scholarly journals Clonal rolling circle amplification for on-chip DNA cluster generation

2017 ◽  
Vol 2 (1) ◽  
Author(s):  
Christian Korfhage ◽  
Evelyn Fricke ◽  
Andreas Meier ◽  
Andreas Geipel ◽  
Mark Baltes ◽  
...  
Keyword(s):  
Single Molecule ◽  
Rolling Circle Amplification ◽  
Nucleic Acid Detection ◽  
Rolling Circle ◽  
Oligonucleotide Hybridization ◽  
Dna Strands ◽  
Multiple Copies ◽  
Cluster Generation ◽  
On Chip ◽  
Micrometer Size

Abstract Generation of monoclonal DNA clusters on a surface is a useful method for digital nucleic acid detection applications (e.g. microarray or next-generation sequencing). To obtain sufficient copies per cluster for digital detection, the single molecule bound to the surface must be amplified. Here we describe ClonalRCA, a rolling-circle amplification (RCA) method for the generation of monoclonal DNA clusters based on forward and reverse primers immobilized on the surface. No primer in the reaction buffer is needed. Clusters formed by ClonalRCA comprise forward and reverse strands in multiple copies tethered to the surface within a cluster of micrometer size. Single stranded circular molecules are used as a target to create a cluster with about 10 000 forward and reverse strands. The DNA strands are available for oligonucleotide hybridization, primer extension and sequencing.

scholarly journals Highly specific imaging of mRNA in single cells by target RNA-initiated rolling circle amplification

2017 ◽  
Vol 8 (5) ◽  
pp. 3668-3675 ◽  
Author(s):  
Ruijie Deng ◽  
Kaixiang Zhang ◽  
Yupeng Sun ◽  
Xiaojun Ren ◽  
Jinghong Li
Keyword(s):  
Single Molecule ◽  
Single Cells ◽  
Rolling Circle Amplification ◽  
Robust Method ◽  
Single Nucleotide ◽  
Rolling Circle ◽  
Target Rna

We report a robust method for the efficient imaging of mRNA with single-nucleotide and near-single-molecule resolution in single cells.

scholarly journals Rolling circle amplification-driven encoding of different fluorescent molecules for simultaneous detection of multiple DNA repair enzymes at the single-molecule level

2020 ◽  
Vol 11 (22) ◽  
pp. 5724-5734
Author(s):  
Chen-chen Li ◽  
Hui-yan Chen ◽  
Juan Hu ◽  
Chun-yang Zhang
Keyword(s):  
Dna Repair ◽  
Single Molecule ◽  
Rolling Circle Amplification ◽  
Simultaneous Detection ◽  
Single Molecule Detection ◽  
Dna Repair Enzymes ◽  
Rolling Circle ◽  
Single Molecule Level ◽  
Repair Enzymes ◽  
Fluorescent Molecules

Integration of single-molecule detection with rolling circle amplification-driven encoding of different fluorescent molecules enables simultaneous detection of multiple DNA repair enzymes.

Mutation detection and single-molecule counting using isothermal rolling-circle amplification

10.1038/898 ◽  
1998 ◽  
Vol 19 (3) ◽  
pp. 225-232 ◽  
Author(s):  
Paul M. Lizardi ◽  
Xiaohua Huang ◽  
Zhengrong Zhu ◽  
Patricia Bray-Ward ◽  
David C. Thomas ◽  
...  
Keyword(s):  
Single Molecule ◽  
Mutation Detection ◽  
Rolling Circle Amplification ◽  
Rolling Circle

scholarly journals Preparation of long single-strand DNA concatemers for high-level fluorescence in situ hybridization

2021 ◽  
Author(s):  
Dongjian Cao ◽  
Sa Wu ◽  
Caili Xi ◽  
Dong Li ◽  
Kaiheng Zhu ◽  
...  
Keyword(s):  
Single Molecule ◽  
Fluorescent Proteins ◽  
Signal To Noise Ratio ◽  
Rolling Circle Amplification ◽  
Simultaneous Detection ◽  
Single Strand ◽  
Detection Methods ◽  
Rolling Circle ◽  
High Level

Abstract Fluorescence in situ hybridization (FISH) is a powerful tool to visualize transcripts in fixed cells and tissues. Despite the recent advances in FISH detection methods, it remains challenging to achieve high-level FISH imaging with a simple workflow. Here, we introduce a method to prepare long single-strand DNA concatemers (lssDNAc) through a controllable rolling-circle amplification (CRCA). Prepared lssDNAcs were used to develop novel AmpFISH workflows. In addition, we present its applications in different scenarios. AmpFISH shows the following advantages: 1) enhanced FISH signal-to-noise ratio (SNR) up to 160-fold compared with single-molecule FISH; 2) simultaneous detection of FISH signals and fluorescent proteins or immunofluorescence (IF) in tissues; 3) simple workflows; and 4) cost-efficiency. In brief, AmpFISH provides convenient and versatile tools for sensitive RNA/DNA detection and to gain useful information on cellular molecules using simple workflows.

scholarly journals Detection of Genetic Variations in Coagulopathy-Related Genes Using Ramified Rolling Circle Amplification

2014 ◽  
Vol 2014 ◽  
pp. 1-11
Author(s):  
James H. Smith ◽  
Miao Cui ◽  
David Y. Zhang ◽  
Thomas P. Beals ◽  
Fei Ye
Keyword(s):  
Clinical Laboratory ◽  
Rolling Circle Amplification ◽  
Experimental System ◽  
Clinical Samples ◽  
Nucleic Acid Detection ◽  
Rolling Circle ◽  
Padlock Probes ◽  
Target Capture ◽  
Automated Platform ◽  
Blind Comparison

We evaluated single nucleotide polymorphism (SNP) detection via a target-capture, C-probe ligation, and RAM assay in a single-blind comparison to clinical samples that had been tested with FDA-cleared tests for up to 4 different vascular disease-related SNPs. In the RAM assay circulizable linear probes (C- or padlock probes) were annealed directly to genomic DNA, processed on a largely automated platform, and ligated C-probes were amplified by real-time RAM. After allele determinations were made with the experimental system, the sample genotypes were unblinded and the experimentally determined genotypes were found to be completely consistent with the FDA-cleared test results. The methods and results presented here show that a combination of C-probes, automated sample processing, and isothermal RAM provides a robust, and specific, nucleic acid detection platform that is compatible with automated DNA sample preparation and the throughput requirements of the clinical laboratory.

Single-Molecule DNA Patterning and Detection by Padlock Probing and Rolling Circle Amplification in Microchannels for Analysis of Small Sample Volumes

2011 ◽  
Vol 83 (9) ◽  
pp. 3352-3357 ◽  
Author(s):  
Yo Tanaka ◽  
Hui Xi ◽  
Kae Sato ◽  
Kazuma Mawatari ◽  
Björn Renberg ◽  
...  
Keyword(s):  
Single Molecule ◽  
Rolling Circle Amplification ◽  
Small Sample ◽  
Rolling Circle

A plasmonic aptasensor for ultrasensitive detection of thrombin via arrested rolling circle amplification

2015 ◽  
Vol 51 (37) ◽  
pp. 7927-7930 ◽  
Author(s):  
Sai Wang ◽  
Sai Bi ◽  
Zonghua Wang ◽  
Jianfei Xia ◽  
Feifei Zhang ◽  
...  
Keyword(s):  
Single Molecule ◽  
Rolling Circle Amplification ◽  
Signal Generation ◽  
Ultrasensitive Detection ◽  
Eye Detection ◽  
Rolling Circle ◽  
Single Molecule Level ◽  
Nanoparticle Growth ◽  
Naked Eye ◽  
Naked Eye Detection

A sensitive signal generation mechanism for gold nanoparticle growth is applied in a plasmonic aptasensor, achieving naked-eye detection of thrombin at the single-molecule level.

scholarly journals ENDO-Pore: High-throughput linked-end mapping of single DNA cleavage events using nanopore sequencing

2021 ◽  
Author(s):  
Oscar E Torres Montaguth ◽  
Stephen J Cross ◽  
Kincaid W.A. Ingram ◽  
Laura Lee ◽  
Fiona M Diffin ◽  
...  
Keyword(s):  
High Throughput ◽  
Single Molecule ◽  
Dna Cleavage ◽  
Cleavage Site ◽  
Rolling Circle Amplification ◽  
Time Resolved ◽  
Rolling Circle ◽  
Precise Position ◽  
And Function

Mapping the precise position of DNA cleavage events plays a key role in determining the mechanism and function of endonucleases. ENDO-Pore is a high-throughput nanopore-based method that allows the time resolved mapping single molecule DNA cleavage events in vitro. Following linearisation of a circular DNA substrate by the endonuclease, a resistance cassette is ligated recording the position of the cleavage event. A library of single cleavage events is constructed and subjected to rolling circle amplification to generate concatemers. These are sequenced and used to produce accurate consensus sequences. To identify the cleavage site(s), we developed CSI (Cleavage Site Investigator). CSI recognizes the ends of the cassette ligated into the cleaved substrate and triangulates the position of the dsDNA break. We firstly benchmarked ENDO-Pore using Type II restriction endonucleases. Secondly, we analysed the effect of crRNA length on the cleavage pattern of CRISPR Cas12a. Finally, we mapped the time-resolved DNA cleavage by the Type ISP restriction endonuclease LlaGI that introduces random double-strand breaks into its DNA substrates.

Padlock Probe-based Rolling Circle Amplification Lateral Flow Assay for Point-of-Need Nucleic Acid Detection

The Analyst ◽  
2021 ◽  
Author(s):  
Sidhartha Jain ◽  
David S. Dandy ◽  
Brian Geiss ◽  
Charles Henry
Keyword(s):  
Food Safety ◽  
Nucleic Acid ◽  
Environmental Monitoring ◽  
Rolling Circle Amplification ◽  
Cost Effective ◽  
Clinical Diagnostics ◽  
Nucleic Acid Amplification ◽  
Nucleic Acid Detection ◽  
Padlock Probe ◽  
Rolling Circle

Sensitive, reliable and cost-effective detection of pathogens has wide ranging applications in clinical diagnostics and therapeutics, water and food safety, environmental monitoring, biosafety and epidemiology. Nucleic acid amplification tests (NAATs)...

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