Rolling circle amplification: A (random) primer on the enrichment of an infinite linear DNA template

2019 ◽  
Vol 2 (1) ◽  
Author(s):  
Rachel E. Kieser ◽  
Bruce Budowle
2016 ◽  
Vol 52 (40) ◽  
pp. 6701-6704 ◽  
Author(s):  
Qing Sun ◽  
Wilfred Chen

We report here the generation of artificial cellulosomes onto a DNA scaffold using the self-labeling HaloTag for DNA conjugation. Rolling circle amplification multiplexing templates were used to increase the complexity of this system with higher efficiency observed.


RSC Advances ◽  
2015 ◽  
Vol 5 (6) ◽  
pp. 4019-4025 ◽  
Author(s):  
Fulin Zhou ◽  
Baoxin Li ◽  
Jiyuan Ma

Linear single-labeled DNA probes are used in this RCA-based fluorescence strategy for DNA detection, which could effectively avoid the fluorescence quenching between neighboring signal probes using hairpin probe as signal probe.


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.


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>


IMA Fungus ◽  
2021 ◽  
Vol 12 (1) ◽  
Author(s):  
Shan Shen ◽  
Shi-Liang Liu ◽  
Ji-Hang Jiang ◽  
Li-Wei Zhou

Abstract“Sanghuang” refers to a group of important traditionally-used medicinal mushrooms belonging to the genus Sanghuangporus. In practice, species of Sanghuangporus referred to in medicinal studies and industry are now differentiated mainly by a BLAST search of GenBank with the ITS barcoding region as a query. However, inappropriately labeled ITS sequences of “Sanghuang” in GenBank restrict accurate species identification and, to some extent, the utilization of these species as medicinal resources. We examined all available 271 ITS sequences related to “Sanghuang” in GenBank including 31 newly submitted sequences from this study. Of these sequences, more than half were mislabeled so we have now corrected the corresponding species names. The mislabeled sequences mainly came from strains utilized by non-taxonomists. Based on the analyses of ITS sequences submitted by taxonomists as well as morphological characters, we separate the newly described Sanghuangporus subbaumii from S. baumii and treat S. toxicodendri as a later synonym of S. quercicola. Fourteen species of Sanghuangporus are accepted, with intraspecific distances up to 1.30% (except in S. vaninii, S. weirianus and S. zonatus) and interspecific distances above 1.30% (except between S. alpinus and S. lonicerinus, and S. baumii and S. subbaumii). To stabilize the concept of these 14 species of Sanghuangporus, their taxonomic information and reliable ITS reference sequences are provided. Moreover, ten potential diagnostic sequences are provided for Hyperbranched Rolling Circle Amplification to rapidly confirm three common commercial species, viz. S. baumii, S. sanghuang, and S. vaninii. Our results provide a practical method for ITS barcoding-based species identification of Sanghuangporus and will promote medicinal studies and commercial development from taxonomically correct material.


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.


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