scholarly journals Suppression of rolling circle amplification by nucleotide analogs in circular template for three DNA polymerases

2016 ◽  
Vol 80 (8) ◽  
pp. 1555-1561 ◽  
Author(s):  
Suming Tang ◽  
Hua Wei ◽  
Tianyu Hu ◽  
Jiquan Jiang ◽  
Jinglin Chang ◽  
...  
Biosensors ◽  
2021 ◽  
Vol 11 (7) ◽  
pp. 222
Author(s):  
Chenxin Fang ◽  
Ping Ouyang ◽  
Yuxing Yang ◽  
Yang Qing ◽  
Jialun Han ◽  
...  

A microRNA (miRNA) detection platform composed of a rolling circle amplification (RCA) system and an allosteric deoxyribozyme system is proposed, which can detect miRNA-21 rapidly and efficiently. Padlock probe hybridization with the target miRNA is achieved through complementary base pairing and the padlock probe forms a closed circular template under the action of ligase; this circular template results in RCA. In the presence of DNA polymerase, RCA proceeds and a long chain with numerous repeating units is formed. In the presence of single-stranded DNA (H1 and H2), multi-component nucleic acid enzymes (MNAzymes) are formed that have the ability to cleave substrates. Finally, substrates containing fluorescent and quenching groups and magnesium ions are added to the system to activate the MNAzyme and the substrate cleavage reaction, thus achieving fluorescence intensity amplification. The RCA–MNAzyme system has dual signal amplification and presents a sensing platform that demonstrates broad prospects in the analysis and detection of nucleic acids.


Biosensors ◽  
2021 ◽  
Vol 11 (10) ◽  
pp. 352
Author(s):  
Kuankuan Zhang ◽  
Hua Zhang ◽  
Haorui Cao ◽  
Yu Jiang ◽  
Kang Mao ◽  
...  

Environmental contaminants are a global concern, and an effective strategy for remediation is to develop a rapid, on-site, and affordable monitoring method. However, this remains challenging, especially with regard to the detection of various contaminants in complex water environments. The application of molecular methods has recently attracted increasing attention; for example, rolling circle amplification (RCA) is an isothermal enzymatic process in which a short nucleic acid primer is amplified to form a long single-stranded nucleic acid using a circular template and special nucleic acid polymerases. Furthermore, this approach can be further engineered into a device for point-of-need monitoring of environmental pollutants. In this paper, we describe the fundamental principles of RCA and the advantages and disadvantages of RCA assays. Then, we discuss the recently developed RCA-based tools for environmental analysis to determine various targets, including heavy metals, organic small molecules, nucleic acids, peptides, proteins, and even microorganisms in aqueous environments. Finally, we summarize the challenges and outline strategies for the advancement of this technique for application in contaminant monitoring.


2019 ◽  
Vol 11 (15) ◽  
pp. 2082-2088 ◽  
Author(s):  
Zhanmin Liu ◽  
Yanming Wang ◽  
Liping Li ◽  
Junhai Li ◽  
Yuanyuan Yuan

MicroRNA-378 (miRNA-378) is widely regarded as a novel noninvasive serum biomarker for early detection of gastric cancer.


2021 ◽  
Author(s):  
Thanyarat Chaibun ◽  
Jiratchaya Puenpa ◽  
Tatchanun Ngamdee ◽  
Nimaradee Boonapatcharoen ◽  
Pornpat Athamanolap ◽  
...  

Abstract This protocol describes the rolling circle amplification (RCA) and electrochemical detection of SARS-CoV-2. The procedure consists of 3 parts, which are the amplification, hybridization and detection steps. In the presence of RNA template, the circular DNA template will be ligated to produce a Padlock DNA, which serves as a template for amplification by phi29 DNA polymerase to produce RCA amplicons. The RCA amplicon is a concatemer containing multiple repeats of sequences that are complementary to the circular template. The RCA amplicons are hybridized with redox active probes and detected by electrochemical biosensor using differential pulse voltammetry (DPV). Due to its isothermal nature, RCA can be performed using a simple water bath or heating block. Overall, the whole assay takes approximately 45 min. The assay enables rapid, quantitative results to be obtained for detection of SARS-CoV-2, either in the laboratory or more importantly, in a field setting.


Biomics ◽  
2020 ◽  
Vol 12 (4) ◽  
pp. 469-474
Author(s):  
A.R. Gilvanov ◽  
A.R. Sakhabutdinova ◽  
R.R. Garafutdinov

The nucleic acids amplification is one of the key methods for molecular biology research and clinical diagnostics. The isothermal amplification methods, for example rolling circle amplification, are a good alternative for a widely spread polymerase chain reaction. Strand-displacement DNA polymerases are required for isothermal amplification. In this work, we studied the influence of temperature on the formation of specific and non-specific amplification products by 9°Nm, Vent exo-, Hemo KlenTaq DNA polymerases during rolling circle amplification. The temperature values for the most effective formation of non-specific products and specific concatemeric products were determined. The obtained data will allow the development of more specific isothermal amplification methods with DNA polymerases used.


2017 ◽  
Author(s):  
Bo Tian ◽  
Peter Svedlindh ◽  
Mattias Strömberg ◽  
Erik Wetterskog

In this work, we demonstrate for the first time, a ferromagnetic resonance (FMR) based homogeneous and volumetric biosensor for magnetic label detection. Two different isothermal amplification methods, <i>i.e.</i>, rolling circle amplification (RCA) and loop-mediated isothermal amplification (LAMP) are adopted and combined with a standard electron paramagnetic resonance (EPR) spectrometer for FMR biosensing. For RCA-based FMR biosensor, binding of RCA products of a synthetic Vibrio cholerae target DNA sequence gives rise to the formation of aggregates of magnetic nanoparticles. Immobilization of nanoparticles within the aggregates leads to a decrease of the net anisotropy of the system and a concomitant increase of the resonance field. A limit of detection of 1 pM is obtained with an average coefficient of variation of 0.16%, which is superior to the performance of other reported RCA-based magnetic biosensors. For LAMP-based sensing, a synthetic Zika virus target oligonucleotide is amplified and detected in 20% serum samples. Immobilization of magnetic nanoparticles is induced by their co-precipitation with Mg<sub>2</sub>P<sub>2</sub>O<sub>7</sub> (a by-product of LAMP) and provides a detection sensitivity of 100 aM. The fast measurement, high sensitivity and miniaturization potential of the proposed FMR biosensing technology makes it a promising candidate for designing future point-of-care devices.<br>


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