Sensitive fluorescent detection of exosomal microRNA based on enzymes-assisted dual-signal amplification

A ultrasensitive, easy operated and robust assay of S1 nuclease in real samples and ATP has been successfully achieved with the dual-amplification strategy based on rolling circle replication and Exo III-aided recycling.

Nuclease

Rolling circle replication

Replication

Exonuclease III

10.1039/c5ra13301g

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Citations (7)

A one-pot isothermal Cas12-based assay for the sensitive detection of microRNAs

Nature Biomedical Engineering (2023)

He Yan Yunjie Wen Zimu Tian Nathan Hart Song Han

Extracellular Vesicles

Rolling circle replication

Cancer Biomarkers

10.1038/s41551-023-01033-1

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Citations (155)

Accurate Detection of Target MicroRNA in Mixed Species of High Sequence Homology Using Target-Protection Rolling Circle Amplification

ACS Omega (2021)

Bin Zhang Shuo Li Yifu Guan Ying Yuan

The close relationships of miRNAs with human diseases highlight the urgent needs for miRNA detection. However, the accurate detection of a target miRNA in mixed miRNAs of high sequence homology presents a great challenge. Herein, a novel method called target-protection rolling circle amplification (TP-RCA) is proposed for this purpose. The protective probe is designed so that it can form a fully complementary duplex with the target miRNA and can also mismatch duplexes with other nontarget miRNAs. These duplexes are treated with a single strand-specific nuclease. Consequently, only the target miRNA in a perfect-match duplex can resist the cleavage of nuclease, whereas the nontarget miRNAs in mismatched duplexes will be digested completely. The protected target miRNA can be detected using RCA reactions. MicroRNA let-7 family members (let-7a–let-7f) and nuclease CEL I were used as proof-of-concept models to evaluate the feasibility of the TP-RCA method under different experimental conditions. The experimental results show that the TP-RCA method can unambiguously detect the target let-7 species in mixtures of let-7 family members even though they may differ by only a single nucleotide. This TP-RCA method significantly improves the detection specificity of miRNAs.

Nuclease

Duplex (building)

Rolling circle replication

Homology

Cleavage (geology)

10.1021/acsomega.0c05279

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Citations (8)

Biochemical method for mapping mutational alterations in DNA with S1 nuclease: the location of deletions and temperature-sensitive mutations in simian virus 40.

Proceedings of the National Academy of Sciences (1975)

Thomas Shenk Craig Rhodes Peter Rigby Paul Berg

S1 nuclease (EC 3.1.4.X), a single-strand-specific nuclease, can be used to accurately map the location of mutational alterations in simian virus 40 (SV40) DNA. Deletions of between 32 and 190 base pairs, which are at or below the limit of detectability by conventional electron microscopic analysis of heteroduplex DNAs, have been located in this way. To map a deletion, a mixture of unit length, linear DNA, prepared from the SV40 deletion mutant and its wild-type parent, are denatured and reannealed to form heteroduplexes. S1 nuclease can cut such heteroduplexes at the nonbase-paired region to produce fragments whose lengths correspond to the position of the deletion. Similarly, specific fragments are produced when S1 nuclease cleaves a heteroduplex formed from the DNAs of SV40 temperature-sensitive mutants and either their revertants or wild-type parents. Thus, the positions of the nonhomology between these DNAs can be determined.

Heteroduplex

Nuclease

10.1073/pnas.72.3.989

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Citations (264)

Applications of heteroduplex analysis for mutation detection in disease genes

Human Mutation (1995)

Damjan Glavač Michael Dean

Double-stranded heteroduplex molecules that form between a mutant and wild-type DNA strand are often distinguished from homoduplex molecules upon gel electrophoresis. This method, heteroduplex analysis (HA), can be performed rapidly without radioisotopes or specialized equipment. Modifications and enhancements of the HA method have been developed that increase the sensitivity of detection of single-base pair alterations. © 1995 Wiley-Liss, Inc.1 This article is a US Government work and, as such, is in the public domain in the United States of America.

Heteroduplex

10.1002/humu.1380060402

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Citations (54)