Small RNA (ribonucleic acid) detection kit and quantitative method based on unbiased recognition and isothermal amplification

Abstract

The invention discloses a small RNA (ribonucleic acid) detection kit and quantitative method based on unbiased recognition and isothermal amplification. Through nucleic acid complementary hybridization, target small RNA specifically identifies a 3-WJ primer and a 3-WJ template, a stable three-way cross structure is formed, and the 3-WJ primer starts an SDA (strand displacement amplification) reaction along with the phosphorothioate-modified 3-WJ template and produces a large number of single-chain SDA products with restriction enzyme cutting sites. The single-chain DNA opens stem-loop structures of molecular beacons to enable fluorescence to recover. A double-chain complementary structure formed by the molecular beacons and the single-chain SDA products contains nucleic acid nickase recognition sites, the sites are recognized by nickase after the double-chain structure is formed, the cut molecular beacons fall off from the double-chain structure to produce fluorescence signals, and the released SDA products and new molecular beacons can form hybrid double chains and produce more fluorescence signals. The quantitative method and the kit are high in sensitivity, and cascade amplification of the SDA reaction is realized skillfully by the aid of the phosphorothioate-modified template.

Description

technical field [0001] The invention belongs to the technical field of small RNA detection, and in particular relates to a small RNA detection kit and quantitative method based on unbiased identification and constant temperature amplification. Background technique [0002] Small RNAs are a class of endogenous non-protein-coding RNA molecules widely present in eukaryotes, with a length of about 20-30 bases, mainly including small interfering RNAs (siRNAs), microRNAs (miRNAs) and piwi-interacting RNA (piRNA). Small RNA guides AGO to approach its target molecules (DNA or RNA) by binding to specific Argonaute family proteins (AGO proteins), and specifically reduces the expression of target genes through RNA-induced silencing complexes. Small RNAs participate in the regulation of cell growth, development, differentiation, proliferation, apoptosis and other life processes, affect almost all signaling pathways, participate in various physiological and pathological processes, and p...

AI Technical Summary

Problems solved by technology

Although the 3' end methylation of these small RNAs does not change the nucleotide sequence, it brings great challenges to the existing detection technologies based on enzyme excision, enzyme polymerization, and enzyme ligation. These enzyme reactions need to be combined with The 3' end of the small molecule RNA acts, and the methylation of the 3' end will affect the recognition ability of the enzyme, inhibit the efficiency of the enzyme reaction, and eventually lead to inaccurate detection results

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