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

A detection kit and constant temperature amplification technology, which is applied in the direction of microbial measurement/testing, biochemical equipment and methods, etc., can solve the problems of affecting enzyme recognition ability and inaccurate detection results, and achieve rapid enzyme synergistic cascade constant temperature amplification. Increased response, increased sensitivity, and simple design effects

Active Publication Date: 2015-07-22
XI AN JIAOTONG UNIV
View PDF2 Cites 11 Cited by
  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

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 reactio...

Method used

the structure of the environmentally friendly knitted fabric provided by the present invention; figure 2 Flow chart of the yarn wrapping machine for environmentally friendly knitted fabrics and storage devices; image 3 Is the parameter map of the yarn covering machine
View more

Image

Smart Image Click on the blue labels to locate them in the text.
Viewing Examples
Smart Image
  • Small RNA (ribonucleic acid) detection kit and quantitative method based on unbiased recognition and isothermal amplification
  • Small RNA (ribonucleic acid) detection kit and quantitative method based on unbiased recognition and isothermal amplification
  • Small RNA (ribonucleic acid) detection kit and quantitative method based on unbiased recognition and isothermal amplification

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0047] A small RNA detection kit based on unbiased recognition and constant temperature amplification, including:

[0048] A composition that forms a three-way hybrid structure with the target small RNA, including 10nM 3-WJ primer and 10nM 3-WJ template:

[0049] The 3-WJ primer consists of two parts, the 5' end part is complementary to the 3' end part of the target small RNA, and the 3' end part is complementary to the middle segment of the 3-WJ template;

[0050] The 3-WJ template is composed of three parts, the 3' end part is complementary to the 5' end part of the target small RNA, the middle part is complementary to the 3' end part of the 3-WJ primer, and the 5' end part contains two nuclease recognition sites The SDA template region of the point; one of the enzyme cleavage sites has been thio-modified;

[0051] The nucleotide sequence of the 3-WJ primer is (5'to 3'):

[0052] GTG CTC ACT CAT CCA AAA (SEQ.ID.NO.1)

[0053] Nucleotide sequence (5'to 3') of the thio-modi...

the structure of the environmentally friendly knitted fabric provided by the present invention; figure 2 Flow chart of the yarn wrapping machine for environmentally friendly knitted fabrics and storage devices; image 3 Is the parameter map of the yarn covering machine
Login to view more

PUM

No PUM Login to view more

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...

Claims

the structure of the environmentally friendly knitted fabric provided by the present invention; figure 2 Flow chart of the yarn wrapping machine for environmentally friendly knitted fabrics and storage devices; image 3 Is the parameter map of the yarn covering machine
Login to view more

Application Information

Patent Timeline
no application Login to view more
IPC IPC(8): C12Q1/68
Inventor 赵永席陈锋赵越
Owner XI AN JIAOTONG UNIV
Who we serve
  • R&D Engineer
  • R&D Manager
  • IP Professional
Why Eureka
  • Industry Leading Data Capabilities
  • Powerful AI technology
  • Patent DNA Extraction
Social media
Try Eureka
PatSnap group products

Browse by: Latest US Patents, China's latest patents, Technical Efficacy Thesaurus, Application Domain, Technology Topic.

© 2024 PatSnap. All rights reserved.Legal|Privacy policy|Modern Slavery Act Transparency Statement|Sitemap