expression regulation in animals and plants. miRNA and siRNA have similar lengths, and both are cut by Dicer enzyme. But the difference is that the mature miRNA is processed once by the original pri-miRNA to form pre-miRNA (microRNA precursor), and then processed to form mature miRNA. These processing processes require Dorsha enzyme and Dicer enzyme. In terms of biological function, the complete complementary pairing of miRNA and target messenger RNA will lead to the degradation of the messenger RNA of th...

AI Technical Summary

Problems solved by technology

But before this, it is necessary to connect the padlock probe to form a circle as the template for amplification, so the efficiency of the ligation reaction is also an important factor affecting the detection sensitivity.

In previous studies, T4 DNA ligase was widely used, but the ligation efficiency was not very satisfactory in the ligation reaction where miRNA was used as a template to ligate padlock probes

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