Unlock instant, AI-driven research and patent intelligence for your innovation.

A Nucleic Acid Isothermal Exponential Amplification Technology Based on Symmetrical Ring Dumbbell Template and Its Application in MicroRNA Detection

A circular, dumbbell-shaped technology, applied in DNA/RNA fragmentation, recombinant DNA technology, microbial assay/inspection, etc., can solve the problems of reducing amplification specificity, loss of amplification efficiency, etc. The effect of good performance and shortening of detection time

Active Publication Date: 2019-02-12
SUN YAT SEN UNIV
View PDF2 Cites 0 Cited by
  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

The primers hybridized at the 3' end can be amplified normally, but the primers hybridized at the 5' end cannot be amplified. Therefore, the amplification reaction loses 50% of the amplification efficiency; the non-specific amplification at the 3' end will also greatly reduce the amplification efficiency. Enhanced specificity

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
  • A Nucleic Acid Isothermal Exponential Amplification Technology Based on Symmetrical Ring Dumbbell Template and Its Application in MicroRNA Detection
  • A Nucleic Acid Isothermal Exponential Amplification Technology Based on Symmetrical Ring Dumbbell Template and Its Application in MicroRNA Detection
  • A Nucleic Acid Isothermal Exponential Amplification Technology Based on Symmetrical Ring Dumbbell Template and Its Application in MicroRNA Detection

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0042] Embodiment 1 Nucleic acid constant temperature exponential amplification technical scheme based on symmetrical circular dumbbell-shaped template

[0043] 1. Preparation and characterization of circular dumbbell-shaped amplification template

[0044] (1) Synthesis of circular dumbbell-shaped amplification template

[0045]A series of linear probes with different toehold lengths were designed according to the miRNA sequence to be tested, and named T according to the number of toehold bases. N . A monophosphate group is modified at the 5' end of the probe, and the 3' and 5' ends of the probe are connected by T4 DNA ligase to form a circular probe, which is then hydrolyzed by exonuclease I and exonuclease III to react. Linear probe to obtain circular dumbbell-shaped amplification template.

[0046] (2) Characterization of circular dumbbell-shaped amplification template

[0047] The circular dumbbell-shaped amplification template prepared in (1) was characterized by elec...

Embodiment 2

[0073] The technical solution of the present invention is further described below by taking the detection of let-7a in the let-7 family of miRNAs as an example.

[0074] 1. Synthesis of circular dumbbell-shaped amplification template

[0075] let-7a sequence: 5'-UGA GGU AGU AGG UUG UAU AGUU-3' (SEQ ID NO. 7).

[0076] A series of linear probes with different toehold lengths were designed for the let-7a sequence, which were named T according to the number of toehold bases. 9 ~T 14 . The base sequence of the probe is as follows: (underlined is the toehold region, and the bold part is the nickase recognition sequence) T 14 :5′-(PO 4 )TTA ACT ATA C AA CCT ACT ACC TCA CCC CGT ATA GTTAAC TAT AC A ACC TAC TAC CTC A CC CCG TAT AG-3' (SEQ ID NO. 1);

[0077] T 13 :5′-(PO 4 )TTA ACT ATA CA A CCT ACT ACC TCA CCC TGT ATA GTTAAC TAT ACA ACC TAC TAC CTC A CC CTG TAT AG-3' (SEQ ID NO. 2);

[0078] T 12 :5′-(PO 4 )TTA ACT ATA CAA CCT ACT ACC TCA CCT TGT ATA GTTAA...

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 nucleic acid isothermal exponential amplification technology based on a symmetric ring-like dumbbell template and application of the nucleic acid isothermal exponential amplification technology to microRNA detection. The nucleic acid isothermal exponential amplification technology based on the symmetric ring-like dumbbell template has the advantages that the symmetric ring-like dumbbell template is designed based on a toehold exchange principle to solve the problem of amplification efficiency loss in amplification reaction, so that specificity of the amplification reaction is guaranteed while 100% quadratic exponential amplification is obtained, and rapider, more efficient and higher-selectivity nucleic acid isothermal exponential amplification is achieved and applied to sensitive detection of short-chain nucleic acid.

Description

technical field [0001] The invention belongs to the technical field of molecular detection, and in particular relates to a nucleic acid constant temperature exponential amplification technology based on a symmetrical circular dumbbell template and its application in microRNA detection. Background technique [0002] MicroRNA (miRNA) is a class of non-coding small RNAs (18-24 bases) that regulate gene expression. It plays an important role in the growth, development, differentiation and reproduction of animals and plants. About 30% of human genes are regulated by miRNA, and the expression level of miRNA is closely related to major human diseases. Quantitative detection of miRNA is helpful to deeply understand its mechanism of action, which is of great significance to the diagnosis and treatment of diseases and the development of related gene drugs. Due to the short sequence of miRNA, the expression level in cells or body fluids is very low, and it is easy to degrade, and the...

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
Patent Type & Authority Patents(China)
IPC IPC(8): C12N15/11C12Q1/6844
CPCC12Q1/6844C12Q2531/119C12Q2525/207C12Q2525/307
Inventor 戴宗陈俊邹小勇
Owner SUN YAT SEN UNIV